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  • 1
    ISSN: 1432-1424
    Keywords: mTALH ; Cl− channel ; protein kinase A ; channel activation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Cl− channels from basolaterally-enriched rabbit outer renal medullary membranes are activated either by increases in intracellular Cl− activity or by intracellular protein kinase A (PKA). Phosphorylation by PKA, however, is not obligatory for channel activity since channels can be activated by intracellular Cl− in the absence of PKA. The PKA requirement for activation of Cl− channels in certain secretory epithelia is, in contrast, obligatory. In the present studies, we examined the effects of PKA and intracellular Cl− concentrations on the properties of Cl− channels obtained either from basolaterally-enriched vesicles derived from highly purified suspensions of mouse medullary thick ascending limb (mTALH) segments, or from apical membrane vesicles obtained from two secretory epithelia, bovine trachea and rabbit small intestine. Our results indicate that the Cl− channels from mTALH suspensions were virtually identical to those previously described from rabbit outer renal medulla. In particular, an increase in intracellular (trans) Cl− concentration from 2 to 50 mm increased both channel activity (P o) and channel conductance (g Cl, pS). Likewise, trans PKA increased mTALH Cl− channel activity by increasing the activity of individual channels when the trans solutions were 2 mm Cl. Under the latter circumstance, PKA did not activate quiescent channels, nor did it affect g Cl. Moreover, when mTALH Cl− channels were inactivated by reducing cis Cl− concentrations to 50 mm, cis PKA addition did not affect P o. These results are consistent with the view that these Cl− channels originated from basolateral membranes of the mTALH. Cl− channels from apical vesicles from trachea and small intestine were completely insensitive to alterations in trans Cl− concentrations and demonstrated markedly different responses to PKA. In the absence of PKA, tracheal Cl− channels inactivated spontaneously after a mean time of 8 min; addition of PKA to trans solutions reactivated these channels. The intestinal Cl− channels did not inactivate with time. Trans PKA addition activated new channels with no effect on basal channel activity. Thus the regulation of Cl− channel activity by both intracellular Cl− and by PKA differ in basolateral mTALH Cl− channels compared to apical Cl− channels from either the tracheal or small intestine.
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  • 2
    ISSN: 1432-1424
    Keywords: rental tubule transport ; medullary thick ascending limb ; intracellular voltage recording ; ADH ; K+ conductance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Cellular impalements were used in combination with standard transepithelial electrical measurements to evaluate some of the determinants of the spontaneous lumen-positive voltage,V e , which attends net Cl− absorption,J Cl net , and to assess how ADH might augment bothJ Cl met andV e in the mouse medullary thick ascending limb of Henle microperfusedin vitro. Substituting luminal 5mm Ba++ for 5mm K+ resulted in a tenfold increase in the apical-to-basal membrane resistance ratio,R c /R bl , and increasing luminal K+ from 5 to 50mm in the presence of luminal 10−4 m furosemide resulted in a 53-mV depolarization of apical membrane voltage,V a . Thus K+ accounted for at least 85% of apical membrane conductance. Either with or without ADH. 10−4 m luminal furosemide reducedV e andJ Cl net to near zero values and hyperpolarized bothV a andV bl , the voltage across basolateral membranes; however, the depolarization ofV bl was greater in the presence than in the absence of hormone while the hormone had no significant effect on the depolarization ofV a , Thus ADH-dependent increases inV b were referable to greater depolarizations ofV bl in the presence of ADH than in the absence of ADH 68% of the furosemide-induced hyperpolarization ofV a was referable to a decrease in the K+ current across apical membranes, but, at a minimum, only 19% of the hyperpolarization ofV bl could be accounted for by a furosemide-induced reduction in basolateral membrane Cl− current. Thus an increase in intracellular Cl− activity may have contributed to the depolarization ofV bl during net Cl− absorption, and the intracellular Cl− activity was likely greater with ADH than without hormone. Since ADH increases apical K+ conductance and since the chemical driving force for electroneutral Na+,K+,2Cl− cotransport from lumen to cell may have been less in the presence of ADH than in the absence of hormone, the cardinal effects of ADH may have been to increase the functional number of both Ba++-sensitive conductance K+ channels and electroneutral Na+,K+,2Cl− cotransport units in apical plasma membranes.
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 113 (1990), S. 49-56 
    ISSN: 1432-1424
    Keywords: Cl− channels/bilayers ; Cl− channels/vesicles ; thick ascending limb ; rectification ; channel conductance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary This paper provides the results of studies which characterized conductive36Cl− flux in basolaterally enriched membrane vesicles prepared from rabbit renal outer medulla. Conductive36Cl− uptake was studied under two different experimental conditions. In the first,36Cl− flux was driven by an inside positive voltage created with oppositely directed Cl− and gluconate gradients. In the second, an inwardly direct K+ gradient was used to drive36Cl− uptake. By these two methods, voltage-sensitive36Cl− uptake was shown to comprise about 45 and 65%, respectively, of the initial rates of total36Cl− flux. Separate paired studies demonstrated that the conductive36Cl− uptake was inhibited by the Cl− channel blocker diphenylamine-2-carboxylate (DPC) with an IC50 for DPC of 154 μm. The voltagedependent36Cl− uptake had an activation energy of 6.4 kcal/mole. This36Cl− conductance had an anion selectivity sequence of I−〉Cl−≧NO 3 − ≫gluconate.
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 113 (1990), S. 57-65 
    ISSN: 1432-1424
    Keywords: Cl− channels/bilayers ; Cl− channels/vesicles ; thick ascending limb ; rectification ; channel conductance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The present studies examined some of the properties of Cl− channels in renal outer medullary membrane vesicles incorporated into planar lipid bilayers. The predominant channel was anion selective having aP Cl/P K ratio of 10 and a unit conductance of 93 pS in symmetric 320mm KCl. In asymmetric KCl solutions, theI-V relations conformed to the Goldman-Hodgkin-Katz equation. Channel activity was voltage-dependent with a gating charge of unity. This voltage dependence of channel activity may account, at least in part, for the striking voltage dependence of the basolateral membrane Cl− conductance of isolated medullary thick ascending limb segments. The Cl− channels incorporated into the planar bilayers were asymmetrical: thetrans surface was sensitive to changes in ionized Ca2+ concentrations and insensitive to reducing KCl concentrations to 10mm, while thecis side was insensitive to changes in ionized Ca2+ concentrations, but was inactivated by reducing KCl concentrations to 50mm.
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  • 5
    ISSN: 1432-1424
    Keywords: Cl− channels ; Cl−-interactive loci ; PGO ; TNBS ; Anion binding sites
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract A unique property of basolateral membrane Cl− channels from the mTAL is that the Cl− concentration facing the intracellular aspects of these channels is a determinant of channel open time probability (P 0 ). The K 1/2 for maximal activation of P 0 by Cl− facing intracellular domains of these channels is 10 mm Cl−. The present experiments evaluated the nature of these Cl−-interactive sites. First, we found that the impermeant anion isethionate, when exposed to intracellular Cl− channel faces, could augment P 0 with a K 1/2 in the range of 10 mm isethionate without affecting conductance (g Cl, pS). Second, pretreatment of the solutions facing the intracellular aspects of the channels with either 1 mm phenylglyoxal (PGO), an arginine-specific reagent, or the lysine/terminal amine reagent trinitrobenzene sulfonic acid (TNBS, 1 mm), prevented the activation of P 0 usually seen when the Cl− concentration of solutions facing intracellular channel domains was raised from 2 to 50 mm. However, when the Cl− channel activity was increased by first raising the Cl− concentration bathing intracellular channel faces from 2 to 50 mm, subsequent addition of either PGO or TNBS to solutions bathing intracellular Cl− channel faces had no effect on P 0 . We conclude that the intracellular aspects of these Cl− channels contain Cl−-interactive loci (termed [Cl] i ) which are accessible to impermeant anions in intracellular fluids and which contain arginineand lysine-rich domains which can be inactivated, at low ambient Cl− or isethionate concentrations, by interactions with PGO or TNBS.
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  • 6
    ISSN: 1432-1424
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The present studies were designed to assess the ways in which antidiuretic hormone (ADH) alters water and solute permeation across isolated, rabbit cortical collecting tubules. In earlier work, it was observed: that ADH produced a tenfold increment inP f (cm per sec), the osmotic water permeability coefficient, and a fourfold increment inP D w (cm per sec), the diffusional water permeability coefficient; that small hydrophilic solutes such as urea, thiourea and acetamide (each having oil/water partition coefficients≦0.0008) had vanishingly low permeation coefficients and unity reflection coefficients, even in the presence of ADH; that lumen to bath osmosis involved a transcellular route; and, that the disparity betweenP f andP D w, either with or without ADH, could be rationalized in terms of cellular diffusion constraints, i.e., that water transport across luminal membranes was diffusional. The present experiments evaluated the effects of ADH on diffusion of moderately lipophilic solutes (e.g., butyramide, isobutyramide, and antipyrine, each solute having an oil/water partition≧0.0008) across luminal membranes of rabbit cortical collecting tubules, and the effects of ADH on the apparent activation energies (E A, kcal per moel) for water and solute permeation across these tubules. Three major results were obtained: (1) ADH produced a 60–100% increase in the permeation rates for these solutes. (2) The ADH-dependent apparentE A for water permeation was 9.35±0.92 kcal per mole, and the ADH-dependent apparentE A for permeation of moderately lipophilic solutes was in the range 15.8–19.6 kcal per mole. (3) The ADH-independentE A values for these transport processes were statistically indistinguishable from the ADH-dependentE A values. When viewed in the context of transport mechanisms for water and solute permeation across synthetic lipid bilayer membrane systems, these results are consistent with the possibility that diffusion of water and moderately lipophilic solutes across mammalian collecting tubules may involve parallel sites in luminal plasma membranes: routes for water diffusion which are either aqueous and/or disorganized, particularly with respect to synthetic lipid bilayer lamellae; and, discrete hydrophobic regions for diffusion of moderately lipophilic solutes. ADH may act by increasing the number of both types of sites within luminal plasma membranes.
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  • 7
    ISSN: 1432-1424
    Keywords: renal tubule transport ; medullary thick ascending limb ; ADH ; K+ conductance ; Na+,K+,Cl− cotransport ; cell conductance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary This paper reports experiments designed to assess the relations between net salt absorption and transcellular routes for ion conductance in single mouse medullary thick ascending limbs of Henle microperfusedin vitro. The experimental data indicate that ADH significantly increased the transepithelial electrical conductance, and that this conductance increase could be rationalized in terms of transcellular conductance changes. A minimal estimate (G c min ) of the transcellular conductance, estimated from Ba++ blockade of apical membrane K+ channels, indicated thatG c min was approximately 30–40% of the measured transepithelial conductance. In apical membranes, K+ was the major conductive species; and ADH increased the magnitude of a Ba++-sensitive K+ conductance under conditions where net Cl− absorption was nearly abolished. In basolateral membranes, ADH increased the magnitude of a Cl− conductance; this ADH-dependent increase in basal Cl− conductance depended on a simultaneous hormone-dependent increase in the rate of net Cl− absorption. Cl− removal from luminal solutions had no detectable effect onG e , and net Cl− absorption was reduced at luminal K+ concentrations less than 5mm; thus apical Cl− entry may have been a Na+,K+,2Cl− cotransport process having a negligible conductance. The net rate of K+ secretion was approximately 10% of the net rate of Cl− absorption, while the chemical rate of net Cl− absorption was virtually equal to the equivalent short-circuit current. Thus net Cl− absorption was rheogenic; and approximately half of net Na+ absorption could be rationalized in terms of dissipative flux through the paracellular pathway. These findings, coupled with the observation that K+ was the principal conductive species in apical plasma membranes, support the view that the majority of K+ efflux from cell to lumen through the Ba++-sensitive apical K+ conductance pathway was recycled into cells by Na+,K+,2Cl− cotransport.
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 109 (1989), S. 65-72 
    ISSN: 1432-1424
    Keywords: ADH ; cAMP-dependent protein kinase ; mTALH K+ channels
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary ADH, acting through cAMP, increases the potassium conductance of apical membranes of mouse medullary thick ascending limbs of Henle. The present studies tested whether exposure of renal medullary apical membranes in vitro to the catalytic subunit of cAMP-dependent protein kinase resulted in an increase in potassium conductance. Apical membrane vesicles prepared from rabbit outer renal medulla demonstrated bumetanide-and chloride-sensitive22Na+ uptake and barium-sensitive, voltage-dependent86Rb+-influx. When vesicles were loaded with purified catalytic subunit of cAMP-dependent protein kinase (150 mU/ml), 1mm ATP, and 50mm KCl, the barium-sensitive86Rb+ influx increased from 361±138 to 528±120pm/mg prot · 30 sec (P〈0.01). This increase was inhibited completely when heat-stable protein kinase inhibitor (1 μg/ml) was also present in the vesicle solutions. The stimulation of86Rb+ uptake by protein kinase required ATP rather than ADP. It also required opening of the vesicles by hypotonic shock, presumably to allow the kinase free access to the cytoplasmic face of the membranes. We conclude that cAMP-dependent protein kinase-mediated phosphorylation of apical membranes from the renal medulla increases the potassium conductance of these membranes. This mechanism may account for the ADH-mediated increase in potassium conductance in the mouse mTALH.
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  • 9
    Electronic Resource
    Electronic Resource
    Palo Alto, Calif. : Annual Reviews
    Annual Review of Physiology 63 (2001), S. 631-645 
    ISSN: 0066-4278
    Source: Annual Reviews Electronic Back Volume Collection 1932-2001ff
    Topics: Medicine , Biology
    Notes: Abstract Cl- transport in the loop of Henle is responsible for reclamation of 25-40% of the filtered NaCl load and for the formation of dilute urine. Our understanding of the physiologic and molecular mechanisms responsible for Cl- reabsorption in both the thin ascending limb and thick ascending limb of Henle's loop has increased greatly over the last decade. Plasma membrane Cl- channels are known to play an integral role in transcellular Cl- transport in both the thin and thick ascending limbs. This review focuses on the functional characteristics and molecular identities of these Cl- channels, as well as the role of these channels in the pathophysiology of disease.
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  • 10
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Annals of the New York Academy of Sciences 235 (1974), S. 0 
    ISSN: 1749-6632
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Natural Sciences in General
    Type of Medium: Electronic Resource
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