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  • Articles  (163)
  • heart  (84)
  • calcium  (81)
  • Springer  (163)
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  • 1
    ISSN: 1436-6215
    Keywords: Strontium ; oraler Strontium-Test ; Calcium ; Absorption ; gesunde Probanden ; Strontium ; oral strontium test ; calcium ; absorption ; healthy volunteers
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Medicine
    Description / Table of Contents: Summary Intestinal strontium absorption has been discussed recently as an indirect measure for calcium uptake. Prerequisite for the clinical use of an oral strontium test is the availability of a reliable procedure including controlled strontium supply, sample pretreatment and analysis as well as the assessment of normal values. In the present study, a group of young females (n=33; 24.0 ± 2.7 y; BMI 21.5 ± 1.9) received an oral dose of 2.27 mmol strontium in a standardized breakfast that contained 0.625 mmol calcium. Before and 220 min after the bolus serum strontium concentrations were determined by means of atomic absorption spectrophotometry (coefficient of variation: within day 4.8 %, n=10; day-to-day 9.5 %, n=8). The error of the method was 2.7 %. Calculation of the fractional strontium absorption rate considered the respective distribution volume (extracellular fluid; either estimated using body weight or determined by means of bioimpedance analysis [BIA]). Average absorption rates were 13.3 ± 3.1 % and, considering BIA measurement 13.6 ± 2.6 %, respectively. Smoking, exercise and, use of oral contraceptives showed no effects. Our oral strontium test is characterized by excellent reliability, easy handling and low costs and, thus, is suitable for routine use.
    Notes: Zusammenfassung Die Erfassung der Strontiumabsorption wird heute als indirektes Verfahren zur Beurteilung der intestinalen Calciumabsorption diskutiert. Voraussetzung für die klinische Anwendung ist ein vertrauenswürdiges Testverfahren inclusive kontrollierter Strontiumgabe, Probenaufarbeitung und -analyse sowie die Erfassung von Normalwerten. Für unsere Studien wurde ein Kollektiv junger Frauen (n=33, 24,0 ± 2,7 Jahre; BMI 21,5 ± 1,9) herangezogen. Die Probandinnen erhielten eine Bolusgabe von 2,27 mmol Strontium zusammen mit einem Standardfrühstück (ca. 0,625 mmol Calcium). Vor und 220 min nach der Bolusgabe erfolgte die Bestimmung des Serum-Strontiumgehaltes mittels Atomabsorptionsspektrometrie. Der Variationskoeffizient der Methode lag innerhalb eines Tages bei 4,8 % (n=10) und von Tag zu Tag 9,5 % (n=8). Der Fehler der Methode betrug 2,7 %. Die Berechnung der fraktionellen Strontiumabsorptionsrate erfolgte unter Berücksichtigung des entsprechenden Verteilungsraumes (Extrazellulärflüssigkeit; Schätzverfahren über Körpergewicht bzw. Bioimpedanz-Analyse [BIA]). Die Strontiumabsorptionsrate lag im Mittel bei 13,3 ± 3,1 %, unter Berücksichtigung der BIA-Werte bei 13,6 ± 2,6 %. Rauchen, sportliche Aktivität bzw. Einnahme oraler Kontrazeptiva zeigten keinen Einfluß. Das hier vorgestellte Testverfahren ist aufgrund seiner hohen Vertrauenswürdigkeit und relativ einfacher Handhabung für Routine-untersuchungen geeignet.
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  • 2
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    Cellular and molecular life sciences 52 (1996), S. 583-586 
    ISSN: 1420-9071
    Keywords: Crustacea ; heart ; muscle ; oscillator ; tetrodotoxin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The heart beat of early juveniles of the littoral isopodLigia exotica occurred at a frequency of 250 to 350/min, associated with rhythmic activity of the heart muscle. Each burst was composed of a slow depolarizing potential with superimposed spike potentials. The spike potential was eliminated by perfusion with TTX-containing or Na+-free saline. In TTX-saline, the slow potential was unchanged in frequency and amplitude. By current injection into the heart muscle, the rhythm of the slow potential was phase-shifted and its frequency was changed in a membrane potential-dependent manner. These results show that the heart ofLigia early juveniles acts as an endogenous muscle oscillator generating oscillatory slow potentials and Na+-dependent spikes.
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  • 3
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    Cellular and molecular life sciences 52 (1996), S. 831-833 
    ISSN: 1420-9071
    Keywords: Paramecium caudatum ; thigmotaxis ; Ja-value ; CNR ; calcium ; ruthenium red ; LaCl3 ; caffeine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract To understand the role of calcium ions in thigmotaxis inParamecium caudatum, the effects of caffeine, ruthenium red and lanthanum (LaCl3) on thigmotaxis were examined. Thigmotaxis in the CNR mutant, which lacks voltage-dependent Ca2+-channels in the ciliary membrane, was also examined. Ruthenium red and LaCl3 suppressed thigmotaxis inP. caudatum, while caffeine enhanced it. The CNR mutant showed hardly any thigmotaxis. It can be thought that an increase in Ca2+ influx and the intraciliary concentration of Ca2+ ions induces thigmotaxis inParamecium.
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  • 4
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    European journal of clinical pharmacology 16 (1979), S. 211-215 
    ISSN: 1432-1041
    Keywords: fluoride ; bioavailability ; calcium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology , Medicine
    Notes: Summary The effect of milk products on the gastrointestinal absorption of fluoride from sodium fluoride tablets was studied in five healthy subjects. Two different diets were tested: (1) 250 ml standardized milk (3% fat) and (2) 500 ml of milk, 3 pieces of white bread with cheese and 150 ml of yoghurt. The 100% bioavailability of sodium fluoride tablets during fasting was greatly decreased by coadministration of milk products: with Diet 1 the absolute bioavailability calculated from combined plasma and urine data was in the range 50–79% and with Diet 2 it ranged from 50–71%. It is suggested that the decreased bioavailability produced by dairy products should be taken into account when establishing fluoride dosage regimens for prophylaxis of caries.
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  • 5
    ISSN: 1573-4919
    Keywords: regucalcin ; calcium-binding protein ; insulin ; calcium ; gene expression ; rat liver
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The effect of refeeding on the expression of Ca2+-binding protein regucalcin mRNA in the liver of fasted rats was investigated. When rats were fasted overnight, the hepatic regucalcin mRNA level was reduced about 70% of that in feeding rats. Refeeding produced a remarkable elevation of hepatic regucalcin mRNA level (about 150–170% of fasted rats). Liver regucalcin concentration was appreciably increased by refeeding, although it was not altered by fasting. The oral administration of glucose (2 g/kg body weight) to fasted rats caused a significant increase in hepatic regucalcin mRNA level. Moreover, hepatic regucalcin mRNA level was clearly elevated by a single subcutaneous administration of insulin (10 and 100 U/kg) to fasted rats. The hormonal effect was not further enhanced by the simultaneous administration of calcium chloride (250 mg Ca/kg) to fasted rats, although calcium administration stimulated regucalcin mRNA expression in the liver. The present study suggests that the expression of hepatic regucalcin mRNA stimulated by refeeding is significantly involved in the action of insulin and/or calcium as stimulating factors.
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  • 6
    ISSN: 1573-4919
    Keywords: calcium ; calcium channels ; smooth muscle ; sarcolemma ; coronary artery
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Tension generation and Ca2+ flux in smooth muscle varies depending upon the diameter of a vessel and its location. The purpose of the present investigation was to determine if the biochemical characteristics of the Na+−Ca2+ exchanger and the Ca2+ channel differ in sarcolemmal membrane preparations isolated from a large conduit vessel (thoracic aorta) or from large and small coronary arteries. We also investigated the possibility of differences between sarcolemmal membranes isolated from coronary arteries dissected from the right and left ventricles. The purification of the sarcolemmal membranes was of a similar magnitude amongst the different groups. Contamination of the sarcolemmal membranes with other membranous organelles was negligible and similar amongst the groups. The Km and Vmax of Na+-dependent Ca2+ uptake in sarcolemmal vesicles was similar amongst the groups. Calcium channel characteristics were examined by measuring [3H]PN200-110 binding to sarcolemmal vesicles. The right coronary artery membranes from both large and small caliber vessels exhibited a higher Kd and the small right coronary artery sarcolemmal preparation had a lower maximal binding density for [3H] PN200-110. The results suggest that the right coronary artery, and in particular the small diameter right coronary artery, possesses altered Ca2+ channel characteristics in isolated sarcolemmal membranes.
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  • 7
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    Molecular and cellular biochemistry 154 (1996), S. 123-132 
    ISSN: 1573-4919
    Keywords: rat pancreas ; cholecystokinin-octapeptide ; magnesium ; calcium ; secretion ; cyclic AMP
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract This study investigates the effect of magnesium (Mg2+) on the secretory responses and the mobilization of calcium (Ca2+) and Mg2+ evoked by cholecystokinin-octapeptide (CCK-8) in the exocrine rat pancreas. In the isolated intact perfused pancreas CCK-8 (10−10 M) produced marked increases in juice flow and total protein output in zero and normal (1.1 mM) extracellular Mg2+ [Mg2+]o compared to a much reduced secretory response in elevated (5 mM and 10 mM) [Mg2+]o Similar effects of perturbation of [Mg2+]o on amylase secretion and 45Ca2+ uptake (influx) were obtained in isolated pancreatic segments. In pancreatic acinar cells loaded with the fluorescent bioprobe fura-2 acetomethylester (AM), CCK-8 evoked marked increases in cytosolic free Ca2+ concentration [Ca2+]i in zero and normal [Mg2+]o compared to a much reduced response in elevated [Mg2+]o Pretreatment of acinar cells with either dibutyryl cyclic AMP (DB2 cAMP) or forskolin had no effect on the CCK-8 induced changes in [Ca2+]i. In magfura-2-loaded acinar cells CCK-8 (10−8 M) stimulated an initial transient rise in intracellular free Mg2+ concentration [Mg2+]i followed by a more prolonged and sustained decrease. This response was abolished when sodium Na+ was replaced with N-methyl-D-glucamine (NMDG). Incubation of acinar cells with 10 mM Mg2+ resulted in an elevation in [Mg2+]i. Upon stimulation with CCK-8, [Mg2+]i. decreased only slightly compared with the response obtained in normal [Mg2+]o. CCK-8 caused a net efflux of Mg2+ in pancreatic segments; this effect was abolished when extracellular sodium [Na+]o was replaced with either NMDG or choline. The results indicate that Mg2+ can regulate CCK-8-evoked secretory responses in the exocrine pancreas possibly via Ca2+ mobilization. Moreover, the movement of Mg2+ in pancreatic acinar cells is dependent upon extracellular Na+.
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  • 8
    ISSN: 1573-4919
    Keywords: nitric oxide ; carbon monoxide ; ischemia ; heart ; intracellular signaling ; cGMP ; SOD
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract To examine the intracellular signaling mechanism of NO in ischemic myocardium, isolated working rat hearts were made ischemic for 30 min followed by 30 min of reperfusion. A separate group of hearts were pre-perfused with 3 mM L-arginine in the presence or absence of 650 μM of protoporphyrin, a heme oxygenase inhibitor for 10 min prior to ischemia. The release of NO was monitored using an on-line amperometric sensor placed into the right atrium. The aortic flow and developed pressure were examined to determine the effects of L-arginine on ischemic/reperfusion injury. Induction for the expression of heme oxygenase was studied by Northern hybridization. For signal transduction experiments, sarcolemmal membranes were radiolabeled by perfusing the isolated hearts with [3H] myoinositol and [14C] arachidonic acid. Biopsies were processed to determine the isotopic incorporation into various phosphoinositols as well as phosphatidic acid and diacylglycerol. cGMP was assayed by radioimmunoassay and SOD content was determined by enzymatic analysis. The release of NO was diminished following ischemia and reperfusion and was augmented by L-arginine. L-arginine reduced ischemic/reperfusion injury as evidenced by the enhanced myocardial functional recovery. Protoporphyrin modulated the effects of L-arginine. cGMP, which was remained unaffected by ischemia and reperfusion, was stimulated significantly after L-arginine treatment. The NO-mediated augmentation of cGMP was reduced by protoporphyrin suggesting that part of the effects may be mediated by CO generated through the heme oxygenase pathway. Reperfusion of ischemic myocardium resulted in significant accumulation of radiolabeled inositol phosphate, inositol bisphosphate, and inositol triphosphate. Isotopic incorporation of [3H] inositol into phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate was increased significantly during reperfusion. Reperfusion of the ischemic heart prelabeled with [14C] arachidonic acid resulted in modest increases in [14C] diacylglycerol and [14C] phosphatidic acid. Pretreatment of the heart with L-arginine significantly reversed this enhanced phosphodiesteratic breakdown during ischemia and early reperfusion. However, at the end of the reperfision the inhibitory effect of L-arginine on the phosphodiesterases seems to be reduced. In L-arginine treated hearts, SOD activity was progressively decreased with the duration of reperfusion time. The results suggests for the first time that NO plays a significant role in transmembrane signaling in the ischemic myocardium. This signaling appears to be on- and off- nature, and linked with SOD content of the tissue. The signaling is transmitted via cGMP and opposes the effects of phosphodiesterases by inhibiting the ischemia/reperfusion-induced phosphodiesteratic breakdown. Our results also suggest that NO activates heme oxygenase which further stimulates the production of cGMP presumably by CO signaling. Thus, NO not only potentiates cGMP mediated intracellular signaling, it also functions as a retrograde messenger for CO signaling in heart.
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  • 9
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    Molecular and cellular biochemistry 157 (1996), S. 211-216 
    ISSN: 1573-4919
    Keywords: renin ; angiotensin ; local renin-angiotensin system ; heart ; vessel wall ; angiotensin-converting enzyme
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The existence of a local cardiovascular renin-angiotensin system (RAS) is often invoked to explain the long-term beneficial effects of RAS inhibitors in heart failure and hypertension. The implicit assumption is that all components of the RAS are synthesized in situ, so that local angiotensin II formation may occur independently of the circulating RAS. Evidence for this assumption however is lacking. The angiotensin release from isolated perfused rat hearts or hindlimbs depends on the presence of renal renin. When calculating the in vivo angiotensin production at tissue sites in humans and pigs, taking into account the extensive regional angiotensin clearance by infusing radiolabeled angiotensin I or II, it was found that angiotensin production correlated closely with plasma renin activity. Moreover, in pigs the cardiac tissue levels of renin and angiotensin were directly correlated with their respective plasma levels, and both in tissue and plasma the levels were undetectably low after nephrectomy. Similarly, rat vascular renin and angiotensin decrease to low or undetectable levels within 48 h after nephrectomy. Aortic renin has a longer half life than plasma renin, suggesting that renin may be bound by the vessel wall. In support of this assumption, both renin receptors and renin-binding proteins have been described. Like ACE, renin was enriched in a purified membrane fraction prepared from cardiac tissue. Binding of renin to cardiac or vascular membranes may therefore be part of a mechanism by which renin is taken up from plasma. It appears that the concept of a local RAS needs to be reassessed. Local angiotensin formation in heart and vessel wall does occur, but depends, at least under normal circumstances, on the uptake of renal renin from the circulation. Tissues may regulate their local angiotensin concentrations by varying the number of renin receptors and/or renin-binding proteins, the ACE level, the amount of metabolizing enzymes and the angiotensin receptor density.
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  • 10
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    Molecular and cellular biochemistry 146 (1995), S. 179-186 
    ISSN: 1573-4919
    Keywords: Ca2+-ATPase ; calcium ; nuclear DNA ; DNA fragmentation ; regucalcin ; regenerating rat liver
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The alteration of calcium content, Ca2+-ATPase activity, DNA content and DNA fragmentation in the nuclei of regenerating rat liver was investigated. Liver was surgically removed about 70% of that of sham-operated rats. the reduced liver weight by partial hepatectomy was completely restored at 3 days after the surgery. Regenerating liver significantly increased Ca2+-ATPase activity and DNA content in the nuclei between 1 and 5 days after hepatectomy. The nuclear calcium content was clearly increased from 2 days after hepatectomy. The increase of Ca2+-ATPase activity in regenerating liver was clearly inhibited by the presence of trifluoperazine (10 μM), staurosporine (2.5 μM) and dibucaine (10 μM), which are inhibitors of calmodulin and protein kinase, in the enzyme reaction mixture. However, the nuclear enzyme activity in normal rat liver was not significantly altered by these inhibitors. Meanwhile, the increase of nuclear DNA content in regenerating liver was completely blocked by the administration of trifluoperazine (2.5 mg/100 g body weight), suggesting an involvement of calmodulin. Now, the nuclear DNA fragmentation was significantly decreased in regenerating liver, suggesting that this decrease is partly contributed to the increase in nuclear DNA content. The present study clearly demonstrates that regenerating liver enhances nuclear Ca2+-ATPase activity and induces a corresponding elevation of nuclear calcium content. This Ca2+-signaling system may be involved in the regulation of nuclear DNA functions in regenerating rat liver.
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  • 11
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    Molecular and cellular biochemistry 163-164 (1996), S. 145-149 
    ISSN: 1573-4919
    Keywords: heart ; glycogen ; adenylyl cyclase ; catecholamine heart failure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The role of glycogen content in the heart for the development of isoprenaline-induced myocardial lesions (IML) was studied in Wistar rats and in two inbred rat strains: In IR rats (resistant to the development of IML) and in IS rats (sensitive to IML development). Glycogen content in the heart can be dramatically lowered or increased by various interventions. IML develop during the period of very low heart glycogen content (about 0.6 mg.g−1) induced by isoprenaline administration. In animals with increased resistance to IML, either due to genetic factors or induced by isoprenaline pretreatment a high glycogen content in the heart is found (up to 7.5 mg.g−1). The increase of resistance to IML development and increased glycogen content induced by isoprenaline pretreatment were accompanied by lower basal or ISO-, guanylylimidodiphosphate- (Gpp/NH/p) and forskolin-stimulated activities of adenylyl cyclase. On the other hand, these parameters did not differ between IR and IS rats in spite of the presence of significant differences in the resistance to the development of IML and in heart glycogen content in these two rats strains. These results suggest that genetically determined differences between two inbred rat strains in the resistance of the heart to the development of IML and in the heart glycogen content are caused by factors which are independent of the receptor-adenylyl cyclase complex and are therefore different from those involved in the increase of resistance and glycogen content due to isoprenaline pretreatment.
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  • 12
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    Molecular and cellular biochemistry 148 (1995), S. 67-72 
    ISSN: 1573-4919
    Keywords: regucalcin ; calcium ; protease ; calpain ; rat liver cytosol
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The increasing effect of regucalcin, isolated from rat liver cytosol, on neutral proteolytic activity in the hepatic cytosol was characterized. The proteolytic activity was markedly elevated by the addition of regucalcin (0.1–0.5 μM) in the absence of Ca2+. This increase was not significantly altered by the presence of diisopropylfluorophsophate (DPF;2.5 mM)—although DFP caused a significant decrease in the proteolytic activity. Regucalcin (0.25 μM) additively enhanced the dithiothreitol (DTT; 1.0 mM)—increased proteolytic activity, while the regucalcin or DTT effect was completely abolished by NEM (5 mM), indicating that regucalcin may act on the SH group in proteases. Also, regucalcin (0.25 μM) enhanced the effect of Ca2+ (10 μM) increasing liver proteolytic activity, suggesting that regucalcin does not influence on the active sites for Ca2+ in proteases. Moreover, the proteolytic activity of regucalcin (0.25 μM) was significantly decreased by the presence of calpastatin (24 μg/ml), an inhibitor of Ca2+-activated neutral protease (calpain). Now, regucalcin (0.25 μM) increased about 7-fold the activity ofm-calpain isolated from rabbit skeletal muscle. These observations demonstrate that regucalcin directly activates cysteinyl-proteases. Regucalcin may have a role as a potent proteolytic activator in the cytoplasm of liver cells.
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  • 13
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    Molecular and cellular biochemistry 148 (1995), S. 89-94 
    ISSN: 1573-4919
    Keywords: ICa(L) ; cyclic nucleotides ; PK-A ; PK-G ; isoproterenol ; embryo ; chick ; heart
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract In order to assess the interaction between the cAMP-dependent and the cGMP-dependent phosphorylation pathways on the slow Ca2+ current (ICa(L)), whole-cell voltage-clamp experiments were conducted on embryonic chick heart cells. Addition of 8Br-cGMP to the bath solution reduced the basal (unstimulated) ICa(L). Intracellular application of the catalytic subunit of PK-A (PK-A(cat); 1.5 μM) via the patch pipette rapidly potentiated ICa(L) by 215±16% (n=4); subsequent addition of 1 mM 8Br-cGMP to the bath reduced the amplitude of ICa(L) towards the initial control values (123±29%). Intracellular application of PK-G (25 nM pre-activated by 10−7 M cGMP), rapidly inhibited the basal ICa(L) by 64±6% (n=8). Heat-denatured PK-G was ineffective. Subsequent additions of relatively high concentrations of 8Br-cAMP (1 mM) or isoproterenol (ISO, 1–10 μM) did not significantly remove the PK-G blockade of ICa(L). The results of the present study suggest that: (a) 8Br-cGMP can inhibit the basal or stimulated (by PK-A(cat)) ICa(L) in embryonic chick myocardial cells. (b) PK-G applied intracellularly inhibits the basal ICa(L).
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  • 14
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    Molecular and cellular biochemistry 149-150 (1995), S. 71-75 
    ISSN: 1573-4919
    Keywords: pyruvate ; pyruvate dehydrogenase kinase ; heart
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Sensitivity of rat heart pyruvate dehydrogenase kinase (PDHK) to pyruvate inhibition was tested under various conditions using pyruvate dehydrogenase complex (PDC) in mitochondria (mPDC) and in a high speed precipitate of whole tissue homogenates (hPDC). In the latter preparation pyruvate in the range of concentration 1–10 mM caused increasing inhibition of PDHK when the enzyme was prepared from animals fedad libitum but had no effect when the enzyme was prepared from 48 h starved animals. Similar behaviour was observed inmPDC from fed and starved animals when rotenone was present in the incubation medium. When carbonyl cyanide m-chlorophenylhydrazone (CCCP) instead of rotenone was present, pyruvate at 1 mM concentration stimulated PDHK from hearts of fed animals but was without effect at 10 mM. WhenmPDC orhPDC from hearts of starved animals was incubated at 30°C for 30 min, inhibition of PDHK by pyruvate was restored.
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  • 15
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    Molecular and cellular biochemistry 149-150 (1995), S. 203-212 
    ISSN: 1573-4919
    Keywords: calcium ; mitochondria ; FAD-glycerol 3-phosphate dehydrogenase ; pyruvate dehydrogenase ; oxoglutarate dehydrogenase ; isocitrate dehydrogenase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract In mammalian cells, increases in calcium concentration cause increases in oxidative phosphorylation. This effect is mediated by the activation of four mitochondrial dehydrogenases by calcium ions; FAD-glycerol 3-phosphate dehydrogenase, pyruvate dehydrogenase, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase. FAD-glycerol 3-phosphate dehydrogenase, being located on the outer surface of the inner mitochondrial membrane, is exposed to fluctuations in cytoplasmic calcium concentration. The other three enzymes are located within the mitochondrial matrix. While the kinetic properties of all of these enzymes are well characterised, the molecular basis for their regulation by calcium is not. This review uses information derived from calcium binding studies, analysis of conserved calcium binding motifs and comparison of amino acid sequences from calcium sensitive and non-sensitive enzymes to discuss how the recent cloning of several subunits from the four dehydrogenases enhances our understanding of the ways in which these enzymes bind calcium. FAD-glycerol 3-phosphate dehydrogenase binds calcium ions through a domain which is part of the polypeptide chain of the enzyme. In contrast, it is possible that the calcium sensitivity of the other dehydrogenases may involve separate calcium binding subunits.
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  • 16
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    Molecular and cellular biochemistry 157 (1996), S. 163-170 
    ISSN: 1573-4919
    Keywords: G-proteins ; adenylyl cyclase ; heart ; aorta ; spontaneously hypertensive rats
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The present studies were undertaken to examine if adenylyl cyclase activity and the levels of G-proteins (Gsα and Giα) are altered in cardiovascular tissues in hypertension. Adenylyl cyclase activity and its responsiveness to stimulatory and inhibitory hormones as well as the expression of G-proteins (Gs and Gi) were determined at protein and mRNA levels by using specific antibodies and cDNA probes in hearts and aorta from 12 week old spontaneously hypertensive rats (SHR) and their age-matched control Wistar Kyoto (WKY) rats. The stimulatory effects of guanine nucleotides, isoproterenol, glucagon etc. on adenylyl cyclase activity were decreased in SHR rats as compared to the WKY rats, whereas, the inhibitory hormones inhibited enzyme activity to a grater extent in SHR rats as compared to WKY rats. Furthermore, the levels of Giα-2 and Giα-3 proteins and Giα-2 and Giα-3 mRNA as determined by immunoblotting and Northern blotting techniques respectively were higher in SHR as compared to WKY rats. However, the levels of Gsa were unaltered in SHR. To further investigate if these alterations are the cause or effect of hypertension, the SHRs at various ages of the development of blood pressure (3–5 days, 2, 4 and 8 weeks) and their age-matched WKY were used for G-protein expression and adenylyl cyclase activity. The increased expression of Giα−2 and Giα−3 protein and mRNA levels in hearts and aorta were observed as early as in 2-weeks old SHR as compared to WKY, when the blood pressure was still normal. However, the levels of Gsα in SHR were not different from WKY rats. In addition, the altered responsiveness of adenylyl cyclase to hormone stimulation and inhibition was also observed as early as in 2 week old SHR. These results suggest that the increased expression of Giα−2 and Giα−3 and decreased levels of cAMP precedes the development of blood pressure and may be one of the contributing factors in the pathogenesis of hypertension.
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  • 17
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    Molecular and cellular biochemistry 158 (1979), S. 91-95 
    ISSN: 1573-4919
    Keywords: smooth muscle ; vascular ; ATPase ; calcium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Densities of sarcoplasmic reticulum (SR) Ca2+-pump were compared in proximal and distal segments of pig left coronary artery using two biochemical methods: acylphosphate formation and immunoreactivity in Western blots, and a functional assay based on contraction to SR Ca2+-pump inhibitors. In the microsomes prepared from smooth muscle, the level of the 115 kDa SR Ca2+-pump acylphosphate was 7.1 ± 0.3 -fold greater in distal than in proximal segments. Similarly in Western blots using these microsomes, the reactivity of the 115 kDa band to an anti-SR Ca2+-pump antibody was 5.3 ± 0.8 -fold greater in distal than in proximal segments. Endothelium free coronary artery rings contracted to the SR Ca2+-pump inhibitors Cyclopiazonic acid (CPA, EC50 = 0.19 ± 0.06 μM) and thapsigargin (EC50 = 0.0095 ± 0.0035 μM). With 10 μM CPA, the force of contraction per tissue wet weight was 4.2 ± 0.5-fold greater in distal than in proximal rings, and with 1 μM thapsigargin it was 4.0 ± 1.0 -fold greater. The contractions produced by 60 mM KCl were used as a control. In contrast to the CPA and thapsigargin, the force per mg tissue weight produced by 60 mM KCl did not differ significantly between the proximal and distal segments. Thus, the results from the two biochemical methods and those from the contractility data were all consistent with the smooth muscle in the distal segments of the coronary artery containing a higher density of the SR Ca2+-pump than the proximal segments.
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  • 18
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    Molecular and cellular biochemistry 159 (1996), S. 7-14 
    ISSN: 1573-4919
    Keywords: coronary circulation ; heart ; hydrogen peroxide ; nitric oxide ; oxidative stress ; vasodilation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Oxidative stress mediated by hydrogen peroxide (H2O2) increases coronary flow (CF) in Langendorff-perfused rat hearts. We investigated the possible role of nitric oxide (NO) in H2O2-induced vasolidation. A dose-response study was conducted to find a concentration of H2O2 which increased CF without influencing left ventricular developed (LVDP) or end-diastolic (LVEDP) pressures. 80 (n = 10),100 (n = 7), 120 (n = 7),140 (n = 7),160 (n = 7), and 180 (n = 10) μM H2O2 was infused for 10 min, followed by recovery for 50 min. 80 μM H2O2 increased CF to a maximum of 143 ± 4 (mean ± S.E.M) percent of initial value after 15 min observation (p 〈 0.001 compared to buffer only), with no effect on LVDP or LVEDP. Another series of hearts were perfused with N-nitro-L-Arginine methylester (L-NAME, 1 μM), methylene blue (MB, 50 μM), or haemoglobin (Hb, 10 μM), without (n = 7 in each) or with (n = 10 in each) 80 μM H2O2 for 10 min. L-NAME, MB, and Hb alone increased CF, but attenuated the H2O2-induced increase of CF. LVDP was depressed when L-NAME, MB, or Hb were given in conjunction with 80 μM H2O2. In summary, H2O2 concentration-dependently increased LVEDP and depressed LVDP. The H2O2-induced increase of CF was independent of concentration. Inhibition of NO synthesis, action, or soluble guanylate cyclase attenuated the H2O2-induced increase of CF, and depressed LVDP when given together with H2O2. H2O2 induces a NO-dependent vasodilation, and inhibition of NO is detrimental to left ventricular function after H2O2-mediated oxidative stress.
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  • 19
    ISSN: 1573-4919
    Keywords: stress ; heart ; dietary fatty acids ; blood pressure ; rats ; docosahexaenoic acid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Epidemiological studies suggest that n-3 polyunsaturated fatty acids (PUFA) are involved in the prevention of cardiovascular disease. Stress is known to increase the incidence of CVD and the present study was realised to evaluate some physiological and biochemical effects of dietary docosahexaenoic acid (DHA) in male Wistar rats subjected to a psycho social stress. Rats were fed for 8 weeks a semi-purified diet containing 10% of either sunflower seed oil or the same oil supplemented with DHA. This food supply represented 50% of their daily requirement. The remaining 50% were supplied as 45 mg food pellets designed to induce stress in rats by an intermittent-feeding schedule process. The control group (n = 12) was fed the equivalent food ration as a single daily feeding. The physiological cardiovascular parameters were recorded by telemetry through a transmitter introduced in the abdomen. At the end of the experimentation, the heart and adrenals were withdrawn and the fatty acid composition and the catecholamine store were determined. Dietary DHA induced a pronounced alteration of the fatty acid profile of cardiac phospholipids (PL). The level of all the n-6 PUFAs was reduced while 22:6 n-3 was increased. The stress induced a significant increase in heart rate which was not observed in DHA-fed group. The time evolution of the systolic blood pressure was not affected by the stress and was roughly similar in the stressed rats of either dietary group. Conversely, the systolic blood pressure decreased in the unstressed rats fed DHA. Similar data were obtained for the diastolic blood pressure. The beneficial effect of DHA was also observed on cardiac contractility, since the dP/dtmax increase was prevented in the DHA-fed rats. The stress-induced modifications were associated with an increase in cardiac noradrenaline level which was not observed in DHA-fed rats. The fatty acid composition of adrenals was significantly related to the fatty acid intake particularly the neutral lipid fraction (NL) which incorporated a large amount of DHA. Conversely, n-3 PUFAs were poorly incorporated in adrenal phospholipids. Moreover the NL/PL ratio was significantly increased in the DHA fed rats. The amount of adrenal catecholamines did not differ significantly between the groups. These results show that a supplementation of the diet with DHA induced cardiovascular alterations which could be detected in conscious animals within a few weeks. These alterations were elicited by a reduced heart rate and systolic and diastolic blood pressure.
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  • 20
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    Molecular and cellular biochemistry 179 (1998), S. 99-110 
    ISSN: 1573-4919
    Keywords: diaphragm ; oxygen-derived free radicals ; respiratory muscle fatigue ; nitric oxide ; sarcoplasmic reticulum ; calcium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract It is now recognized that respiratory muscle fatigue contributes to the development of respiratory failure in some patients with lung disease. This observation has prompted an examination into the mechanisms of development of muscle fatigue, with the understanding that an elucidation of these processes may lead to new therapeutic approaches to the treatment of these patients. A series of recent studies examining this issue have, moreover, discovered that oxygen-derived free radicals generated during strenuous contraction may modulate respiratory muscle contractile function and contribute to the development of muscle fatigue. The data supporting this concept include: (a) direct (e.g. EPR, ESR studies) and indirect (evidence of lipid peroxidation, protein carbonyl formation, glutathione oxidation) evidence that there is heightened free radical production in contracting muscle, (b) evidence that pharmacologic depletion of muscle antioxidant stores increases degree of muscle fatigue present after a period of exercise, and (c) evidence that administration of agents that act as free radical scavengers retard the development muscle fatigue. Free radicals may produce these changes in muscle force generating capacity by interacting with and altering the function of a number of intracellular-biophysical processes (i.e. sarcolemmal action potential propagation, sarcoplasmic reticulum calcium handling, mitochondrial function, contractile protein interactions).
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  • 21
    ISSN: 1573-4919
    Keywords: creatine kinase ; transgenic mice ; energy transfer ; mathematical model ; compartmentation ; heart ; energy metabolism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract A mathematical model of the compartmentalized energy transfer in cardiac cells is described and used for interpretation of novel experimental data obtained by using phosphorus NMR for determination of the energy fluxes in the isolated hearts of transgenic mice with knocked out creatine kinase isoenzymes. These experiments were designed to study the meaning and importance of compartmentation of creatine kinase isoenzymes in the cells in vivo. The model was constructed to describe quantitatively the processes of energy production, transfer, utilization, and feedback between these processes. It describes the production of ATP in mitochondrial matrix space by ATP synthase, use of this ATP for phosphocreatine production in the mitochondrial creatine kinase reaction coupled to the adenine nucleotide translocation, diffusional exchange of metabolites in the cytoplasmic space, and use of phosphocreatine for resynthesis of ATP in the myoplasmic creatine kinase reaction. It accounts also for the recently discovered phenomenon of restricted diffusion of adenine nucleotides through mitochondrial outer membrane porin pores (VDAC). Practically all parameters of the model were determined experimentally. The analysis of energy fluxes between different cellular compartments shows that in all cellular compartments of working heart cells the creatine kinase reaction is far from equilibrium in the systolic phase of the contraction cycle and approaches equilibrium only in cytoplasm and only in the end-diastolic phase of the contraction cycle. Experimental determination of the relationship between energy fluxes by a 31P-NMR saturation transfer method and workload in isolated and perfused heart of transgenic mice deficient in MM isoenzyme of the creatine kinase, MM -/- showed that in the hearts from wild mice, containing all creatine kinase isoenzymes, the energy fluxes determined increased 3-4 times with elevation of the workload. By contrast, in the hearts in which only the mitochondrial creatine kinase was active, the energy fluxes became practically independent of the workload in spite of the preservation of 26% of normal creatine kinase activity. These results cannot be explained on the basis of the conventional near-equilibrium theory of creatine kinase in the cells, which excludes any difference between creatine kinase isoenzymes. However, these apparently paradoxical experimental results are quantitatively described by a mathematical model of the compartmentalized energy transfer based on the steady state kinetics of coupled creatine kinase reactions, compartmentation of creatine kinase isoenzymes in the cells, and the kinetics of ATP production and utilization reactions. The use of this model shows that: (1) in the wild type heart cells a major part of energy is transported out of mitochondria via phosphocreatine, which is used for complete regeneration of ATP locally in the myofibrils - this is the quantitative estimate for PCr pathway; (2) however, in the absence of MM-creatine kinase in the myofibrils in transgenic mice the contraction results in a very rapid rise of ADP in cytoplasmic space, that reverses the mitochondrial creatine kinase reaction in the direction of ATP production. In this way, because of increasing concentrations of cytoplasmic ADP, mitochondrial creatine kinase is switched off functionally due to the absence of its counterpart in PCr pathway, MM-creatine kinase. This may explain why the creatine kinase flux becomes practically independent from the workload in the hearts of transgenic mouse without MM-CK. Thus, the analysis of the results of studies of hearts of creatine kinase-deficient transgenic mice, based on the use of a mathematical model of compartmentalized energy transfer, show that in the PCr pathway of intracellular energy transport two isoenzymes of creatine kinase always function in a coordinated manner out of equilibrium, in the steady state, and disturbances in functioning of one of them inevitably result in the disturbances of the other component of the PCr pathway. In the latter case, energy is transferred from mitochondria to myofibrils by alternative metabolic pathways, probably involving adenylate kinase or other systems.
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  • 22
    ISSN: 1573-4919
    Keywords: heart ; oxidative phosphorylation ; dynamic responses ; metabolic wave ; creatine kinase ; compartmentation ; mitochondria ; adenine nucleotides ; oxygen consumption ; NMR stunning
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Although usually steady-state fluxes and metabolite levels are assessed for the study of metabolic regulation, much can be learned from studying the transient response during quick changes of an input to the system. To this end we study the transient response of O2 consumption in the heart during steps in heart rate. The time course is characterized by the mean response time of O2 consumption which is the first statistical moment of the impulse response function of the system (for mono-exponential responses equal to the time constant). The time course of O2 uptake during quick changes is measured with O2 electrodes in the arterial perfusate and venous effluent of the heart, but the venous signal is delayed with respect to O2 consumption in the mitochondria due to O2 diffusion and vascular transport. We correct for this transport delay by using the mass balance of O2, with all terms (e.g. O2 consumption and vascular O2 transport) taken as function of time. Integration of this mass balance over the duration of the response yields a relation between the mean transit time for O2 and changes in cardiac O2 content. Experimental data on the response times of venous [O2] during step changes in arterial [O2] or in perfusion flow are used to calculate the transport time between mitochondria and the venous O2 electrode. By subtracting the transport time from the response time measured in the venous outflow the mean response time of mitochondrial O2 consumption (tmito) to the step in heart rate is obtained. In isolated rabbit heart we found that tmito to heart rate steps is 4-12 s at 37°C. This means that oxidative phosphorylation responds to changing ATP hydrolysis with some delay, so that the phosphocreatine levels in the heart must be decreased, at least in the early stages after an increase in cardiac ATP hydrolysis. Changes in ADP and inorganic phosphate (Pi) thus play a role in regulating the dynamic adaptation of oxidative phosphorylation, although most steady state NMR measurements in the heart had suggested that ADP and Pi do not change. Indeed, we found with 31P-NMR spectroscopy that phosphocreatine (PCr) and Pi change in the first seconds after a quick change in ATP hydrolysis, but remarkably they do this significantly faster (time constant ~2.5 s) than mitochondrial O2 consumption (time constant 12 s). Although it is quite likely that other factors besides ADP and Pi regulate cardiac oxidative phosphorylation, a fascinating alternative explanation is that the first changes in PCr measured with NMR spectroscopy took exclusively place in or near the myofibrils, and that a metabolic wave must then travel with some delay to the mitochondria to stimulate oxidative phosphorylation. The tmito slows with falling temperature, intracellular acidosis, and sometimes also during reperfusion following ischemia and with decreased mitochondrial aerobic capacity. In conclusion, the study of the dynamic adaptation of cardiac oxidative phosphorylation to demand using the mean response time of cardiac mitochondrial O2 consumption is a very valuable tool to investigate the regulation of cardiac mitochondrial energy metabolism in health and disease.
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  • 23
    ISSN: 1573-4919
    Keywords: heart ; heat stress ; ischemia ; reperfusion ; hemodynamics ; creatine kinase ; phospholipids ; arachidonic acid ; fattt acids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Heat stress pretreatment of the heart is known to protect this organ against an ischemic/reperfusion insult 24 h later. Degradation of membrane phospholipids resulting in tissue accumulation of polyunsaturated fatty acids, such as arachidonic acid, is thought to play an important role in the multifactorial process of ischemia/reperfusion-induced damage. The present study was conducted to test the hypothesis that heat stress mitigates the postischemic accumulation of arachidonic acid in myocardial tissue, as a sign of enhanced membrane phospholipid degradation. The experiments were performed on hearts isolated from rats either 24 h after total body heat treatment (42°C for 15 min) or 24 h after sham treatment (control). Hearts were made ischemic for 45 min and reperfused for another 45 min. Heat pretreatment resulted in a significant improvement of postischemic hemodynamic performance of the isolated rat hearts. The release of creatine kinase was reduced from 30 ± 14 (control group) to 17 ± 5 units/g wet wt per 45 min (heat-pretreated group) (p 〈 0.05). Moreover, the tissue content of the inducible heat stress protein HSP70 was found to be increased 3-fold 24 h after heat treatment. Preischemic tissue levels of arachidonic acid did not differ between heat-pretreated and control hearts. The postischemic ventricular content of arachidonic acid was found to be significantly reduced in heat-pretreated hearts compared to sham-treated controls (6.6 ± 3.3. vs. 17.8 ± 12.0 nmol/g wet wt). The findings suggest that mitigation of membrane phospholipid degradation is a potential mechanism of heat stress-mediated protection against the deleterious effects of ischemia and reperfusion on cardiac cells.
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  • 24
    ISSN: 1573-4919
    Keywords: heart cells ; taurine ; β-alanine ; taurine-Na+ cotransport ; CBDMB ; Na+-Ca2+ exchanger ; calcium ; nucleus ; confocal
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Recent studies in heart cells have shown taurine to induce a sustained increase of both intracellular Ca2+ and Na+. These results led us to believe that the increase in Na+ by taurine could be due to Na+ entry through the taurine-Na+ cotransporter which in turn favours transarcolemmal Ca2+ influx through Na+-Ca2+ exchange. Therefore, we investigated the effect of β-alanine, a blocker of the taurine-Na+ cotransporter and low concentrations of CBDMB (a pyrazine derivative, 5-(N-4chlorobenzyl)-2′,4′-dimethylbenzamil), a Na+-Ca2+ exchanger blocker on taurine-induced [Ca]i increase in embryonic chick heart cells. Using Fura-2 Ca2+ imaging and Fluo-3 Ca2+ confocal microscopy techniques, taurine (20 mM) as expected, induced a sustained increase in [Ca]i at both the cytosolic and the nuclear levels. Preexposure to 500 μM of the blocker of the taurine-Na+ cotransporter, β-alanine, prevented the amino acid-induced increase of total [Ca]i. On the other hand, application of β-alanine did not reverse the action of taurine on total [Ca]i. However, low concentrations of the Na+-Ca2+ exchanger blocker, CBDMB, reversed the taurine-induced sustained increase of cytosolic and nuclear free calcium (in presence or absence of β-alanine). Thus, the effect of taurine on [Ca]i in heart cells appears to be due to Na+ entry through the taurine-Na+ cotransporter which in turn favours transarcolemmal Ca2+ influx through the Na+-Ca2+ exchanger.
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  • 25
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    Molecular and cellular biochemistry 190 (1999), S. 185-190 
    ISSN: 1573-4919
    Keywords: calcium ; calcium wave ; calcium oscillation ; inositol 1,4,5-trisphosphate receptor ; ryanodine receptor ; excitation-concentration coupling
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract After the seminal work of Ebashi and coworkers which established the essential role of the intracellular Ca2+ concentration ([Ca2+]i) in the regulation of skeletal muscle contraction, we have witnessed an explosive elongation of the list of cell functions that are controlled by the [Ca2+]i. In numerous instances, release of intracellular Ca2+ stores plays important roles in Ca2+ signalling which displays significant variation in spatio-temporal pattern. There are two families of Ca2+ release channels, ryanodine receptors and inositol 1,4,5-trisphosphate (IP3) receptors. These Ca2+ release channels are structurally and functionally similar. In particular, the activity of both types of channels is regulated by the [Ca2+]i. The [Ca2+]i dependence of the Ca2+ release channel activity provides both types of channels with properties of a Ca2+ signal amplifier. This function of the ryanodine receptor is important in striated muscle excitation-contraction coupling, whereas that of the IP3 receptor seems to be the basis of the generation of Ca2+ waves. Thus the wide variety of Ca2+ signalling patterns seem to be critically dependent on the [Ca2+]i dependence of the Ca2+ release channels.
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  • 26
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    Molecular and cellular biochemistry 190 (1999), S. 39-45 
    ISSN: 1573-4919
    Keywords: microcalorimetry ; calcium ; troponin C ; calmodulin ; parvalbumin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Results of microcalorimetric titrations of calcium-binding proteins with calcium or magnesium have been reviewed and evaluated. Results were analyzed mostly in terms of heat capacity changes, which is most closely related to the structural changes of the molecule on metal binding. Two high-affinity sites of rabbit skeletal troponin C are distinguishable in terms of their affinity to calcium and associated enthalpy changes. Heat capacity changes on calcium binding to one of the two high-affinity sites is negative and is in the range ascribed to the ligand binding. In contrast, that to the other of the high-affinity sites is large and positive, indicating that a substantial area of hydrophobic groups become exposed to the solvent. In frog skeletal troponin C, the anomalous positive heat capacity changes occur in one of the low-affinity calcium-specific sites, so that this may be involved in the regulation of contraction. Unlike skeletal troponin C, both of the two high-affinity sites of cardiac troponin C show negative heat capacity changes. In calmodulin, heat capacity changes are positive but small, indicating that calcium binding may induce clustering of the hydrophobic residues on the surface of the molecule. In parvalbumins, heat capacity changes are negative, characteristic of most ligand binding.
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  • 27
    ISSN: 1573-4919
    Keywords: amiodarone ; arrhymias ; mitochondrial oxidative phosphorylation ; rat ; heart
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract This study was carried out in order to determine if the efficiency of amiodarone, a class III antiarrhythmic agent, is associated with changes in mitochondrial oxidative phosphorylation. A population of 30 rats were treated with amiodarone (100 mg/kg/day) for 5 days. A second population receiving only vehicle was used as control. The hearts were perfused according to the working mode. After 15 min of normoxic perfusion, the left main coronary artery was ligated and the ligation was maintained for 20 min. The ligation was removed and reperfusion continued for a further 30 min. The electrocardiogram was monitored continuously. At the end of perfusion, the ischemic and non ischemic areas were visually separated and mitochondria were harvested from each area. Their oxidative and energy metabolism were assessed with palmitoylcarnitine as substrate in 2 respiration media differing in their free calcium concentration (0 or 0.34 μm). In normoxic conditions, amiodarone treatment increased the cardiac metabolic efficiency (mechanical work to oxygen consumption ratio). The local ischemia decreased the aortic and coronary flows without modifying the cardiac metabolic efficiency. Amiodarone treatment maintained the aortic flow at a significantly higher value; the duration of severe arrhythmias was significantly decreased by the drug. The reperfusion of the ischemic area allowed the partial recovery of fluid dynamics. The coronary flow was restored to 89% of the pre ischemic value. Conversely, the aortic flow never exceeded that measured at the end of ischemia, partly due to the important development of severe arrhythmias. The recovery of aortic flow and metabolic efficiency during reperfusion was improved by amiodarone treatment; ventricular tachycardia and fibrillation duration were reduced. In the mitochondria issued from the normoxic area, the energy metabolism was not altered by the amiodarone treatment, but the presence of calcium in the respiration medium modified the oxidative phosphorylation. The divalent cation slightly decreased the state III respiration rate and increased noticeably the state IV respiration rate. This was associated with an important mitochondrial AMP production and maintenance of ADP in the respiration medium. This energy wasting was reported to decrease the mitochondrial metabolic efficiency. After an ischemia-reperfusion sequence, mitochondrial oxidation phosphorylation was reduced and amiodarone treatment amplified this decrease. This was presumably due to an increased mitochondrial calcium accumulation. Thus, the beneficial properties of amiodarone during reperfusion are supposed to be due to a protection against the deleterious effect of excess matrix calcium on mitochondrial energy metabolism.
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  • 28
    ISSN: 1573-4919
    Keywords: heart ; ANF ; phenylephrine ; etomoxir ; metabolism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Long-term, serum supplemented cultures of rat adult ventriculocytes were utilized to study the trophic effects of the α-agonist phenylephrine and of the carnitine palmitoyltransferase I inhibitor etomoxir. Cell protein and the rate of incorporation of phenylalanine were measured, corrected for cellular DNA content and utilized as an index for hypertrophy and of anabolic acitivity of the cells, respectively. The mRNA level of ANF was utilized as an index for the pathological phenotypic change (i.e., switch to fetal gene program), and that of the Na-channel — a constantly expressed gene in normal and hypertrophic cardiomyocytes — served as an internal control. Both mRNAs were quantified at various stages in culture by competitive reverse transcriptase PCR. The size of control myocytes steadily increased for over 3 weeks. The cells were completely redifferentiated and reached a maximum of anabolic activity 2 weeks after plating. Secretion and mRNA levels of ANF were increased severalfold after 7–8 days. Addition of 10 μM phenylephrine considerably speeded up cell growth. Maximum anabolic activity and complete redifferentiation were reached already after 1 week. Levels of mRNA and of ANF release increased 30–40 fold. Interestingly, induction of ANF gene transcription lagged behind the redifferentiation of the cells. Ten μM etomoxir inhibited the oxidation of palmitic acid and stimulated that of exogenous glucose by adult cardiomyocytes. In spite of its clear effect on fuel utilization, etomoxir had no direct hypertrophic effect on the myocytes in culture and did not inhibit the stimulatory action of α-agonists. Reactivation of the fetal gene program, as visualized by ANF production, was not reversed by etomoxir.
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  • 29
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    Molecular and cellular biochemistry 149-150 (1995), S. 263-265 
    ISSN: 1573-4919
    Keywords: NO synthase ; toxic metals ; signal transduction ; calcium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract This study was designed to evaluate thein vitro effects of transition heavy metal cations on activity of constitutive isoform of nitric oxide synthase (cNOS) in rat brain. NOS activity was determined in the cytosolic fractions of rat cerebral hemispheres by conversion of3H-L-arginine to3H-L-citrulline. Different concentrations of mercury (Hg2+), nickel (Ni2+), manganese (Mn2+), zinc (Zn2+), cadmium (Cd2+), lead (Pb2+) and calcium (Ca2+) were tested on NOS activity. While all the cations caused inhibition, there were differences in the apparent inhibition constants (Ki) among the cations. With the exception of calcium ion no other cation required preincubation with the enzyme preparation. These results indicate that while calcium ion modulate cNOS activity at regulatory site(s), inhibitory influence of toxic heavy metal cations may be exerted on the catalytic site(s) either by direct binding to it or by interfering with the electron transfer during catalysis.
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  • 30
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    Molecular and cellular biochemistry 159 (1996), S. 139-148 
    ISSN: 1573-4919
    Keywords: cardiolipin ; heart ; phospholipid biosynthesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Cardiolipin is one of the principle phospholipids in the mammalian heart comprising as much as 15–20% of the entire phospholipid phosphorus mass of that organ. Cardiolipin is localized primarily in the mitochondria and appears to be essential for the function of several enzymes of oxidative phosphorylation. Thus, cardiolipin is essential for production of energy for the heart to beat. Cardiac cardiolipin is synthesized via the cytidine-5′-diphosphate-1,2-diacyl-sn-glycerol pathway. The properties of the four enzymes of the cytidine-5′-diphosphate-1,2-diacyl-sn-glycerol pathway have been characterized in the heart. The rate-limiting step of this pathway is catalyzed by the phosphatidic acid: cytidine-5′-triphosphate cytidylyltransferase. Several regulatory mechanisms that govern cardiolipin biosynthesis in the heart have been uncovered. Current evidence suggests that cardiolipin biosynthesis is regulated by the energy status (adenosine-5′-triphosphate and cytidine-5′-triphosphate level) of the heart. Thyroid hormone and unsaturated fatty acids may regulate cardiolipin biosynthesis at the level of three key enzymes of the cytidine-5′-diphosphate-1,2-diacyl-sn-glycerol pathway, phosphatidylglycerolphosphate synthase, phosphatidylglycerolphosphate phosphatase and cardiolipin synthase. Newly synthesized phosphatidic acid and phosphatidylglycerol may be preferentially utilized for cardiolipin biosynthesis in the heart. In addition, separate pools of phosphatidylglycerol, including an exogenous (extra-mitochondrial) pool not derived from de novo phosphatidylglycerol biosynthesis, may be utilized for cardiac cardiolipin biosynthesis. In several mammalian tissues a significant number of studies on polyglycerophospholipid biosynthesis have been documented, including detailed studies in the lung and liver. However, in spite of the important role of cardiolipin in the maintenance of mitochondrial function and membrane integrity, studies on the control of cardiolipin biosynthesis in the mammalian heart have been largely neglected. The purpose of this review will be to briefly discuss cardiolipin and cardiolipin biosynthesis in some selected model systems and focus primarily on current studies involving the regulation of cardiolipin biosynthesis in the heart. (Mol Cell Biochem 159: 139–148, 1996)
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  • 31
    ISSN: 1573-4919
    Keywords: respiration ; ADP diffusion ; heart ; skeletal muscle ; liver ; brain ; in vivo regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Very recent experimental data, obtained by using the permeabilized cell technique or tissue homogenates for investigation of the mechanisms of regulation of respiration in the cells in vivo, are shortly summarized. In these studies, surprisingly high values of apparent Km for ADP, exceeding that for isolated mitochondria in vitro by more than order of magnitude, were recorded for heart, slow twitch skeletal muscle, hepatocytes, brain tissue homogenates but not for fast twitch skeletal muscle. Mitochondrial swelling in the hypo-osmotic medium resulted in the sharp decrease of the value of Km for ADP in correlation with the degree of rupture of mitochondrial outer membrane, as determined by the cytochrome c test. Very similar effect was observed when trypsin was used for treatment of skinned fibers, permeabilized cells or homogenates. It is concluded that, in many but not all types of cells, the permeability of the mitochondrial outer membrane for ADP is controlled by some cytoplasmic protein factor(s). Since colchicine and taxol were not found to change high values of the apparent Km for ADP, the participation of microtubular system seems to be excluded in this kind of control of respiration but studies of the roles of other cytoskeletal structures seem to be of high interest. In acute ischemia we observed rapid increase of the permeability of the mitochondrial outer membrane for ADP due to mitochondrial swelling and concomitant loss of creatine control of respiration as a result of dissociation of creatine kinase from the inner mitochondrial membrane. The extent of these damages was decreased by use of proper procedures of myocardial protection showing that outer mitochondrial membrane permeability and creatine control of respiration are valuable indices of myocardial preservation. In contrast to acute ischemia, chronic hypoxia seems to improve the cardiac cell energetics as seen from better postischemic recovery of phosphocreatine, and phosphocreatine overshoot after inotropic stimulation. In general, adaptational possibilities and pathophysiological changes in the mitochondrial outer membrane system point to the central role such a system may play in regulation of cellular energetics in vivo.
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  • 32
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    Molecular and cellular biochemistry 160-161 (1996), S. 159-166 
    ISSN: 1573-4919
    Keywords: reactive oxygen species ; 8-hydroxy-2′-deoxyguanosine ; heart ; nitric oxide ; aging ; rat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Biochemical and structural changes occurring in the myocardium with aging are mainly resulting from the association of a general tissue atrophy with the hypertrophy of the remaining myocytes. Whilst hypertrophy seems to be a compensatory process to the loss of cardiomyocytes and to a mild systolic hypertensive condition that accompanies elderly people, atrophy should be the modification more closely related to aging ‘per se.’ In support to the free radical theory of aging, several signs of oxidative damage have been shown in the aged heart, such as lipofuscin accumulation, decreased phospholipid unsaturation index, greater formation of both hydrogen peroxide and 8-hydroxy-2′deoxyguanosine. As a compensatory reaction, the activities of the main oxygen-radical scavenger enzymes are stimulated in the mitochondria of aged rat heart. Endothelium-mediated vasoregulation is more susceptible to oxidative stress in aged with respect to young rats, suggesting that also the vasculature can be negatively influenced by the oxygen free radicals generated during aging. The possible primary role of oxygen free radicals in the development of myocardial atrophy is also discussed.
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  • 33
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    Molecular and cellular biochemistry 168 (1997), S. 95-100 
    ISSN: 1573-4919
    Keywords: aluminium ; calcium ; brain ; neurodegenerative disease
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The present study investigates the possible effects of chronic aluminium exposure on the various aspects of calcium homeostasis in the primate central nervous system. Aluminium administration caused a marked decline in the activity of Ca2+ ATPase in the monkey brain. The total calcium content was also significantly raised following aluminium exposure. Concomittant to the increase in the calcium content, the levels of lipid peroxidation were also augmented in the aluminium treated animals, thereby further accentuating the aluminium induced neuronal damage. In addition, aluminium had an inhibitory effect on the depolarization induced 45Ca2+ uptake via the voltage operated channels. The results presented herein, indicate that the toxic effects of aluminium could be mediated through modifications in the intracellular calcium homeostasis with resultant altered neuronal function.
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  • 34
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    Molecular and cellular biochemistry 163-164 (1996), S. 261-275 
    ISSN: 1573-4919
    Keywords: apoptosis ; necrosis ; myocyte ; heart
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Since mammalian cardiac myocytes essentially rely on aerobic energy metabolism, it has been assumed that cardiocytes die in a catastrophic breakdown of cellular homeostasis (i.e. necrosis), if oxygen supply remains below a critical limit. Recent observations, however, indicate that a process of gene-directed cellular suicide (i.e. apoptosis) is activated in terminally differentiated cardiocytes of the adult mammalian heart by ischemia and reperfusion, and by cardiac overload as well. Apoptosis or programmed cell death is an actively regulated process of cellular self destruction, which requires energy and de novo gene expression, and which is directed by an inborn genetic program. The final result of this program is the fragmentation of nuclear DNA into typical “nucleosomal ladders”, while the functional integrity of the cell membrane and of other cellular organelles is still maintained. The critical step in this regulated apoptotic DNA fragmentation is the proteolytic inactivation of poly-[ADPribose]-polymerase (PARP) by a group of cysteine proteases with some structural homologies to interleukin-1β-converting enzyme (ICE-related proteases [IRPs] such as apopain, yama and others). PARP catalyzes the ADP-ribosylation of nuclear proteins at the sites of spontaneous DNA strand breaks and thereby facilitates the repair of this DNA damage. IRP-mediated destruction of PARP, the ‘supervisor of the genome’, can be induced by activation of membrane receptors (e.g. FAS or APOI) and other signals, and is inhibited by activation of ‘anti-death genes’ (e.g. bcl-2). Overload-triggered myocyte apoptosis appears to contribute to the transition to cardiac failure, which can be prevented by therapeutic hemodynamic unloading. In myocardial ischemia, the activation of the apoptotic program in cardiocytes does not exclude their final destiny to catastrophic necrosis with release of cytosolic enzymes, but might be considered as an adaptive process in hypoperfused ventricular zones, sacrificing some jeopardized myocytes to regulated apoptosis, which may by less arrhythmogenic than necrosis with the primary disturbance of membrane function.
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  • 35
    ISSN: 1573-4919
    Keywords: nitric oxide ; endotoxin ; cardiomyocytes ; guanosine 3′, 5′-cyclic monophosphate ; calcium ; ADP-ribosylation ; phosphorylation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract To evaluate the effects of the in vivo endotoxin treatment of the rat on (1) the contractile responses in the subsequently isolated papillary muscle to adrenergic and cholinergic agonists and (2) the biochemical parameters (cyclic GMP, nitric oxide synthesis, protein phosphorylation and ADP-ribosyslation) in the subsequently isolated cardiomyocytes. Following the in vivo endotoxin treatment (4 mg/kg i.p., 18 h), contractile responses to increasing amounts of isoprenaline or to increasing amounts of oxotremorine in the presence of a fixed amount of isoprenaline were determined in isolated papillary strips. Activities of nitric oxide synthase, guanylyl cyclase, as well as phosphorylation of phospholamban and troponin-inhibitory subunit, and pertussis toxin-catalyzed and endogenous ADP-ribosylations were determined in the intact cardiomyocytes and subcellular fractions. The increase in the force of contraction by isoprenaline was reduced, while its inhibition by oxotremorine was greater in the endotoxin-treated papillary strips. The activities of both nitric oxide synthase, primarily of the inducible form of the enzyme, and cytosolic guanylyl cyclase were higher while the phosphorylations of both phospholamban and troponin-inhibitory subunit were of lesser magnitude in the cardiomyocytes following the in vivo endotoxin treatment. Pertussis toxin-catalyzed ADP-ribosylation of the 41 kDa polypeptide, which is the alpha subunit of Gi, was also decreased. The results of the present study support the postulate that alterations in both the cyclic AMP and cyclic GMP signalling cascade contribute to the myocardial dysfunction caused by endotoxin and cytokines.
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  • 36
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    Molecular and cellular biochemistry 147 (1995), S. 57-68 
    ISSN: 1573-4919
    Keywords: angiogenesis ; capillaries ; heart ; exercise ; bradycardia ; vasodilation ; mechanical factors ; growth factors
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Growth of capillaries in the heart occurs under physiological circumstances during endurance exercise training, exposure to high altitude and/or cold, and changes in cardiac metabolism or heart rate elicited by modification of thyroid hormone levels. Capillary growth in all these conditions can be linked with increased coronary blood flow, decreased heart rate, or both. This paper brings evidence that, although increased blood flow due to long-term administration of coronary vasodilators results in capillary growth, a long-term decrease in heart rate induced by electrical bradycardial pacing in rabbits and pigs, or by chronic administration of a bradycardic drug, alinidine, in rats, stimulates capillary growth with little or no change in coronary blood flow. Decreased heart rate results in increased capillary wall tension, increased end-diastolic volume and increased force of contraction, and thus stretch of the capillary wall. This could lead to release of various growth factors possibly stored in the capillary basement membrane. Correlation was found between capillary density (CD) and the levels of low molecular endothelial cell stimulating angiogenic factor (ESAF) both in rabbit and pig hearts with CD increased by pacing. There was no relation between expression of mRNA for basic fibroblast growth factor and CD in sham-operated and paced rabbit hearts. In contrast, mRNA for TGFß was increased in paced hearts, and the possible role of this factor in the regulation of capillary growth induced by bradycardia is discussed.
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  • 37
    ISSN: 1573-4919
    Keywords: taurine ; heart cells ; calcium ; sodium ; confocal microscopy ; nucleus ; fluo-3 ; sodium green ; Ca2+ overload
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The effect of taurine on the different types of ionic currents appears to depend on [Ca]o and [Ca]i and may also vary accordingly to tissue or cell type studied. Using microfluorometry and Ca2+ imaging techniques, short-term exposure (5–10 min) of single heart cells to taurine was found to increase total intracellular free Ca2+ in a concentration-dependent manner. However, long-term exposure of heart myocytes to taurine was found to decrease both nuclear and cytosolic Ca2+ without significantly changing either nuclear or cytosolic Na+ levels, as measured by 3-dimensional Ca2+ and Na+ confocal imaging techniques. Long- term exposure to taurine was found to prevent cytosolic and nuclear increases of Ca2+ induced by permanent depolarization of heart cells with high [K]o. This preventive effect of taurine on nuclear Ca2+ overload was associated with an increase of both cytosolic and nuclear free Na+. Thus, the effect of long-term exposure to taurine on intranuclear Ca2+ overload in heart cells seems to be mediated via stimulation of sarcolemmal and nuclear Ca2+ outflow through the Na+-Ca2+ exchanger.
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  • 38
    ISSN: 1573-4919
    Keywords: heart ; DNA ; library ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The availability of high quality cDNA libraries is often crucial to the successful identification and characterization of genes. The concepts and potential pitfalls of constructing cDNA libraries are presented. Various applications requiring high quality cDNA libraries are outlined, including large-scale single pass sequencing of cDNA clones to generate expressed sequence tags (ESTs) and differential screening of cDNA libraries. The usefulness of combining such approaches for the discovery of novel disease-related and cardiovascular-based ESTs (CVBest) is discussed.
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  • 39
    ISSN: 1573-4919
    Keywords: mitochondria ; calcium ; permeability transition ; vasopressin ; glucagon ; thapsigargin ; protein kineses and phosphatases ; rat hepatocytes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Ca2+ functions as an intracellular signal to transfer hormonal messages to different cellular compartments, including mitochondria, where it activates intramitochondrial Ca2+-dependent enzymes. However, excessive mitochondrial Ca2+ uptake can promote the mitochondrial permeability transition (MPT), a process known to be associated with cell injury. The factors controlling mitochondrial Ca2+ uptake and release in intact cells are poorly understood. In this paper, we investigate mitochondrial Ca2+ accumulation in intact hepatocytes in response to the elevation of cytosolic Ca2+ levels ([Ca2+]c) induced either by a hormonal stimulus (vasopressin), or by thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump. After stimulation, cells were rapidly permeabilized for the determination of the mitochondrial Ca2+ content (Ca2+_m) and to analyze the susceptibility of the mitochondria to undergo the MPT. Despite very similar levels of [Ca2+]c elevation, vasopressin and thapsigargin had markedly different effects on mitochondrial Ca2+ accumulation. Vasopressin caused a rapid (〈 90 sec), but modest (〈 2 fold) increase in Ca2+m that was not further increased during prolonged incubations, despite a sustained [Ca2+]c elevation. By contrast, thapsigargin induced a net Ca2+ accumulation in mitochondria that continued for up to 30 min and reached Ca2+_m levels 10–20 fold over basal. Accumulation of mitochondrial Ca2+ was accompanied by a markedly increased susceptibility to undergo the MPT. Both mitochondrial Ca2+ accumulation and MPT activation were modulated by treatment of the cells with inhibitors of protein kineses and phosphatases. The results indicate that net mitochondrial Ca2+ uptake in response to hormonal stimulation is regulated by processes that depend on protein kinase activation. These controls are inoperative when the cytosol is flooded by Ca2+ through artificial means, enabling mitochondria to function as a Ca2+ sink under these conditions. (Mol Cell Biochem 174: 173–179, 1997)
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  • 40
    ISSN: 1573-4919
    Keywords: heart ; calcium ; magnesium ; contractility ; dietary ; L-type calcium current
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract This study employs both dietary and physiological studies to investigate the relationship between calcium (Ca2+) and magnesium (Mg2+) signalling in the mammalian myocardium. Rats maintained on a low Mg2+ diet (LMD; 39 mg Kg-1 Mg2+ in food) consumed less food and grew more slowly than control rats fed on a control Mg2+ diet (CMD; 500 mg Kg-1 Mg2+ in food). The Mg2+ contents of the heart and plasma were 85 ± 3% and 34 ± 6.5%, respectively relative to the control group. In contrast, Ca2+ contents in the heart and plasma were 177 ± 5% and 95 ± 3%. The levels of potassium (K+) was raised in the plasma (129 ± 16%) and slightly decreased in the heart (88 ± 6%) compared to CMD. Similarly, sodium (Na+) contents were slightly higher in the heart and lowered in the plasma of low Mg2+ diet rats compared to control Mg2+ diet rat. Perfusion of the isolated Langendorff's rat heart with a physiological salt solution containing low concentrations (0-0.6 mM) of extracellular magnesium [Mg2+]0 resulted in a small transient increase in the amplitude of contraction compared to control [Mg2+]0 (1.2 mM). In contrast, elevated [Mg2+]0 (2-7.2 mM) caused a marked and progressive decrease in contractile force compared to control. In isolated ventricular myocytes the L-type Ca2+ current (ICa,L was significantly (p 〈 0.001) attenuated in cells dialysed with 7.1 mM Mg2+ compared to cells dialysed with 2.9 µM Mg2+. The results indicate that hypomagnesemia is associated with decrease levels of Mg2+ and elevated levels of Ca2+ in the heart and moreover, internal Mg2+ is able to modulate the Ca2+ current through the L-type Ca2+ channel which in turn may be involved with the regulation of contractile force in the heart.
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  • 41
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    Molecular and cellular biochemistry 158 (1996), S. 161-169 
    ISSN: 1573-4919
    Keywords: heart ; ischemia ; mitochondria ; oxidative phosphorylation ; energy wasting
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The purpose of this study was to evaluate the oxidative capacities and the rate of energy synthesis in isolated mitochondria extracted from normal and post-ischemic myocardium. Isolated rat hearts were perfused according to the working mode with a Krebs Heinseleit buffer containing glucose (11 mM), insulin (10 IU/1) and caprylic acid (25 μM). After a 15 min perfusion in normoxic conditions, the hearts were subjected to a 20 min local zero-flow ischemia followed by a 20 min reperfusion. During the perfusion, the aortic and coronary flows, the aortic pressure and the electrocardiogram were monitored. At the end of the reperfusion period, the non-ischemic and ischemic zones (NIZ and IZ, respectively) were separated and the mitochondria were harvested from each zone. The oxygen uptake and the rate of energy production of the NIZ and IZ mitochondria were then assessed with palmitoylcarnitine as substrate in 2 buffers differing in their free calcium concentration (0.041 and 0.150 μM). Ischemia provoked a 50% reduction of coronary and aortic flows. The reperfusion of the IZ allowed the partial recovery of coronary flow, but the aortic flow decreased beneath its ischemic value because of the occurrence of severe arrhythmias, stunning and probably hibernation. The IZ mitochondria displayed a lower rate of oxygen consumption, whatever the buffer free calcium concentration. Conversely, their rate of energy production was increased, indicating that their metabolic efficiency was improved as compared to NIZ mitochondria. This might be due to the mitochondrial calcium overload persisting during reperfusion, to the activation of the inner membrane Na+/Ca2+ exchange and to a significant mitochondrial swelling. On the other hand, the presence of an elevated free calcium concentration in the respiration buffer provoked some energy wasting characterized by a constant AMP production. This was attributed to some accumulation of acetate and the activation of the energy-consuming acetylCoA synthetase. In conclusion, ischemia and reperfusion did not alter the membrane integrity of the mitochondria but improved their metabolic efficiency. Nevertheless, these in vitro results can not reflect the mitochondrial function in the reperfused myocardium. The mitochondrial calcium overload reported to last during reperfusion in the cardiomyocytes might mimic the free calcium-induced reduction of metabolic efficiency observed in vitro in the present study. The resulting energy wasting might be responsible for the contractile abnormalities noticed in the reperfused myocardium.
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  • 42
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    Molecular and cellular biochemistry 177 (1997), S. 209-214 
    ISSN: 1573-4919
    Keywords: regucalcin ; calmodulin ; calcium ; cyclic AMP phosphodiesterase ; rat kidney cytosol
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The effect of regucalcin, a novel Ca2+-binding protein, on Ca2+/ calmodulin-dependent cyclic adenosine monophosphate (AMP) phosphodiesterase activity in the cytosol of rat renal cortex was investigated. Regucalcin with physiologic concentration (10-7 M) in rat kidney had no effect on cyclic AMP phosphodiesterase activity in the absence of CaCl2 and calmodulin. However, the activatory effect of both CaCl2 (10 µM) and calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was markedly inhibited by the addition of regucalcin (10-8 to 10-6 M) in the enzyme reaction mixture. The inhibitory effect of regucalcin on the enzyme activity was also seen in the presence of CaCl2 (5-50 µM) or calmodulin (5-50 U/ml) with increasing concentrations. The presence of trifluoperazine (10 µM), an antagonist of calmodulin, caused a partial inhibition of Ca2+ /calmodulin-dependent cyclic AMP phosphodiesterase activity. This inhibition was further enhanced by the addition of regucalcin (10-7 M). The inhibitory effect of regucalcin (10-7 M) was not seen in the presence of 20 µM trifluoperazine. Moreover, the activatory effect of calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was not entirely seen, when calmodulin was added 10 min after incubation in the presence of CaCl2 (10 µM) and regucalcin (10-7 M). The present results demonstrates that regucalcin has an inhibitory effect on Ca2+ /calmodulin-dependent cyclic AMP phosphodiesterase activation in the cytosol of rat renal cortex.
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  • 43
    ISSN: 1573-4919
    Keywords: heart ; postnatal development ; sarcoplasmic reticulum ; phospholamban ; calcium transport ; spontaneously hypertensive rats ; growth
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract This comparative study investigates the relationship between sarcoplasmic reticulum (SR) calcium(Ca2+)-ATPase transport activity and phospholamban (PLB) phosphorylation in whole cardiac homogenates of spo`ntaneously hypertensive rats (SHR) and their parent, normotensive Wistar Kyoto (WKY) strain during early postnatal development at days 1, 3, 6, 12 and at day 40 to ascertain any difference in SR Ca2+ handling before the onset of hypertension. At day 1, the rate of homogenate oxalate-supported Ca2+ uptake was significantly higher in SHR than in WKY (0.25 ± 0.02 vs 0.12 ± 0.01 nmoles Ca2+/mg wet ventricular weight/min, respectively; p 〈 0.001). This interstrain difference disappeared with further developmental increase in SR Ca2+ transport. Western Blot analysis and a semiquantitative ELISA did not reveal any difference in the amount of immunoreactive PLB (per mg of total tissue protein) between strains at any of the ages studied. In addition, levels of phosphorylated PLB formed in vitro in the presence of radiolabelled ATP and catalytic (C) subunit of protein kinase A did not differ between SHR and WKY at days 1, 3, 6 and 12. At day 40, C subunit-catalyzed formation of 32P-PLB was reduced by 66% (p 〈 0.001) in SHR when compared to age-matched WKY In the early postnatal period between day 1 and 12 SR Ca2+-transport values were linearly related to the respective 32P-PLB levels of both SHR and WKY rats. The results indicate that cardiac SR of SHR can sequester Ca2+ at a much higher rate immediately after birth compared to WKY rats. The disappearance of this interstrain difference with further development suggests that some endogenous neuroendocrine or nutritional factor(s) from the hypertensive mother may exert an influence upon the developing heart in utero resulting in a transiently advanced maturation of the SR Ca2+ transport function in SHR pups at the time of birth.
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  • 44
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    Molecular and cellular biochemistry 180 (1998), S. 53-57 
    ISSN: 1573-4919
    Keywords: diabetes ; cardiomyopathy ; lipids ; lipoprotein lipase ; calcium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract It has been established that diabetes results in a cardiomyopathy, and increasing evidence suggests that an altered substrate supply and utilization by cardiac myocytes could be the primary injury in the pathogenesis of this specific heart muscle disease. For example, in diabetes, glucose utilization is insignificant, and energy production is shifted almost exclusively towards β-oxidation of free fatty acids (FFA). FFA's are supplied to cardiac cells from two sources: lipolysis of endogenous cardiac triglyceride (TG) stores, or from exogenous sources in the blood (as free acid bound to albumin or as TG in lipoproteins). The approximate contribution of FFA from exogenous or endogenous sources towards β-oxidation in the diabetic heart is unknown. In an insulin-deficient state, adipose tissue lipolysis is enhanced, resulting in an elevated circulating FFA. In addition, hydrolysis of the augmented myocardial TG stores could also lead to high tissue FFA. Whatever the source of FFA, their increased utilization may have deleterious effects on myocardial function and includes the abnormally high oxygen requirement during FFA metabolism, the intracellular accumulation of potentially toxic intermediates of FFA, a FFA-induced inhibition of glucose oxidation, and severe morphological changes. Therapies that target these metabolic aberrations in the heart during the early stages of diabetes could potentially delay or impede the progression of more permanent sequelae that could ensue from otherwise uncontrolled derangements in cardiac metabolism.
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  • 45
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    Molecular and cellular biochemistry 179 (1998), S. 135-145 
    ISSN: 1573-4919
    Keywords: calcium ; non-lysosomal proteases ; muscle damage ; neutrophils ; muscle regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract It is well established that periods of increased contractile activity result in significant changes in muscle structure and function. Such morphological changes as sarcomeric Z-line disruption and sarcoplasmic reticulum vacuolization are characteristic of exercise-induced muscle injury. While the precise mechanism(s) underlying the perturbations to muscle following exercise remains to be elucidated, it is clear that disturbances in Ca2+ homeostasis and changes in the rate of protein degradation occur. The resulting elevation in intracellular [Ca2+] activates the non-lysosomal cysteine protease, calpain. Because calpain cleaves a variety of protein substrates including cytoskeletal and myofibrillar proteins, calpain-mediated degradation is thought to contribute to the changes in muscle structure and function that occur immediately following exercise. In addition, calpain activation may trigger the adaptation response to muscle injury. The purpose of this paper is to: (i) review the chemistry of the calpain-calpastatin system; (ii) provide evidence for the involvement of the non-lysosomal, calcium-activated neutral protease (calpain) in the response of skeletal muscle protein breakdown to exercise (calpain hypothesis); and (iii) describe the possible involvement of calpain in the inflammatory and regeneration response to exercise.
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  • 46
    ISSN: 1573-4919
    Keywords: aortic cells ; steady state R-type Ca2+ channel ; ET-1 ; insulin ; calcium ; G-protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract In single rabbit aortic smooth muscle cells, and at a concentration known to induce a maximum sustained increase of intracellular Ca2+ via activation of the steady-state voltage dependent R-type Ca2+ channels, endothelin-1 (10-7 M) and insulin (80 μU/ml) were found to induce a sustained increase in cytosolic free Ca2+ ([Ca]i) levels that was significantly attenuated by pre-treatment with either pertussis toxin (PTX), cholera toxin (CTX) or removal of extracellular Ca2+. However, both PTX and CTX failed to inhibit the sustained depolarization-evoked sustained Ca2+ influx and [Ca]i elevation via activation of the R-type Ca2+ channels. Moreover, ET-1 and insulin-evoked sustained increases in Ca2+ influx were not attenuated by the selective PKC inhibitor, bisindolylmaleimide (BIS), or the specific L-type Ca2+ channel blocker, nifedipine, but were completely reversed by the R-type Ca2+ channel blocker, (-) PN 200-110 (isradipine). These data suggest that both insulin and ET-1 activate the nifedipine-insensitive but isradipine-sensitive steady state voltage dependent R-type Ca2+ channels present on rabbit VSMCs and these channels are directly coupled to PTX and CTX sensitive G protein(s).
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  • 47
    ISSN: 1573-4919
    Keywords: hyperosmolality ; hyperglycemia ; calcium ; smooth muscle cells ; diabetes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Hyperglycemia and/or hyperosmolality may disturb calcium homeostasis in vascular smooth muscle cells (SMCs), leading to altered vascular contractility in diabetes. To test this hypothesis, the KCl induced increases in [Ca2+]i in primarily cultured vascular SMCs exposed to different concentrations of glucose were examined. With glucose concentration in solutions kept at 5.5 mM, KCl induced a fast increase in [Ca2+]i which then slowly declined (type 1 response) in 83% of SMCs from non-diabetic rats. In 9% of non-diabetic SMCs KCl induced a slow increase in [Ca2+]i (type 2 response). Interestingly, under the same culture conditions KCl induced type 1 and type 2 responses in 47 and 35% of SMCs from diabetic rats. When SMCs from non-diabetic or diabetic rats were cultured in 36 mM glucose, KCl induced a fast increase in [Ca2+]i which, however, maintained at a high level (type 3 response). The sustained level of [Ca2+]i in the presence of KCl was significantly higher in cells cultured with 36 mM glucose than that in non-diabetic cells cultured with 5.5 mM glucose. Furthermore, the hyperglycemia-induced alterations in calcium mobilization were similarly observed in cells cultured in high concentration of mannitol (30.5 mM) or L-glucose, indicating that hyperosmolality was mainly responsible for the abnormal calcium mobilization in diabetic SMCs.
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  • 48
    ISSN: 1573-4919
    Keywords: CGRP-1 receptor ; HEK-293 cells ; calcium ; cholera toxin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Calcitonin gene-related peptide (CGRP) is a neuropeptide with diverse biological properties including potent vasodilating activity. Recently, we reported the cloning of complementary DNAs (cDNAs) encoding the human and porcine CGRP receptors which share significant amino acid sequence homology with the human calcitonin receptor, a member of the recently described novel subfamily of G-protein-coupled 7TM receptors. Activation of this family of receptors has been shown to result in an increase in intracellular cAMP accumulation and calcium release. In this study, we demonstrate that HEK-293 cells expressing recombinant CGRP receptors (HEK-293HR or PR) respond to CGRP with increased intracellular calcium release (EC50 = 1.6 nM) in addition to the activation of adenylyl cyclase (EC50 = 1.4 nM). The effect of CGRP on adenylyl cyclase activation and calcium release was inhibited by CGRP (8-37), a CGRP receptor antagonist. Both effects were mediated by cholera toxin-sensitive G-proteins, but these two signal transduction pathways were independent of each other. While cholera toxin pretreatment of HEK-293PR cells resulted in permanent activation of adenylyl cyclase, the same pretreatment resulted in an inhibition of CGRP-mediated [Ca2+]i release. Pertussis toxin was without effect on CGRP-mediated responses. In addition, CGRP-mediated calcium release appears to be due to release from a thapsigargin-sensitive intracellular calcium pool. These results show that the recombinant human as well as porcine CGRP receptor can independently increase both cAMP production and intracellular calcium release when stably expressed in the HEK-293 cell line.
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  • 49
    ISSN: 1573-4919
    Keywords: endothelin-1 ; ventricular cardiomyocytes ; contraction ; calcium ; heart failure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Endothelin (ET-1) is found at elevated concentrations in the plasma of patients with heart failure and in animal models of cardiomyopathy. The peptide is a potent positive inotropic agent, the effects of which are mediated by increases in cytosolic Ca2+ in cardiomyocytes. The object of this study was to investigate at the cellular level, the actions of ET-1 on contractile function and on Ca2+ currents in heart-failed ventricular myocardium. Male New Zealand White rabbits (8 wks) were treated with twice weekly injections of epirubicin (4 mg/kg/wk, n=7) or with saline (n=7) for 6 wks, followed by a washout period of 2 wks. Ventricular cardiomyocytes were isolated from rabbit hearts using Langendorff perfusion with collagenase; contractile function was examined using a video microscopy method, and L-type Ca2+ currents were recorded using a whole-cell patch-clamp technique. ET-1 produced a concentration-dependent increase in contractile response (% increase from basal value) to a maximum at 1 nM ET-1 of 69 ± 11% (mean ± S.D.) in control cardiomyocytes and 33 ± 6% in heart-failed cells. However, there was no significant change in the EC50 obtained with ET-1 for healthy (0.31 ± 0.1 nM) and for failed cardiomyocytes (0.24 ± 0.1 nM). The effects of ET-1 on L-type Ca2+ channels were similar with a peak amplitude at 1 nM ET-1 of −3.26 ± 0.8 ⋬ in control cardiomyocytes and −3.32 ± 0.9 nA in heart-failed cells. The attenuation of the contractile response to ET-1 in heart-failed cells may reflect a desensitization of ET receptors as a consequence of elevated circulating levels of ET and was not reflected by alteration of transmembrane Ca2+ conductance. It is probable, therefore, that multiple signalling pathways are involved in the actions of ET on ventricular myocardium.
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  • 50
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    Molecular and cellular biochemistry 160-161 (1996), S. 83-87 
    ISSN: 1573-4919
    Keywords: phospholipase D ; hydrolysis ; transphosphatidylation ; sarcolemma ; sarcoplasmic reticulum ; heart
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Myocardial phospholipase D (PLD) is located in different subcellular membranes, including sarcolemma (SL) and sarcoplasmic reticulum (SR). In this study, the kinetics of PLD-dependent hydrolytic and transphosphatidylation activities were examined in SL and SR fractions isolated from rat heart by measuring the formation of phosphatidic acid and phosphatidylethanol, respectively. The results showed that, compared to SR PLD, SL PLD had a higher Vmax, i.e. 373 vs. 70 nmol/mg protein/h for the hydrolytic activity and 415 vs. 60 nmol/mg protein/h for the transphosphatidylation activity. In comparison with the SR enzyme, SL PLD had a lower Km value for the hydrolytic activity (0.46 vs. 0.65 mM), but a higher Km for the transphosphatidylation activity (225 vs. 179 mM). These distinctive kinetic parameters suggest that SL PLD and SR PLD may be isoforms of the enzyme and/or have different membrane domain. Therefore, SL- and SR-localized PLD activities may be under independent control mechanism(s) and play distinct roles in normal conditions and pathological processes.
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  • 51
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    Molecular and cellular biochemistry 163-164 (1996), S. 125-130 
    ISSN: 1573-4919
    Keywords: cardiac myocytes ; early after depolarisations ; delayed after depolarisations ; calcium ; sarcoplasmic reticulum ; noradrenaline
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract We investigated the effect of 10−8 M noradrenaline (NA) on [Ca2+], and electrical activity of single myocytes of guinea-pig ventricular myocardium loaded with Indo 1-AM. Membrane potential was recorded by means of the patch electrode and patch amplifier set to the current clamp mode. Cells were stimulated at a rate of 30/min by 3 ms pulses of the current injected through the recording electrode. Superfusion of NA resulted in slight shortening of action potentials (APs), increase in rate of rise and amplitude of the respective Ca2+ transients, and appearance of secondary Ca2+ transients of two kinds: 1. appearing before repolarisation of AP and decay of the preceding Ca2+ transient were completed and 2. appearing between the APs. We named them early after-transients (EAT) and delayed after-transients (DAT), respectively. Without any additional intervention EATS caused some prolongation of APs duration and DATs resulted in subthreshold delayed after-depolarisations (DADS). When sarcolemmal K+ conductance was decreased by tetraethylammonium (TEA) in the patch electrode or 20 μM BaCl2 in the Tyrode solution, EATs initiated early after depolarizations (EADs) and DATs initiated suprathreshold DADs triggering full-sized APs. Superfusion of 30.0 mM Na+ (replaced with LiCl) resulted in reduction of AP duration by -70% and appearance of DATs. Also, the frequent multiple oscillations of Ca 2+ concentration were often observed. Neither DATs nor the oscillations had any affect on electrical activity of the cells. Their electrogenicity could not be increased by TEA or 20.0 μM Ba2+. EATs and DATs and their respective EADs and DADs could not be initiated by NA or low Na+ superfusion in the cells pretreated with 2 × 10−7 M thapsigargin, a selective blocker of Ca2+-ATPase of sarcoplasmic reticulum (SR). We conclude that in contrast to the current hypothesis, EADs can be initiated by Ca2+ released early in the cardiac cycle from the overloaded SR, and that electrogenicity of both types of Ca2+ oscillations critically depends on the sarcolemmal K+ conductance.
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  • 52
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    Molecular and cellular biochemistry 168 (1997), S. 1-12 
    ISSN: 1573-4919
    Keywords: remodeling ; heart ; vascular ; matrix metalloproteinase ; tissue inhibitor of metalloproteinase ; collagenase ; gelatinase ; collagen ; elastin ; proteoglycans ; growth factor ; structure function ; cancer ; angiogenesis ; cardiomyopathy ; ischemia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Extracellular structural remodeling is the compensatory response of the tissue following pathological stage. Myocardial infarction, which leads to adverse remodeling, thinning of the ventricle wall, dilatation and heart failure, is one of the leading causes of death. Remodeling implies an alteration in the extracellular matrix and in the spatial orientation of cells and intracellular components. The extracellular matrix is responsible for cardiac cell alignment and myocardial structural integrity. Substances that break down the extracellular matrix, specialized proteinases as well as inhibitors of proteinases, appear to be normally balanced in maintaining the integrity of the myocardium. Myocardial infarction leads to an imbalance in proteinase/ antiproteinase activities causing alterations in the stability and integrity of the extracellular matrix and adverse tissue remodeling. To explore mechanisms involved in this process and, in particular, to focus on matrix metalloproteinases, their inhibitors, and activators, an understanding of proteinase and antiproteinase is needed. This review represents new and significant information regarding the role of activated matrix proteinases antiproteinases in remodeling. Such information will have a significant impact both on the understanding of the basic cell biology of extracellular matrix turnover, as well as on potential avenues for pharmacological approaches to the treatment of ischemic heart disease and failure.
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  • 53
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    Molecular and cellular biochemistry 187 (1998), S. 47-55 
    ISSN: 1573-4919
    Keywords: Mimosa pudica ; apyrase ; arabinogalactan ; calcium ; circular dichroism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Mimosa pudica Linn leaves with pulvini contain unique isoforms (I and II) of apyrase enzyme (EC 3.6.1.5). The activity of isoform I depends on divalent cation Mn2+. This isoform is associated noncovalently with the polysaccharide, containing mainly of galactose and arabinose sugars. The apparent molecular mass of these 2 isoforms are 36 and 34 Kd respectively. The association of the polysaccharide with the isoform I has been found to be Ca2+ dependent which is endogenously present in this isoform. Removal of Ca2+ and polysaccharide from the enzyme (isoform I) leads to an inactivation. The enzyme activity can be restored when both Ca2+ and endogenous polysaccharide fraction were added at an optimal molar ratio of Ca2+:protein of 7:1. The endogenous polysaccharide can be replaced by the standard arabinogalactan. No other sugar or polysaccharide except the arabinogalactan can restore the apyrase activity. Calcium mediates a conformational change in the protein which helps in association of polysaccharide as evidenced from fluorometric and far UV-CD studies to restore the enzymic activity. Neither any interaction of the polysaccharide with the protein is detected in absence of Ca2+ nor the enzyme activity could be recovered under such condition.
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  • 54
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    Molecular and cellular biochemistry 187 (1998), S. 1-10 
    ISSN: 1573-4919
    Keywords: oxidant ; cardiovascular system ; signal transduction ; calcium ; mitogen activated protein kinases ; nuclear transcription factors ; tyrosine kinase ; protein kinase C ; superoxide ; hydrogen peroxide ; ischemia-reperfusion ; atherosclerosis ; phospholipases ; apoptosis ; antioxidant
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Although oxidants such as superoxide (O2.-) and hydrogen peroxide (H2O2) play a role in host-mediated destruction of foreign pathogens yet excessive generation of oxidants may lead to a variety of pathological complications in the cardiovascular system. An important mechanism by which oxidants cause dysfunction of the cardiovascular system appears to be due to the increase in intracellular free Ca2+ concentration. Oxidants cause cellular Ca2+ mobilization by modulating activities of a variety of regulators such as Na+/H+ and Na+/Ca2+ exchangers, Na+/K+ ATPase and Ca2+ ATPase and Ca2+ channels that are associated with Ca2+ transport in the plasma membrane and the sarco(endo)plasmic reticular membrane of myocardial cells. Recent research have suggested that the increase in Ca2+ level by oxidants plays a pivotal role in indicing several protein kinases such as protein kinase C, tyrosine kinase and mitogen activated protein kinases. Oxindant-mediated alteration of different signal transduction systems and their interations eventually regulate a variety of pathological conditoins such as atherosclerosis, apoptosis and necrosis in the myocardium
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  • 55
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    Molecular and cellular biochemistry 172 (1997), S. 121-127 
    ISSN: 1573-4919
    Keywords: inositol phosphates ; anion exchange HPLC ; heart
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The pathways of release and metabolism of inositol phosphates in intact heart tissue are different from those observed in isolated cardiomyocytes in culture. Thus, it is essential that methods are available for the quantitation of inositol phosphates in intact tissue preparations. This manuscript describes methods which allow the quantitation of inositol phosphates in different heart preparations including isolated atria and intact perfused heart. The availability of such methods should facilitate study of the role of inositol phosphates in cardiac control mechanisms under physiological and pathological conditions.
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  • 56
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    Keywords: heart ; vascular endothelium ; vascular smooth muscle ; confocal microscopy ; pH ; calcium ; sodium ; voltage probe ; heart ; endothelin-1 ; Angiotensin II ; PAF ; nucleus ; mitochondria ; SR ; cardiomyopathy ; cells interaction ; R-type Ca2+ channel ; excitation-contraction coupling ; dystrophic mouse
    Source: Springer Online Journal Archives 1860-2000
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    Notes: Abstract In recent years, fluorescence microscopy imaging has become an important tool for studying cell structure and function. This non invasive technique permits characterization, localisation and qualitative quantification of free ions, messengers, pH, voltage and a pleiad of other molecules constituting living cells. In this paper, we present results using various commercially available fluorescent probes as well as some developed in our laboratory and discuss the advantages and limitations of these probes in confocal microscopy studies of the cardiovascular system.
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  • 57
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    Keywords: cyclosporin A ; mitochondrial permeability transition ; reperfusion injury ; cyclophilin ; oxidative stress ; calcium
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    Notes: Abstract When loaded with high (pathological) levels of Ca2+, mitochondria become swollen and uncoupled as the result of a large non-specific increase in membrane permeability. This process, known as the mitochondrial permeability transition (MPT), is exacerbated by oxidative stress and adenine nucleotide depletion. These conditions match those that a heart experiences during reperfusion following a period of ischaemia. The MPT is caused by the opening of a non-specific pore that can be prevented by sub-micromolar concentrations of cyclosporin A (CsA). A variety of conditions that increase the sensitivity of pore opening to [Ca2+], such as thiol modification, oxidative stress, increased matrix volume and chaotropic agents, all enhance the binding of matrix cyclophilin (CyP) to the inner mitochondrial membrane in a CsA-sensitive manner. In contrast, ADP, membrane potential and low pH decrease the sensitivity of pore opening to [Ca2+] without affecting CyP binding. We present a model of pore opening involving CyP binding to a membrane target protein followed by Ca2+-dependent triggering of a conformational change to induce channel opening. Using the ischaemic/reperfused rat heart we have shown that the mitochondrial pore does not open during ischaemia, but does do so during reperfusion. Recovery of heart during reperfusion is improved in the presence of 0.2 µM CsA, suggesting that the MPT may be critical in the transition from reversible to irreversible reperfusion injury. (Mol Cell Biochem 174: 167–172, 1997)
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  • 58
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    Keywords: annexin V ; cardiac myocyte ; growth ; hypertrophy ; heart
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    Notes: Abstract Recently it was shown that annexin V is the most prominent member of the annexin family in the adult heart [1]. Amongst others, annexin V has been suggested to play a role in developmental processes. The aim of the present study was to explore whether in the heart annexin V content and localization change during maturational and hypertrophic growth, in order to obtain indications that annexin V is involved in cardiac growth processes. First, in the intact rat heart annexin V content and localization were studied during perinatal development. It was clearly demonstrated that annexin V content in total heart transiently increased in the first week after birth, from 0.79 ± 0.06 µg/mg protein at l day before birth to a peak value of 1.24 ± 0.08 µg/mg protein 6 days after birth, whereafter annexin V protein levels declined to a value of 0.70 ± 0.06 µg/mg protein at 84 days after birth (p 〈 0.05). Differences in annexin V content were also observed between myocytes isolated from neonatal and adult hearts [0.81 ± 0.09 and 0.17 ± 0.08 µg/mg protein, respectively (p 〈 0.05)]. Moreover, during cardiac maturational growth the subcellular localization of annexin V might change from a cytoplasmic to a more prominent sarcolemmal localization. Second, in vivo hypertrophy induced by aortic coarctation resulted in a marked degree of hypertrophy (22% increase in ventricular weight), but was not associated with a change in annexin V localization or content. The quantitative results obtained with intact hypertrophic rat hearts are supported by findings in neonatal ventricular myocytes, in which hypertrophy was induced by phenylephrine (10-5 M). In the latter model no changes in annexin V content could be observed either. In conclusion, the marked alterations in annexin V content during the maturational growth in the heart suggest a possible involvement of this protein in this process. In contrast, the absence of changes in annexin V content and localization in hypertrophied hearts compared to age matched control hearts suggests that annexin V does not play a crucial role in the maintenance of the hypertrophic phenotype of the cardiac muscle cell. This notion is supported by observations in phenylephrine-induced hypertrophied neonatal cardiomyocytes.
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  • 59
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    Keywords: diabetes ; Ca2+-Mg2+-ATPase ; calcium ; liver plasma membrane
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The alteration in calcium transport in the liver of rats with streptozocin(STZ)-diabetic state was investigated. STZ (6 mg/100 g body weight) was subcutaneously administered in rats, and 1 or 2 weeks later they were sacrificed by bleeding. STZ administration caused a remarkable elevation of serum glucose concentration. Liver calcium content was significantly increased by STZ administration. Hepatic plasma membrane (Ca2+-Mg2+)-ATPase activity was markedly elevated by STZ administration. This increase was completely abolished by the presence of staurosporine (10-7-10-5 M), an inhibitor of protein kinase C, in the enzyme reaction mixture, suggesting an involvement of protein kinase C signalling. Moreover, the STZ-induced increase in liver plasma membrane (Ca2+-Mg2+)-ATPase activity was significantly raised by the presence of okadaic acid (10-5 and 10-4 M). Meanwhile, the STZ-increased (Ca2+-Mg2+)-ATPase activity was not appreciably altered by the presence of anti-regucalcin IgG in the reaction mixture, indicating that the activatory protein regucalcin does not participate in the elevation of the enzyme activity. The present study demonstrates that STZ-induced diabetes causes the increase in hepatic plasma membrane (Ca2+-Mg2+)-ATPase activity of rats.
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  • 60
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    Keywords: heart ; denervation ; catcholamines ; β-adrenoceptors
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Surgical ablation of extrinsic cardiac nerve fibers results in a chronically denervated state of the left ventricle of the heart. The present study was performed to elucidate the effect of a period of five weeks of chronic denervation on cardiac catecholamine levels in general and dopamine in particular. Moreover, the possible effect on cardiac β-adrenoceptor subtypes was investigated. Experiments were performed on adult dogs. In addition to adrenaline and noradrenaline the tissue levels of dopamine were found to be severely depressed. A significant shift from β1- to β2-adrenoceptor subtype was observed, while the total β-adrenoceptor density remained unaffected. The present findings indicate that catecholamine synthesis in chronically denervated hearts is impaired upstream of dopamine and that a shift in β-adrenoceptor subtype occurs already within a relatively short period of five weeks of denervation, and suggest that the lack of endogenous catecholamines influence the relative expression levels of the two subtypes of β-adrenoceptors present in cardiac tissue.
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  • 61
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    Keywords: mitochondrial respiration ; skinned fibers ; permeabilized cell ; heart ; skeletal muscle ; regulation ; cytoskeleton ; myopathies
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    Notes: Abstract In this chapter we describe in details the permeabilized cell and skinned fiber techniques and their applications for studies of mitochondrial function in vivo. The experience of more than 10 years of research in four countries is summarized. The use of saponin in very low concentration (50-100 μg/ml) for permeabilisation of the sarcolemma leaves all intracellular structures, including mitochondria, completely intact. The intactness of mitochondrial function in these skinned muscle fibers is demonstrated in this work by multiple methods, such as NADH and flavoprotein fluorescence studies, fluorescence imaging, confocal immunofluorescence microscopy and respiratory analysis. Permeabilized cell and skinned fiber techniques have several very significant advantages for studies of mitochondrial function, in comparison with the traditional methods of use of isolated mitochondria: (1) very small tissue samples are required; (2) all cellular population of mitochondria can be investigated; (3) most important, however, is that mitochondria are studied in their natural surrounding. The results of research by using this method show the existence of several new phenomenon - tissue dependence of the mechanism of regulation of mitochondrial respiration, and activation of respiration by selective proteolysis. These phenomena are explained by interaction of mitochondria with other cellular structures in vivo. The details of experimental studies with use of these techniques and problems of kinetic analysis of the results are discussed. Examples of large-scale clinical application of these methods are given.
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  • 62
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    Keywords: creatine kinase ; heart ; skeletal muscle ; mitochondria ; respiration ; energy metabolism
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract After discussing approaches to the modelling of mitochondrial regulation in muscle, we describe a model that takes account, in a simplified way, of some aspects of the metabolic and physical structure of the energy production/usage system. In this model, high-energy phosphates (ATP and phosphocreatine) and low energy metabolites (ADP and creatine) diffuse between the mitochondrion and the myofibrillar ATPase, and can be exchanged at any point by creatine kinase. Creatine kinase is not assumed to be at equilibrium, so explicit account can be taken of substantial changes in its activity of the sort that can now be achieved by transgenic technology in vivo. The ATPase rate is the input function. Oxidative ATP synthesis is controlled by juxtamitochondrial ADP concentration. To allow for possible functional ‘coupling’ between the components of creatine kinase associated with the mitochondrial adenine nucleotide translocase and the myofibrillar ATPase, we define parameters ϕ and ψ that set the fraction of the total flux carried by ATP rather than phosphocreatine out of the mitochondrial unit and into the ATPase unit, respectively. This simplification is justified by a detailed analysis of the interplay between the mitochondrial outer membrane porin proteins, mitochondrial creatine kinase and the adenine nucleotide translocase. As both processes of possible ‘coupling’ are incorporated into the model as quantitative parameters, their effect on the energetics of the whole cell model can be explicitly assessed. The main findings are as follows: (1) At high creatine kinase activity, the hyperbolic relationship of oxidative ATP synthesis rate to spatially averaged ADP concentration at steady state implies also a near-linear relationship to creatine concentration, and a sigmoid relation to free energy of ATP hydrolysis. At high creatine kinase activity, the degree of functional coupling at either the mitochondrial or ATPase end has little effect on these relationships. However, lowering the creatine kinase activity raises the mean steady state ADP and creatine concentrations, and this is exaggerated when ϕ or ψ is near unity (i.e. little coupling). (2) At high creatine kinase activity, the fraction of flow at steady state carried in the middle of the model by ATP is small, unaffected by the degree of functional coupling, but increases with ADP concentration and rate of ATP turnover. Lowering the creatine kinase activity raises this fraction, and this is exaggerated when ϕ or ψ is near unity. (3) Both creatine and ADP concentrations show small gradients decreasing towards the mitochondrion (in the direction of their net flux), while ATP and phosphocreatine concentration show small gradients decreasing towards the myosin ATPase. Unless ϕ = ψ ≈ 0 (i.e. complete coupling), there is a gradient of net creatine kinase flux that results from the need to transform some of the ‘adenine nucleotide flux’ at the ends of the model into ‘creatine flux’ in the middle; the overall net flux is small, but only zero if ϕ = ψ. A reduction in cytosolic creatine kinase activity decreases ADP concentration at the mitochondrial end and increases it at the ATPase end. (4) During work-jump transitions, spatial average responses exhibit exponential kinetics similar to those of models of mitochondrial control that assume equilibrium conditions for creatine kinase. (5) In response to a step increase in ATPase activity, concentration changes start at the ATPase end and propagate towards the mitochondrion, damped in time and space. This simplified model embodies many important features of muscle in vivo, and accommodates a range of current theories as special cases. We end by discussing its relationship to other approaches to mitochondrial regulation in muscle, and some possible extensions of the model.
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  • 63
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    Molecular and cellular biochemistry 184 (1998), S. 393-400 
    ISSN: 1573-4919
    Keywords: ATP synthase ; phosphorylation potential ; cytosolic pH ; reperfusion damage ; calcium ; free radicals
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract A short period of ischemia followed by reperfusion produces a state of affairs in which the cells' potential for surviving longer ischemia is enhanced. This is called ischemic preconditioning. The effects of preconditioning are also related to the reperfusion damage which ensues upon tissue oxygenation. The role of the cellular energy state in reperfusion damage remains an enigma, although ischemic preconditioning is known to trigger mechanisms which contribute to the prevention of unnecessary ATP waste. In some species up to 80% of ATP hydrolysis in ischemia can be attributed to mitochondrial F1-F0-ATPase (ATP synthase), and a role for its inhibitor protein (IF1) in ATP preservation has been proposed. Although originally regarded as limited to large animals with a slow heart beat, inhibition by IF1 is probably a universal phenomenon. Coincidentally with ATPase inhibition, the decline in cellular ATP slows down, but even so the difference in ATP concentration between preconditioned and non-conditioned hearts is still small at the final stages of a long ischemia, when the beneficial effect of preconditioning is observable, although the energy state during reperfusion remains low in hearts which do not recover.
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  • 64
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    Keywords: calcium ; Ca2+-ATPase ; DNA fragmentation ; liver nuclei ; liver injury ; carbon tetrachloride
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The alteration in calcium transport in the liver nuclei of rats orally administered carbon tetrachloride (CCl4) was investigated. Rats received a single oral administration of CCl4(5, 10, and 25%, 1.0ml/100 g body weight), and 5, 24 and 48 h later the animals were sacrificed. The administration of CCl4 (25%) caused a remarkable elevetion of calcium content in the liver tissues and the nuclei of rats. Liver nuclear Ca2+-ATPase activity was markedly decreased by CCl4 (25%) administration. The presence of dibutyryl cyclic AMP(10-4 and 10-3 M) or inositol 1,4,5-trisphosphate (10-6 and 10-5 M) in the enzyme reaction mixture caused a significant decrease in Ca2+-ATPase activity in the liver nuclei obtained from normal rat, while the enzyme activity was significantly increased by calmodulin (1.0 and 2.0 μg/ml). These signaling factor's effects were completely impaired in the liver nuclei obtained from CCl4 (25%)-administered rats. DNA fragmentation in the liver nuclei obtained from CCl4 -administered rats was significantly decreased by the presence of EGTA (2 mM) in the reaction mixture, suggesting that the endogenous calcium activates nuclear DNA fragmentation. The present study demonstrates that calcium transport system in the liver nuclei is impaired by liver injury with CCl4 administration in rats.
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  • 65
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    Molecular and cellular biochemistry 189 (1998), S. 9-17 
    ISSN: 1573-4919
    Keywords: sarcoplasmic reticulum ; calcium uptake ; ryanodine ; thapsigargin ; cyclopiazonic acid ; heart
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Thapsigargin is a natural product that specifically inhibits all known SERCA calcium pumps with high affinity. We investigated the effects of thapsigargin on cardiac sarcoplasmic reticulum (SR) by measuring the oxalate-supported calcium uptake rate in the unfractionated homogenate and in the isolated SR fraction. The uptake rate in both the isolated SR and unfractionated homogenate are stimulated about two-fold by preincubation with high concentrations of ryanodine, which closes the SR efflux channel. Thapsigargin stoichiometrically and completely inhibited the calcium uptake rate in the isolated SR, both in the presence and absence of SR channel blockade. In contrast, thapsigargin nearly completely inhibited the homogenate calcium uptake only in the absence of SR channel blockade; in the presence of blockade, about 20% of the uptake activity was insensitive to thapsigargin. This result unmasks a thapsigargin-insensitive, ryanodine-sensitive component of calcium uptake in the heart. This activity is in an oxalate-permeable pool and is inhibited by cyclopiazonic acid, another inhibitor of the SERCA calcium pumps. There was no TG-insensitive activity in the rat EDL muscle homogenate. The absence of thapsigargin-insensitive uptake activity in the isolated SR can be attributed to its inactivation during the isolation of the SR. The oxalate permeability and ryanodine sensitivity suggest that the TG-insensitive calcium uptake activity is closely related to the classical SR. The different thapsigargin sensitivities suggests the existence of two kinds of intracellular calcium pumps in the heart.
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  • 66
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    Molecular and cellular biochemistry 194 (1999), S. 159-164 
    ISSN: 1573-4919
    Keywords: free radicals ; ischemia-reperfusion ; sarcoplasmic reticulum ; Ca2+-Mg2+-ATPase ; calcium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Reactive oxygen species (ROS, free radicals) produced during cardiac ischemia and reperfusion can damage the contractile functions of arteries. The sarcoplasmic reticulum (SR) Ca2+ pump in coronary artery smooth muscle is very sensitive to ROS. Here we show that contractions of de-endothelialized rings from porcine left coronary artery produced by the hormone Angiotensin II and by the SR Ca2+ pump inhibitors cyclopiazonic acid and thapsigargin correlate negatively with the tissue weight. In contrast, the contractions due to membrane depolarization by high KCl correlate positively. Peroxide also produces a small contraction which correlates negatively with the tissue weight. When artery rings are treated with peroxide and washed, their ability to contract with Angiotensin II, cyclopiazonic acid and thapsigargin decreases. Thus, the SR Ca2+ pump may play a more important role in the contractility of the smaller segments of the coronary artery than in the larger segments. These results are consistent with the hypothesis that ROS which damage the SR Ca2+ pump affect the contractile function of the distal segments more adversely than of the proximal segments.
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    Molecular and cellular biochemistry 194 (1999), S. 173-177 
    ISSN: 1573-4919
    Keywords: calcium ; ATPase ; central nervous system ; phencyclidine ; inhibition ; in vitro ; in vivo
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Phencyclidine (PCP) is a potent psychotomimetic drug of abuse and has profound effect on the functioning of the central nervous system (CNS). Many of the CNS functions are known to be mediated by calcium (Ca2+). In the present study we have investigated the effects of PCP on Ca2+ ATPase activity in rat brain both in vitro and in vivo. For in vitro studies, synaptic membrane fractions prepared from normal rat brain were incubated with PCP at different concentrations (25-100 μM) before the addition of substrate. For n vivo studies, rats were treated with a single moderate dose of PCP (10 mg/kg, IP) and animals were sacrificed at 1,2, 6 and 12 h after treatment. Ca2+ ATPase activity in synaptic membrane fractions was assayed by estimation of inorganic phosphate. PCP inhibited the Ca2+ ATPase in vitro in a concentration dependent manner with significant effect at 50 and 100 μM. A significant time-dependent reduction of the Ca2+ ATPase activity was evident in vivo. As early as 2 h after the treatment of rats with PCP the ATPase activity was significantly reduced. The reduction of Ca2+ ATPase observed even at 12 h after treatment suggesting a prolonged presence of the drug in the brain tissue. Further, kinetic studies in vitro indicated PCP to be a competitive inhibitor of Ca2+ ATPase with respect to the substrate, ATP. The present findings indicate that PCP inhibits synaptic membrane Ca2+ ATPase thus altering cellular Ca2+ homeostasis in CNS which may partially explain the pharmacological effects of the drug and/or its neurotoxicity.
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  • 68
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    Keywords: ultraviolet radiation ; oxidative stress ; calcium ; phospholipase A2 ; thrombin ; V79 fibroblast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract V79 fibroblasts were treated with ultraviolet (UV) C radiation alone as well as in conjunction with chronic oxidative stress. The effects of these treatments on calcium signaling were observed at 30 min post-irradiation. In the absence of extracellular calcium, thrombin released calcium from internal stores of UVC-irradiated V79 fibroblasts even after exposure to neomycin. In neomycin-treated control and chronic oxidative stress cells, no calcium release by thrombin was observed after chelation of external calcium. Calcium release by thrombin from internal stores of UV-irradiated and neomycin-treated cells was completely abolished by pretreatment with N-acetyl cysteine and dexamethasone. Cellular total soluble thiol content which is a good indicator of cellular reduced glutathione (GSH) level was significantly elevated 30 min after ultraviolet radiation, indicating an adaptive response after oxidative stress. Chronic oxidative stress alone resulted in a much smaller increase in GSH but chronic oxidative stress in conjunction with UVC produced a very prominent elevation in GSH levels. Our data suggest that thrombin can cause calcium release from internal stores of ultraviolet-irradiated fibroblasts which is independent of phosphatidylinositol bisphosphate hydrolysis and is directly related to the level of oxidative stress. Involvement of phopholipase A2 and a role for its products as possible mediators of calcium release from intracellular stores, is strongly indicated.
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  • 69
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    Molecular and cellular biochemistry 196 (1999), S. 133-139 
    ISSN: 1573-4919
    Keywords: ischemia ; heme oxygenase ; immunohistochemistry ; heart ; pig
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Myocardial adaptation to ischemia involves up-regulated expression of a number of genes implicated in conferring cytoprotection. We have previously shown that myocardial ischemia followed by reperfusion leads to a co-ordinated expression of mRNAs encoding heme oxygenase-1 (HO-1) and ubiquitin in pigs. HO-1 participates in biological reaction leading to the formation of the antioxidant, bilirubin and the putative cellular messenger, carbon monoxide. In the present study, we examined the expression and cellular localization of HO-1 in the heart during myocardial stunning in anesthetized pigs. After thoracotomy, the LAD was occluded for 10 min and reperfused for 30 min (group I, n = 4), again occluded for 10 min and reperfused for 30 min (group II, n = 6), 90 min (group III, n = 4), 210 min (group IV, n = 5) and for 390 min (group V, n = 4). Myocardial tissue specimens were collected in 10% formalin as well as in liquid nitrogen and processed for immunohistochemistry and mRNA expression analysis, respectively. In the distribution territory of the LAD (experimental, E), systolic wall thickening was significantly decreased (39 ± 6%) as compared to that of the area perfused by left circumflex coronary artery (LCx, control) in group I and remained depressed in all subsequent groups. Northern blot analysis revealed that the expression of a single mRNA species of 1.8 kb encoding HO-1 was significantly induced in E as compared to control in groups II and III with maximum mRNA levels in group II (1.9 ± 0.4 fold vs. control). Immunoreactive HO-1 was localized in the cytoplasm of cardiomyocytes as well as in the perivascular regions in all groups. Semiquantitative analysis of HO-1 staining showed significantly enhanced levels of HO-1 in perivascular region in E as compared to respective controls derived from groups III and IV. These results suggest that myocardial adaptive response to ischemia involves up-regulation of HO-1 in cells of perivascular region indicating that this enzyme may participate in regulating vascular tone via CO and thereby, contributing in pathophysiologically important defense mechanism(s) in the heart.
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    Molecular and cellular biochemistry 197 (1999), S. 25-29 
    ISSN: 1573-4919
    Keywords: regucalcin ; anti-regucalcin antibody ; protein phosphatase ; calcium ; rat liver cytosol
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The effect of anti-regucalcin monoclonal antibody on neutral phoshatase activity in rat liver cytosol was investigated. Phosphotyrosine, phosphoserine, and phosphothreonine were used as the substrate toward phosphatase asssy. Liver cytosolic phosphatase activity with three phosphoaminoacids was significantly increased in the presence of anti-regucalcin antibody (100 and 200 ng/ml) in the enzyme reaction mixture with calcium chloride (0.1 mM) or EGTA (1.0 mM). The effect of anti-regucalcin antibody was completely abolished in the presence of exogenous regucalcin (1.0 μM), indicating the involvement of endogenous regucalcin. The anti-regucalcin anti body- increased phosphatase activity was not significantly altered in the presence of trifluoperazine (20 μM), an antagonist of calmodulin, or akadaic acid (10 μM), an inhibitor of protein phosphatase, although these inihibitors caused a slight decrease in liver cytosolic phosphatase activity. The effect of endogenous regucalcin might be not related to calmodulin, and it was insensitive to okadaic acid. The present findings suggest that endogenous regucalcin is involved in the regulation of protein phasphatase in rat liver cytoplasm.
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  • 71
    ISSN: 1573-4919
    Keywords: isolated cardiac mitochondria ; cyclosporin A ; calcium ; magnesium ; oxidative phosphorylation ; high energy phosphate production ; Krebs cycle intermediates
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract This study was designed to determine the effect of calcium and ADP-Mg on the oxidative phosphorylation in isolated cardiac mitochondria. The influence of cyclosporin A was also evaluated. The mitochondria were extracted from rat ventricles. Their oxidative phosphorylations were determined in two respiration media with different free Ca2+ concentrations. Respiration was determined with palmitoylcarnitine and either ADP- or ADP-Mg. With elevated free Ca2+concentrations and ADP-Mg, the transition state III to state IV respiration did not occurred. The ADP:O ratio was reduced. The phenomenon was not observed in the other experimental conditions (low free Ca2+ concentration with either ADP- or ADP-Mg or elevated free Ca2+ concentration with ADP-). Uncoupling was allied with a constant AMP production, which maintained an elevated ADP level in the respiration medium and prevented the return to state IV respiration. It was also observed in a respiration medium devoid of free Ca2+ when the mitochondria were pre-loaded with Ca2+. Uncoupling was inhibited by cyclosporin A. Furthermore, the Krebs cycle intermediates released from14C-palmitoylcarnitine oxidation revealed that succinate was increased by elevated free Ca2+ and ADP-Mg. Succinate is a FAD-linked substrate with low respiration efficiency. Its accumulation could account for the decreased ADP:O ratio. The Ca2+- and ADP-Mg-induced uncoupling might be partly responsible for the mechanical abnormalities observed during low-flow ischemia. (Mol Cell Biochem 000: 000-000, 1999)
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  • 72
    ISSN: 1573-4919
    Keywords: heart ; atrium ; rat ; function ; sex/gender ; isometric contraction ; force ; calcium sensitivity ; myofibrillar ATPase ; adrenergic agonists ; isoproteronol ; phenylephrine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract A number of investigations in humans and animals suggest that there may be intrinsic sex-associated differences in cardiac function. Using left atrial preparations from male and female rat hearts, we examined differences in myocardial function and response to adrenergic agonists. Contractile parameters were measured in isolated atria by conventional isometric methods in the absence or presence of isoproterenol or phenylephrine. Responsiveness to Ca2+ was measured in detergent-skinned atrial fibers and actomyosin ATPase activity was measured in isolated myofibrils. Tetanic contractions were generated by treating the atrium with ryanodine followed by high frequency stimulation. Developed force was greater and maximal rates of contraction and relaxation were more rapid in the female atrium. The relationship between Ca2+ concentration and force in both intact atria and detergent-skinned atrial fibers in females fell to the left of that for males. At low Ca2+ concentrations, skinned fibers from female atria generated more force and myofibrils from female atria had higher myosin ATPase activity than males. Tetanic contraction in the presence of high extracellular Ca2+ was greater in female atria. Male atrium had larger inotropic responses to isoproterenol and to phenylephrine, but drug-elicited cAMP and inositol phosphate production did not differ between sexes. The results demonstrate sex-related differences in atrial function that can be partially explained by greater myofibrillar Ca2+-sensitivity in females. A potential contribution of sarcolemmal Ca2+ influx is suggested by greater tetanic contraction in ryanodine-treated female atrium. The larger response of males to adrenergic stimulation does not appear to be explained by higher production of relevant second messengers. Future studies will investigate the role of sex hormones in these sexually dimorphic responses and may indicate a need for gender-specific therapeutic interventions for myocardial dysfunction.
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  • 73
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    Keywords: Salmonella typhimurium ; diarrhoea ; porins ; calcium ; protein kinase C ; free radicals
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Attachment of Salmonella typhimurium to epithelial surfaces elicit significant alterations in different cell signalling events which lead to the development of disease. The present investigation was conducted to evaluate the effect of immunization of rats with porins, on gut physiologic markers following challenge with S. typhimurium. Male albino Wistar rats were immunized with purified porins and challenged by intragastric infection with S. typhimurium. Electrolyte transport, levels of different second messengers and inflammatory mediators were studied. A net absorption of transepithelial fluxes of Na+ and Cl- in immunized-challenged group and secretion in infected group was found. Ca2+ and 3-O-methyl-D-glucose fluxes did not show any change. Significant increase in the levels of [Ca+]i, cAMP, membrane form of protein kinase C, prostaglandins, NADPH oxidase, Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, total oxygen free radicals, reactive nitrogen intermediates, citrulline and lipid peroxidation was found in the infected group. However, in the immunized-challenged group, the values of all the parameters were found to be almost the same as that of control as well as immunized groups. Na+, K+-ATPase and calmodulin levels were unaltered in all the groups of animals. The results of this study thus suggest that immunization of rats with purified Salmonella porins followed by subsequent challenge with the organism might be helpful for the prevention of multiple physiologic derangements in isolated ideal cells.
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  • 74
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    Molecular and cellular biochemistry 151 (1995), S. 39-47 
    ISSN: 1573-4919
    Keywords: 7B2 ; calcium ; protein aggregation ; secretogranins ; protein sorting
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract To study the behavior of the neuroendocrine polypeptide 7B2 in the presence of calcium, various fragments of this molecule were produced inEscherichia coli as fusion proteins to glutathione S-transferase (GST). Addition of millimolar concentrations of Ca2+ to purified preparations of hybrid molecules carrying the N-terminal segment of 7B2 induced precipitation in a manner dependent on protein and cation concentrations. This precipitation occurred at pH 7.5 but not at pH 5.2. It was augmented by 4 and 8 mM ATP, and reduced by 12 and 24 mM ATP. ADP had a similar but weaker effect. Calcium failed to cause precipitation of GST alone or of GST fused to the C-terminal peptide 7B2156–186. However, when the latter protein was mixed with a GST protein carrying a short fragment of the N-terminal region of 7B2, both proteins were precipitated by calcium. Except for the pH dependence, the behavior of 7B2 fusion proteins in the presence of calcium and adenosine nucleotides are reminiscent of those exhibited by chromogranins and secretogranins, which, like 7B2, are acidic proteins found in the secretory granules of a variety of neuroendocrine cells. As suggested for other granins, this property may underlie the segregation of 7B2 fragments into secretory granules.
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  • 75
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    Molecular and cellular biochemistry 151 (1995), S. 55-60 
    ISSN: 1573-4919
    Keywords: regucalcin ; calcium ; gene expression ; kidney damage ; rat kidney cortex
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The alteration of Ca2+-binding protein regucalcin mRNA expression in the kidney cortex of rats administered cisplatin and cephaloridine, which can induce kidney damage, was investigated. Cisplatin (0.25, 0.5 and 1.0 mg/100 g body weight) or cephaloridine (25, 50 and 100 mg/100 g) was intraperitoneally administered in rats, and 1, 2 and 3 days later they were sacrificed. The alteration in serum findings after the administration of cisplatin (1.0 mg/100 g) or cephaloridine (50 and 100 mg/100 g) demonstrated chemically induced kidney damage; blood urea nitrogen (BUN) concentration increased markedly and serum inorganic phosphorus or calcium concentration decreased significantly. Moreover, the administration of cisplatin (1.0 mg/100 g) or cephaloridine (100 mg/100 g) caused a remarkable increase of calcium content in the kidney cortex of rats, indicating kidney damage. The expression of regucalcin mRNA in the kidney cortex was markedly reduced by the administration of cisplatin or cephaloridine in rats, when the mRNA levels were analyzed by Northern blotting using rat liver regucalcin cDNA (0.9 kb). The mRNA decreases were seen with the used lowest dose of cisplatin or cephaloridine. The present study clearly demonstrates that the mRNA expression of Ca2+-binding protein regucalcin in the kidney cortex of rats is decreased by chemically induced kidney damage.
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  • 76
    ISSN: 1573-4919
    Keywords: hyperthyroid heart ; high energy phosphates ; oxidative metabolism ; cardiac work ; calcium ; 31P-NMR spectroscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The effect of calcium activation on energy production was investigated in isolated perfused hearts from rats treated with triiodothyronine (T3) during 15 days (0.2 mg/kg/day) and in hearts of rats allowed to recover after T3-treatment during 15 days. Changes in phosphorylated compound concentrations were followed in the isolated hearts perfused with a glucose-pyruvate medium by 31P-NMR spectroscopy, when the external calcium concentration was increased from 0.5–1, 1.5 and 2 mM. As expected, T3-treatment resulted in the hypertrophy of the heart (50% increase in HW/BW) that was nearly reversible 15 days after discontinuation of the treatment. When compared to controls, creatine, phosphocreatine (PCr) and glycogen contents were lower (58, 24 and 17% decrease respectively) in the hypertrophied hearts and higher (10, 14 and 18% respectively) after regression of hypertrophy. Intracellular pH, ATP, inorganic phosphate concentrations and the phosphorylation potential were not altered under T3-treatment and after regression of hypertrophy, while calculated free ADP concentration was lower in hypertrophied hearts (control: 40±2 μM, T3-treatment: 21±1 μM, regression: 37±1 μM). Increasing the calcium concentration induced a similar increase in left ventricular developed pressure in the three groups of hearts, with inorganic phosphate concentration increasing with cardiac work. The PCr concentration slightly decreased while the ATP concentration did not change. In spite of different initial PCr concentrations, the evolutions of PCr and Pi concentrations for each stepwise increase in external calcium were similar in the three groups. It is concluded that, in spite of the well-known decrease in efficiency induced by the drug, the mechanisms of PCr (ATP) production ramain able to respond to an acute moderate increase in energy demand provoked by a physiological stimulus. This adaptation also persists after the treatment when the energy metabolism balance is apparently improved.
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  • 77
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    Molecular and cellular biochemistry 151 (1995), S. 149-155 
    ISSN: 1573-4919
    Keywords: SERCA2 ; ATPase ; calcium ; transport ; vascular
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Pig coronary artery cultured smooth muscle cells were skinned using saponin. In the presence of an ATP-regenerating system and oxalate, the skinned cells showed an ATP-dependent azide insensitive Ca2+-uptake which increased linearly with time for 〉1 h. The Ca2+-uptake occurred with Km values of 0.20±0.03 μM for Ca2+ and 400±34 μM for MgATP2−. Thapsigargin and cyclopiazonic acid inhibited this uptake with IC50 values of 0.13±0.02 and 0.56±0.04 μM, respectively. These properties of SR Ca2+-pump are similar to those reported for membrane fractions isolated from fresh smooth muscle of coronary artery and other arteries. However, optimum pH of the uptake in the skinned cells (6.2) was lower than that reported previously using isolated membranes (6.4–6.8).
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  • 78
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    Molecular and cellular biochemistry 154 (1996), S. 113-121 
    ISSN: 1573-4919
    Keywords: heart cells ; nucleus ; calcium ; R-type channel ; excitation-contraction coupling ; pacemaker activity ; Fura-2 ; Fluo-3 ; confocal microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract In the present study, Fluo-3 Ca2+ measurement and confocal microscopy techniques were used in order to localize cytosolic [ ]c and nuclear [ ]n free Ca2+ distribution in resting and spontaneously contracting single heart cells from 10-day-old chick embryos. In resting single cells, the concentration of Ca2+ in the cytoplasm was lower than that in the nucleus. Increasing cytosolic free Ca2+ from 100–1600 nM gradually increased [Ca2+]n with a maximum capacity near 1200 nM. Results from Fura-2 microfluorometry and Fluo-3 confocal microscopy suggest a potential cross talk between the increase of cytosolic free Ca2+ and the uptake and release of Ca2+ by the nucleus during spontaneous contraction of single myocytes. Calcium waves in spontaneously contracting cells were found to spread from one cell to the next with the nucleus acting as a fluorescent beacon in which Ca2+ levels remained elevated for several milliseconds even after cytosolic Ca2+ had returned to near basal values. These results strongly suggest that the nucleus plays a negative and positive feedback role in controlling cytosolic free Ca2+ concentration during excitation-contraction coupling in heart cells.
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  • 79
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    Molecular and cellular biochemistry 154 (1996), S. 165-170 
    ISSN: 1573-4919
    Keywords: dexamethasone ; glycohydrolases ; cathepsins ; serum ; heart ; kidney
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Glucocorticoids have been used in the treatment of a number of diseases where immunological intolerance plays a predominant role. Since immunological intolerance points to the involvement of lysosomal enzymes and glucocorticoids are known to affect their activities, we have attempted to study the effect of these steroids on cardiac and renal enzymes. Dexamethasone, a glucocorticoid, is administered subcutaneously to male Wistar rats at a dosage of 2.5 mg/kg/week on alternate days for two weeks. After withdrawing the steroid, the animals are monitored for one week to oversee the recovery process. Total and free activities of glycohydrolases and cathepsins in serum, heart and kidney are assayed on the days 4, 8, 12, 16 of dexamethasone administration and also on days 4 and 8 following discontinuation of the steroid. During dexamethasone administration, a significant decrease in both the free and total activities of β-glucuronidase, β-N-acetyl glucosaminidase, β-galactosidase, α-galactosidase, α-mannosidase, cathepsin B and cathepsin D are observed in heart and kidney, but the enzyme levels are shown to increase in serum. On withdrawal of the steroid, the activities of β-glucuronidase, β-N-acetyl glucosaminidase, β-galactosidase are found to be increased in heart and kidney, whereas, the activity of α-mannosidase remains within normal values. Thus, it could be seen that dexamethasone alters the pattern of glycohydrolases and cathepsins, which are involved in protein degradation.
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  • 80
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    Molecular and cellular biochemistry 156 (1996), S. 17-24 
    ISSN: 1573-4919
    Keywords: antioxidant enzymes ; heart ; buthionine sulfoximine ; GSH-deficiency
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The role of glutathione (GSH) in myocardial antioxidant defense was investigated in Swiss-Webster mice either performing swim exercise to exhaustion or rested in both the GSH adequate (GSH-A) and GSH deficient (GSH-D) states. GSH deficiency was accomplished by injecting mice with L-buthionine [S,R]sulfoximine (BSO; 2 nmol/kg body wt, i.p.) and providing BSO (20 mM) in drinking water for 12 days. GSH and glutathione disulfide (GSSG) contents in the GSH-D hearts were decreased to 10 and 8%, respectively, of those in the GSH-A mice. This decrease was associated with a significant decline of the total glutathione level in the liver, skeletal muscle and plasma. Myocardial GSH peroxidase and GSH sulfur-transferase activities decreased significantly following GSH deficiency, whereas superoxide dismutase activity was significantly elevated. GSH deficiency did not affect exercise endurance performance. However, exhaustive exercise decreased GSH content in the myocardium of the GSH-A and GSH-D mice by 22 and 44% (p 〈 0.05), respectively. The GSH:GSSG ratio was not altered significantly following exercise because of a concomitant decrease in GSSG (p 〈 0.05). γ-Glutamyltranspeptidase activity was significantly increased after exercise, especially in the GSH-D hearts (72%; p 〈 0.05). GSH content after exercise correlated negatively with exercise time in both GSH-A and GSH-D mice (p 〈 0.05). These data indicate that GSH is actively used in the myocardium during prolonged exercise at moderate intensity and that GSH deficiency is tolerated by the heart, possibly compensated for by an increased GSH uptake from the plasma.
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  • 81
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    Keywords: ischemia ; heart ; endothelium ; lactate ; urate ; Iysolecithin ; carnitine ; long-chain acylcarnitine (LCAC)
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    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Rat Langendorff hearts perfused with media that do not contain erythrocytes or fluorocarbon as oxygen carriers are borderline aerobic during 5 Hz pacing. This follows from the release of catabolic products measured: lactate, urate and lysophosphatidylcholine (IysoPC). Addition of L-carnitine to the perfusion medium reduced the level of these compounds, while the release of long-chain acylcarnitine (LCAC) increased. Previously, we found (Biochem Biophys Acta 847:62–66,1985) that micromolar LCAC protects membranes during reperfusion after ischemia, Therefore, the observed inverse relation between LCAC and the other compounds measured suggests that LCAC is the basis of an acute relief of imminent ischemia by carnitine addition. LCAC may be released from various cell types, including vascular endothelium, as demonstrated. The cationic amphiphilic nature of LCAC is responsible for protection of membrane functions in imminent ischemia. (Mol Cell Biochem 156: 87-91, 1996)
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  • 82
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    Keywords: calcium ; calcium transport ; brain ; calcium-regulating hormone ; calcium-antagonist ; energy dependency
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The characterization of calcium accumulation in the brain of rats administered orally calcium chloride solution was investigated. Rats received a single oral administration of calcium (15–50 mg/100 g body weight), and they were sacrificed by bleeding-between 15 and 120 min after the administration. The administration of calcium (50 mg/100 g) produced a significant increase in serum calcium concentration and a corresponding elevation of brain calcium content, indicating that the transport of calcium into the brain is associated with the elevation of serum calcium levels. The increase in brain calcium content by calcium administration was not appreciably altered by the pretreatment with Ca2+ channel blockers (verapamil or diltiazem with the doses of 1.5 and 3.0 mg/100 g). In thyroparathyroidectomized rats, the administration of calcium (50 mg/100 g) caused a significant increase in brain calcium content, indicating that calcium-regulating hormones do not participate in the brain calcium transport. Now, brain calcium content was clearly elevated by fasting (overnight), although serum calcium level was not significantly altered. Calcium administration to fasted rats induced a further elevation of brain calcium content as compared with that of control (fasted) rats. The fasting-induced increase in brain calcium content was appreciably restored by refeeding. This restoration was also seen by the oral administration of glucose (0.4 g/100 g) to fasted rats. The present study demonstrates that serum calcium is transported to brain, and that the increased brain calcium is released promptly. The release of calcium from brain may be involved in energy metabolism, and this release may be weakened by the reduction of glucose supply into brain. The finding suggests a physiological significance of energy-dependent mechanism in the regulation of brain calcium.
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  • 83
    ISSN: 1573-4919
    Keywords: hydroxyl radical ; oxidant ; hydrogen peroxide ; smooth muscle tissue ; mitochondria ; calcium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract We sought to investigate the mechanism(s) by which the oxidant H2O2 stimulates Ca2+ release from mitochondria of bovine pulmonary vascular smooth muscle tissue and to test the hypothesis that hydroxyl radical is involved in this phenomenon. Treatment of the smooth muscle tissue with 1 mM H2O2 dramatically stimulated hydroxyl radical generation as measured by methane (CH4) production by GLC using dimethylsulfoxide (DMSO) as the substrate. Pretreatment of the mitochondria with the hydroxyl radical scavanger dimethylthiourea (DMTU) prevented the increase in CH4 production caused by H2O2. In the absence of EGTA, H2O2 caused stimulation of Ca2+ release from mitochondria occurred with a lag time of about 4 min. Addition of EGTA to Ca2+ loaded mitochondria resulted an immediate loss of Ca2+ and that has been found to be augmented by H2O2. The release of Ca2+ by H2O2 did not appear to occur with concommitant increase in sucrose entry into, K+ release from, and swelling of mitochondria when the Ca2+ cycling was prevented by EGTA. These observations suggested that H2O2-mediated Ca2+ release from bovine pulmonary vascular smooth muscle tissue mitochondria occurred (i) through the involvement of hydroxyl radical; (ii) via specific pathway(s); and (iii) did not appear to happen primarily via nonspecific ‘pore’ formation.
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  • 84
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    Molecular and cellular biochemistry 160-161 (1996), S. 137-141 
    ISSN: 1573-4919
    Keywords: cardiomyocytes ; ischemia ; heart ; reoxygenation ; ATP ; LDH ; hexosaminidase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The LDH release pattern from cardiomyocytes under ‘ischemia-like’ conditions shows two phases. In the initial slow phase, reoxygenation immediately stops further enzyme release. Accelerated LDH release, which occurs concomitantly with Iysosomal enzyme release, characterizes the second phase of ‘ischemia.’ Reoxygenation at this stage does not put a stop to further enzyme release. Reoxygenation during the first phase of ‘ischemia’ rapidly restored ATP level, while in the second phase, ATP levels remained low even after 6 h of reoxygenation. This study as well as previous data seem to suggest that irreversible cellular damage leading to cell death, occurs by synergistic action of many effectors, each of which does not necessarily cause irreversible damage.
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  • 85
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    Keywords: heart ; cell culture ; neurons ; cardiomyocytes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract In this study, we used laser confocal scanning microscopy and immunofluorescent markers to describe the establishment of long-term cultures of adult guinea-pig cardiomyocytes and their cocultures with adult intrinsic cardiac neurons. We have also investigated the effect of plating density on the adaptation of the myocytes in culture. Providing that the preparation of freshly isolated cardiomyocytes consists mostly (〉 80%) of rod-shaped, Ca-tolerant, and quiescent cells and these are plated under optimal conditions and density (105/cm2), these myocytes have the following characteristics: (1) they remain elongated with regular ultrastructural characteristics and quiescent for several days; (2) within 10-14 days, they reestablish their intercellular contacts and resume contractile activity, which becomes synchronous all through the confluent layers; (3) they retain their regular myofibrilar striation all through the adaptation to culture conditions without any sign of dedifferentiation or redifferentiation; (4) these characteristics are lost when the cells are plated at too low (〈 104/cm2) or too high (2 × 105/cm2) a density and they exhibit signs of dedifferentiation; (5) the adult ventricular myocytes appear to retain their ability to express atrial natriuretic peptide (ANP), as indicated by immunoreactivity to anti-ANP antibody; (6) this activity seems to be directly related to the surface area of the myocytes in contact with the substrate (i.e. to the ‘stretch’ of the myocytes); (7) the intrinsic cardiac neurons grow intricate networks of neurites, which form a free-ending type of contact with the cocultured myocytes.abstract typed in here; if it is more than one paragraph use Long-term cultures of adult guinea-pig ventricular myocytes, alone or in their cocultures with cardiac neurons in which both are fully active functionally, provide a valuable experimental model which opens new possibilities for studying the cellular and molecular regulation of myocardial function under acute or chronic effects of various intrinsic and/or extrinsic factors, including neuroregulation.
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    Molecular and cellular biochemistry 174 (1997), S. 79-85 
    ISSN: 1573-4919
    Keywords: heart ; mitochondrial respiration in skinned fibers ; creatine kinase ; ischemia ; preservation ; cardioplegia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The skinned fibers technique was applied for studies of the effects of global acute ischemia (1 h at 37°C) and long time (15 h) hypothermic (4°C) preservation of isolated rat hearts under different conditions (immersion or low-flow perfusion) on mitochondrial function in the cells in vivo. Skinned fibers were obtained by using saponin for permeabilization of the sarcolemma in separated fiber bundles cut from left ventricle. The experimental protocol of the respiration rate determination included a cytochrome c test to check the intactness of the outer mitochondrial membrane. The apparent Km for ADP and the effect of creatine on the mitochondrial activity were also evaluated in these permeabilized fibers, taken from different groups of hearts. The preservation of low-flow perfused hearts resulted only in a slight decrease of creatine (20 mM) stimulated respiration at 0.1 mM ADP. The fibers from ischemic hearts or from hearts preserved by immersion showed a decrease of the apparent Km for ADP, and a complete loss of the stimulatory effect of creatine. In these fibers, we could observe that the outer mitochondrial membrane was damaged. In conclusion, the results of this study show that assessment of mitochondrial parameters sensitive to organelles swelling – intactness of outer membrane and functionally coupled creatine kinase reaction – are the most sensitive indicators of early hypoxic or ischemic damage to mitochondria. Their determination in biopsy samples could be used for evaluation of the efficiency of the cardiac protection in heart surgery. (Mol Cell Biochem 174: 79–85, 1997)
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    Molecular and cellular biochemistry 173 (1997), S. 169-175 
    ISSN: 1573-4919
    Keywords: regucalcin ; calcium-binding protein ; calcium ; nuclear RNA synthesis ; regenerating ratliver
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The effect of regucalcin, a Ca2+-bindingf protein isolated from rat livercytosol, on ribonucleic acid (RNA) synthesis in the nuclei of normal ratliver and of regenerating rat liver was investigated. The liver weight at 1day after partial hepatectomy was increased about 50% of that ofsham-operated (control) rats. Calcium chloride (1.0-20 µM Ca2+ asfinal concentration) was added into the reaction mixture of nuclear RNAsynthesis. RNA synthesis was established by incorporation of [3H]-uridine5'-triphosphate (UTP) into the nuclear RNA. Addition of Ca2+ (5 and 10µM) caused a significant increase of RNA synthesis in the nuclei fromcontrol rat liver. Such effect of Ca2+ was potentiated in the nuclei ofregenerating liver; nuclear RNA synthesis was increased about 2 fold by the1.0 and 2.5 µM Ca2+ addition. The stimulatory effect of Ca2+ wassignificantly inhibited by the presence of a-amanitin (10-8 M), an inhibitorof RNA polymerase II. The presence of regucalcin (0.25 and 0.5 µM)significantly inhibited RNA synthesis in the nuclei from control rat liverand from regenerating rat liver. The inhibitory effect of regucalcin wasremarkable in the presence of EGTA (0.5 mM), and it was weakened by theaddition of Ca2+ (5 µM). Such regucalcin effect was not seen in thepresence of a-amanitin. The presence of anti-regucalcin IgG in the reactionmixture significantly increased RNA synthesis in the nuclei from control ratliver, indicating that the endogenous regucalcin may be involved in nuclearRNA synthesis. The present resuits demonstrate that regucalcin can inhibitnuclear RNA synthesis in rat liver. Regucalcin may have an inhibitory rolein the regulation of liver nuclear RNA synthesis.
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    Molecular and cellular biochemistry 149-150 (1995), S. 175-182 
    ISSN: 1573-4919
    Keywords: rat pancreas ; cholecystokinin ; magnesium ; calcium ; acetylcholine ; amylase secretion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Application of either acetylcholine (ACh, 10−5 M) or cholecystokininoctapeptide (CCK-8, 10−8 M) to the isolated rat pancreas elicited large increases in amylase secretion, radiolabelled45Ca2+ influx and cytosolic free calcium [Ca2+]i levels in zero and normal (1.1 mM) extracellular magnesium [Mg2+]o. Elevated [Mg2+]o significantly (p〈0.001) reduced the secretagogueevoked secretory responses and Ca2+ mobilisation. Stimulation of pancreatic segments with either ACh (10−5 and 10−6 M) or CCK-8 (10−8 and 10−10 M) resulted in marked elevation in Mg2+ concentration in effluent samples (net efflux). On removal of either ACh or CCK-8, Mg2+ concentration returned to resting level. In pancreatic acinar cells loaded the flourescent dye magfura, ACh and CCK-8 evoked marked reduction in cytosolic free Mg2+ concentration [Mg2+]i compared to the resting value of 0.82±0.03 mM (n=50) in normal medium in the absence of secretagogues. In elevated [Mg2+]o (10 mM) medium, [Mg2+]i rises to 0.98±0.04 mM (n=6). Addition of CCK-8 led to only a small reduction in [Mg2+ i in elevated [Mg2+]o. In Mg2+ loaded pancreatic acinar cells, Mg2+ is released in a time dependent manner and this efflux of Mg2+ was sensitive to sodium, extracellular amiloride (1 mM), dinitrophenol (10 mM) and lidocaine (1 mM). The results indicate that Mg2+ is acting as an intracellular messenger to regulate the mobilisation of Ca2+ which in turn mediates enzyme secretion.
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  • 89
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    Molecular and cellular biochemistry 175 (1997), S. 153-162 
    ISSN: 1573-4919
    Keywords: heart ; trimetazidine ; lipid ; metabolism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Trimetazidine (TMZ) is an anti-ischemic compound devoid of hemodynamic effects. It was recently suggested to induce cardiomyocyte protection by a mechanism involving lipid metabolism. The effects of TMZ were evaluated in rats on cardiac lipid composition, and in cultured rat cardiomyocytes on phospholipid metabolism. Rats were treated with TMZ for 4 weeks, and the fatty acid compositions were determined. Treatment with TMZ induced a significant decrease in phospholipid linoleic acid, balanced by a small increase in oleic and stearic acids. These changes were not correlated to alterations in plasma fatty acid composition. Cultured ventricular myocytes were treated with TMZ, 16 and l before experimentation. The time-dependent incorporation of radio labelled precursors of membrane phospholipids (3-inositol, 14C-ethanolamine, 14 C-choline, 14C-arachidonic acid, 10 µmol/L) was investigated. The cells were harvested 30, 60, 105 or 150 min after precursor addition. In TMZ-cells, arachidonic ac id (AA) incorporation was increased in the phospholipids, but not in other lipid fractions. This increase elicited a net increase in the total AA uptake. The incorporation of 3-inositol in the phospholipids was strongly stimulated by TMZ, although the uptake of inositol was not altered. The difference was significant within 30 min, and after 150 min the phospholipid labelling in TMZ cells was higher by 70%. A similar result was obtained with ethanolamine as precursor, which turnover increased by 50% in TMZ-treated cells. Conversely, the incorporation of choline was not significantly affected by the presence of TMZ. In conclusion TMZ appears to interfere with the metabolism of phospholipids in cardiac myocytes in a manner which could indicate an increase of membrane phospholipid turnover. (Mol Cell Biochem 175: 153–162, 1997)
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  • 90
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    Molecular and cellular biochemistry 176 (1997), S. 317-326 
    ISSN: 1573-4919
    Keywords: calcium ; metabolism ; glucose ; hypoxia ; rat brain
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract In a previous communication we reported that glucose deprivation from KHRB medium resulted in a marked stimulation of Ca2+ uptake by brain tissue, suggesting a relationship between glucose and Ca2+ homeostasis in brain tissue [17]. Experiments were carried out to investigate the significance of glucose in Ca2+ transport in brain cells. The replacement of glucose with either D-methylglucoside or 2-deoxyglucose, non-metabolizable analogues of glucose, resulted in stimulation of Ca2+ uptake just as by glucose deprivation. These data show that glucose metabolism rather than glucose transfer was necessary to stimulate Ca2+ uptake in brain tissue. Inhibition of glucose metabolism with either NaF, NaCN, or iodoacetate resulted in stimulation of Ca2+ uptake similar to that produced by glucose deprivation. These results lend further support for the concept that glucose metabolism is essential for Ca2+ homeostasis in brain. Anoxia promotes glucose metabolism through glycolytic pathway to keep up with the demand for ATP by cellular processes (the Pasteur effect). Incubation of brain slices under nitrogen gas did not alter Ca2+ uptake by brain tissue, as did glucose deprivation and the inhibitors of glucose metabolism. We conclude that glucose metabolism resulting in the synthesis of ATP is essential for Ca2+ homeostasis in brain. Verapamil and nifedipine which block voltage-gated Ca2+ channels, did not alter Ca2+ uptake stimulated by glucose deprivation, indicating that glucose deprivation-enhanced Ca2+ uptake was not mediated by Ca2+ channels. Tetrodotoxin which specifically blocks Na+ channels, abolished Ca2+ uptake enhanced by glucose deprivation, but had no effect on Ca2+ uptake in presence of glucose (controls). These results suggest that stimulation of Ca2+ uptake by glucose deprivation may be related to Na+ transfer via Na-Ca exchange in brain.
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  • 91
    ISSN: 1573-4919
    Keywords: Glut4 translocation ; heart ; fatty Zucker rat ; insulin ; word ; subcellular fractionation ; ELISA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Impaired cardiac glucose metabolism and glucose transport have been shown in the insulin resistant fatty Zucker rat. The aim of the present study was to examine the translocation of the insulin-sensitive glucose transporter (Glut4) in the heart of the fatty Zucker rat under in vivo conditions. Insulin was injected into both lean (FA/?) and fatty (fa/fa) Zucker rats via the tail vein. The time course of cardiac Glut4 translocation was studied by determining the subcellular distribution of Glut4 using a newly developed ELISA quantitation method. Insulin (10 U/kg) caused a 30% and 37% increase in plasma membrane Glut4 content at 20 min after injection in lean and fatty rats respectively. The plasma membrane Glut4 contents in the basal and insulin-stimulated states were significantly lower in the fatty rat when compared to the lean control. The dose effect of insulin (2.5-10 U/kg) on Glut4 mobilization to the plasma membrane was similar in both phenotypes. The time course of Glut4 mobilization to the plasma membrane (5-30 min), which was similar in both lean and fatty Zucker rats, showed that maximal translocation was reached at 5 min post insulin injection and persisted throughout the remaining 25 min. However, in fatty Zucker rats, Glut4 content in the intracellular membrane remained unchanged at all insulin doses and all time points studied. Collectively, these results show that Glut4 recruitment to the plasma membrane is responsive to insulin in the fatty Zucker rat heart and that the maximal response was similar to that in lean Zucker rats. However, the recruitment of Glut4 to the plasma membrane was not associated with changes in the intracellular membrane Glut4 content.
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  • 92
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    Molecular and cellular biochemistry 184 (1998), S. 101-105 
    ISSN: 1573-4919
    Keywords: heart ; microtubules ; desmin ; ADP ; porin ; mitochondria-associated proteins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract It has been well established that the cytoskeleton is an essential modulator of cell morphology and motility, intracytoplasmic transport and mitosis, however cytoskeletal linkage to the organelles has not been unequivocally demonstrated. Indeed, cytoskeleton appears to be essential in determining and modulating gene phenotype as a function of cellular environment. According to recent studies, the organization of the cytoskeleton network together with associated protein(s) could be essential in regulating mitochondrial function and particularly the permeability of the mitochondrial outer membrane to ADP. The aim of this chapter is to summarize the main properties of the cytoskeletal environment of mitochondria and the possible role(s) of this network in mitochondrial function in myocytes.
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  • 93
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    Molecular and cellular biochemistry 194 (1999), S. 117-123 
    ISSN: 1573-4919
    Keywords: glycogenin ; differential display ; heart ; development ; cloning
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Glycogenin is a self-glycosylating protein required to initiate glycogen biosynthesis. Utilizing the differential display technique to analyze changes in gene expression during early postnatal cardiac development, we have isolated and cloned a 484 bp cDNA fragment that corresponds to the 3′ end of rat glycogenin. Northern blot analysis on neonatal cardiac tissues demonstrated hybridization to a 1.7-1.8 kb transcript, which was highly expressed at 3 days and at progressively reduced levels at 1, 2, 3 and 4 weeks of age. A 1624 bp fragment of rat glycogenin was cloned by RT-PCR that includes a 1002 bp open reading frame encoding a 333 amino acid protein. At the nucleotide level, rat glycogenin exhibited 87.2 and 83.6% identity with human and rabbit glycogenin over the open reading frame. The deduced amino acid sequence showed 86.7 and 83.4% identity with human and rabbit sequences, respectively. Given the significance of glycogenin in glycogen biosynthesis, the results of this study suggest a possible molecular basis for the regulation of glycogen during early postnatal cardiac development. In addition, the nucleotide and amino acid sequences of rat glycogenin may be used to investigate the physiological and pathophysiological roles of glycogenin in rat tissues.
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  • 94
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    Molecular and cellular biochemistry 201 (1999), S. 159-167 
    ISSN: 1573-4919
    Keywords: phospholipases A1, A2 and C ; Ureaplasma urealyticum ; calcium ; plasma membrane ; phospholipids ; pH ; detergents
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The presence of endogenous phospholipase A (PL-A) activity of U. urealyticum hydrolyzing the acyl ester bond and phospholipase C (PL-C) activity hydrolyzing the phosphodiester bond is primarily localized in the membranes of ureaplasmas. Characterization of the membrane PL-A and PL-C activity in exponential growing cells of serovars 3, 4, and 8 was investigated. The pH optimum was about 8.5-9 for phospholipase A1 (PL-A1) in the three serovars. A more acidic pH optimum of 6 was observed for phospholipase A2 (PL-A2) enzymes in serovars 3 and 4. However, a very significant stimulation of PL-A2 activity in serovar 8 occurred around pH 7. The specific activity of PL-A2 was always 50-100 fold higher than PL-A1 activity in the pH range studied. Ca2+ ions only slightly stimulated PL-A1 activity in all 3 serovars. PL-A2 activity was stimulated about 6-fold from 0.5-0.8 mM Ca2+ ion concentrations for serovar 3 and 12-fold for serovar 8. Only lower concentrations (0.2-0.4 mM) of calcium stimulated PL-A2 activity in serovar 4. EDTA inhibition corresponded to Ca2+ stimulation for PL-A2 activity for serovars 3 and 8. A general stimulation of PL-A2 activity by diethyl ether was evident but the degree of stimulation varied with the serovar. Sodium deoxycholate enhanced PL-A activity of serovars 4 and 3, but partially inhibited that of serovar 8. PL-A activity in the three serovars were not significantly affected by p-hydroxymercuribenzoate, a marker of -SH groups in the enzyme. All 3 serovars were inactivated by heat. A broad pH optimum for PL-C activity was evident around 7-8. Diethyl ether enhanced PL-C activity of serovar 8. Sodium deoxycholate and heat were inhibitory to PL-C activity. The results demonstrate that the major characteristics of ureaplasma membrane bound PL-A and PL-C are basically similar to those of other mollicutes and bacteria. However, the major differences in the specific characteristics of specially PL-A1 and PL-A2 suggest that the ureaplasma phospholipases are unique enzymes different from the phospholipases of bacteria. Both the PL-A and PL-C enzymes function over the broad range at which ureaplasma can grow, pH 5-9 essential for survival. The ureaplasma PL-As are also markedly different from one serovar to another. This variation in specific activity could contribute significantly to differences in virulence among serovars in specific host milieus. There is significant variation from acidic pH of the vagina and alveolar surface of the lung to a more neutral pH of the endometrium and placenta. There are marked differences in calcium concentrations under specific circumstances in various host tissues. Thus the differences in specific activity among the phospholipases of the serovars of U. urealyticum may be of physiological importance in interactions with host tissues and pathogenesis of disease.
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  • 95
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    Molecular and cellular biochemistry 201 (1999), S. 151-158 
    ISSN: 1573-4919
    Keywords: heart ; adenylate deaminase ; phosphorylation ; enzyme control
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The properties of piglet cardiac AMP deaminase were determined and its regulation by pH, phosphate, nucleotides and phosphorylation is described. AMP deaminase purified from the ventricles of newborn piglet hearts displayed hyperbolic kinetics with a Km of 2 mM for 5′-AMP. The enzyme had a pH optimum of 7.0 and was strongly inhibited by inorganic phosphate. ATP decreased the Km of the native enzyme 3-fold, but did not significantly block the inhibitory effects of phosphate. Kinetic parameters were not significantly altered in the presence of adenosine, cyclic AMP and NAD+, whereas, the Km was decreased by 50% in the presence of NADH. Piglet cardiac AMP deaminase was phosphorylated by protein kinase C, resulting in a 2-fold increase in Vmax with no change in Km. However, incubation with cAMP-dependent protein kinase did not affect enzyme kinetics. The 80-85 kD protein subunit of piglet cardiac AMP deaminase immunoreacted with antisera raised against human erythrocyte AMP deaminase, rabbit heart AMP deaminase and human recombinant AMP deaminase 3 (isoform E). These results are discussed in relation to in situ AMP deaminase activity in neonatal piglet heart myocytes.
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  • 96
    ISSN: 1352-8661
    Keywords: heart ; ejection fraction ; MR studies ; volume
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine , Physics
    Notes: Abstract To evaluate the reproducibility of measurements of left ventricular (LV) dimensions, function, and myocardial mass, segmentedk-space gradient-recalled-echo (GRE) magnetic resonance (MR) imaging was performed on two occasions on 12 healthy volunteers. To compare the MR data, all volunteers underwent a two-dimensional echocardiography with determination of LV dimensions and function. The left ventricle was imaged during breath-hold by consecutive, contiguous short-axis views at end-diastole and end-systole. An average of eight short-axis views was needed to encompass the whole left ventricle. This fast MR sequence limited the total acquisition time to 12 min. LV volumes and masses were calculated after manual delineation of epicardial and endocardial surfaces by two observers in a blinded fashion. Interstudy variability varied between 4.1% and 10.3% for LV end-diastolic volume and end-systolic volume, respectively. Differences in interobserver variability were smaller and varied between 3.6% and 7.3% for LV ejection fraction and end-diastolic volume, respectively. Intraobserver variabilities ranged between 2.0% and 7.0% for LV ejection fraction and end-systolic volume, respectively. These variability percentages agree very well with other studies in literature using other MR sequences. No significant differences in LV dimensions or function were found between MR imaging and echocardiography. In conclusion, this MR sequence allows fast and reproducible LV quantification.
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  • 97
    ISSN: 1352-8661
    Keywords: magnetic resonance ; heart ; rapid imaging ; fast spin-echo
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine , Physics
    Notes: Abstract Purpose: To implement and evaluate two robust methods for T1-and T2-weighted snapshot imaging of the heart with data acquisition within a single heart beat and suppression of blood signal. Methods: Both Tl-and T2-weighted diastolic images of the heart can be obtained with half Fourier single-shot turbo spin echo (HASTE) and turbo fast low-angle shot (turboFLASH) sequences, respectively, in less than 350 ms. Signal from flowing blood in the ventricles and large vessels can be suppressed by a preceding inversion recovery preparing pulse pair (PRESTO). Fifteen volunteers and five patients have been evaluated quantitatively for signal-to-noise ratio (SNR) contrast-to-noise ratio (CNR) and flow void and qualitatively for image quality, artifacts, and black-blood effect. Results: Both PRESTO-HASTE and PRESTO-turboFLASH achieved consistently good image quality and blood signal suppression. In contrast to gradient-echo (GRE) echo-planar imaging techniques, (EPI) HASTE and turboFLASH are much less sensitive to local susceptibility differences in the thorax, resulting in a more robust imaging technique without the need for time-consuming system tuning. Compared to standard spin-echo sequences with cardiac triggering, HASTE and turboFLASH have significantly shorter image acquisition times and are not vulnerable to respiratory motion artifacts. Conclusion: PRESTO-HASTE and PRESTO-turboFLASH constitute suitable methods for fast and high-quality cardiac magnetic resonance imaging (MRI).
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  • 98
    ISSN: 1573-0646
    Keywords: somatostatin ; angiogenesis ; somatostatin receptors ; signal transduction ; xanthines ; calcium ; proteins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology , Medicine
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  • 99
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    Bulletin of experimental biology and medicine 83 (1977), S. 155-158 
    ISSN: 1573-8221
    Keywords: ouabain ; cyclic AMP ; calcium ; mitochondria
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The action of ouabain and cyclic AMP on the Ca-accumulating capacity and outflow of Ca2+ ions from loaded rat heart mitochondria was studied by the tetracycline probe method. In the course of the investigations no effect of ouabain on these processes was found. Cyclic AMP did not act on Ca binding by the mitochondrial membrane but it induced rapid liberation of Ca2+ from organelles loaded with these ions.
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  • 100
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    Bulletin of experimental biology and medicine 81 (1976), S. 639-641 
    ISSN: 1573-8221
    Keywords: blood plasma ; magnesium ; calcium ; strontium ; cation-exchange method
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Adsorption of Mg++, Ca++, and Sr++ ions from physiological solutions and from solutions containing canine blood plasma and physiological saline in different proportions was studied by a cation-exchange method on KU-2×8 resin in the balanced salt form. Equilibrium constants were calculated from the experimental results and used to determine the molar fractions (or percentage content) of “free” and “bound” ions of the corresponding metals.
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