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  • Articles  (348,962)
  • Articles: DFG German National Licenses  (348,962)
  • Springer  (348,874)
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  • Chemistry and Pharmacology  (348,962)
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  • Articles  (348,962)
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  • 1
    Electronic Resource
    Electronic Resource
    SBIC News (2001)
    Springer
    JBIC 6 (2001), S. 661-662 
    Details
    ISSN: 1432-1327
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/PL00010670
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  • 2
    Electronic Resource
    Electronic Resource
    Meetings of interest (2001)
    Springer
    JBIC 6 (2001), S. 659-660 
    Details
    ISSN: 1432-1327
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/PL00010669
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  • 3
    Electronic Resource
    Electronic Resource
    Cell Shrinkage is Essential in Lysophosphatidic Acid Signaling in Ehrlich Ascites Tumor Cells (2000)
    Springer
    The journal of membrane biology 173 (2000), S. 19-29 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Lysophosphatidic acid — [Ca2+]i— Whole cell currents — pHi— F-actin cytoskeleton
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The present study aimed at elucidating the initial intracellular lysophosphatidic acid (LPA)-induced signaling events, in order to investigate the sequence in which LPA affects the intracellular concentration of free, cytosolic Ca2+, [Ca2+] i , ion channels, the F-actin cytoskeleton, cell volume and the Na+/H+ exchanger. We found that stimulation of Ehrlich cells with LPA induced a transient, concentration-dependent increase in [Ca2+] i , which is due to Ca2+ release from intracellular Ins(1,4,5)P3-sensitive stores as well as an influx of Ca2+. The EC50 values for LPA-induced Ca2+ mobilization were estimated at 0.03 nm and 0.4 nm LPA in the presence and absence of extracellular Ca2+, respectively. The LPA-induced increase in [Ca2+] i resulted in (i) co-activation of Ca2+-activated, charybdotoxin (ChTX)-sensitive K+ and niflumic acid-sensitive Cl− currents; (ii) a subsequent cell shrinkage and increased polymerization of F-actin, and (iii) activation of a Na+/H+ exchange, resulting in a concentration-dependent intracellular alkalinization. The EC50 value for the LPA-induced rate of alkalinization was estimated at 0.37 nm LPA. When cell shrinkage was prevented, the LPA-induced activation of the Na+/H+ exchanger was impaired. In conclusion, the initial signaling events induced by LPA involves activation of volume regulatory mechanisms.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002320001003
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  • 4
    Electronic Resource
    Electronic Resource
    Regulation of Lens rCx46-formed Hemichannels by Activation of Protein Kinase C, External Ca2+ and Protons (2000)
    Springer
    The journal of membrane biology 173 (2000), S. 39-46 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Ca2+— Connexin46 — Hemichannel activation — Inactivation — pH — OAG/TPA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Rodent lens connexin46 (rCx46) formed active voltage-dependent hemichannels when expressed in Xenopus oocytes. Time-dependent macroscopic currents were evoked upon depolarization. The observed two activation time constants were weakly voltage-dependent and in the order of hundreds of milliseconds and seconds, respectively. Occasionally, the macroscopic steady-state current and the corresponding current-voltage curve showed inactivation at high depolarizing voltages (〉+50 mV). To account for the fast recovery from inactivation (〈2 msec) favored by hyperpolarization, a four-state kinetic model (C 1 closed ↔C 2 closed ↔O open ↔I inactivated ) is proposed. In the absence of inactivation, the macroscopic conductance decreased and inactivation became visible at voltages positive of +50 mV when the rCx46-expressing oocytes were treated with the protein-kinase-C-activators OAG or TPA, high external concentrations of Ca2+ or H+. However, the underlying mechanisms of OAG, H+ or Ca2+ action were different. While OAG did not alter the voltage-dependent activation of the rCx46-hemichannels, an increase in the external Ca2+ or H+ level shifted the voltage threshold for activation to more positive voltages. In contrast to Ca2+, protons were not effective in the physiological concentration range. We propose that under physiological conditions only external Ca2+ and intracellular PKC-dependent processes regulate rCx46 in the lens.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002320001005
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  • 5
    Electronic Resource
    Electronic Resource
    Effects of Cationic Substitutions on Delayed Rectifier Current in Type I Vestibular Hair Cells (2000)
    Springer
    The journal of membrane biology 173 (2000), S. 139-148 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Semicircular canal — Utricle — Gerbil — Potassium channel — Anomalous mole fraction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The resting potassium current (I KI ) in gerbil dissociated type I vestibular hair cells has been characterized under various ionic conditions in whole cell voltage-clamp. When all K+ in the patch electrode solution was replaced with Na+, (Na+) in or Cs+, (Cs+) in , large inward currents were evoked in response to voltage steps between −90 and −50 mV. Activation of these currents could be described by a Hodgkin-Huxley-type kinetic scheme, the order of best fit increasing with depolarization. Above ∼−40 mV currents became outward and inactivated with a monoexponential time course. Membrane resistance was inversely correlated with external K+ concentration. With (Na+) in , currents were eliminated when K+ was removed from the external solution or following extracellular perfusion of 4-aminopyridine, indicating that currents flowed through I KI channels. Also, reduction of K+ entry through manipulation of membrane potential reduced the magnitude of the outward current. Under symmetrical Cs+, 0 K+ conditions I KI is highly permeable to Cs+. However, inward currents were reduced when small amounts of external K+ were added. Higher concentrations of K+ resulted in larger currents indicating an anomalous mole fraction effect in mixtures of external Cs+ and K+.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002320001015
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  • 6
    Electronic Resource
    Electronic Resource
    Two Types of Pharmacologically Distinct Ca2+ Currents with Voltage-dependent Similarities in Zona Fasciculata Cells Isolated from Bovine Adrenal Gland (2000)
    Springer
    The journal of membrane biology 173 (2000), S. 149-163 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Adrenal cells — Ca2+ channel types — Voltage-dependence — Pharmacology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Voltage-activated Ca2+ currents, in zona fasciculata cells isolated from calf adrenal gland, were characterized using perforated patch-clamp recording. In control solution (Ca2+: 2.5 mm) a transient inward current was followed, in 40% of the cells, by a sustained one. In 20 mm Ba2+, 61% of the cells displayed an inward current, which consisted of transient and sustained components. The other cells produced either a sustained or a transient inward current. These different patterns were dependent upon time in culture. Current-voltage relationships show that both the transient and sustained components activated, peaked and reversed at similar potentials: −40, 0 and +60 mV, respectively. The two components, fully inactivated at −10 mV, were separated by double-pulse protocols from different holding potentials where the transient component could be inactivated or reactivated. The decaying phase of the sustained component was fitted by a double exponential (time constants: 1.9 and 20 sec at +10 mV); that of the transient component was fitted by a single exponential (time constant: 19 msec at +10 mV). Steady-state activation and inactivation curves of the two components were superimposed. Their half activation and inactivation potentials were similar, about −15 and −34 mV, respectively. The sustained component was larger in Ba2+ than in Sr2+ and Ca2+. Ni2+ (20 μm) selectively blocked the transient component while Cd2+ (10 μm) selectively blocked the sustained one. (±)Bay K 8644 (0.5 μm) increased the sustained component and nitrendipine (0.5–1 μm) blocked it selectively. The sustained component was inhibited by calciseptine (1 μm). Both components were unaffected by ω-conotoxin GVIA and MVIIC (0.5 μm). These results show that two distinct populations of Ca2+ channels coexist in this cell type. Although the voltage dependence of their activation and inactivation are comparable, these two components of the inward current are similar to T- and L-type currents described in other cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002320001016
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  • 7
    Electronic Resource
    Electronic Resource
    Activation and Inactivation of Mechanosensitive Currents in the Chick Heart (2000)
    Springer
    The journal of membrane biology 173 (2000), S. 237-254 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Stretch — Voltage clamp — Mechanotransduction — Patch clamp — Modeling
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The behavior of MS channels in embryonic chick ventricular myocytes activated by direct mechanical stimulation is strongly affected by inactivation. The amplitude of the current is dependent not only on the amplitude of the stimulus, but also the history of stimulation. The MS current inactivation appears to be composed of at least two contributions: (i) rearrangement of the cortical tension transducing elements and (ii) blocking action of an autocrine agent released from the cell. With discrete mechanical stimuli, the MS current amplitude in the second press of a double press protocol was always smaller than the amplitude of the first MS current. Occasionally, a large MS current occurred when the cell was first stimulated, but subsequently the cell became unresponsive. For a series of stimuli of varying amplitudes, the order in which they were applied to the cell affected the size of the observed MS current for a given stimulus magnitude. When continuous sinusoidal stimulation was applied to the cells, the MS current envelope either reached a steady state, or inactivated. With sinusoidal stimulation, the MS response could be enhanced or restored by simple perfusion of fluid across the cell. This suggests that mechanical stimulation of the cells produces an autocrine inhibitor of MS channels as well as resulting in cortical rearrangement.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002320001023
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  • 8
    Electronic Resource
    Electronic Resource
    Current Oscillations Under Voltage-Clamp Conditions: An Interplay of Series Resistance and Negative Slope Conductance (2000)
    Springer
    The journal of membrane biology 173 (2000), S. 31-37 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Current oscillations — Negative slope conductance — Series resistance — Tonoplast —Mesembryanthemum crystallinum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Using the patch-clamp technique, we observed profound oscillations of the whole-vacuole outward current across the tonoplast of Mesembryanthemum crystallinum L. (common ice plant). These current oscillations showed a clear voltage dependence and appeared at membrane potentials more positive than 90–100 mV. This paper describes the oscillations in terms of two separate mechanisms. First, the Mesembryanthemum vacuolar membrane shows a negative slope conductance at membrane potentials more positive than 100–120 mV. The fact that the oscillations and the negative slope conductance show a similar threshold potential suggests that (part of) the same mechanism is involved in both phenomena. The second mechanism involved is the voltage drop across the series resistance. As a result, the potential actually experienced by the vacuolar membrane deviates from the command potential defined by the patch-clamp amplifier. This deviation depends in an Ohmic manner on the current magnitude. We suggest that the interplay of the negative slope conductance and the voltage drop across the series resistance can cause a positive feedback which is responsible for the current oscillations.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002320001004
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  • 9
    Electronic Resource
    Electronic Resource
    Lysine 77 is a Key Residue in Aggregation of Equinatoxin II, a Pore-forming Toxin from Sea Anemone Actinia equina (2000)
    Springer
    The journal of membrane biology 173 (2000), S. 47-55 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Pore-forming toxin — Sea anemone — Secondary structure — Chemical modification — Sulfhydryl reagents — Pore size — Ion selectivity —Actinia equina
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Among eighteen point mutants of equinatoxin II produced in E. coli, containing a single cystein substitution at variable position, EqtIIK77C was chosen for its peculiar properties. It was almost 100 times less hemolytic than the wild-type, but its hemolytic activity could be restored by chemical modification of the thiol group, provided that a positive charge was reintroduced. This indicates that a positive charge at this position is necessary for toxin activity. The mutant formed larger pores as compared to the wild type, but displayed the same cation selectivity. The pores reverted to normal size upon reintroduction of the positive charge. The conformation of EqtIIK77C and its binding to lipid membranes, either vesicles or red blood cells, was almost normal. However the kinetics of calcein release from lipid vesicles was substantially slower than that of the wild-type. Taken together with the different size of the pore formed, this is an indication that mutation of Lys77 → Cys influences the normal development of the aggregate which is required for assembling the functional pore.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002320001006
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  • 10
    Electronic Resource
    Electronic Resource
    Silent Calcium Channels in Skeletal Muscle Fibers of the Crustacean Atya lanipes (2000)
    Springer
    The journal of membrane biology 173 (2000), S. 9-17 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Crustacean muscle — L-type calcium channels — Excitation-contraction coupling — Calcium-induced calcium release
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The superficial (tonic) abdominal flexor muscles of Atya lanipes do not generate Ca2+ action potentials when depolarized and have no detectable inward Ca2+ current. These fibers, however, are strictly dependent on Ca2+ influx for contraction, suggesting that they depend on Ca2+-induced Ca2+ release for contractile activation. The nature of the communication between Ca2+ channels in the sarcolemmal/tubular membrane and Ca2+ release channels in the sarcoplasmic reticulum in this crustacean muscle was investigated. The effects of dihydropyridines on tension generation and the passive electrical response were examined in current-clamped fibers: Bay K 8644 enhanced tension about 100% but did not alter the passive electrical response; nifedipine inhibited tension by about 70%. Sr2+ and Ba2+ action potentials could be elicited in Ca2+-free solutions. The spikes generated by these divalent cations were abolished by nifedipine. As the Sr2+ or Ba2+ concentrations were increased, the amplitudes of the action potentials and their maximum rate of rise, V max , increased and tended towards saturation. Three-microelectrode voltage-clamp experiments showed that even at high (138 mm) extracellular Ca2+ concentration the channels were silent, i.e., no inward Ca2+ current was detected. In Ca2+-free solutions, inward currents carried by 138 mm Sr2+ or Ba2+ were observed. The currents activated at voltages above −40 mV and peaked at about 0 mV. This voltage-activation profile and the sensitivity of the channels to dihydropyridines indicate that they resemble L-type Ca2+ channels. Peak inward current density values were low, ca.−33 μA/cm2 for Sr2+ and −14 μA/cm2 for Ba2+, suggesting that Ca2+ channels are present at a very low density. It is concluded that Ca2+-induced Ca2+ release in this crustacean muscle operates with an unusually high gain: Ca2+ influx through the silent Ca2+ channels is too low to generate a macroscopic inward current, but increases sufficiently the local concentration of Ca2+ in the immediate vicinity of the sarcoplasmic reticulum Ca2+ release channels to trigger the highly amplified release of Ca2+ required for tension generation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002320001002
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  • 11
    Electronic Resource
    Electronic Resource
    Calcium Channel Current in Rat Dental Pulp Cells (2000)
    Springer
    The journal of membrane biology 178 (2000), S. 21-30 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Dental pulp — Ca2+ current — Dihydropyridine — Lanthanum — Conotoxin — In vitro
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Voltage-gated Ca2+ currents in early-passage rat dental pulp cells were studied using whole-cell patch-clamp techniques. With Ba2+ as the charge carrier, two prominent inwardly-directed currents, I f and I s , were identified in these cells that could be distinguished on the basis of both kinetics and pharmacology. I f was activated by membrane depolarizations more positive than −30 mV, and displayed fast inactivation kinetics, while I s was activated by steeper depolarizations and inactivated more slowly. At peak current, time constants of inactivation for I f and I s were ∼17 vs.∼631 msec. Both I f and I s could be blocked by lanthanum. By contrast, only I s was sensitive to either Bay-K or nifedipine, a specific agonist and antagonist, respectively, of L-type Ca2+ channels. I s was also blocked by the peptide omega-Conotoxin GVIA. Taken together, results suggested that I f was mediated by divalent cation flow through voltage-gated T-type Ca2+ channels, whereas I s was mediated by L- and N-type Ca2+ channels in the pulp cell membrane. The expression of these prominent, voltage-gated Ca2+ channels in a presumptive mineral-inductive phenotype suggests a functional significance vis a vis differentiation of dental pulp cells for the expression and secretion of matrix proteins, and/or formation of reparative dentin itself.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002320010011
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  • 12
    Electronic Resource
    Electronic Resource
    Characterization of the Increase in [Ca2+] i During Hypotonic Shock and the Involvement of Ca2+-activated K+ Channels in the Regulatory Volume Decrease in Human Osteoblast-like Cells (2000)
    Springer
    The journal of membrane biology 178 (2000), S. 11-20 
    Details
    ISSN: 1432-1424
    Keywords: Key words: K(Ca) channels — [Ca2+]i— Volume regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The calcium indicator fura-2 was used to study the effect of hypotonic solutions on the intracellular calcium concentration, [Ca2+] i , in a human osteoblast-like cell line. Decreasing the tonicity of the extracellular solution to 50% leads to an increase in [Ca2+] i from ∼150 nm up to 1.3 μm. This increase in [Ca2+] i was mainly due to an influx of extracellular Ca2+ since removing of extracellular Ca2+ reduced this increase to ∼250 nm. After cell swelling most of the cells were able to regulate their volume to the initial level within 800 sec. The whole-cell recording mode of the patch-clamp technique was also used to study the effect of an increase in [Ca2+] i on membrane currents in these cells. An increase in [Ca2+] i revealed two types of Ca2+-activated K+ channels, K(Ca) channels. Current through both channel types could not be observed below voltage of +80 mV with [Ca2+] i buffered to 100 nm or less. With patch-electrodes filled with solutions buffering [Ca2+] i to 10 μm both channels types could be readily observed. The activation of the first type was apparently voltage-independent since current could be observed over the entire voltage range used from −160 to +100 mV. In addition, the current was also blocked by charybdotoxin (CTX). The second type of K(Ca) channels in these cells could be activated with depolarizations more positive than −40 mV from a holding potential of −80 mV. This type was blocked by CTX and paxilline. Adding paxilline to the extracellular solution inhibited regulatory volume decrease (RVD), but could not abolish RVD. We conclude that two K(Ca) channel types exist in human osteoblasts, an intermediate conductance K(Ca) channel and a MaxiK-like K(Ca) channel. MaxiK channels might get activated either directly or by an increase in [Ca2+] i elicited through hypotonic solutions. In combination with the volume-regulated Cl− conductance in the same cells this K+ channel seems to play a vital role in volume regulation in human osteoblasts.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002320010010
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  • 13
    Electronic Resource
    Electronic Resource
    Candidate Inhibitor of the Volume-Sensitive Kinase Regulating K-Cl Cotransport: The Myosin Light Chain Kinase Inhibitor ML-7 (2000)
    Springer
    The journal of membrane biology 178 (2000), S. 31-41 
    Details
    ISSN: 1432-1424
    Keywords: Key words: K-Cl cotransport — Mammalian red blood cells — ML-7 — Cell volume regulation — Signal transduction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. K-Cl cotransport, KCC, is activated by swelling in many cells types, and promotes volume regulation by a KCl efflux osmotically coupled to water efflux. KCC is probably activated by swelling-inhibition of a kinase, permitting dephosphorylation, and activation of the cotransporter by a phosphatase. The myosin light chain kinase (MLCK) inhibitor ML-7 inhibits transporters activated by shrinkage. In red blood cells from three mammalian species, ML-7 stimulated KCC in a volume-dependent manner. Relative stimulation was greatest in more shrunken cells. Stimulation was reduced by moderate cell swelling and abolished by further swelling. The half-maximal stimulation is at ∼20 μm ML-7, 50-fold greater than the IC50 for inhibition of MLCK in vitro. Stimulation of KCC by ML-7 did not require cell Ca, while MLCK does. Therefore the target of ML-7 in stimulating KCC in red cells is probably not MLCK. The evidence favors stimulation of KCC by ML-7 by inhibiting the volume-sensitive kinase. Qualitatively similar effects of ML-7 on KCC in red cells from three mammalian species suggest a general mechanism.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002320010012
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  • 14
    Electronic Resource
    Electronic Resource
    Effects of NS1608 on MaxiK Channels in Smooth Muscle Cells from Urinary Bladder (2000)
    Springer
    The journal of membrane biology 173 (2000), S. 57-66 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Bladder — Smooth muscle cells — MaxiK channels — NS1608 — Hyperpolarization — Muscle relaxation — Urge incontinence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Using the patch-clamp technique, we have characterized membrane currents in single detrusor smooth muscle cells from rat and human urinary bladder. From the voltage- and Ca2+-dependence of the current as well as the single channel conductance we conclude that rat and human urinary bladder smooth muscle cells express MaxiK channels. In smooth muscle cells from rat urinary bladder we tested the action of NS1608 on current through these MaxiK channels. Application of 10 μm NS1608 increased the amplitude of the current and this increase could be explained by a shift in the activation voltage of the MaxiK channels ∼100 mV towards more negative potentials. Charybdotoxin as well as paxilline, well known blockers of MaxiK channels, were able to reduce current through MaxiK channels in our cell preparation. In addition, application of 10 μm NS1608 hyperpolarized the membrane potential of the investigated cells. This hyperpolarization could be antagonized by the application of paxilline. We conclude that application of NS1608 results in the opening of MaxiK channels under physiological conditions that leads to a hyperpolarization of the cells. This hyperpolarization in turn could relax urinary bladder smooth muscle cells. MaxiK channels in these cells could therefore play a role in directly controlling muscle tone by regulating the membrane potential. This opens up the possibility of MaxiK channels being targets for the treatment of urge incontinence.
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    URL: http://dx.doi.org/10.1007/s002320001007
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  • 15
    Electronic Resource
    Electronic Resource
    Histamine Increases [Ca2+] in and Activates Ca-K and Nonselective Cation Currents in Cultured Human Capillary Endothelial Cells (2000)
    Springer
    The journal of membrane biology 173 (2000), S. 107-116 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Endothelial cell — Histamine — Nonselective cation channel — BKCA — Ca signaling — Patch clamp
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. We characterized the effects of histamine on intracellular Ca2+ and activation of ionic currents in human capillary endothelial cells. Histamine produced both a transient and sustained increase in intracellular Ca2+. The transient response was mediated largely through intracellular Ca2+ release and the sustained response was due to extracellular Ca2+ entry. The increase in intracellular Ca2+ by histamine was not affected by the H2 blocker cimetidine. But was entirely blocked by the H1 antagonist diphenhydramine showing that the histamine response in these cells is mediated through the H1 receptor. A transient ionic current is coactivated with the histamine-induced increase in intracellular Ca2+ and this current has several properties of a nonselective, Ca2+ permeable, cation channel (NSC). The magnitude of the NSC current does not strictly correlate with intracellular Ca2+ levels. A Ca2+-activated K+ current (BKCA) is activated by the increase in intracellular Ca2+ and this current is blocked by the selective BKCA blocker iberiotoxin.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002320001012
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  • 16
    Electronic Resource
    Electronic Resource
    Cyanide Inhibition of Chloride Conductance Across Toad Skin (2000)
    Springer
    The journal of membrane biology 173 (2000), S. 117-125 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Mitochondria-rich cells — Tight junctions — Electrophysiology — cAMP — Dinitrophenol
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The effect of cyanide (CN−) on voltage-activated or cAMP-induced passive chloride conductance (G Cl ) was analyzed in isolated toad skin. Comparatively low concentrations of CN− inhibited G Cl almost completely and fully reversibly, regardless of whether it was applied from the mucosal or serosal side. The IC50 was 180 ± 12 μm for voltage-activated G Cl and 305 ± 30 μm for the cAMP-inducted conductance. At [CN] 〈100 μm, the initial inhibition frequently declined partly in the continuous presence of CN−. Inhibition was independent of the presence of Ca2+. Inhibition was stronger at more alkaline pH, which suggests that dissociated CN− is the effective inhibitor. The onset of the inhibition of voltage-activated or cAMP-induced G Cl by CN− occurred with half-times of 34 ± 10 sec, whereas reversibility upon washout was twice as fast (18 ± 7 sec). If [CN−] 〈200 μm was applied under inactivating conditions (serosa −30 mV), the reduction of G Cl was stronger upon subsequent voltage-activation than under steady-state activated conditions. This effect was essentially complete less than 30 sec after apical addition of CN−, but G t recovered thereafter partially in the continuous presence of CN−. Dinitrophenol inhibited G Cl similarly, while omission of oxygen did not affect it. These observations, as well as the time course of inhibition and the full reversibility, suggest that interference of CN− with oxidative phosphorylation and subsequent metabolic depletion is not the reason for the inhibition of G Cl . We propose that the inhibition is directly on G Cl , presumably by competition with Cl− at a rate-limiting site in the pathway. Location and molecular nature of this site remain to be identified.
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    URL: http://dx.doi.org/10.1007/s002320001013
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  • 17
    Electronic Resource
    Electronic Resource
    Serotonergic Agonists Inhibit Calcium-Activated Potassium and Voltage-Dependent Sodium Currents in Rat Taste Receptor Cells (2000)
    Springer
    The journal of membrane biology 173 (2000), S. 127-138 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Gustation — Signal transduction — 8OH-DPAT — TFMPP — Piperazine — 5HT1A receptor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Recently we reported that rat taste receptor cells respond to the neurotransmitter serotonin with an inhibition of a calcium-activated potassium current [17]. In the present study, this observation is confirmed and extended by studying the effects of an array of serotonergic agonists on membrane properties, calcium-activated potassium current, and voltage-dependent sodium current in taste receptor cells using the patch-clamp recording technique in the whole-cell configuration. Serotonergic inhibition of calcium-activated potassium current was mimicked by the agonists N-(3-trifluoromethylphenyl)piperazine and by (±)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene. Both produced reversible inhibition of K Ca as well as significantly increasing the input resistance of the cell. The agonists 1-(1-naphthyl)piperazine and buspirone (both serotonin receptor 1A agonists) were similarly effective in reducing K Ca . Outward current was unaffected by application of phenylbiguanide, a serotonin receptor 3 agonist, though current was affected by subsequent application of (±)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene. Two agonists—N-(3-trifluoromethylphenyl)piperazine and (±)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene—were also tested on voltage-dependent sodium currents; both were effective and reversible in reducing its magnitude at a variety of applied potentials. These data are consistent with the notion that serotonin effects in rat taste receptor cells are mediated by serotonin 1A receptors, though other receptor subtypes may be additionally expressed. Serotonin may affect the taste cell electrical properties during active stimulation in a paracrine fashion.
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    Erratum (2000)
    Springer
    The journal of membrane biology 173 (2000), S. 264-264 
    Details
    ISSN: 1432-1424
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002320001025
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    Functional Characterization of Glycosylation-Deficient Human P-Glycoprotein Using A Vaccinia Virus Expression System (2000)
    Springer
    The journal of membrane biology 173 (2000), S. 203-214 
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    ISSN: 1432-1424
    Keywords: Key words: Multidrug resistance — P-glycoprotein — Drug transport — Glycosylation — Vaccinia virus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. P-glycoprotein (P-gp), the product of human MDR1 gene, which functions as an ATP-dependent drug efflux pump, is N-linked glycosylated at asparagine residues 91, 94, and 99 located within the first extracellular loop. We report here the biochemical characterization of glycosylation-deficient (Gly−) P-gp using a vaccinia virus based transient expression system. The staining of HeLa cells expressing Gly− P-gp (91, 94, and 99N→Q), with P-gp specific monoclonal antibodies, MRK-16, UIC2 and 4E3 revealed a 40 to 50% lower cell-surface expression of mutant P-gp compared to the wild-type protein. The transport function of Gly− P-gp, assessed using a variety of fluorescent compounds indicated that the substrate specificity of the pump was not affected by the lack of glycosylation. Additional mutants, Gly− D (91, 94, 99N→D) and Gly−Δ (91, 94, 99 N deleted) were generated to verify that the reduced cell surface expression, as well as total expression, were not a result of the glutamine substitutions. Gly− D and Gly−Δ Pgps were also expressed to the same level as the Gly− mutant protein. 35S-Methionine/cysteine pulse-chase studies revealed a reduced incorporation of 35S-methionine/cysteine in full length Gly− P-gp compared to wild-type protein, but the half-life (∼3 hr) of mutant P-gp was essentially unaltered. Since treatment with proteasome inhibitors (MG-132, lactacystin) increased only the intracellular level of nascent, mutant P-gp, the decreased incorporation of 35S-methionine/cysteine in Gly− P-gp appears to be due to degradation of improperly folded mutant protein by the proteasome and endoplasmic reticulum-associated proteases. These results demonstrate that the unglycosylated protein, although expressed at lower levels at the cell surface, is functional and suitable for structural studies.
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    URL: http://dx.doi.org/10.1007/s002320001020
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    Thermal Stability of the Plasma Membrane Calcium Pump. Quantitative Analysis of Its Dependence on Lipid-Protein Interactions (2000)
    Springer
    The journal of membrane biology 173 (2000), S. 215-225 
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    ISSN: 1432-1424
    Keywords: Key words: Membrane proteins — Ca2+-ATPase — PMCA — Thermal inactivation — Micellar phase — Protein-amphiphiles interactions
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Thermal stability of plasma membrane Ca2+ pump was systematically studied in three micellar systems of different composition, and related with the interactions amphiphile-protein measured by fluorescence resonance energy transfer. Thermal denaturation was characterized as an irreversible process that is well described by a first order kinetic with an activation energy of 222 ± 12 kJ/mol in the range 33–45°C. Upon increasing the mole fraction of phospholipid in the mixed micelles where the Ca2+ pump was reconstituted, the kinetic coefficient for the inactivation process diminished until it reached a constant value, different for each phospholipid species. We propose a model in which thermal stability of the pump depends on the composition of the amphiphile monolayer directly in contact with the transmembrane protein surface. Application of this model shows that the maximal pump stability is attained when 80% of this surface is covered by phospholipids. This analysis provides an indirect measure of the relative affinity phospholipid/detergent for the hydrophobic transmembrane surface of the protein (K LD ) showing that those phospholipids with higher affinity provide greater stability to the Ca2+ pump. We developed a method for directly measure K LD by using fluorescence resonance energy transfer from the membrane protein tryptophan residues to a pyrene-labeled phospholipid. K LD values obtained by this procedure agree with those obtained from the model, providing a strong evidence to support its validity.
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    Influence of a Transmembrane Protein on the Permeability of Small Molecules Across Lipid Membranes (2000)
    Springer
    The journal of membrane biology 173 (2000), S. 187-201 
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    ISSN: 1432-1424
    Keywords: Key words: Permeability — Gramicidin A — Partition coefficients — Lipid bilayers — Membrane transport — Linear free-energy relationships
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The influence of the nonchannel conformation of the transmembrane protein gramicidin A on the permeability coefficients of neutral and ionized α-X-p-methyl-hippuric acid analogues (XMHA) (X = H, OCH3, CN, OH, COOH, and CONH2) across egg-lecithin membranes has been investigated in vesicle efflux experiments. Although 10 mol% gramicidin A increases lipid chain ordering, it enhances the transport of neutral XMHA analogues up to 8-fold, with more hydrophilic permeants exhibiting the greatest increase. Substituent contributions to the free energies of transfer of both neutral and anionic XMHA analogues from water into the bilayer barrier domain were calculated. Linear free-energy relationships were established between these values and those for solute partitioning from water into decadiene, chlorobutane, butyl ether, and octanol to assess barrier hydrophobicity. The barrier domain is similar for both neutral and ionized permeants and substantially more hydrophobic than octanol, thus establishing its location as being beyond the hydrated headgroup region and eliminating transient water pores as the transport pathway for these permeants, as the hydrated interface or water pores would be expected to be more hydrophilic than octanol. The addition of 10 mol% gramicidin A alters the barrier domain from a decadiene-like solvent to one possessing a greater hydrogen-bond accepting capacity. The permeability coefficients for ionized XMHAs increase with Na+ or K+ concentration, exhibiting saturability at high ion concentrations. This behavior can be quantitatively rationalized by Gouy-Chapman theory, though ion-pairing cannot be conclusively ruled out. The finding that transmembrane proteins alter barrier selectivity, favoring polar permeant transport, constitutes an important step toward understanding permeability in biomembranes.
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    URL: http://dx.doi.org/10.1007/s002320001019
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    Effect of Calcium and Calcium Channel Blockers on Transient Outward Current of F76 and D1 Neuronal Soma Membranes in the Subesophageal Ganglia of Helix aspersa (2000)
    Springer
    The journal of membrane biology 173 (2000), S. 179-185 
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    ISSN: 1432-1424
    Keywords: Key words: Transient outward current — Calcium — Di- and trivalent ions — Channel gating
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Twin-electrode voltage-clamp techniques were used to study the effect of calcium and calcium channel blockers on the transient outward current in isolated F76 and D1 neurones of Helix aspersa subesophageal ganglia in vitro (soma only preparation with no cell processes). On lowering extracellular Ca2+ concentration from 10 to 2 mm or removing extracellular calcium from the bathing medium, the threshold for this current shifted in a negative direction by 11.5 and 20 mV, respectively. On the other hand, increasing the extracellular Ca2+ concentration from 10 to 20 and to 40 mm shifted the steady-state inactivation curves in positive directions on the voltage axis by 7 and 15 mV, respectively. Upon application of calcium channel blockers, Co2+, La3+, Ni2+ and Cd2+, transient potassium current amplitude was reduced in a voltage-dependent manner, being more effective at voltages close to the threshold. The current was elicited even at a holding potential of −34 mV. The specific calcium channel blockers, amiloride and nifedipine did not shift the activation and steady-state inactivation curves and did not reduce the transient outward current amplitude. It was concluded that the transient outward current is not dependent on intracellular Ca2+ but that it is modulated by Ca2+ and di- and trivalent ions extracellularly. The effects of these ions are very unlikely to be due to a surface charge effect because the addition of La3+ (200 μm) completely reverses the shift in a hyperpolarizing direction when the extracellular Ca2+ concentration was reduced from 10 to 1 mm and additionally shifts the kinetics further still in a depolarizing direction. The responses seen here are consistent with a specific effect of di- and trivalent ions on the transient outward current channels leading to a modification of gating.
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    Surface Antigen CD98(4F2): Not a Single Membrane Protein, But a Family of Proteins with Multiple Functions (2000)
    Springer
    The journal of membrane biology 173 (2000), S. 165-177 
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    ISSN: 1432-1424
    Keywords: Key words: CD98 — 4F2—Amino acid transport — Cell activation — Adhesion — Proliferation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
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    URL: http://dx.doi.org/10.1007/s002320001017
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    Nitric Oxide Activates or Inhibits Skeletal Muscle Ryanodine Receptors Depending on Its Concentration, Membrane Potential and Ligand Binding (2000)
    Springer
    The journal of membrane biology 173 (2000), S. 227-236 
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    ISSN: 1432-1424
    Keywords: Key words: Ryanodine receptors — Nitric oxide — Regulatory thiols — Oxidation — Skeletal Muscle — Calcium channels
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. We show that rabbit skeletal RyR channels in lipid bilayers can be activated or inhibited by NO, in a manner that depends on donor concentration, membrane potential and the presence of channel agonists. 10 μm S-nitroso-N-acetyl-penicillamine (SNAP) increased RyR activity at −40 mV within 15 sec of addition to the cis chamber, with a 2-fold increase in frequency of channel opening (F o ). 10 μm SNAP did not alter activity at +40 mV and did not further activate RyRs previously activated by 2 mm cis ATP at +40 or −40 mV. In contrast to the increase in F o with 10 μm SNAP, 1 mm SNAP caused a 2-fold reduction in F o but a 1.5-fold increase in mean open time (T o ) at −40 mV in the absence of ATP. 1 mm SNAP or 0.5 mm sodium nitroprusside (SNP) induced ∼3-fold reductions in F o and T o at +40 or −40 mV when channels were activated by 2 mm cis ATP or in channels activated by 6.5 μm peptide A at −40 mV (peptide A corresponds to part of the II–III loop of the skeletal dihydropyridine receptor). Both SNAP-induced activation and SNAP/SNP-induced inhibition were reversed by 2 mm dithiothreitol. The results suggest that S-Nitrosylation or oxidation of at least three classes of protein thiols by NO each produced characteristic changes in RyR activity. We propose that, in vivo, initial release of NO activates RyRs, but stronger release increases [NO] and inhibits RyR activity and contraction.
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    Transient and Permanent Fusion of Vesicles in Zea mays Coleoptile Protoplasts Measured in the Cell-attached Configuration (2000)
    Springer
    The journal of membrane biology 174 (2000), S. 15-20 
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    ISSN: 1432-1424
    Keywords: Key words: Capacitance — Exocytosis — Endocytosis — Transient and permanent membrane fusion — Maize coleoptile
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Exocytosis in protoplasts from Zea mays L. coleoptiles was studied using patch-clamp techniques. Fusion of individual vesicles with the plasma membrane was monitored as a step increase of the membrane capacitance (C m ). Vesicle fusion was observed as (i) An irreversible step increase in C m . (ii) Occasionally, irreversible C m steps were preceded by transient changes in C m , suggesting that the electrical connection between the vesicle with the plasma membrane opens and closes reversibly before full connection is achieved. (iii) Most frequently, however, stepwise transient changes in C m did not lead to an irreversible C m step. Within one patch of membrane capacitance steps due to transient and irreversible fusions were of similar amplitude. This suggests that the exocytosis events do not result from the fusion of vesicles with different sizes but are due to kinetically different states in a fusion process of the same vesicle type. The dwell time histogram of the transient fusion events peaked at about 100 msec. Fusion can be described with a circular three-state model for the fusion process of two fused states and one nonfused state. It predicts that energy input is required to drive the system into a prevailing direction.
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    H+- and K+-Dependence of Ca2+ Uptake in Lung Lamellar Bodies (2000)
    Springer
    The journal of membrane biology 174 (2000), S. 41-51 
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    ISSN: 1432-1424
    Keywords: Key words: pH gradient — Ca2+/H+-exchange — Ca2+-activated K+ channels — ATP-dependent Ca2+ uptake — Vacuolar type H+-ATPase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Lung lamellar bodies maintain an acidic interior by an energy-dependent process. The acidic pH may affect the packaging of surfactant phospholipids, processing of surfactant proteins, or surfactant protein A-dependent lipid aggregation. The electron-probe microanalysis of lamellar body elemental composition has previously suggested that lamellar bodies contain high levels of calcium some of which may be in ionic form. In this study, we investigated the Ca2+ uptake characteristics in isolated lung lamellar bodies. The uptake of Ca2+ was measured by monitoring changes in the fluorescence of Fluo-3, a Ca2+ indicator dye. The uptake of Ca2+ in lamellar bodies was ATP-dependent and increased with increasing concentrations of Ca2+. At 100 nm Ca2+, the uptake was almost completely inhibited by bafilomycin A1, a selective inhibitor of vacuolar type H+-ATPase, or by NH4Cl, which raises the lamellar body pH, suggesting that the pH gradient regulates the uptake. The uptake of Ca2+ increased as the Ca2+ concentration was increased, but the relative contribution of bafilomycin A1-sensitive uptake decreased. At 700 nm, it comprised only 20% of the total uptake. These results suggest the presence of additional mechanism(s) for uptake at higher Ca2+ concentrations. At 700 nm Ca2+, the rate and extent of uptake were lower in the absence of K+ than in the presence of K+. The inhibitors of Ca2+-activated K+-channels, tetraethylammonium, Penitrem A, and 4-aminopyridine, also inhibited the K+-dependent Ca2+ uptake at 700 nm Ca2+. Thus the uptake of Ca2+ in isolated lung lamellar bodies appears to be regulated by two mechanisms, (i) the H+-gradient and (ii) the K+ transport across the lamellar body membrane. We speculate that lamellar bodies accumulate Ca2+ and contribute to regulation of cytosolic Ca2+ in type II cells under resting and stimulated conditions.
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    Maturational Changes in Rabbit Renal Basolateral Membrane Vesicle Osmotic Water Permeability (2000)
    Springer
    The journal of membrane biology 174 (2000), S. 53-58 
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    ISSN: 1432-1424
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. We have recently demonstrated that while the osmotic water permeability (P f ) of neonatal proximal tubules is higher than that of adult tubules, the P f of brush-border membrane vesicles from neonatal rabbits is lower than that of adults. The present study examined developmental changes in the water transport characteristics of proximal tubule basolateral membranes by determining aquaporin 1 (AQP1) protein abundance and the P f in neonatal (10–14 days old) and adult rabbit renal basolateral membrane vesicles (BLMV). At 25°C the P f of neonatal BLMV was significantly lower than the adult BLMV at osmotic gradients ranging from 40 to 160 mOsm/kg water. The activation energies for osmotic water movement were identical in the neonatal and adult BLMV (8.65 ± 0.47 vs. 8.86 ± 1.35 kcal · deg−1· mol−1). Reflection coefficients for sodium chloride and sodium bicarbonate were identical in both the neonatal and adult BLMV and were not different from one. Mercury chloride (0.5 mm) reduced osmotic water movement by 31.3 ± 5.5% in the adult BLMV, but by only 4.0 ± 4.0% in neonatal vesicles (P 〈 0.01). Adult BLMV AQP1 abundance was higher than that in the neonate. These data demonstrate that neonatal BLMV have a lower P f and AQP1 protein abundance than adults and that a significantly greater fraction of water traverses the basolateral membrane lipid bilayer and not water channels in neonates compared to adults. The lower P f of the neonatal BLMV indicates that the basolateral membrane is not responsible for the higher transepithelial P f in the neonatal proximal tubule.
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    Effect of Dicyclohexylcarbodiimide (DCCD) on Transport Parameters in the Frog Cornea Epithelium (2000)
    Springer
    The journal of membrane biology 174 (2000), S. 97-103 
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    ISSN: 1432-1424
    Keywords: Key words: Dicyclohexylcarbodiimide — Frog cornea (R. catesbeiana) — Na+/K+-ATPase — K+ conductance — Short-circuit current — Microelectrode technique
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Dicyclohexylcarbodiimide (DCCD) is a carboxyl group modifier and it is an inhibitor of various ATPases. Present experiments, using an in vitro preparation, were designed to study whether DCCD affected the transporters of the bullfrog cornea epithelium, specifically, the Na+/K+ ATPase pump located in the basolateral membrane. For this purpose, corneas were impaled with microelectrodes and experiments were done under short-circuit current (I sc ) conditions. Addition of DCCD to a concentration of 10−4 m to the tear solution gave a marked decrease in I sc ; a marked depolarization of the intracellular potential, V o ; and a significant decrease in the apical membrane fractional resistance, fR o . There were small and variable although significant changes in the transepithelial conductance, g t . The effects may be explained by a decrease in the basolateral membrane K+ conductance, in combination with a partial inhibition of the Na+/K+-ATPase pump located in the basolateral membrane. There is also evidence for an increase in the apical membrane Cl− conductance.
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    Single-Channel Characterization of a Nonselective Cation Channel from Human Placental Microvillous Membranes. Large Conductance, Multiplicity of Conductance States, and Inhibition by Lanthanides (2000)
    Springer
    The journal of membrane biology 174 (2000), S. 59-70 
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    ISSN: 1432-1424
    Keywords: Key words: Syncytiotrophoblast — Epithelia — Channel reconstitution — Substates — Mechanosensitivity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The rate-limiting step for the maternofetal exchange of low molecular-weight solutes in humans is constituted by transport across a single epithelial layer (syncytiotrophoblast) of the placenta. Other than the well-established presence of a large-conductance, multisubstate Cl− channel, the ionic channels occurring in this syncytial tissue are, for the most part, unknown. We have found that fusion of apical plasma membrane-enriched vesicle fractions with planar lipid bilayers leads, mainly (96% of 353 reconstitutions), to the reconstitution of nonselective cation channels. Here we describe the properties of this novel placental conductance at the single-channel level. The channel has a large (〉200 pS) and variable conductance, is cation selective (P Cl /P K ≅ 0.024), is reversibly inhibited (presumably blocked) by submillimolar La3+, has very unstable kinetics, and displays a large number (〉10) of current sublevels with a ``promiscuous'' connectivity pattern. The occurrence of both ``staircaselike'' and ``all-or-nothing'' transitions between the minimum and maximum current levels was intriguing, particularly considering the large number of conductance levels spanned at a time during the concerted current steps. Single-channel data simulated according to a multistate linear reaction scheme, with rate constants that can vary spontaneously in time, reproduce many aspects of the recorded subconductance behavior. The channel's sensitivity to lanthanides is reminiscent of stretch-sensitive channels which, in turn, suggests a physiological role for this ion channel as a mechanotransducer during syncytiotrophoblast-volume regulation.
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    Cation Permeability and Selectivity of a Root Plasma Membrane Calcium Channel (2000)
    Springer
    The journal of membrane biology 174 (2000), S. 71-83 
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    ISSN: 1432-1424
    Keywords: Key words: Calcium (Ca2+) — Permeation — Planar lipid bilayer — Potassium (K+) —rca channel — Wheat (Triticum aestivum L.)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Calcium channels in the plasma membrane of root cells fulfill both nutritional and signaling roles. The permeability of these channels to different cations determines the magnitude of their cation conductances, their effects on cell membrane potential and their contribution to cation toxicities. The selectivity of the rca channel, a Ca2+-permeable channel from the plasma membrane of wheat (Triticum aestivum L.) roots, was studied following its incorporation into planar lipid bilayers. The permeation of K+, Na+, Ca2+ and Mg2+ through the pore of the rca channel was modeled. It was assumed that cations permeated in single file through a pore with three energy barriers and two ion-binding sites. Differences in permeation between divalent and monovalent cations were attributed largely to the affinity of the ion binding sites. The model suggested that significant negative surface charge was present in the vestibules to the pore and that the pore could accommodate two cations simultaneously, which repelled each other strongly. The pore structure of the rca channel appeared to differ from that of L-type calcium channels from animal cell membranes since its ion binding sites had a lower affinity for divalent cations. The model adequately accounted for the diverse permeation phenomena observed for the rca channel. It described the apparent submillimolar K m for the relationship between unitary conductance and Ca2+ activity, the differences in selectivity sequences obtained from measurements of conductance and permeability ratios, the changes in relative cation permeabilities with solution ionic composition, and the complex effects of Ca2+ on K+ and Na+ currents through the channel. Having established the adequacy of the model, it was used to predict the unitary currents that would be observed under the ionic conditions employed in patch-clamp experiments and to demonstrate the high selectivity of the rca channel for Ca2+ influx under physiological conditions.
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    Modulation of the Calmodulin-induced Inhibition of Sarcoplasmic Reticulum Calcium Release Channel (Ryanodine Receptor) by Sulfhydryl Oxidation in Single Channel Current Recordings and [3H]Ryanodine Binding (2000)
    Springer
    The journal of membrane biology 174 (2000), S. 105-120 
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    ISSN: 1432-1424
    Keywords: Key words: Skeletal muscle — Sarcoplasmic reticulum — Calcium release channel — Ryanodine receptor — Calmodulin — Sulfhydryl oxidation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The modulation of the calmodulin-induced inhibition of the calcium release channel (ryanodine receptor) by two sulfhydryl oxidizing compounds, 4-(chloromercuri)phenyl–sulfonic acid (4-CMPS) and 4,4′-dithiodipyridine (4,4′-DTDP) was determined by single channel current recordings with the purified and reconstituted calcium release channel from rabbit skeletal muscle sarcoplasmic reticulum (HSR) and [3H]ryanodine binding to HSR vesicles. 0.1 μm CaM reduced the open probability (P o ) of the calcium release channel at maximally activating calcium concentrations (50–100 μm) from 0.502 ± 0.02 to 0.137 ± 0.022 (n= 28), with no effect on unitary conductance. 4-CMPS (10–40 μm) and 4,4′-DTDP (0.1–0.3 mm) induced a concentration dependent increase in P o (〉 0.9) and caused the appearance of longer open states. CaM shifted the activation of the calcium release channel by 4-CMPS or 4,4′-DTDP to higher concentrations in single channel recordings and [3H]ryanodine binding. 40 μm 4-CMPS induced a near maximal (P o 〉 0.9) and 0.3 mm 4,4′-DTDP a submaximal (P o = 0.74) channel opening in the presence of CaM, which was reversed by the specific sulfhydryl reducing agent DTT. Neither 4-CMPS nor 4,4′-DTDP affected Ca-[125I]calmodulin binding to HSR. 1 mm MgCl2 reduced P o from 0.53 to 0.075 and 20–40 μm 4-CMPS induced a near maximal channel activation (P o 〉 0.9). These results demonstrate that the inhibitory effect of CaM or magnesium in a physiological concentration is diminished or abolished at high concentrations of 4-CMPS or 4,4′-DTDP through oxidation of activating sulfhydryls on cysteine residues of the calcium release channel.
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    Mechanically Induced Calcium Movements in Astrocytes, Bovine Aortic Endothelial Cells and C6 Glioma Cells (2000)
    Springer
    The journal of membrane biology 174 (2000), S. 121-134 
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    ISSN: 1432-1424
    Keywords: Key words: Magnetic — Ion channels — Fluorescence — Integrins — Manganese — Calcium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Forces applied to resting primary astrocytes, bovine aortic endothelial cells and C6 glioma cells with collagen-coated magnetite particles produce a fast transient change of intracellular Ca2+. It peaks in the micromolar range as measured by Fura-2. This mechanical response adapts within seconds so that repeated stimulation causes smaller responses requiring 〉10 min for recovery. When cytoplasmic Ca2+ is high after treating with ATP, cyclopiazonic acid and thapsigargin, stimulation causes a transient decrease in Ca2+. In these three cell types, no influx of ions is required for Ca2+ elevation showing the response is not caused by activation of plasmalemmal mechanosensitive channels. Approximately half the cells tested showed similar behavior, while the other half, such as fibroblasts, required extracellular Ca2+. The Ca2+ response is not temperature sensitive suggesting the possible involvement of intracellular mechanosensitive channels. We tested a number of second messenger reagents and were only able to block the response in BAECs, but not C6 glioma cells, with Xestospongin C, a blocker of IP3-activated channels. Despite the lack of a causal involvement of plasmalemmal mechanosensitive channels, mechanical stimulation immediately activates a persistent Mn2+ influx pathway. This Mn2+ pathway may be mechanosensitive channels, Ca2+-activated cation channels or depletion-activated Ca2+ channels.
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    The Melibiose Carrier of Escherichia coli: Cysteine Substitutions for Individual Residues in Helix XI (2000)
    Springer
    The journal of membrane biology 174 (2000), S. 135-140 
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    ISSN: 1432-1424
    Keywords: Key words: Melibiose carrier — Cotransporter — Cysteine mutagenesis — Helix XI
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The melibiose carrier from Escherichia coli is a sugar-cation cotransport system. Previously evidence was obtained that this integral membrane protein consists of 12 transmembrane helices. Starting with the cysteine-less melibiose carrier, cysteine has been substituted individually for amino acids 374–396, which includes all of the residues in the proposed helix XI. The carriers with cysteine substitutions were studied for their transport activity and the effect of the water soluble sulfhydryl reagent p-chloromercuribenzenesulfonic acid (PCMBS). Studies were carried out on both intact cells and inside out vesicles. Cysteine substitution caused loss of transport activity in seven of the mutants (K377C, G379C, A383C, F385C, L391C, G395C and Y396C). PCMBS produced more than 50% inhibition in six of the mutants (S380C, A381C, A384C, F387C, A388C and L391C). Preincubation of the cells with melibiose protected five of these residues from the inhibitory action of PCMBS. It was concluded that the residues whose cysteine derivatives were inhibited by PCMBS probably faced the aqueous channel.
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    Polyamine Triggering of Exocytosis in Paramecium Involves an Extracellular Ca2+/(Polyvalent Cation)-Sensing Receptor, Subplasmalemmal Ca-Store Mobilization and Store-Operated Ca2+-Influx via Unspecific Cation Channels (2000)
    Springer
    The journal of membrane biology 174 (2000), S. 141-156 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Ca2+— Calcium — Exocytosis —Paramecium— Secretion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The polyamine secretagogue, aminoethyldextran (AED), causes a cortical [Ca2+] transient in Paramecium cells, as analyzed by fluorochrome imaging. Our most essential findings are: (i) Cortical Ca2+ signals also occur when AED is applied in presence of the fast Ca2+ chelator, BAPTA. (ii) Extracellular La3+ application causes within seconds a rapid, reversible fluorescence signal whose reversibility can be attributed to a physiological [Ca2+] i transient (while injected La3+ causes a sustained fluorescence signal). (iii) Simply increasing [Ca2+] o causes a similar rapid, short-lived [Ca2+] i transient. All these phenomena, (i–iii), are compatible with activation of an extracellular ``Ca2+/(polyvalent cation)-sensing receptor'' known from some higher eukaryotic systems, where this sensor (responding to Ca2+, La3+ and some multiply charged cations) is linked to cortical calcium stores which, thus, are activated. In Paramecium, such subplasmalemmal stores (``alveolar sacs'') are physically linked to the cell membrane and they can also be activated by the Ca2+ releasing agent, 4-chloro-m-cresol, just like in Sarcoplasmic Reticulum. Since this drug causes a cortical Ca2+ signal also in absence of Ca2+ o we largely exclude a ``Ca2+-induced Ca2+ release'' (CICR) mechanism. Our finding of increased cortical Ca2+ signals after store depletion and re-addition of extracellular Ca2+ can be explained by a ``store-operated Ca2+ influx'' (SOC), i.e., a Ca2+ influx superimposing store activation. AED stimulation in presence of Mn2+ o causes fluorescence quenching in Fura-2 loaded cells, indicating involvement of unspecific cation channels. Such channels, known to occur in Paramecium, share some general characteristics of SOC-type Ca2+ influx channels. In conclusion, we assume the following sequence of events during AED stimulated exocytosis: (i) activation of an extracellular Ca2+/polyamine-sensing receptor, (ii) release of Ca2+ from subplasmalemmal stores, (iii) and Ca2+ influx via unspecific cation channels. All three steps are required to produce a steep cortical [Ca2+] signal increase to a level required for full exocytosis activation. In addition, we show formation of [Ca2+] microdomains (≤0.5 μm, ≤33 msec) upon stimulation.
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    Block of Voltage-dependent Calcium Channel by the Green Mamba Toxin Calcicludine (2000)
    Springer
    The journal of membrane biology 174 (2000), S. 157-165 
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    ISSN: 1432-1424
    Keywords: Key words: Calcium channel blockers — Peptide toxins — Pharmacology — Partial pore block — Calcium channel chimeras — Channel gating
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. A number of peptide toxins derived from marine snails and various spiders have been shown to potently inhibit voltage-dependent calcium channels. Here, we describe the effect of calcicludine, a 60 amino-acid peptide isolated from the venom of the green mamba (Dendroaspis angusticeps), on transiently expressed high voltage-activated calcium channels. Upon application of calcicludine, L-type (α1 C ) calcium channels underwent a rapid, irreversible decrease in peak current amplitude with no change in current kinetics, or any apparent voltage-dependence. However, even at saturating toxin concentrations, block was always incomplete with a maximum inhibition of 58%, indicating either partial pore block, or an effect on channel gating. Block nonetheless was of high affinity with an IC50 value of 88 nm. Three other types of high voltage activated channels tested (α1 A , α1 B , and α1 E ) exhibited a diametrically different response to calcicludine. First, the maximal inhibition observed was around 10%, furthermore, the voltage-dependence of channel activation was shifted slightly towards more negative potentials. Thus, at relatively hyperpolarized test potentials, calcicludine actually upregulated current activity of (N-type) α1 B channels by as much as 50%. Finally, the use of several chimeric channels combining the major transmembrane domains of α1 C and α1 E revealed that calcicludine block of L-type calcium channels involves interactions with multiple structural domains. Overall, calcicludine is a potent and selective inhibitor of neuronal L-type channels with a unique mode of action.
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    Partners in Protection: Interdependence of Cytoskeleton and Plasma Membrane in Adaptations to Applied Forces (2000)
    Springer
    The journal of membrane biology 174 (2000), S. 85-95 
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    ISSN: 1432-1424
    Keywords: Key words: Mechanoprotection — Cytoskeleton — Ion channels — Membrane
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. In mechanically active environments mammalian cells must cope with potentially injurious forces to survive, but the most proximal mechanosensors are largely unknown. How mechanoprotective responses to applied forces are generated and regulated is still a mystery. We consider recent evidence that suggests cellular mechanoprotective adaptations involve a coordinated remodeling of the cell membrane and the associated cytoskeleton. The plasma membrane ``protects'' the cytoskeleton by maintenance of intracellular ionic balance and can modulate force-induced cytoskeletal rearrangements by stretch-activated (e.g., Ca2+) ion channels and mechanosensitive enzymes (e.g., Phospholipase A2 and Phospholipase C). Conversely, the cytoskeleton protects the plasma membrane by providing structural support, reinforcement of the cortical framework at sites of force application, modulation of mechanosensitive ion channels and by potentially contributing to the membrane resealing process after mechanical rupture. We suggest that the plasma membrane and the cytoskeleton are partners in the cytoprotective response to physical forces.
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    Nonselective Cation and BK Channels in Apical Membrane of Outer Sulcus Epithelial Cells (2000)
    Springer
    The journal of membrane biology 174 (2000), S. 167-179 
    Details
    ISSN: 1432-1424
    Keywords: Key words: BK channel — Nonselective cation channel — Cation absorption — Inner ear epithelium — Patch clamp — Vibrating probe — Gerbil
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The outer sulcus epithelium was recently shown to absorb cations from the lumen of the gerbil cochlea. Patch clamp recordings of excised apical membrane were made to investigate ion channels that participate in this reabsorptive flux. Three types of channel were observed: (i) a nonselective cation (NSC) channel, (ii) a BK (large conductance, maxi K or K Ca ) channel and (iii) a small K+ channel which could not be fully characterized. The NSC channel found in excised insideout patch recordings displayed a linear current-voltage (I-V) relationship (27 pS) and was equally conductive for Na+ and K+, but not permeable to Cl− or N-methyl-d-glucamine. Channel activity required the presence of Ca2+ at the cytosolic face, but was detected at Ca2+ concentrations as low as 10−7 m (open probability (P o ) = 0.11 ± 0.03, n= 8). Gadolinium decreased P o of the NSC channel from both the external and cytosolic side (IC50∼ 0.6 μm). NSC currents were decreased by amiloride (10 μm− 1 mm) and flufenamic acid (0.1 mm). The BK channel was also frequently (38%) observed in excised patches. In symmetrical 150 mm KCl conditions, the I-V relationship was linear with a conductance of 268 pS. The Goldman-Hodgkin-Katz equation for current carried solely by K+ could be fitted to the I-V relationship in asymmetrical K+ and Na+ solutions. The channel was impermeable to Cl− and N-methyl-d-glucamine. P o of the BK channel increased with depolarization of the membrane potential and with increasing cytosolic Ca2+. TEA (20 mm), charybdotoxin (100 nm) and Ba2+ (1 mm) but not amiloride (1 mm) reduced P o from the extracellular side. In contrast, external flufenamic acid (100 μm) increased P o and this effect was inhibited by charybdotoxin (100 nm). Flufenamic acid inhibited the inward short-circuit current measured by the vibrating probe and caused a transient outward current. We conclude that the NSC channel is Ca2+ activated, voltage-insensitive and involved in both constitutive K+ and Na+ reabsorption from endolymph while the BK channel might participate in the K+ pathway under stimulated conditions that produce an elevated intracellular Ca2+ or depolarized membrane potential.
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    Acetylcholine Receptor Gating is Influenced by the Polarity of Amino Acids at Position 9′ in the M2 Domain (2000)
    Springer
    The journal of membrane biology 174 (2000), S. 191-197 
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    ISSN: 1432-1424
    Keywords: Key words: Ligand-gated ion channels — Site-directed mutagenesis — Conformational changes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Ligand-gated ion channels contain a conserved leucine at position 9′ (L9′) in the M2 transmembrane domain. We used multiple substitutions at this position in the γ subunit of the mouse acetylcholine receptor (AChR) (γL9′) to examine the role of residue polarity at this position in the gating process at both the macroscopic and single-channel levels. The midpoint of the macroscopic dose-response relationship (EC50) and the channel closing rate constant, α, decreased as the polarity of the residue at that position increased, suggesting a stabilization of the open state of the channel. Both parameters showed similar dependencies on the polarity of the substituted residue. These data support the notion that during AChR gating, the amino acid at the 9′ position moves into a polar environment, and that interactions between this residue and the polar environment determine the stability of the open state. Since this residue is conserved in all other members of the ligand-gated ion channel family, we suggest that a similar mechanism applies to the other members of the family.
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    Topogenic Motifs in P-type ATPases (2000)
    Springer
    The journal of membrane biology 174 (2000), S. 181-190 
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    ISSN: 1432-1424
    Keywords: Key words: P-type ATPases — Topogenic motifs — Membrane insertion — Polytopic membrane proteins — Subunit oligomerization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
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    Mutants of the Lactose Carrier of Escherichia coli Which Show Altered Sugar Recognition Plus a Severe Defect in Sugar Accumulation (2000)
    Springer
    The journal of membrane biology 174 (2000), S. 199-205 
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    ISSN: 1432-1424
    Keywords: Key words: Lactose — Melibiose — Transport — Recognition — Mutant — Accumulation — Permease — Sugar — Symport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Lactose and melibiose are actively accumulated by the wild-type Escherichia coli lactose carrier, which is an integral membrane protein energized by the proton motive force. Mutants of the E. coli lactose carrier were isolated by their ability to grow on minimal plates with succinate plus IPTG in the presence of the toxic lactose analog β-thio-o-nitrophenylgalactoside (TONPG). TONPG-resistant mutants were streaked on melibiose MacConkey indicator plates, and red clones were picked. These melibiose positive mutants were then streaked on lactose MacConkey plates, and white clones were picked. Transport assays indicated that the mutants had altered sugar recognition and a defect in sugar accumulation. The mutants had a poor apparent K m for both lactose and melibiose in transport. One mutant had almost no ability to take up lactose, but melibiose downhill transport was 58% (V max ) of normal. All of the mutants accumulated methyl-α-d-galactopyranoside (TMG) to only 8% or less of normal, and two failed to accumulate. Immunoblot analysis of the mutant lactose carrier proteins indicated that loss of sugar transport activity was not due to loss of expression in the membrane. Nucleotide sequencing of the lacY gene from the mutants revealed changes in the following amino acids of the lactose carrier: M23I, W151L, G257D, A295D and G377V. Two of the mutants (G257D and G377V) are novel in that they represent the first amino acids in periplasmic loops to be implicated with changes in sugar recognition. We conclude that the amino acids M23, W151, G257, A295 and G377 of the E. coli lactose carrier play either a direct or an indirect role in sugar recognition and accumulation.
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    Gating and Permeation in Ion Channels Formed by Gramicidin A and Its Dioxolane-linked Dimer in Na+ and Cs+ Solutions (2000)
    Springer
    The journal of membrane biology 174 (2000), S. 207-212 
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    ISSN: 1432-1424
    Keywords: Key words: Single ion channels — Gramicidin — Gating — Permeation — Dioxolane — Chirality
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The association of two gramicidin A (gA) peptides via H-bonds in lipid bilayers causes the formation of an ion channel that is selective for monovalent cations only. In this study, two gAs were covalently linked with a dioxolane group (SS dimer). Some functional properties of natural gA channels were compared to that synthetic dimer in Na+- or Cs+-containing solutions. The SS dimer remained in the open configuration most of the time, while natural gA channels had a relatively brief mean open time. Single channel conductances to Na+ (g Na ) or Cs+ (g Cs ) in the SS dimer were smaller than in natural gA. However, g Na was considerably more attenuated than g Cs . This probably results from a tight solvation of Na+ by the dioxolane linker in the SS channel. In Cs+ solutions, the SS had frequent closures. By contrast, in Na+ solutions the synthetic dimer remained essentially in the open state. The mean open times of SS channels in different solutions (T open,Na 〉 T open,Cs 〉 T open,H ) were inversely proportional to the single channel conductances (g H 〉 g Cs 〉 g Na ). This suggests that ion occupancy inside the pore stabilizes the open configuration of the gA dimer. The mean closed time of the SS dimer was longer in Cs+ than in H+ solutions. Possible mechanisms for these effects are discussed.
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    Lateral Microheterogeneity of Diphenylhexatriene-Labeled Choline Phospholipids in the Erythrocyte Ghost Membrane as Determined by Time-Resolved Fluorescence Spectroscopy (2000)
    Springer
    The journal of membrane biology 174 (2000), S. 237-243 
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    ISSN: 1432-1424
    Keywords: Key words: Plasmalogen — Membrane domains — Fluorescent phospholipids — Fluorescence lifetime distribution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Choline phospholipids are the major constituents of the outer layer of the erythrocyte membrane. To investigate their lateral membrane organization we determined the fluorescence lifetime properties of diphenylhexatriene analogues of phosphatidylcholine, choline plasmalogen, (the respective enolether derivative), and sphingomyelin inserted into the outer layer of hemoglobin-free ghosts. Fluorescence lifetimes were recorded by time-resolved phase and modulation fluorometry and analyzed in terms of Continuous Lorentzian distributions. To assess the influence of membrane proteins on the fluorescence lifetime of the labeled lipids in the biomembrane, lipid vesicles were used as controls. In general, the lifetime distributions in the ghost membranes are broad compared to vesicles. Phosphatidylcholine and sphingomyelin exhibit very similar lifetime distributions in contrast to an increased plasmalogen lifetime heterogeneity in both systems. Orientational effects of side chain mobilities on the observed lifetimes can be excluded. Fluorescence anisotropies revealed identical values for all three labeled phospholipids in the biomembrane.
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    Substrate Selectivity and pH Dependence of KAAT1 Expressed in Xenopus laevis Oocytes (2000)
    Springer
    The journal of membrane biology 174 (2000), S. 213-224 
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    ISSN: 1432-1424
    Keywords: Key words: Amino acid transport — Cotransport — KAAT1 — Inhibition —Xenopus laevis oocyte
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. When expressed in Xenopus oocytes KAAT1 increases tenfold the transport of l-leucine. Substitution of NaCl with 100 mm LiCl, RbCl or KCl allows a reduced but significant activation of l-leucine uptakes. Chloride-dependence is not strict since other pseudohalide anions such as thyocyanate are accepted. KAAT1 is highly sensitive to pH. It can transport l-leucine at pH 5.5 and 8, but the maximum uptake has been observed at pH 10, near to the physiological pH value, when amino and carboxylic groups are both deprotonated. The pH value mainly influences the V max in Na+ activation curves and l-leucine kinetics. The kinetic parameters are K mNa = 4.6 ± 2 mm, V maxNa = 14.8 ± 1.7 pmol/oocyte/5 min for pH 8.0 and K mNa = 2.8 ± 0.7 mm, V maxNa = 31.3 ± 1.9 pmol/oocyte/5 min for pH 10.0. The kinetic parameters of l-leucine uptake are: K m = 120.4 ± 24.2 μm, V max = 23.2 ± 1.4 pmol/oocyte/5 min at pH 8.0 and K m = 81.3 ± 24.2 μm, V max = 65.6 ± 3.9 pmol/oocyte/5 min at pH 10.0. On the basis of inhibition experiments, the structural features required for KAAT1 substrates are: (i) a carboxylic group, (ii) an unsubstituted α-amino group, (iii) the side chain is unnecessary, if present it should be uncharged regardless of length and ramification.
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    Regulatory Processes on the Cytoplasmic Surface of the Na+/Ca2+ Exchanger from Lobster Exoskeletal Muscle (2000)
    Springer
    The journal of membrane biology 174 (2000), S. 225-235 
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    ISSN: 1432-1424
    Keywords: Key words: Catalytic regulation — Ionic strength —α-chymotrypsin; — Protection from proteolytic digestion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. A partially purified preparation of the lobster muscle Na+/Ca2+ exchanger was reconstituted with, presumably, random orientation in liposomes. Ca2+ efflux from 45Ca-loaded vesicles was studied in exchanger molecules in which the transporter cytoplasmic surface was exposed to the extravesicular (ev) medium. Extravesicular Na+ (Na ev )-dependent Ca2+ efflux depended directly upon the extravesicular Ca2+ concentration ([Ca2+] ev ) with a half-maximal activation at [Ca2+] ev = 0.6 μm. This suggests that the lobster muscle exchanger is catalytically upregulated by cytoplasmic Ca2+, as in most other species. In contrast, at low [Na+] ev , the Ca ev -binding site (i.e., on the cytoplasmic surface) for Ca2+ transported via Ca2+/Ca2+ exchange was half-maximally activated by about 7.5 μm Ca2+. Mild proteolysis of the Na+/Ca2+ exchanger by α-chymotrypsin also upregulated the Na ev -dependent Ca2+ efflux. Following proteolytic digestion in Ca-free medium, the exchanger was no longer regulated by nontransported ev Ca2+. Proteolytic digestion in the presence of 1.9 μm free ev Ca2+, however, induced only a 1.6-fold augmentation of Ca2+ efflux, whereas, after digestion in nominally Ca-free medium, a 2.3-fold augmentation was observed; Ca2+ also inhibited proteolytic degradation of the Na+/Ca2+ exchanger measured by immunoblotting. These data suggest that Ca2+, bound to a high affinity binding site, protects against the activation of the Na+/Ca2+ exchanger by α-chymotrypsin. Additionally, we observed a 6-fold increase in the Na+/Ca2+ exchange rate, on average, when the intra- and extravesicular salt concentrations were increased from 160 to 450 mm, suggesting that the lobster muscle exchanger is optimized for transport at the high salt concentration present in lobster body fluids.
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    Voltage Gating of Gap Junctions in Cochlear Supporting Cells: Evidence for Nonhomotypic Channels (2000)
    Springer
    The journal of membrane biology 175 (2000), S. 17-24 
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    ISSN: 1432-1424
    Keywords: Key words: Voltage dependence — Gap junction — Hybrid coupling — Supporting cell — Inner ear — Hearing loss
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The organ of Corti has been found to have multiple gap junction subunits, connexins, which are localized solely in nonsensory supporting cells. Connexin mutations can induce sensorineural deafness. However, the characteristics and functions of inner ear gap junctions are not well known. In the present study, the voltage-dependence of gap junctional conductance (G j ) in cochlear supporting cells was examined by the double voltage clamp technique. Multiple types of asymmetric voltage dependencies were found for both nonjunctional membrane voltage (V m ) and transjunctional (V j ) voltage. Responses for each type of voltage dependence were categorized into four groups. The first two groups showed rectification that was polarity dependent. The third group exhibited rectification with either voltage polarity, i.e., these cells possessed a bell-shaped G j -V j or G j -V m function. The rectification due to V j had fast and slow components. On the other hand, V m -dependent gating was fast (〈5 msec), but stable. Finally, a group was found that evidenced no voltage dependence, although the absence of V j dependence did not preclude V m dependence and vice versa. In fact, for all groups V j sensitivity could be independent of V m sensitivity. The data show that most gap junctional channels in the inner ear have asymmetric voltage gating, which is indicative of heterogeneous coupling and may result from heterotypic channels or possibly heteromeric configurations. This heterogeneous coupling implies that single connexin gene mutations may affect the normal physiological function of gap junctions that are not limited to homotypic configurations.
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    Large Conductance Ca2+-Activated K+ Channels in Human Meningioma Cells (2000)
    Springer
    The journal of membrane biology 175 (2000), S. 25-33 
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    ISSN: 1432-1424
    Keywords: Key words: Meningioma — Human — Patch clamp — BKCa channels — Cytoskeleton
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Cells from ten human meningiomas were electrophysiologically characterized in both living tissue slices and primary cultures. In whole cells, depolarization to voltages higher than +80 mV evoked a large K+ outward current, which could be blocked by iberiotoxin (100 nm) and TEA (half blocking concentration IC50= 5.3 mm). Raising the internal Ca2+ from 10 nm to 2 mm shifted the voltage of half-maximum activation (V 1/2) of the K+ current from +106 to +4 mV. Respective inside-out patch recordings showed a voltage- and Ca2+-activated (BK Ca ) K+ channel with a conductance of 296 pS (130 mm K+ at both sides of the patch). V 1/2 of single-channel currents was +6, −12, −46, and −68 mV in the presence of 1, 10, 100, and 1000 μm Ca2+, respectively, at the internal face of the patch. In cell-attached patches the open probability (P o ) of BK Ca channels was nearly zero at potentials below +80 mV, matching the activation threshold for whole-cell K+ currents with 10 nm Ca2+ in the pipette. Application of 20 μm cytochalasin D increased P o of BK Ca channels in cell-attached patches within minutes. These data suggest that the activation of BK Ca channels in meningioma cells does not only depend on voltage and internal Ca2+ but is also controlled by the cytoskeleton.
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    Diffusion of Small Solutes in the Lateral Intercellular Spaces of MDCK Cell Epithelium Grown on Permeable Supports (2000)
    Springer
    The journal of membrane biology 175 (2000), S. 9-16 
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    ISSN: 1432-1424
    Keywords: Key words: Caged compound — HEPES — Dextran — Fluid transport — Caged protons — Neuraminidase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The diffusion coefficients of four solutes ranging in molecular weight from 238 to 10,000 in the lateral intercellular spaces (LIS) of cultured kidney cells (MDCK) grown on permeable supports were determined from the spread of fluorescence produced after the release of caged compounds by a pulse from a UV laser. Two types of experiments were performed: measurement of the rate of change of fluorescence after releasing a caged fluorophore, and measurement of the change in fluorescence of a relatively static fluorescent dye produced by the diffusion of an uncaged ligand for the dye. Fluorescence intensity was determined by photon-counting the outputs of a multichannel photomultiplier tube. Diffusion coefficients were determined in free solution as well as in the LIS of MDCK cells grown on permeable supports and the hindrance factor, θ, determined from the ratio of the free solution diffusivity to that in the LIS. The hindrance factors for 3000-MW dextran, 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS, MW 524) and N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES, MW 238) were not significantly different from 1. The diffusion of 10,000-MW dextran was substantially reduced in the LIS with a θ of 5.6 ± 0.3. Enzymatic digestion by neuraminidase of the sialic acid residues of the glycosylation groups in the LIS increased the diffusivity of the 10,000-MW dextran 1.8-fold indicating hindrance by the glycocalyx. We conclude that small solutes, such as Na+ and Cl−, would not be significantly restricted in their diffusion in the LIS and that solute concentration gradients could not develop along the LIS under physiologic conditions.
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    Molecular Properties of Sodium/Dicarboxylate Cotransporters (2000)
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    The journal of membrane biology 175 (2000), S. 1-8 
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    ISSN: 1432-1424
    Keywords: Key words: Succinate — Citrate — Sodium-dependent transport — NaDC family — Kidney--〉
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
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    Direct Comparison of NPPB and DPC as Probes of CFTR Expressed in Xenopus Oocytes (2000)
    Springer
    The journal of membrane biology 175 (2000), S. 35-52 
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    ISSN: 1432-1424
    Keywords: Key words: Cystic fibrosis — Cystic fibrosis transmembrane conductance regulator — Chloride channel blocker — Anion channel — Arylaminobenzoate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Blockers of CFTR with well-characterized kinetics and mechanism of action will be useful as probes of pore structure. We have studied the mechanism of block of CFTR by the arylaminobenzoates NPPB and DPC. Block of macroscopic currents by NPPB and DPC exhibited similar voltage-dependence, suggestive of an overlapping binding region. Kinetic analysis of single-channel currents in the presence of NPPB indicate drug-induced closed time constants averaging 2.2 msec at −100 mV. The affinity for NPPB calculated from single-channel block, K D = 35 μm, exceeds that for other arylaminobenzoates studied thus far. These drugs do not affect the rate of activation of wild-type (WT) channels expressed in oocytes, consistent with a simple mechanism of block by pore occlusion, and appear to have a single binding site in the pore. Block by NPPB and DPC were affected by pore-domain mutations in different ways. In contrast to its effects on block by DPC, mutation T1134F-CFTR decreased the affinity and reduced the voltage-dependence for block by NPPB. We also show that the alteration of macroscopic block by NPPB and DPC upon changes in bath pH is due to both direct effects (i.e., alteration of voltage-dependence) and indirect effects (alteration of cytoplasmic drug loading). These results indicate that both NPPB and DPC block CFTR by entering the pore from the cytoplasmic side and that the structural requirements for binding are not the same, although the binding regions within the pore are similar. The two drugs may be useful as probes for overlapping regions in the pore.
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    Influence of Membrane Physical State on the Lysosomal Proton Permeability (2000)
    Springer
    The journal of membrane biology 175 (2000), S. 53-62 
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    ISSN: 1432-1424
    Keywords: Key words: Lysosomes — Membrane fluidity — Proton permeability — Membrane potential — Proton leakage
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Influence of membrane physical state on the proton permeability of isolated lysosomes was assessed by measuring the membrane potential with 3,3′-dipropylthiadicarbocyanine iodide and monitoring their proton leakage with p-nitrophenol. Changes in the membrane order were examined by the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. Both the membrane potential and proton leakage increased with fluidizing the lysosomal membranes by benzyl alcohol and decreased with rigidifying the membranes by cholesteryl hemisuccinate. The proton permeability increased to the maximum of 42% by the benzyl alcohol treatment and decreased to the minimum of 38.1% by the cholesteryl hemisuccinate treatment. Treating the lysosomes with protonophore CCCP increased the proton permeability by 58%. The effects of the membrane fluidization and rigidification can be reversed by rigidifying the fluidized membranes and fluidizing the rigidified membranes, respectively. The results indicate that the proton permeability of lysosomes increased and decreased with increasing and decreasing their membrane fluidity, respectively. Moreover, the lysosomal proton permeability did not alter further if the changes, either an increase or a decrease, in the fluidity exceeded some amount. The results suggest that the proton permeability of lysosomes can be modulated finitely by the alterations in their membrane physical state.
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    Involvement of Protein Tyrosine Kinase in Osmoregulation of Na+ Transport and Membrane Capacitance in Renal A6 Cells (2000)
    Springer
    The journal of membrane biology 175 (2000), S. 63-77 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Amiloride — Na+ channel — Brefeldin A — ENaC — Genistein — Tyrphostin A23 — Hyposmolality
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Renal A6 cells have been reported in which hyposmolality stimulates Na+ transport by increasing the number of conducting amiloride-sensitive 4-pS Na+ channels at the apical membrane. To study a possible role of protein tyrosine kinase (PTK) in the hyposmolality-induced signaling, we investigated effects of PTK inhibitors on the hyposmolality-induced Na+ transport in A6 cells. Tyrphostin A23 (a PTK inhibitor) blocked the stimulatory action of hyposmolality on a number of the conducting Na+ channels. Tyrphostin A23 also abolished macroscopic Na+ currents (amiloride-sensitive short-circuit current, I Na ) by decreasing the elevating rate of the hyposmolality-increased I Na . Genistein (another type of PTK inhibitor) also showed an effect similar to tyrphostin A23. Brefeldin A (BFA), which is an inhibitor of intracellular translocation of protein, blocked the action of hyposmolality on I Na by diminishing the elevating rate of the hyposmolality-increased I Na , mimicking the inhibitory action of PTK inhibitor. Further, hyposmolality increased the activity of PTK. These observations suggest that hyposmolality would stimulate Na+ transport by translocating the Na+ channel protein (or regulatory protein) to the apical membrane via a PTK-dependent pathway. Further, hyposmolality also caused an increase in the plasma (apical) membrane capacitance, which was remarkably blocked by treatment with tyrphostin A23 or BFA. These observations also suggest that a PTK-dependent pathway would be involved in the hyposmolality-stimulated membrane fusion in A6 cells.
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    Effects of Na+ on the Predominant K+ Channel in the Tonoplast of Chara: Decrease of Conductance by Blocks in 100 Nanosecond Range and Induction of Oligo- or Poly-subconductance Gating Modes (2000)
    Springer
    The journal of membrane biology 175 (2000), S. 87-93 
    Details
    ISSN: 1432-1424
    Keywords: Key words:Chara corallina— Fast Na+ block — Gating — Subconductance — Patch clamp — Open channel noise
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. We present three mechanisms by which Na+ inhibits the open channel currents of the predominant K+ channel in the tonoplast of Chara corallina: (i) Fast block, i.e., short (100 ns range) interruptions of the open channel current which are determined by open channel noise analysis, (ii): Oligo-subconductance mode, i.e., a gating mode which occurs preferentially in the presence of Na+; this mode comprises a discrete number (here 3) of open states with smaller conductances than normal, and (iii): Polysubconductance mode, i.e., a gating mode with a nondiscrete, large number (〉30) of states with smaller conductances than the main open channel conductance. This novel mode has also been observed only in the presence of Na+.
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    Lipid Phase Fatty Acid Flip-Flop, Is It Fast Enough for Cellular Transport? (2000)
    Springer
    The journal of membrane biology 175 (2000), S. 79-86 
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    ISSN: 1432-1424
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The mechanism by which fatty acids are transported across cell membranes is controversial. The essence of the controversy is whether transport requires membrane protein mediation or whether the membrane's lipid phase provides a pathway so rapid that a protein is not needed. This review focuses on the mechanisms of fatty acid transport across lipid bilayer membranes. These results for lipid membranes are used to help evaluate transport across cell membranes. Within the context of this analysis, a lipid phase mediated process is consistent with results for the transport of fatty acids across erythrocytes but provides a less adequate explanation for fatty acid transport across more complex cells.
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    Kinetic Properties of the Channels Formed by the Bacillus thuringiensis Insecticidal Crystal Protein Cry1C in the Plasma Membrane of Sf9 Cells (2000)
    Springer
    The journal of membrane biology 175 (2000), S. 115-122 
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    ISSN: 1432-1424
    Keywords: Key words:Bacillus thuringiensis— Sf9 cells — Pore-forming toxin — Intracellular K+— Ion channel
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Spectrofluorimetric measurements were conducted to quantify, in real-time, membrane permeability changes resulting from the treatment of Sf9 insect cells (Spodoptera frugiperda, Lepidoptera) with different Bacillus thuringiensis Cry insecticidal proteins. Coumarin-derived CD222 and Merocyanin-540 probes were respectively used to monitor extracellular K+ and membrane potential variations upon Sf9 cells incubation with Cry toxins. Our results establish that Cry1C induces, after a delay, the depolarization of the cell membrane and the full depletion of intracellular K+. These changes were not observed upon Sf9 cells treated with Cry1A family toxins. Both the rate of the K+ efflux and the delay before its onset were dependent on toxin concentration. Both parameters were sensitive to temperature but only the delay was affected by pH. Cry1C-induced K+ efflux was inhibited by lanthanum ions in a dose-dependent manner. This study provides the first kinetic and quantitative characterization of the ion fluxes through the channels formed by a Cry toxin in the plasma membrane of a susceptible insect cell line.
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    Permeability of Boric Acid Across Lipid Bilayers and Factors Affecting It (2000)
    Springer
    The journal of membrane biology 175 (2000), S. 95-105 
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    ISSN: 1432-1424
    Keywords: Key words: Boric acid — Permeability — Uptake — Boron — Liposomes —Arabidopsis thaliana
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Boron enters plant roots as undissociated boric acid (H3BO3). Significant differences in B uptake are frequently observed even when plants are grown under identical conditions. It has been theorized that these differences reflect species differences in permeability coefficient of H3BO3 across plasma membrane. The permeability coefficient of boric acid however, has not been experimentally determined across any artificial or plant membrane. In the experiments described here the permeability coefficient of boric acid in liposomes made of phosphatidylcholine was 4.9 × 10−6 cm sec−1, which is in good agreement with the theoretical value. The permeability coefficient varied from 7 × 10−6 to 9.5 × 10−9 cm sec−1 with changes in sterols (cholesterol), the type of phospholipid head group, the length of the fatty acyl chain, and the pH of the medium. In this study we also used Arabidopsis thaliana mutants which differ in lipid composition to study the effect of lipid composition on B uptake. The chs1-1 mutant which has lower proportion of sterols shows 30% higher B uptake compared with the wild type, while the act1-1 mutant which has an increased percentage of longer fatty acids, exhibited 35% lower uptake than the wild type. Lipid composition changes in each of the remaining mutants influenced B uptake to various extents. These data suggest that lipid composition of the plasma membrane can affect total B uptake.
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    Effect of Ferriprotoporphyrin IX and Non-heme Iron on the Ca2+ Pump of Intact Human Red Cells (2000)
    Springer
    The journal of membrane biology 175 (2000), S. 107-113 
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    ISSN: 1432-1424
    Keywords: Key words: Ferriprotoporphyrin IX — Heme — Non-heme iron — Ca2+-pump — Red blood cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Previous studies have shown that ferriprotoporphyrin IX (FP) and non-heme iron have a marked inhibitory effect on the Ca2+-Mg2+-ATPase activity of isolated red cell membranes, the biochemical counterpart of the plasma membrane Ca2+ pump (PMCA). High levels of membrane-bound FP and non-heme iron have been found in abnormal red cells such as sickle cells and malaria-infected red cells, associated with a reduced life span. It was important to establish whether sublytic concentrations of FP and non-heme iron would also inhibit the PMCA in normal red cells, to assess the possible role of these agents in the altered Ca2+ homeostasis of abnormal cells. Active Ca2+ extrusion by the plasma membrane Ca2+ pump was measured in intact red cells that had been briefly preloaded with Ca2+ by means of the ionophore A23187. The FP and nonheme iron concentrations used in this study were within the range of those applied to the isolated red cell membrane preparations. The results showed that FP caused a marginal inhibition (∼20%) of pump-mediated Ca2+ extrusion and that non-heme iron induced a slight stimulation of the Ca2+ efflux (11–20%), in contrast to the marked inhibitory effects on the Ca2+-Mg2+-ATPase of isolated membranes. Thus, FP and non-heme iron are unlikely to play a significant role in the altered Ca2+ homeostasis of abnormal red cells.
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    Families of Proteins Forming Transmembrane Channels (2000)
    Springer
    The journal of membrane biology 175 (2000), S. 165-180 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Membranes — Transport — Channels — Porins — Toxins — Holins — Bacteriocins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Channel-forming proteins/peptides fall into over 100 currently recognized families, most of which are restricted to prokaryotes or eukaryotes, but a few of which are ubiquitous. These proteins fall into three major currently recognized classes: (i) α-helix-type channels present in bacterial, archaeal and eukaryotic cytoplasmic and organellar membranes, (ii) β-barrel-type porins present in the outer membranes of Gram-negative bacterial cells, mitochondria and chloroplasts, and (iii) protein/peptide toxins targeted to the cytoplasmic membranes of cells other than those that synthesize the toxins. High-resolution 3-dimensional structural data are available for representative proteins/peptides of all three of these channel-forming types. Each type exhibits distinctive features that distinguish them from the other channel protein types and from carriers. Structural, functional, and evolutionary aspects of transmembrane channel-formers are discussed.
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    Very Negative Potential for Half-inactivation of, and Effects of Anions on, Voltage-dependent Sodium Currents in Acutely Isolated Rat Olfactory Receptor Neurons (2000)
    Springer
    The journal of membrane biology 175 (2000), S. 123-138 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Olfactory receptor neuron — Sodium currents — Fluoride — Phosphate — Acetate — Patch clamp
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Previous measurements with CsF pipette solutions using whole-cell patch-clamp techniques in dissociated rat olfactory receptor neurons (ORNs) indicated that the sodium currents had very negative inactivation characteristics with the implication that the cell resting potential must also normally have a very negative value. This study supports the conclusions that such an effect was real and not dependent on either the nature of the pipette anions or the recording situation previously used. For all pipette solutions, sodium currents showed a threshold activation ≈−80 mV and half-maximal activation voltages ≈−55 with half-inactivation potential ≤−100 mV, without being significantly affected by the replacement of F− by other pipette anions (H2PO− 4 and acetate−) or the addition of nucleotides and glutathione (which did cause a very slight positive shift). F−, followed by H2PO− 4 and to a much lesser extent by acetate−, was the most favorable pipette anion for obtaining good seals and whole-cell sodium currents in these extremely small ORNs. These results implied that resting potentials, for viable responsive cells, should be more negative than about −90 mV, as supported by the observation that action potentials could only be evoked from holding potentials more negative than −90 mV.
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    Three Types of Membrane Excitations in the Marine Diatom Coscinodiscus wailesii (2000)
    Springer
    The journal of membrane biology 175 (2000), S. 149-160 
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    ISSN: 1432-1424
    Keywords: Key words: Action potential — Gating model — Parameter identification — Reaction kinetics — Saw-tooth clamp — Voltage clamp
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Three types of electrical excitation have been investigated in the marine diatom Coscinodiscus wailesii. I: Depolarization-triggered, transient Cl− conductance, G Cl (t), followed by a transient, voltage-gated K+ conductance, G K , with an active state a and two inactive states i 1 and i 2 in series (a-i 1-i 2). II: Similar G Cl (t) as in Type-I but triggered by hyperpolarization; a subsequent increase of G K in this type is indicated but not analyzed in detail. III: Hyperpolarization-induced transient of a voltage-gated activity of an electrogenic pump (i 2-a-i 2), followed by G Cl (t) as in Type-II excitations. Type-III with pump gating is novel as such. G Cl (t) in all types seems to reflect the mechanism of InsP− 3 and Ca2+-mediated G Cl (t) in the action potential in Chara (Biskup et al., 1999). The nonlinear current-voltage-time relationships of Type-I and Type-III excitations have been recorded under voltage-clamp using single saw-tooth command voltages (voltage range: −200 to +50 mV, typical slope: ±1 Vs−1). Fits of the corresponding models to the experimental data provided numerical values of the model parameters. The statistical significance of these solutions is investigated. We suggest that the original function of electrical excitability of biological membranes is related to osmoregulation which has persisted through evolution in plants, whereas the familiar and osmotically neutral action potentials in animals have evolved later towards the novel function of rapid transmission of information over long distances.
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    Calcium Mediates the NO-induced Potassium Current in Toad and Rat Olfactory Receptor Neurons (2000)
    Springer
    The journal of membrane biology 175 (2000), S. 139-147 
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    ISSN: 1432-1424
    Keywords: Key words: Nitric oxide — Olfactory receptor neuron — Potassium channel — Calcium — Toad — Rat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Nitric oxide (NO) activates a K+ current in dissociated amphibian olfactory receptor neurons. Using the patch-clamp technique in its whole-cell mode and stimulation with puffs of the NO-donor sodium nitroprusside, we further studied this effect and show that it was sensitive to the K+-channel blockers tetraethylammonium and iberiotoxin, indicating the activation of a Ca2+-dependent K+ conductance. The Ca2+-channel blockers nifedipine and cadmium abolished the NO-induced current, and lowering external Ca2+ reduced it significantly. Ca2+ imaging showed a transient fluorescence increase upon stimulation with NO, and after blockade of K+ currents, an NO-induced inward current could be measured, suggesting that the activation of the Ca2+-dependent K+ conductance is mediated by Ca2+ influx. LY83583, a blocker of the ciliary cAMP-gated channels, did not affect the current, and experiments with focal stimulation indicated that the effect is present in the soma, therefore Ca2+ is unlikely to enter via the transduction channels. Finally, we show that NO exerts an effect with similar characteristics on olfactory receptor neurons from the rat. These data represent the first evidence that NO activates a Ca2+-dependent K+ conductance by causing a Ca2+ influx in a sensory system, and suggest that NO signaling plays a role in the physiology of vertebrate olfactory receptor neurons.
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    Treatment of Cells with Detergent Activates Caspases and Induces Apoptotic Cell Death (2000)
    Springer
    The journal of membrane biology 175 (2000), S. 181-189 
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    ISSN: 1432-1424
    Keywords: Key words: Detergent — Triton X-100 — Nonidet P-40 — n-Octylglucoside — Sodium deoxycholate — Apoptosis — Caspase — Jurkat T lymphoblast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Due to their amphiphilic properties, detergents readily disrupt cellular membranes and cause rapid cytolysis. In this study we demonstrate that treatment of cells with sublytic concentrations of detergents such as Triton X-100, Nonidet P-40, n-octylglucoside and the bile salt sodium deoxycholate induce typical signs of apoptosis including DNA fragmentation and cleavage of poly(ADP-ribose) polymerase molecules. The detergent concentration required for apoptosis was below the critical micellar concentration. Induction of apoptosis was not restricted to human cells but similarly occurred in a variety of other vertebrate cell lines. Unstimulated peripheral blood mononuclear cells were susceptible to apoptosis induction by detergent suggesting that apoptosis in this circumstance is not mediated by CD95. Cell death was not due to influx of calcium from the medium. Apoptosis was blocked and cytolysis prevented by treatment with peptide inhibitors of caspases. These findings suggest a process of apoptosis that is initiated upon nonspecific alterations at the cell membrane level. Physiologic correlates of this process still have to be defined.
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    Evidence for a Calcium-Sensing Receptor in the Vascular Smooth Muscle Cells of the Spiral Modiolar Artery (2000)
    Springer
    The journal of membrane biology 175 (2000), S. 203-212 
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    ISSN: 1432-1424
    Keywords: Key words: Calcium-sensing receptor — Cerebral arteries — Labyrinth — Thapsigargin — Ryanodine — U73122
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The vascular diameter of the gerbilline spiral modiolar artery has been shown to depend on the presence of extracellular Ca2+ but it remained unknown whether the smooth muscle cells of this arteriole contain a Ca2+ sensing receptor (CaSR). The cytosolic Ca2+ concentration ([Ca2+] i ) was monitored as fluo 3 fluorescence and the vascular diameter was measured by video-microscopy in isolated in vitro superfused spiral modiolar arteries. RT-PCR was used to probe for the presence of CaSR transcripts. Increasing the extracellular Ca2+ concentration ([Ca2+] o ) from 1 to 10 mm caused a biphasic increase in [Ca2+] i that was paralleled by a vasoconstriction. The initial rate of this vasoconstriction, 2.01 ± 0.07 μm/sec (n= 131), was inhibited when cytosolic Ca2+ stores were presumably depleted with thapsigargin (IC 50= 3 × 10−9 m, n= 26) or ryanodine (IC 50= 4 × 10−8 m, n= 25) or when PLC was inhibited by 10−6 m U73122 (n= 8). The initial rate of this constriction was not affected by the L-type Ca2+ channel blocker 10−6 m nifedipine (n= 5), by 10−6 m U73343 (n= 6), which is the inactive analogue of U73122, by the T-type Ca2+ channel blocker 10−6 Gd3+ (n= 6) or the Na+/Ca2+ exchanger blocker 10−4 m Ni2+ (n= 5). The agonist rank potency order was Gd3+ 〉 Ni2+ 〉 Ca2+ 〉〉 neomycin = Mg2+. Analysis of RNA isolated from the SMA revealed a RT-PCR product of the appropriate size for the CaSR (448 bp). Sequence analysis of the amplified cDNA fragment revealed a 94–96% amino acid identity compared to other CaSRs. These results demonstrate that the spiral modiolar artery contains a CaSR, which is most likely located in the vascular smooth muscle cells.
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    Skeletal Muscle Ryanodine Receptor Channels Are Activated by the Fungal Metabolite, Gliotoxin (2000)
    Springer
    The journal of membrane biology 175 (2000), S. 223-233 
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    ISSN: 1432-1424
    Keywords: Key words: Ryanodine receptors — Gliotoxin — Calcium regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Interactions between the reactive disulfide fungal metabolite, gliotoxin (GTX), and rabbit skeletal ryanodine receptor (RyR) calcium release channels have been examined. RyRs in terminal cisternae vesicles formed a covalent complex with 100 μm 35S-GTX, which was reversed by 1 mm dithiothreitol (DTT) or 1 mm glutathione. GTX (80–240 μm), added to either cytoplasmic (cis) or luminal (trans) solutions, increased the rate of Ca2+ release from SR vesicles and the frequency of opening of single RyR channels in lipid bilayers. Channel activation was reversed upon addition of 2 mm DTT to the cis solution, showing that the activation was due to an oxidation reaction (2 mm DTT added to the cis solution in the absence of GTX did not affect RyR activity). Furthermore, RyRs were not activated by trans GTX if the cis chamber contained DTT, suggesting that GTX oxidized a site in or near the membrane. In contrast to cis DTT, 2 mm DTT in the trans solution increased RyR activity when added either alone or with 200 μm trans GTX. The results suggest that (i) GTX increases RyR channel activity by oxidizing cysteine residues that are close to the membrane and located on RyR, or associated proteins, and (ii) a disulfide bridge or nitrosothiol, accessible only from the luminal solution, normally suppresses RyR channel activity. Some of the actions of GTX in altering Ca2+ homeostatsis might depend on its modification of RyR calcium channels.
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    K+ Secretion in Strial Marginal Cells is Stimulated via β1-Adrenergic Receptors but not via β2-Adrenergic or Vasopressin Receptors (2000)
    Springer
    The journal of membrane biology 175 (2000), S. 191-202 
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    ISSN: 1432-1424
    Keywords: Key words: Stria vascularis — Receptor — Adrenergic — Beta — Beta-antagonists — Adrenergic — Atenolol
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Pharmacologic tools were used to identify receptors in functional studies by measuring either transepithelial current (I sc ) in strial marginal cells (SMC) or cAMP production in stria vascularis (SV). Further, receptors were identified in SV as transcripts by cloning and sequencing of reverse-transcriptase polymerase chain reaction (RT-PCR) products. Experiments were performed using tissues isolated from gerbils unless specified otherwise. I sc under control conditions was 1090 ± 21 μA/cm2 (n= 213) in gerbil SMC and 2001 ± 95 μA/cm2 (n= 6) in murine SMC. Direct stimulation of adenylate cyclase with 10-5 m forskolin but not with 10−5 m 1,9-dideoxy-forskolin resulted in an increase in the I sc by a factor of 1.14 ± 0.01 (n= 6). The vasopressin-receptor agonist 10−8 m Arg8-vasopressin had no significant effect on I sc in gerbil and murine SMC. The β-adrenergic agonists isoproterenol, norepinephrine and epinephrine stimulated I sc with an EC 50 of (6 ± 2) × 10−7 m (n= 28), (3 ± 1) × 10−6 m (n= 40) and (7 ± 2) × 10−6 m (n= 38), respectively. Isoproterenol stimulated cAMP production in SV with an EC 50 of (5 ± 2) × 10−7 m (n= 8). The β-antagonist 10−4 m propanolol completely inhibited 2 × 10−5 m isoproterenol-induced stimulation of I sc . The β-antagonists atenolol, ICI118551 and CGP20712A inhibited isoproterenol-induced stimulation of I sc with a K DB of 1 × 10−7 m (pK DB = 6.96 ± 0.15, n= 14), 1 × 10−7 m (pK DB = 7.01 ± 0.14, n= 15), 2 × 10−9 m (pK DB = 8.73 ± 0.13, n = 19), respectively. CGP20712A inhibited isoproterenol-induced cAMP production with a K DB of 1 × 10−10 m (pK DB = 9.94 ± 0.55, n= 9). RT-PCR of total RNA isolated from SV using primers specific for the β1-, β2- and β3-adrenergic receptors revealed products of the predicted sizes for the β1- and β2- but not the β3-adrenergic receptor. Sequence analysis confirmed that amplified cDNA fragments encoded gene-specific nucleotide sequences. These results demonstrate that K+ secretion in SMC is under the control of β1-adrenergic receptors but not β2-adrenergic or vasopressin-receptors and that the β1-subtype is the primary β-adrenergic receptor in SV although SV contains transcripts for both β1- and β2-adrenergic receptors.
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    Bidirectional Transepithelial Water Transport: Chloride-Dependent Mechanisms (2000)
    Springer
    The journal of membrane biology 175 (2000), S. 213-221 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Water diffusional permeability — Ion transport — Furosemide — DPC — Norepinephrine — Neuropeptide Y — Vasopressin — Endothelin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. We hypothesized that inhibition and activation of basolateral to luminal chloride transport mechanisms were associated with respective decreases and increases in basolateral to luminal water fluxes. The luminal to basolateral (J W L→B ) and basolateral to luminal (J W B→L ) water fluxes across ovine tracheal epithelia were measured simultaneously. The mean J W L→B (6.5 μl/min/cm2) was larger than J W B→L (6.1 μl/min/cm2). Furosemide reduced J W B→L from 6.0 to 5.6 μl/min/cm2. Diphenylamine-2-carboxylate (DPC) reduced J W B→L from 7.9 to 7.3 μl/min/cm2 and reduced the membrane potential difference by 38%. Furosemide together with DPC decreased J W L→B by 30% and J W B→L by 15%. Norepinephrine increased J W B→L from 4.9 to 6.0 μl/min/cm2. Neuropeptide Y in the presence of norepinephrine decreased J W L→B (6.4 to 5.2 μl/min/cm2) and returned J W B→L to its baseline value. Vasopressin increased J W B→L from 4.1 to 5.1 μl/min/cm2. Endothelin-1 induced a simultaneous increase in J W B→L (7.0 to 7.7 μl/min/cm2) and decrease in J W L→B (7.4 to 6.4 μl/min/cm2); and decreased the membrane resistance. These data indicate that in tracheal epithelia under homeostatic conditions J W B→L has a ∼15% actively coupled component. Consistent with our hypothesis, inhibition and receptor-induced stimulation of chloride effluxes were associated with decreases and increases in J W B→L , respectively. However, as inhibition of transcellular chloride transport always decreased J W L→B more than J W B→L , reducing transepithelial chloride transport did not result in less water being transported into the airway lumen.
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    The Apical Membrane Glycocalyx of MDCK Cells (2000)
    Springer
    The journal of membrane biology 176 (2000), S. 19-29 
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    ISSN: 1432-1424
    Keywords: Key words: pH — Sialic acid — Neuraminidase — Lectin — Microenvironment
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The microenvironment near the apical membrane of MDCK cells was studied by quantitation of the fluorescence of wheat germ agglutin attached to fluorescein (WGA). WGA was shown to bind to sialic acid residues attached to galactose at the α-2,3 position in the glycocalyx on the apical membrane. Young MDCK cells (5–8 days after splitting) showed a patchy distribution of WGA at stable sites that returned to the same locations after removal of sialic acid residues by neuraminidase treatment. Other lectins also showed stable binding to patches on the apical membrane of young cells. The ratio of WGA fluorescence emission at two excitation wavelengths was used to measure near-membrane pH. The near-membrane pH was markedly acidic to the pH 7.4 bathing solution in both young and older cells (13–21 days after splitting). Patches on the apical membrane of young cells exhibited a range of near-membrane pH values with a mean ±sem of 6.86 ± 0.04 (n= 121) while the near-membrane pH of older cells was 6.61 ± 0.04 (n= 120) with a uniform WGA distribution. We conclude that the distribution of lectin binding sites in young cells reflects the underlying nonrandom location of membrane proteins in the apical membrane and that nonuniformities in the pH of patches may indicate regional differences in membrane acid-base transport as well as in the location of charged sugars in the glycocalyx.
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    Furosemide Stimulates K Transport in HCD57 Erythroid Cells (2000)
    Springer
    The journal of membrane biology 175 (2000), S. 235-244 
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    ISSN: 1432-1424
    Keywords: Key words: Furosemide — K transport — Serum-deprivation — Cell shrinkage — HCD57 cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. We examined the influence of serum and furosemide on K movement and cell volume in HCD57 cells, a murine erythroleukemia cell line, which require erythropoietin (EPO) for survival. We found that maintenance of cell volume depends on the concentration of serum in the culture medium. In isotonic medium containing 20% serum, HCD57 cells maintain their steady-state volume. In contrast, the cells shrink progressively as medium serum content is reduced. In serum-free medium, raising external K to 75 mm prevents cell shrinkage and a further increase in K to 145 mm results in swelling, revealing a role for K permeability in the regulation of cell volume. Of particular interest has been a serendipitous finding with furosemide. Below an external K concentration of 2.1 ± 0.3 mm in medium containing 2% serum, furosemide inhibits K uptake, probably stemming from its well known inhibitory action on KCl cotransport. However, above that K concentration, furosemide stimulates K uptake in a dose-dependent manner. Moreover, furosemide potentiates cell shrinkage induced by serum withdrawal. These findings suggest that the transport machinery mediating cellular shrinkage, once primed by serum depletion, becomes receptive to a second stimulus.
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    Electrogenic Properties of the Sodium Pump in a Dynamic Model of Membrane Transport (2000)
    Springer
    The journal of membrane biology 176 (2000), S. 41-52 
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    ISSN: 1432-1424
    Keywords: Key words: Sodium pump — Electrogenic enzymes — Membrane potential — Mathematical models
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The general purpose of this theoretical work is to contribute to understand the physiological role of the electrogenic properties of the sodium pump, by studying a dynamic model that integrates diverse processes of ionic and water transport across the plasma membrane. For this purpose, we employ a mathematical model that describes the rate of change of the intracellular concentrations of Na+, K+ and Cl−, of the cell volume, and of the plasma membrane potential (V m ). We consider the case of a nonexcitable, nonpolarized cell expressing the sodium pump; Na+, K+, Cl− and water channels, and cotransporters of KCl and NaCl in its plasma membrane. We particularly analyze here the conditions under which the physiological V m can be generated in a predominantly electrogenic fashion, as a result of the activity of the sodium pump. A major conclusion of this study is that, for the cell model considered, a low potassium permeability is not a sufficient condition for a predominantly electrogenic generation of the V m by the sodium pump. The presence of an electroneutral exchange of Na+ and K+ represents a necessary additional requirement.
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    Muscarinic Activation of BK Channels Induces Membrane Oscillations in Glioma Cells and Leads to Inhibition of Cell Migration (2000)
    Springer
    The journal of membrane biology 176 (2000), S. 31-40 
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    ISSN: 1432-1424
    Keywords: Key words: BK channels — Muscarine — Acetylcholine — Calcium — Migration — Tumor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Patients with cerebral tumors often present with elevated levels of acetylcholine (ACh) in their cerebrospinal fluid. This motivated us to investigate physiological effects of ACh on cultured human astrocytoma cells (U373) using a combination of videomicroscopy, calcium microspectrofluorimetry and perforated patch-clamp recording. Astrocytoma cells exhibited the typical morphological changes associated with cell migration; polarized cells displayed prominent lamellipodia and associated membrane ruffling at the anterior of the cell, and a long tail region that periodically contracted into the cell body as the cell moved forward. Bath application of the ACh receptor agonist, muscarine, reversibly inhibited cell migration. In conjunction with this inhibition, ACh induced a dose-dependent, biphasic increase in resting intracellular free calcium concentration ([Ca2+] i ) associated with periodic Ca2+ oscillations during prolonged ACh applications. The early transient rise in [Ca2+] i was abolished by ionomycin and thapsigargin but was insensitive to caffeine and ryanodine while the plateau phase was strictly dependent on external calcium. The Ca2+ response to ACh was mimicked by muscarine and abolished by the muscarinic antagonists, atropine or 4-DAMP, but not by pirenzepine. Using perforated patch-clamp recordings combined with fluorescent imaging, we demonstrated that ACh-induced [Ca2+] i oscillations triggered membrane voltage oscillations that were due to the activation of voltage-dependent, Ca2+-sensitive K+ currents. These K+ currents were blocked by intracellular injection of EGTA, or by extracellular application of TEA, quinine, or charybdotoxin, but not by apamin. These studies suggest that activation of muscarinic receptors on glioma cells induce the release of Ca2+ from intracellular stores which in turn activate Ca2+-dependent (BK-type) K+ channels. Furthermore, this effect was associated with inhibition of cell migration, suggesting an interaction of this pathway with glioma cell migration.
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    Apical Na+-Cl− Symport in Rabbit Gallbladder Epithelium: A Thiazide-Sensitive Cotransporter (TSC) (2000)
    Springer
    The journal of membrane biology 176 (2000), S. 53-65 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Hydrochlorothiazide — SITS — Intracellular Cl− activity — Intracellular Na+ activity — Unidirectional Cl− influx — Cl− conductance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Cl− apically enters the epithelium of rabbit gallbladder by a Na+-Cl− symport, sensitive to hydrochlorothiazide (HCTZ). Since HCTZ also activates an apical SITS-sensitive Cl− conductance (G Cl ), the symport inhibition might be merely due to a short circuit of the symport by G Cl rather than to a direct action of HCTZ on the symporter. To examine whether the symport is directly inhibited by HCTZ and whether the symporter belongs to the family of thiazide-sensitive cotransporters (TSC), radiochemical measurements of the apical Cl− uptake, electrophysiological determinations of intracellular Cl− and Na+ activities (a i,Cl and a i,Na ) with selective theta microelectrodes and molecular biology methods were used. The 36Cl− uptake proved to be a measurement of the apical unidirectional Cl− influx (J mc ) and of the symport only (without backflux components), with measuring times of 45 sec under all experiment conditions; its inhibition by HCTZ was unaffected by G Cl activation or abolition. After HCTZ treatment the decrease in a i,Cl (measured as the initial rate or in 3 min) was larger than the decrease in a i,Na . The difference was reduced to one third in a group of epithelia in which the elicited G Cl was reduced to one third; moreover it was abolished in any case when G Cl was abolished with 10−4 m SITS. The SITS-insensitive rate of a i,Cl decrease was equal to that of the a i,Na decrease in any case. Thus the a i,Cl decrease displays a component dependent on G Cl activation and a second component dependent on symport inhibition. Using the RT-PCR technique a cDNA fragment was obtained that was 99% identical to the corresponding region of the rabbit renal TSC isoform. The results indicate that in rabbit gallbladder epithelium HCTZ displays a dual action, namely G Cl activation and Na+-Cl− symport inhibition. This Na+-Cl− symporter is the first TSC found to be functionally expressed in a nonrenal or nonrenal-like epithelium.
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    The Role of Membrane Lateral Tension in Calcium-Induced Membrane Fusion (2000)
    Springer
    The journal of membrane biology 176 (2000), S. 67-75 
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    ISSN: 1432-1424
    Keywords: Key words: Membrane — Lipid — Calcium — Tension — Fusion — Fluorescence — Microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Calcium-induced fusion of liposomes was studied with a view to understand the role of membrane tension in this process. Lipid mixing due to fusion was monitored by following fluorescence of rhodamine-phosphatidyl-ethanolamine incorporated into liposomal membrane at a self-quenching concentration. The extent of lipid mixing was found to depend on the rate of calcium addition: at slow rates it was significantly lower than when calcium was injected instantly. The vesicle inner volume was then made accessible to external calcium by adding calcium ionophore A23187. No effect on fusion was observed at high rates of calcium addition while at slow rates lipid mixing was eliminated. Fusion of labeled vesicles with a planar phospholipid membrane (BLM) was studied using fluorescence microscopy. Above a threshold concentration specific for each ion, Ca2+, Mg2+, Cd2+ and La3+ induce fusion of both charged and neutral membranes. The threshold calcium concentration required for fusion was found to be dependent on the vesicle charge, but not on the BLM charge. Pretreatment of vesicles with ionophore and calcium inhibited vesicle fusion with BLM. This effect was reversible: chelation of calcium prior to the application of vesicle to BLM completely restored their ability to fuse. These results support the hypothesis that tension in the outer monolayer of lipid vesicle is a primary reason for membrane destabilization promoting membrane fusion. How this may be a common mechanism for both purely lipidic and protein-mediated membrane fusion is discussed.
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    HCO3 − Transport in a Mathematical Model of the Pancreatic Ductal Epithelium (2000)
    Springer
    The journal of membrane biology 176 (2000), S. 77-100 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Pancreatic duct cells — Mathematical model — HCO−3 secretion — Cl− secretion — Cystic fibrosis transmembrane conductance regulator
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. We have used computer modeling to investigate how pancreatic duct cells can secrete a fluid containing near isotonic (∼140 mm) NaHCO3. Experimental data suggest that NaHCO3 secretion occurs in three steps: (i) accumulation of HCO− 3 across the basolateral membrane of the duct cell by Na(HCO3) n cotransporters, Na+/H+ exchangers and proton pumps; (ii) secretion of HCO− 3 across the luminal membrane on Cl−/HCO− 3 antiporters operating in parallel with Cl− channels; and (iii) diffusion of Na+ through the paracellular pathway. Programming the currently available experimental data into our computer model shows that this mechanism for HCO− 3 secretion is deficient in one important respect. While it can produce a relatively large volume of a HCO− 3-rich fluid, it can only raise the luminal HCO− 3 concentration up to about 70 mm. To achieve secretion of 140 mm NaHCO3 by the model it is necessary to: (i) reduce the conductive Cl− permeability and increase the conductive HCO− 3 permeability of the luminal membrane of the duct cell, and (ii) reduce the activity of the luminal Cl−/HCO− 3 antiporters. Under these conditions most of the HCO− 3 is secreted via a conductive pathway. Based on our data, we propose that HCO− 3 secretion occurs mainly by the antiporter in duct segments near the acini (luminal HCO− 3 concentration up to ∼70 mm), but mainly via channels further down the ductal tree (raising luminal HCO− 3 to ∼140 mm).
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    Ubiquitination and Endocytosis of Plasma Membrane Proteins: Role of Nedd4/Rsp5p Family of Ubiquitin-Protein Ligases (2000)
    Springer
    The journal of membrane biology 176 (2000), S. 1-17 
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    ISSN: 1432-1424
    Keywords: Key words: Ubiquitin — Endocytosis — Ubiquitin protein ligase — Nedd4/Rsp5p — Plasma membrane proteins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. In addition to its well-known role in recognition by the proteasome, ubiquitin-conjugation is also involved in downregulation of membrane receptors, transporters and channels. In most cases, ubiquitination of these plasma membrane proteins leads to their internalization followed by targeting to the lysosome/vacuole for degradation. A crucial role in ubiquitination of many plasma membrane proteins appears to be played by ubiquitin-protein ligases of the Nedd4/Rsp5p family. All family members carry an N-terminal Ca2+-dependent lipid/protein binding (C2) domain, two to four WW domains and a C-terminal catalytic Hect-domain. Nedd4 is involved in downregulation of the epithelial Na+ channel, by binding of its WW domains to specific PY motifs of the channel. Rsp5p, the unique family member in S. cerevisiae, is involved in ubiquitin-dependent endocytosis of a great number of yeast plasma membrane proteins. These proteins lack apparent PY motifs, but carry acidic sequences, and/or phosphorylated-based sequences that might be important, directly or indirectly, for their recognition by Rsp5p. In contrast to polyubiquitination leading to proteasomal recognition, a number of Rsp5p targets carry few ubiquitins per protein, and moreover with a different ubiquitin linkage. Accumulating evidence suggests that, at least in yeast, ubiquitin itself may constitute an internalization signal, recognized by a hypothetical receptor. Recent data also suggest that Nedd4/Rsp5p might play a role in the endocytic process possibly involving its C2 domain, in addition to its role in ubiquitinating endocytosed proteins.
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    Analysis of the Structure and Electrophysiological Actions of Halitoxins: 1,3 Alkyl-pyridinium Salts from Callyspongia ridleyi (2000)
    Springer
    The journal of membrane biology 176 (2000), S. 119-131 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Calcium permeant ion channel — Pore former — Halitoxin — Sensory neurone — Lipid bilayer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. We have chemically characterized a preparation of halitoxins, (1,3 alkyl-pyridinium salts) isolated from the marine sponge Callyspongia ridleyi. At concentrations of 50 and 5 μg/ml the halitoxin preparation caused irreversible membrane potential depolarization, decreased input resistance and inhibited evoked action potentials when applied to cultured dorsal root ganglion neurones. Under whole cell voltage clamp the halitoxins produced an increase in cation conductance that was attenuated by replacing sodium with N-methyl-d-glucamine. Fura-2 fluorescence ratiometric calcium imaging was used to directly measure calcium flux into neurones after exposure to halitoxins. Calcium influx, evoked by the halitoxins, persisted when the neurones were bathed in medium containing the voltage-activated calcium channel antagonists cadmium and nickel. Experiments on undifferentiated F-11 cells showed little or no calcium influx in response to depolarizing concentrations of potassium and indicated that halitoxins evoked massive calcium influx in the absence of voltage-activated calcium channels. The halitoxins also produced transient increases in intracellular calcium when F-11 cells were bathed in calcium-free medium suggesting that the toxins could release calcium from intracellular stores. The pore-forming action of the halitoxins was identified when the toxins were applied to artificial lipid bilayers composed of phosphatidylcholine and cholesterol. Halitoxins evoked channel-like activity in the lipid bilayers, with estimated unitary conductances of between 145pS and 2280pS, possibly indicating that distinct channels could be produced by the different components in the preparation of halitoxins.
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    Thermodynamics of Fatty Acid Transfer (2000)
    Springer
    The journal of membrane biology 176 (2000), S. 101-109 
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    ISSN: 1432-1424
    Keywords: Key words:
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. There is continuing controversy about the mechanism for transfer of fatty acids (FA) between plasma and the interior of cells and vice versa. One view is that this is a spontaneous process. The generally accepted view is that each step of the process is facilitated by a specialized protein. Whether uptake is spontaneous or facilitated, the components of the uptake system, e.g., albumin, water, FA, plasma membrane, and putative transport proteins of the plasma membrane, must behave according to the rules of the physical chemistry of the system. We review these features to illustrate the constraints they impose on the design of experiments to adduce the mechanism of uptake. Analysis of the literature in the context of the physical chemistry of the uptake system indicates that arguments for a facilitated mechanism of uptake for FA are not supported by any data extant. By contrast, comparison of the rates for individual steps of the pathway traversed by FA moving from albumin to the inside of a cell (or vesicles of a model system) with rates of uptake of FA of tissues in the steady state shows that the rates of the former are sufficient to account for the rate of the latter.
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    Glycoside Binding and Translocation in Na+-Dependent Glucose Cotransporters: Comparison of SGLT1 and SGLT3 (2000)
    Springer
    The journal of membrane biology 176 (2000), S. 111-117 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Na+/glucose cotransporters —Xenopus oocytes — Electrophysiology — Kinetics — Glucose derivatives
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Using cotransporters as drug delivery vehicles is a topic of continuing interest. We examined glucose derivatives containing conjugated aromatic rings using two isoforms of the Na+/glucose cotransporter: human SGLT1 (hSGLT1) and pig SGLT3 (pSGLT3, SAAT1). Our studies indicate that there is similarity between SGLT1 and SGLT3 in the overall architecture of the vestibule leading to the sugar-binding site but differences in translocation pathway interactions. Indican was transported by hSGLT1 with higher affinity (K0.5 0.06 mm) and 2-naphthylglucose with lower affinity (K0.5 0.5 mm) than α-methyl-d-glucopyranoside (αMDG, 0.2 mm). Both were poorly transported (maximal velocities, I max , 14% and 8% of αMDG). Other compounds were inhibitors (K i s 1–13 mm). In pSGLT3, indican and 2-naphthylglucose were transported with higher affinity than αMDG (K0.5s 0.9, 0.2 and 2.5 mm and relative I max s of 80, 25 and 100%). Phenylglucose and arbutin were transported with higher I max s (130 and 120%) and comparable K0.5s (8 and 1 mm). Increased affinity of indican relative to αMDG suggests that nitrogen in the pyrrole ring is favorable in both transporters. Higher affinity of 2-naphthylglucose for pSGLT3 than hSGLT1 suggests more extensive hydrophobic/aromatic interaction in pSGLT3 than in hSGLT1. Our results indicate that bulky hydrophobic glucosides can be transported by hSGLT1 and pSGLT3, and discrimination between them is based on steric factors and requirements for H-bonding. This provides information for design of glycosides with potential therapeutic value.
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    Temperature Dependence of Pyrethroid Modification of Single Sodium Channels in Rat Hippocampal Neurons (2000)
    Springer
    The journal of membrane biology 177 (2000), S. 23-39 
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    ISSN: 1432-1424
    Keywords: Key words: Sodium channel — Temperature — Hippocampal neuron — Pyrethroid — Tetramethrin — Single channel
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Pyrethroid modulation of sodium channels is unique in the sense that it is highly dependent on temperature, the potency being augmented by lowering the temperature. To elucidate the mechanisms underlying the negative temperature dependence of pyrethroid action, single sodium channel currents were recorded from cultured rat hippocampal neurons using the inside-out configuration of patch-clamp technique, and the effects of the pyrethroid tetramethrin were compared at 22 and 12°C. Tetramethrin-modified sodium channels opened with short closures and/or transitions to subconductance levels at 22 and 12°C. The time constants of the burst length histograms for tetramethrin-modified channels upon depolarization to −60 mV were 7.69 and 14.46 msec at 22 and 12°C, respectively (Q10= 0.53). Tetramethrin at 10 μm modified 17 and 23% of channels at 22 and 12°C, respectively, indicating that the sensitivity of the sodium channel of rat hippocampal neurons to tetramethrin was almost the same as that of tetrodotoxin-sensitive sodium channels of rat dorsal root ganglion neurons and rat cerebellar Purkinje neurons. The time constants for burst length in tetramethrin-modified sodium channels upon repolarization to −100 mV from −30 mV were 8.26 and 68.80 msec at 22 and 12°C (Q10= 0.12), respectively. The prolongation of tetramethrin-modified whole-cell sodium tail currents upon repolarization at lower temperature was ascribed to a prolongation of opening of each channel. Simple state models were introduced to interpret behaviors of tetramethrin-modified sodium channels. The Q10 values for transition rate constants upon repolarization were extremely large, indicating that temperature had a profound effect on tetramethrin-modified sodium channels.
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    Adaptation by Corneal Epithelial Cells to Chronic Hypertonic Stress Depends on Upregulation of Na:K:2Cl Cotransporter Gene and Protein Expression and Ion Transport Activity (2000)
    Springer
    The journal of membrane biology 177 (2000), S. 41-50 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Hypertonicity — Cell proliferation — Volume regulation — Potassium influx — Na-K-2Cl cotransporter mRNA and protein expression — Cornea
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. We examined the ability of SV40-immortalized human and rabbit corneal epithelial cells (HCEC and RCEC, respectively) to adapt to chronic hypertonic stress. Under isotonic conditions, in the presence of 50 μm bumetanide, proliferation measured as 3H-thymidine incorporation declined in RCEC and HCEC by 8 and 35%, respectively. After 48 hr exposure to 375 mOsm medium, RCEC proliferation fell by 19% whereas in HCEC it declined by 45%. Light scattering behavior demonstrated that both cell lines mediate nearly complete regulatory volume increase (RVI) responses to an acute hypertonic (375 mOsm) challenge, which in part depend on bumetanide-sensitive Na-K-2Cl cotransporter (NKCC) activity. Following exposing RCEC for 48 hr to 375 mOsm medium, their RVI response to an acute hypertonic challenge was inhibited by 17%. However, in HCEC this response declined by 68%. During exposure to 375 mOsm medium for up to 24 hr, only RCEC upregulated NKCC gene and protein expression as well as bumetanide-sensitive 86Rb influx. These increases are consistent with the smaller declines in RVI and proliferation capacity occurring during this period in RCEC than in HCEC. Therefore, adaptation by RCEC to chronic hypertonic stress is dependent on stimulation of NKCC gene and protein expression and functional activity. On the other hand, under isotonic conditions, HCEC RVI and proliferation are more dependent on NKCC activity than they are in RCEC.
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    Glycosylation Influences Gating and pH Sensitivity of I sK (2000)
    Springer
    The journal of membrane biology 177 (2000), S. 65-79 
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    ISSN: 1432-1424
    Keywords: Key words:IsK— minK — KvLQT1 — Lec mutant — Glycosylation — External pH
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The KvLQT1 and minK subunits that coassemble to form I sK channels, contain potential N-glycosylation sites. To examine the role of glycosylation in channel function, a Chinese hamster ovary cell line deficient in glycosylation (Lec-1) and its parental cell line (Pro-5) were transiently transfected with human KvLQT1 (hKvLQT1) cDNA, alone and in combination with the rat (rminK) or human minK (hminK) cDNA. Functional KvLQT1 and I sK currents were expressed in both cell lines, although amplitudes were larger in Pro-5 than Lec-1 cells transfected with hKvLQT1 and hKvLQT1/hminK. For I sK , but not KvLQT1, the voltage-dependence of activation was shifted to more positive voltages and the activation kinetics were slower in the Lec-1 compared to the Pro-5 cells. The effect of extracellular acidification on recombinant KvLQT1 and I sK currents was investigated in Pro-5 and Lec-1 cells. Changing external pH (pH o ) from 7.4 to 6.0 significantly decreased the amplitude and increased the half-activation voltage (V 1/2) of KvLQT1 currents in Pro-5 and Lec-1 cells. In Pro-5 cells, decreasing pH o reduced I sK amplitude without increasing V 1/2, whether rminK or hminK was coexpressed with hKvLQT. In contrast, changing pH o from 7.4 to 6.0 did not significantly change I sK amplitude in Lec-1 cells. Thus, oligosaccharides attached to the minK subunit affect not only the gating properties, but also the pH sensitivity of I sK .
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    The Swelling-Activated Anion Conductance in the Mouse Renal Inner Medullary Collecting Duct Cell Line mIMCD-K2 (2000)
    Springer
    The journal of membrane biology 177 (2000), S. 51-64 
    Details
    ISSN: 1432-1424
    Keywords: Key words:ICl,swell— mIMCD-K2 — PKC — CLC conductance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Swelling-activated Cl− currents (I Cl,swell ) have been characterized in a mouse renal inner medullary collecting duct cell line (mIMCD-K2). Currents activated by exposing the cells to hypotonicity exhibited characteristic outward rectification and time- and voltage-dependent inactivation at positive potentials and showed an anion selectivity of I− 〉 Br− 〉 Cl− 〉 Asp−. NPPB (100 μm) inhibited the current in a voltage independent manner, as did exposure to 10 μm tamoxifen and 500 μm niflumic acid (NFA). In contrast, DIDS (100 μm) blocked the current with a characteristic voltage dependency. These characteristics of I Cl,swell in mIMCD-K2 cells are essentially identical to those of heterologously expressed cardiac CLC-3. A defining feature of CLC-3 is that activation of PKC by PDBu inhibits the conductance. In mIMCD-K2 cells preincubation with PDBu (100 nm) prevented the activation of I Cl,swell by hypotonicity. However, PDBu inhibition of I Cl,swell was reversed after PDBu withdrawal, but this was refractory to subsequent PDBu inhibition. Activation of either the cystic fibrosis transmembrane conductance regulator (CFTR) or Ca2+ activated Cl− conductance (CaCC), which are coexpressed in mIMCD-K2 cells prior to PDBu treatment, abolished the PDBu inhibition of I Cl,swell . Control of I Cl,swell by PKC therefore depends on the physiological status of the cell. In intact mIMCD-K2 layers in Ussing chambers, forskolin stimulation of an inward short-circuit current (due to transepithelial Cl− secretion via apical CFTR) was inhibited by cell swelling upon hypotonic exposure at the basolateral surface. Activation of I Cl,swell is therefore capable of regulating transepithelial Cl− secretion and suggests that I Cl,swell is located at the basolateral membrane. PDBu exposure prior to or during hypotonic challenge was ineffective in reversing the swelling-activated inhibition of Cl− secretion, but tamoxifen (100 μm) abolished the hypotonic inhibition of forskolin-stimulated short-circuit current (I sc ). RT-PCR analysis confirmed expression of mRNA for members of the CLC family, including both CLC-2 and 3, in the mIMCD-K2 cell line.
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    URL: http://dx.doi.org/10.1007/s002320001099
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    Surviving in a Matrix: Membrane Transport in Articular Chondrocytes (2000)
    Springer
    The journal of membrane biology 177 (2000), S. 95-108 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Cartilage — Chondrocyte — Membrane transport — Homeostasis — Metabolites
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002320001103
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    Lithium and Protein Kinase C Modulators Regulate Swelling-Activated K-Cl Cotransport and Reveal a Complete Phosphatidylinositol Cycle in Low K Sheep Erythrocytes (2000)
    Springer
    The journal of membrane biology 177 (2000), S. 81-93 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Lithium — K-Cl Cotransport — Phosphatidylinositol — Erythrocytes — Protein kinase C — Cell Swelling
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. K-Cl cotransport (COT), a ouabain-insensitive, Cl-dependent bidirectional K flux, is ubiquitously present in all cells, plays a major role in ion and volume homeostasis, and is activated by cell swelling and a variety of chemical interventions. Lithium modulates several cation transport pathways and inhibits phospholipid turnover in red blood cells (RBCs). Lithium also inhibits K-Cl COT by an unknown mechanism. To test the hypothesis whereby Li inhibits swelling-activated K-Cl COT by altering either its osmotic response, its regulation, or by competing with K for binding sites, low K (LK) sheep (S) RBCs were loaded with Li by Na/Li exchange or the cation ionophore nystatin. K-Cl COT was measured as the Cl-dependent, ouabain-insensitive K efflux or Rb influx. The results show that Li altered the cell morphology, and increased both cell volume and diameter. Internal (Li i ) but not external (Li o ) Li inhibited swelling-activated K-Cl COT by 85% with an apparent K i of ∼7 mm. In Cl, Li i decreased K efflux at relative cell volumes between 0.9 and 1.2, and at external pHs between 7.2 and 7.4. Li i reduced the V max and increased the K m for K efflux in Cl. Furthermore, Li i increased the production of diacylglycerol in a bimodal fashion, without significant effects on the phosphatidylinositol concentration, and revealed the presence of a complete PI cycle in LK SRBCs. Finally, phorbol ester treatment and PD89059, an inhibitor of mitogen-activated protein kinase (ERK2) kinase, caused a time-dependent inhibition of K-Cl COT. Hence, Li i appears to inhibit K-Cl COT by acting at an allosteric site on the transporter or its putative regulators, and by modulation of the cellular phospholipid metabolism and a PKC-dependent regulatory pathway, causes an altered response of K-Cl COT to pH and volume.
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    Inositol 1,4,5-Trisphosphate But Not Ryanodine-Receptor Agonists Induces Calcium Release from Rat Liver Golgi Apparatus Membrane Vesicles (2000)
    Springer
    The journal of membrane biology 177 (2000), S. 243-249 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Golgi Apparatus — Ca2+ release — Inositol 1,4,5 trisphosphate — Ryanodine — Caffeine— Cyclic ADP ribose
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. We investigated the direct effect of inositol 1,4,5-trisphosphate (IP3) and ryanodine receptor agonists on Ca2+ release from vesicles of a rat liver Golgi apparatus (GA) enriched fraction, which were actively loaded with 45Ca2+. Results in GA were compared with those obtained in a rat liver endoplasmic reticulum (ER) enriched fraction. The addition of IP3 at concentrations ranging from 100 nm to 100 μm, in the presence of thapsigargin, a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+-ATPases, promoted a rapid decrease in the Ca2+ content of GA vesicles. The amount of Ca2+ released from the vesicles was a function of IP3 concentration, reaching about 60% in both GA and ER fractions at 100 μm IP3. Calcium release was inhibited by heparin, an antagonist of IP3 receptors. Calcium exhibited a bell-shaped effect on IP3-dependent Ca2+ released from GA vesicles: it activated Ca2+ release at concentrations up to 1 μm, and inhibited it at higher concentrations. In contrast to that found in the endoplasmic reticulum fraction, none of the ryanodine receptor agonists tested (cyclic ADP-ribose, caffeine and ryanodine) significantly induced Ca2+ release from GA fraction vesicles in the presence of thapsigargin. Our results indicate the presence of an IP3-sensitive Ca2+ release mechanism in the Golgi apparatus membrane analogous to that of the ER. However, a Ca2+ release mechanism sensitive to ryanodine receptor agonists like that of ER is not evident in the GA membrane.
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    Membrane Phospholipid Asymmetry and Signal Transduction (2000)
    Springer
    The journal of membrane biology 178 (2000), S. 1-10 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Phosphoinositides — Excitation-contraction — Ischemia — Cytosolic phospholipase A2
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002320010009
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    Properties of Hexadecaprenyl Monophosphate/Dioleoylphosphatidylcholine Vesicular Lipid Bilayers (2000)
    Springer
    The journal of membrane biology 177 (2000), S. 259-271 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Phospholipid membrane — Polyprenyl monophosphate — Membrane permeability — Vesicle morphology — Transmission electron microscopy — Prebiotic lipid vesicles
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. In our study we investigated hemispherical phospholipid bilayer membranes and phospholipid vesicles made from hexadecaprenyl monophosphate (C80-P), dioleoylphosphatidylocholine (DOPC) and their mixtures by voltammetric and transmission electron microscopy (TEM) techniques. The current-voltage characteristics, the membrane conductance-temperature relationships and the membrane breakdown voltage have been measured for different mixtures of C80-P/DOPC. The membrane hydrophobic thickness and the activation energy of ion migration across the membrane have been determined. Hexadecaprenyl monophosphate decreased in comparison with DOPC bilayers, the membrane conductance, increased the activation energy and the membrane breakdown voltage for the various value of C80-P/DOPC mole ratio, respectively. The TEM micrographs of C80-P, DOPC and C80-P/DOPC lipid vesicles showed several characteristic structures, which have been described. The data indicate that hexadecaprenyl monophosphate modulates the surface curvature of the membranes by the formation of aggregates in liquid-crystalline phospholipid membranes. We suggest that the dynamics and conformation of hexadecaprenyl monophosphate in membranes depend on the transmembrane electrical potential. The electron micrographs indicate that polyprenyl monophosphates with single isoprenyl chains form lipid vesicular bilayers. The thickness of the bilayer, evaluated from the micrographs, was 11 ± 1 nm. This property creates possibility of forming primitive bilayer lipid membranes by long single-chain polyprenyl phosphates in abiotic conditions. It can be the next step in understanding the origin of protocells.
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    Erratum (2000)
    Springer
    The journal of membrane biology 178 (2000), S. 71-71 
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    ISSN: 1432-1424
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002320010023
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    Characteristics of the Quenching of 9-Aminoacridine Fluorescence by Liposomes Made from Plant Lipids (2000)
    Springer
    The journal of membrane biology 178 (2000), S. 43-48 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Surface charge density — Electrostatic attraction — Sorption — Cations — 9-Aminoacridine — Liposome — Lipid composition
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Several laboratories have determined the surface charge density of membranes utilizing methods based on vesicle-induced quenching of the fluorescence of 9-aminoacridine and its relief by other cations. However, the computational methods by which surface charge density were calculated have not been verified in a model system. In this study, the quenching of 9-aminoacridine fluorescence by liposomes made from varying amounts of digalactosyldiacylglyceride and phosphatidic acid and relief of quenching by salts was examined. Quenching of 9-aminoacridine fluorescence increased with increasing amounts of phosphatidic acid added, independent of the composition of the added liposomes. In certain instances, the computational methods did not yield the surface charge density of the liposomes expected from their composition. However, when the effects of background ionic strength on surface potential were considered, there was a positive correlation between expected and calculated values. Therefore, the data support the contention that changes in the fluorescence of 9-aminoacridine can be used to calculate surface charge density of membranes.
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    Spontaneous Gating of Olfactory Cyclic-Nucleotide-Gated Channels (2000)
    Springer
    The journal of membrane biology 178 (2000), S. 49-54 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Olfaction — Receptor neuron — Cilia — Cyclic-nucleotide-gated channel — Spontaneous gating — Electrophysiology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. In vertebrates, cilia on the olfactory receptor neurons have a high density of cyclic-nucleotide-gated (CNG) channels. During transduction of odorous stimuli, cyclic AMP is formed. cAMP gates the CNG channels and this initiates the neuronal depolarization. Here it is shown that the ciliary CNG channels also open spontaneously. In the absence of odorants and second messengers, olfactory cilia have a small basal conductance to cations. Part of this conductance is similar to the cAMP-activated conductance in its sensitivity to channel inhibitors and divalent cations. The basal conductance may help to stabilize the neuronal membrane potential while limiting the sensitivity of odorant detection.
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    Slow Gating of Gap Junction Channels and Calmodulin (2000)
    Springer
    The journal of membrane biology 178 (2000), S. 55-70 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Cell communication — Connexins — Gap junctions — Calmodulin — Channel gating — CO2
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Certain COOH-terminus mutants of connexin32 (Cx32) were previously shown to form channels with unusual transjuctional voltage (V j ) sensitivity when tested heterotypically in oocytes against Cx32 wild type. Junctional conductance (G j ) slowly increased by severalfold or decreases to nearly zero with V j positive or negative, respectively, at mutant side, and V j positive at mutant side reversed CO2-induced uncoupling. This suggested that the CO2-sensitive gate might be a V j -sensitive slow gate. Based on previous data for calmodulin (CaM) involvement in gap junction function, we have hypothesized that the slow gate could be a CaM-like pore plugging molecule (cork gating model). This study describes a similar behavior in heterotypic channels between Cx32 and each of four new Cx32 mutants modified in cytoplasmic-loop and/or COOH-terminus residues. The mutants are: ML/NN+3R/N, 3R/N, ML/NN and ML/EE; in these mutants, N or E replace M105 and L106, and N replace R215, R219 and R220. This study also reports that inhibition of CaM expression strongly reduces V j and CO2 sensitivities of two of the most effective mutants, suggesting a CaM role in slow and chemical gating.
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    Role of Actin Cytoskeleton in Regulation of Ion Transport: Examples from Epithelial Cells (2000)
    Springer
    The journal of membrane biology 178 (2000), S. 73-87 
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    ISSN: 1432-1424
    Keywords: Key words: Actin — Epithelial cells — Phospholipids — Vesicle trafficking — Signal transduction — Ion transport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
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    URL: http://dx.doi.org/10.1007/s002320010016
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    Isoform-Specific Function and Distribution of Na/K Pumps in the Frog Lens Epithelium (2000)
    Springer
    The journal of membrane biology 178 (2000), S. 89-101 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Na/K ATPase — Lens —α-Isoforms — Whole cell patch clamp — Na+-dependence — K+-dependence — pH
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Epithelial cells from the anterior and equatorial surfaces of the frog lens were isolated and used the same day for studies of the Na/K ATPase. RNase protection assays showed that all cells express α1- and α2-isoforms of the Na/K pump but not the α3-isoform, however the α2-isoform dominates in anterior cells whereas the α1-isoform dominates in equatorial cells. The whole cell patch-clamp technique was used to record functional properties of the Na/K pump current (I P ), defined as the current specifically inhibited by dihydro-ouabain (DHO). DHO-I P blockade data indicate the α1-isoform has a dissociation constant of 100 μm DHO whereas for the α2-isoform it is 0.75 μm DHO. Both α1- and α2-isoforms are half maximally activated at an intracellular Na+-concentration of 9 mm. The α1-isoform is half maximally activated at an extracellular K+-concentration of 3.9 mm whereas for the α2-isoform, half maximal activation occurs at 0.4 mm. Lastly, transport by the α1-isoform is inhibited by a drop in extracellular pH, which does not affect transport by the α2-isoform. Under normal physiological conditions, I P in equatorial cells is approximately 0.23 μA/μF, and in anterior cells it is about 0.14 μA/μF. These current densities refer to the area of cell membrane assuming a capacitance of around 1 μF/cm2. Because cell size and geometry are different at the equatorial vs. anterior surface of the intact lens, we estimate Na/K pump current density per area of lens surface to be around 10 μA/cm2 at the equator vs. 0.5 μA/cm2 at the anterior pole.
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    Activation of A3 Adenosine Receptor Induces Calcium Entry and Chloride Secretion in A6 Cells (2000)
    Springer
    The journal of membrane biology 178 (2000), S. 103-113 
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    ISSN: 1432-1424
    Keywords: Key words: A3 receptor — Cl-IB-MECA — A6 cells — Renal — Chloride — Calcium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. We have previously demonstrated that in A6 renal epithelial cells, a commonly used model of the mammalian distal section of the nephron, adenosine A1 and A2A receptor activation modulates sodium and chloride transport and intracellular pH (Casavola et al., 1997). Here we show that apical addition of the A3 receptor-selective agonist, 2-chloro-N6-(3-iodobenzyl)-adenosine-5′-methyluronamide (Cl-IB-MECA) stimulated a chloride secretion that was mediated by calcium- and cAMP-regulated channels. Moreover, in single cell measurements using the fluorescent dye Fura 2-AM, Cl-IB-MECA caused an increase in Ca2+ influx. The agonist-induced rise in [Ca2+] i was significantly inhibited by the selective adenosine A3 receptor antagonists, 2,3-diethyl-4,5-dipropyl-6-phenylpyridine-3-thiocarboxylate-5-carboxylate (MRS 1523) and 3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS 1191) but not by antagonists of either A1 or A2 receptors supporting the hypothesis that Cl-IB-MECA increases [Ca2+] i by interacting exclusively with A3 receptors. Cl-IB-MECA-elicited Ca2+ entry was not significantly inhibited by pertussis toxin pretreatment while being stimulated by cholera toxin preincubation or by raising cellular cAMP levels with forskolin or rolipram. Preincubation with the protein kinase A inhibitor, H89, blunted the Cl-IB-MECA-elicited [Ca2+] i response. Moreover, Cl-IB-MECA elicited an increase in cAMP production that was inhibited only by an A3 receptor antagonist. Altogether, these data suggest that in A6 cells a G s /protein kinase A pathway is involved in the A3 receptor-dependent increase in calcium entry.
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    Expression and Molecular Characterization of Rat Renal d-Mannose Transport in Xenopus Oocytes (2000)
    Springer
    The journal of membrane biology 178 (2000), S. 127-135 
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    ISSN: 1432-1424
    Keywords: Key words: Mannose transport — Renal reabsorption — Oocyte expression —Xenopus laevis— Membrane vesicles — Rat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Renal reabsorption appears to play a major role in d-mannose homeostasis. Here we show that in rat kidney, the transport of d-mannose by brush border membrane vesicles from tubular epithelial cells involves an uphill and rheogenic Na-dependent system, which is fully inhibited by d-mannose itself, incompletely inhibited by d-glucose, d-fructose, phloridzin, and phloretin, and noninhibited by l-mannose or disaccharides. In addition, this system exhibits both low capacity (112.9 ± 15.6 pmol/mg/second) and high affinity (0.18 ± 0.04 mm), with a 2:1 stoichiometry for the Na:d-mannose interaction, and low affinity for sodium (16.6 ± 3.67 mm). We also show expression of d-mannose transport by Xenopus laevis oocytes injected with rat renal polyA+ RNA. Kinetic analysis of the expressed transport was performed after RNA enrichment by fractionation through a sucrose density gradient and was shown to be identical to that measured in membrane vesicles. The RNA species encoding the expressed transport has a small mean size, 1 kb approximately, and shows no homology with the SGLT family of Na-dependent d-glucose transporters, as shown by low stringent RT-PCR and northern analysis. The expressed transport is specific for d-mannose, since in spite of a significant inhibition by d-glucose and d-fructose, neither of these two substrates was transported above the level of the water-injected oocytes.
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    Cell pH and H+ Secretion by S3 Segment of Mammalian Kidney: Role of H+-ATPase and Cl− (2000)
    Springer
    The journal of membrane biology 178 (2000), S. 115-125 
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    ISSN: 1432-1424
    Keywords: Key words: H+-ATPase — Subcellular vesicles — Bafilomycin — Exocytosis — Cell pH regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The role of H+-ATPase in proximal tubule cell pH regulation was studied by microperfusion techniques and by confocal microscopy. In a first series of experiments, proximal S3 segments of rabbit kidney were perfused ``in vitro'' while their cell pH was measured by fluorescence microscopy after loading with BCECF. In Na+- and Cl−-free medium, cell pH fell by a mean of 0.37 ± 0.051 pH units, but after a few minutes started to rise again slowly. This rise was of 0.17 ± 0.022 pH units per min, and was significantly reduced by bafilomycin and by the Cl− channel blocker NPPB, but not by DIDS. In a second series of experiments, subcellular vesicles of proximal tubule cells of S3 segments of mouse kidney were studied by confocal microscopy after visualization by acridine orange or by Lucifer yellow. After superfusion with low Na+ solution, which is expected to cause cell acidification, vesicles originally disposed in the basolateral and perinuclear cell areas, moved toward the apical area, as detected by changes in fluorescence density measured by the NIH Image program. The variation of apical to basolateral fluorescence ratios during superfusion with NaCl Ringer with time was 0.0018 ± 0.0021 min−1, not significantly different from zero (P 〉 0.42). For superfusion with Na+0 Ringer, this variation was 0.081 ± 0.015 min−1, P 〈 0.001 against 0. These slopes were markedly reduced by the Cl− channel blocker NPPB, and by vanadate at a concentration that has been shown to disrupt cytoskeleton function. These data show that the delayed alkalinization of proximal tubule cells in Na+-free medium is probably due to a vacuolar H+-ATPase, whose activity is stimulated in the presence of Cl−, and dependent on apical insertion of subcellular vesicles. The movement of these vesicles is also dependent on Cl− and on the integrity of the cytoskeleton.
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    Ion Permeation and Selectivity of Wild-Type Recombinant Rat CNG (rOCNC1) Channels Expressed in HEK293 Cells (2000)
    Springer
    The journal of membrane biology 178 (2000), S. 137-150 
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    ISSN: 1432-1424
    Keywords: Key words: Recombinant rat cyclic nucleotide-gated channel (rOCNC1) — Selectivity — Permeation — Cation — Patch clamp
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The permeation properties of adenosine 3′, 5′-cyclic monophosphate (cAMP)-activated recombinant rat olfactory cyclic nucleotide-gated channels (rOCNC1) in human embryonic kidney (HEK 293) cells were investigated using inside-out excised membrane patches. The relative permeability of these rOCNC1 channels to monovalent alkali cations and organic cations was determined from measurements of the changes in reversal potential upon replacing sodium in the bathing solution with different test cations. The permeability ratio of Cl− relative to Na+ (P Cl /P Na ) was about 0.14, confirming that these channels are mainly permeable to cations. The sequence of relative permeabilities of monovalent alkali metal ions in these channels was P Na ≥P K 〉 P Li 〉 P Cs ≥P Rb , which closely corresponds to a high-strength field sequence as previously determined for native rat olfactory receptor neurons (ORNs). The permeability sequence for organic cations relative to sodium was P NH3OH 〉 P NH4 〉 P Na 〉 P Tris 〉 P Choline 〉 P TEA , again in good agreement with previous permeability ratios obtained in native rat ORNs. Single-channel conductance sequences agreed surprisingly well with permeability sequences. These conductance measurements also indicated that, even in asymmetric bi-ionic cation solutions, the conductance was somewhat independent of current direction and dependent on the composition of both solutions. These results indicate that the permeability properties of rOCNC1 channels are similar to those of native rat CNG channels, and provide a suitable reference point for exploring the molecular basis of ion selectivity in recombinant rOCNC1 channels using site-directed mutagenesis.
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    Charged Residues in Surface-located Loops Influence Voltage Gating of Porin from Haemophilus influenzae Type b (2000)
    Springer
    The journal of membrane biology 178 (2000), S. 185-193 
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    ISSN: 1432-1424
    Keywords: Key words:Haemophilus influenzae type b — Porin — Voltage gating — Ion channel
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Porin of Haemophilus influenzae type b (341 amino acids; M r 37782) determines the permeability of the outer membrane to low molecular mass compounds. Purified Hib porin was subjected to chemical modification of lysine residues by succinic anhydride. Electrospray ionization mass spectrometry identified up to 12 modifications per porin molecule. Tryptic digestion of modified Hib porin followed by reverse phase chromatography and matrix assisted laser desorption ionization time-of-flight mass spectrometry mapped the succinylation sites. Most modified lysines are positioned in surface-located loops, numbers 1 and 4 to 7. Succinylated porin was reconstituted into planar lipid bilayers, and biophysical properties were analyzed and compared to Hib porin: there was an increased average single channel conductance compared to Hib porin (1.24+/−0.41 vs. 0.85+/−0.40 nanosiemens). The voltage-gating activity of succinylated porin differed considerably from that of Hib porin. The threshold voltage for gating was decreased from 75 to 40 mV. At 80 mV, steady-state conductance for succinylated porin was 50–55% of the instantaneous conductance. Hib porin at 80 mV showed a decrease to 89–91% of the instantaneous current levels. We propose that surface-located lysine residues are determinants of voltage gating for porin of Haemophilus influenzae type b.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002320010026
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  • 97
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    Tight Junction Dynamics: Oscillations and the Role of Protein Kinase C (2000)
    Springer
    The journal of membrane biology 178 (2000), S. 151-161 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Tight junction — Protein kinase C — Calcium — Oscillations — Paracellular conductance — Feedback control
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. The present study aimed to characterize the role of protein kinase C (PKC) on the dynamics of tight junction (TJ) opening and closing in the frog urinary bladder. The early events of TJ dynamics were evaluated by the fast Ca++ switch assay (FCSA), which consisted in opening the TJs by removing basolateral Ca++ ([Ca++] bl ), and closing them by returning [Ca++] bl to normal values. Changes in TJ permeability can be reliably gauged through changes of transepithelial electrical conductance (G) determined in the absence of apical Na+. The FCSA allows the appraisal of drugs and procedures acting upon the mechanism controlling the TJs. The time courses of TJ opening and closing in an FCSA were shown to follow single exponential time courses. PKC inhibition by H7 (100 μm) caused a reduction of the rate of junction opening in response to removing [Ca++] bl , without affecting junction closing, indicating that PKC is a key element in the control of TJ opening dynamics in this preparation. H7 at 250 μm almost completely inhibits TJ opening in response to basolateral Ca++ withdrawal. Subsequent H7 removal caused a prompt inhibition release characterized by a sharp G increase which, however, once started cannot be stopped by H7 reintroduction, Ca++ being necessary to allow TJ recovery. A step rise of apical Ca++ concentration ([Ca++] ap ) causes a reduction of the rate of TJ opening in a FCSA, an effect that is believed to be mediated by apical Ca++ entering the open TJs. The specific condition of having Ca++ only in the apical solution and the TJs located midway between the Ca++ source (apical solution) and the Ca++-binding sites presumably located at the zonula adhaerens, might configure a situation in which a control feedback loop is set up. A rise of [Ca++] ap during the phase of G increase in an FCSA causes a transient recovery of G followed by a subsequent escape phase where G increases again. Oscillations of G also appear in response to a rise of apical Ca++. Both escape and oscillations result from the properties of the TJ regulatory feedback loop. In conclusion, the present results indicate that PKC plays a key role in TJ opening in response to extracellular Ca++ withdrawal without major effect on the reverse process. In addition, PKC inhibition by H7 not only prevents TJ opening in response to basolateral Ca++ removal but induces a prompt blockade of TJ oscillations induced by apical Ca++, oscillations which reappear again when H7 is removed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002320010022
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  • 98
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    Activation of Metabotropic Glutamate Receptors Inhibits Synapsin I Phosphorylation in Visceral Sensory Neurons (2000)
    Springer
    The journal of membrane biology 178 (2000), S. 195-204 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Synaptic proteins — mGluRs — Nodose ganglia — Baroreceptors — Synapsin — Synaptic transmission
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Activation of glutamate metabotropic receptors (mGluRs) in nodose ganglia neurons has previously been shown to inhibit voltage-gated Ca++ currents and synaptic vesicle exocytosis. The present study describes the effects of mGluRs on depolarization-induced phosphorylation of the synaptic-vesicle-associated protein synapsin I. Depolarization of cultured nodose ganglia neurons with 60 mm KCl resulted in an increase in synapsin I phosphorylation. Application of mGluR agonists 1-aminocyclopentane-1s-3r-dicarboxylic acid (t-ACPD) and L(+)-2-Amino-4-phosphonobutyric acid (L-AP4) either in combination or independently inhibited the depolarization induced phosphorylation of synapsin I. Application of the mGluR antagonist (RS)-α-Methyl-4-carboxyphenylglycine (MCPG) blocked t-ACPD-induced inhibition of synapsin phosphorylation but not the effects of L-AP4. In addition, application of either t-ACPD or L-AP4 in the absence of KCl induced depolarization had no effect on resting synapsin I phosphorylation. RT-PCR analysis of mGluR subtypes in these nodose ganglia neurons revealed that these cells only express group III mGluR subtypes 7 and 8. These results suggest that activation of mGluRs modulates depolarization-induced synapsin I phosphorylation via activation of mGluR7 and/or mGluR8 and that this process may be involved in mGluR inhibition of synaptic vesicle exocytosis in visceral sensory neurons of the nodose ganglia.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002320010027
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  • 99
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    Electronic Resource
    Temperature Sensitivity of the Tonoplast Ca2+-Activated K+ Channel in Chara: The Influence of Reversing the Sign of Membrane Potential (2000)
    Springer
    The journal of membrane biology 178 (2000), S. 215-224 
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    ISSN: 1432-1424
    Keywords: Key words: Ca2+-sensitive K+ channel — Temperature — Thermodynamics —Chara— Voltage sensitivity — Lipid phase transition
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. A detailed temperature dependence study of a well-defined plant ion channel, the Ca2+-activated K+ channel of Chara corallina, was performed over the temperature range of their habitats, 5–36°C, at 1°C resolution. The temperature dependence of the channel unitary conductance at 50 mV shows discontinuities at 15 and 30°C. These temperatures limit the range within which ion diffusion is characterized by the lowest activation energy (E a = 8.0 ± 1.6 kJ/mol) as compared to the regions below 15°C and above 30°C. Upon reversing membrane voltage polarity from 50 to −50 mV the pattern of temperature dependence switched from discontinuous to linear with E a = 13.6 ± 0.5 kJ/mol. The temperature dependence of the effective number of open channels at 50 mV showed a decrease with increasing temperature, with a local minimum at 28°C. The mean open time exhibited a similar behavior. Changing the sign of membrane potential from 50 to −50 mV abolished the minima in both temperature dependencies. These data are discussed in the light of higher order phase transitions of the Characean membrane lipids and corresponding change in the lipid-protein interaction, and their modulation by transmembrane voltage.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002320010029
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  • 100
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    Electronic Resource
    Characterization of ATP-Sensitive Potassium Channels Functionally Expressed in Pituitary GH3 Cells (2000)
    Springer
    The journal of membrane biology 178 (2000), S. 205-214 
    Details
    ISSN: 1432-1424
    Keywords: Key words: GH3 cells — ATP-sensitive K+ channels — Diazoxide — Glibenclamide — Phospholipase A2— 4,4′-dithiodipyridine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. ATP-sensitive K+ (KATP) channels have been characterized in pituitary GH3 cells with the aid of the patch-clamp technique. In the cell-attached configuration, the presence of diazoxide (100 μm) revealed the presence of glibenclamide-sensitive KATP channel exhibiting a unitary conductance of 74 pS. Metabolic inhibition induced by 2,4-dinitrophenol (1 mm) or sodium cyanide (300 μm) increased KATP channel activity, while nicorandil (100 μm) had no effect on it. In the inside-out configuration, Mg-ATP applied intracellularly suppressed the activity of KATP channels in a concentration-dependent manner with an IC50 value of 30 μm. The activation of phospholipase A2 caused by mellitin (1 μm) was found to enhance KATP channel activity and further application of aristolochic acid (30 μm) reduced the mellitin-induced increase in channel activity. The challenging of cells with 4,4′-dithiodipyridine (100 μm) also induced KATP channel activity. Diazoxide, mellitin and 4,4′-dithiodipyridine activated the KATP channels that exhibited similar channel-opening kinetics. In addition, under current-clamp conditions, the application of diazoxide (100 μm) hyperpolarized the membrane potential and reduced the firing rate of spontaneous action potentials. The present study clearly indicates that KATP channels similar to those seen in pancreatic β cells are functionally expressed in GH3 cells. In addition to the presence of Ca2+-activated K+ channels, KATP channels found in these cells could thus play an important role in controlling hormonal release by regulating the membrane potential.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002320010028
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