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B Nilius

Publications and source records attributed to B Nilius.

At least 163 records · Page 9Linked to original sources
PubMed1993

Control of intracellular calcium by membrane potential in human melanoma cells.

The modulation of intracellular calcium ([Ca2+]i) by the membrane potential was investigated in human melanoma cells by combining the nystatin-perforated patch-clamp technique with Ca2+ measurements. Voltage steps to -100 mV induced a rise in [Ca2+]i and a creeping inward current. These effects were absent in Ca(2+)-free solution and could be blocked by Ni2+ or La3+. Voltage ramps revealed a close correlation between [Ca2+]i and voltage, with the strongest voltage dependence around the resting potential. Long-lasting tail currents, closely correlated with the rise in [Ca2+]i and a reversal potential close to the K+ equilibrium potential, occurred if the membrane potential was clamped back to 0 mV. They were absent if intracellular K+ was replaced by Cs+ and blocked by extracellular tetraethylammonium (5 mM), Ba2+ (1 mM), or a membrane-permeable adenosine 3',5'-cyclic monophosphate analogue. These observations are discussed in relation to cell proliferation. The enhanced expression of K+ channels during cell proliferation provides a positive-feedback mechanism resulting in long-term changes in [Ca2+]i required for the G1-S transition in the cell cycle.

Barium↗
PubMed1992

Shear stress induced membrane currents and calcium transients in human vascular endothelial cells.

We have measured membrane currents induced by shear stress together with intracellular calcium signals in endothelial cells from human umbilical cord veins. In the presence of extracellular calcium (Ca2+]o), shear stress induced an inward current at a holding potential of 0 mV which is accompanied by a rise in intracellular Ca2+ ([Ca2+]i). In the absence of extracellular calcium shear stress was unable to evoke a calcium signal but still induced a membrane current. The voltage dependence of the shear stress induced current was obtained from difference currents evoked by linear voltage ramps before and during application of shear stress. Its reversal potential Erev shifted from -2.3 +/- 0.8 mV (n = 4) in a nominally Ca2+ free solution to +1.5 +/- 1.6 mV at 1.5 mM [Ca2+]o (n = 4) and to +21.9 +/- 4.4 mV (n = 7) at 10 mM [Ca2+]o. From our data we conclude that shear stress opens an ion channel that is 12.5 +/- 2.9 (n = 7) times more permeable for calcium than for sodium or cesium.

Calcium↗
PubMed1992

Thrombin stimulates L-type calcium channels of guinea pig cardiomyocytes in cell-attached patches but not after intracellular dialysis.

The action of the blood clotting enzyme thrombin on single channel and whole cell Ca(2+)-currents was studied in isolated mammalian cardiac myocytes. Thrombin, at a concentration of 10(-8) mol/l, increased the Ca(2+)-channel activity in cell-attached patches. The mean open probability of the channel was enhanced, while the number of sweeps without openings, which reflects the availability of the channel, was significantly reduced. Neither the single channel conductance nor the activation curve were affected by thrombin. Thrombin was added to the bath solution, and its effect is therefore indirect and probably mediated via a second messenger. However, thrombin did not affect whole-cell Ca(2+)-currents, whereas a beta-adrenergic stimulation in the same cell increased the Ca(2+)-current. It is concluded that thrombin affects an intracellular mechanism for Ca2+ channel current regulation, which is still unknown and which is rapidly lost during conventional whole-cell Ca2+ current measurements.

Animals↗
PubMed1992

Potassium channels and regulation of proliferation of human melanoma cells.

1. Ion channels and their possible relation to cell proliferation have been studied in a human melanoma cell line (IGR 1). Membrane currents were recorded by the patch-clamp technique using the cell-attached, cell-free and whole-cell mode. Cell growth was monitored by counting the number of cells at different days after seeding and [3H]thymidine incorporation. 2. A voltage-dependent 10 pS non-inactivating potassium channel (delayed rectifier) is the most commonly observed ion channel in this type of human cell. The channel is active at the normal resting potential and can be blocked by tetraethylammonium chloride (TEA) and also by a membrane-permeable cyclic adenosine monophosphate (8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, cyclic AMP). A second type of potassium channel shows properties similar to voltage-dependent A-type potassium channels with complete inactivation. 3. A voltage-independent, non-selective cation channel with a single-channel conductance of approximately 20 pS could be seen in only 8% of the patches. Its properties of modulation are still unknown. 4. The incidence of the 10 pS, non-inactivated potassium channel was maximal at the fourth day after seeding (in 89% of the patches) and was significantly reduced at the seventh day (in 35% of the patches). 5. [3H]thymidine incorporation is maximal at the third day after seeding and is reduced when cells are grown in the presence of TEA or cyclic AMP. This peak of maximal [3H]thymidine incorporation correlated with the incidence of non-inactivated potassium channels. 6. In the presence of TEA or cyclic AMP, growth of the cells is inhibited. We suppose that due to block of potassium channels, most of the melanoma cells are not able to enter the S-phase in the cell division cycle. 7. It is concluded that delayed rectifier potassium channels are involved in the control of melanoma cell proliferation. A similar finding has been reported for K+ channels in T-lymphocytes and human breast carcinoma cells. It is suggested that potassium channels may be involved in controlling the driving force for a calcium influx thereby interacting with Ca(2+)-dependent cell cycle control proteins.

Calcium↗
PubMed1992

Shear stress-induced calcium transients in endothelial cells from human umbilical cord veins.

1. Changes of the free cytosolic Ca2+ concentration induced by shear stress were measured in Fura-2 acetoxymethyl ester-loaded endothelial cells from human umbilical cord veins. 2. We were able to induce Ca2+ transients in almost every cell by blowing a stream of physiological solution onto a single endothelial cell thereby inducing shear stress between 0 and 50 dyn cm-2. The Ca2+ response could be graded by varying the shear stress, and reached a half-maximal value at a shear stress of 30 dyn cm-2. 3. The shear stress responses critically depended on the extracellular Ca2+ concentration and were absent in a Ca(2+)-free solution. Repetitive application of short pulses of shear stress induced cumulative effects because of the slow decay of the shear stress Ca2+ responses (time constants 82.3 +/- 17.8 s from twenty-five cells). Application of a depolarizing high potassium solution to reduce the driving force for Ca2+ entry decreased the Ca2+ transients in some of the cells. 4. Application of shear stress in the presence of other divalent cations, such as nickel, cobalt or barium, always produced substantial changes in the ratio of the 390/360 nm fluorescence signal, indicating influx of these cations and subsequent quenching of the Fura-2 fluorescence. 5. Shear stress responses in the presence of 10 mM Ca2+ were completely blocked by application of 1 mM La3+. 6. Incubation of the cells with the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) did not alter the shear stress response, but completely blocked histamine-induced Ca2+ transients. 7. Small submaximal shear stress potentiated the Ca2+ transients induced by histamine. 8. We conclude that shear stress-dependent Ca2+ signals are induced by an influx of calcium that is not modulated via protein kinase C and not activated by membrane depolarization. The influx pathway is also permeable to divalent cations such as Ni2+, Co2+ and Ba2+, but is blocked by La3+.

Barium↗
PubMed1991

The conductance of single cardiac sodium channels from guinea pig depends on the intracellular sodium concentration.

Currents through DPI 201-106 modified single sodium channels have been measured in cell-free inside-out patches from guinea-pig ventricular myocytes. Single-channel conductance and reversal potential of the sodium channel have been calculated at different intracellular sodium concentrations [( Na+]i) from microscopic I-V curves, which were obtained by application of linear voltage ramps. The relation between the reversal potential and [Na+]i could be fitted with a modified Goldman-Hodgkin-Katz equation with a relative permeability for K+ over Na+ ions of 0.054. The zero-current conductance of the Na channel as a function of [Na+]i shows a plateau value at low Na concentrations, and increases in a sigmoidal manner at higher concentrations. It is concluded that the Na channel can carry outward currents and that its conductance depends on [Na+]i.

Animals↗
PubMed1991

Substates of cardiac sodium channels are due to both decrease in conductance and changes in selectivity.

Currents through DPI 201-106 modified single cardiac sodium channels in guinea pig ventricular cells were measured using the patch clamp technique in the cell-free configuration to control the sodium concentrations on both sides of the patch membrane. Current-voltage relationships of the single channels were obtained by application of linear voltage ramps from -140 to 100 mV. With 10 mmol/l Na+ at the inner surface of the patch, openings of sodium channels with conductances of 17 pS (selectivity ratios PK/PNa = 0.083 and PK/PNa = 0.58) and 12 pS (selectivity ratios PK/PNa = 0.084 and PK/PNa = 1.832) were obtained. With 30 mmol/l internal sodium, conductances of 20, 10, and 7 pS and selectivity ratios of 0.084, 0.386, and 0.543, respectively, could be measured. It is concluded that substates of sodium channel currents are due to changes in single channel conductance as well as in selectivity, or to changes of both independently of each other which accounts for the variability of conductance levels of cardiac Na channels.

Animals↗
PubMed1990

Pharmacological modification of mechanical and electrical responses of frog heart to thrombin.

The pharmacological modification of the thrombin effect on the mechanical and electrical responses of frog heart was examined in the Straub heart preparation and in single ventricular cells. Associated with the positive inotropic action thrombin increases voltage and duration of action potentials of isolated frog ventricular cells. As found by the patch-clamp technique in the cell-attached mode, thrombin stimulates single L-type Ca2+ channels, presumably mediated by a second messenger. The enhancement of contractility by thrombin depends on the proteolytic activity of the enzyme because enzymatically inactivated thrombin has no effect on frog hearts. The positive inotropic effect of thrombin as well as its stimulation of Ca2+ channel currents were inhibited by the protein kinase C blocker 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H7). However, phorbol 12-myristate 13-acetate (PMA), a known stimulator of protein kinase C, was ineffective in stimulating the inotropic action of thrombin. The inhibition of the thrombin-induced enhancement of contractility by indometacin indicates an involvement of arachidonic acid in the action of thrombin on frog heart.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Block of single cardiac sodium channels by intracellular magnesium.

Currents through single cardiac sodium channels have been measured in inside-out patches from guinea pig ventricular cells. To abolish the fast inactivation, Na channels were modified by DPI 201-106. In symmetrical Na solutions, a diminution of outward sodium currents can be observed that depends on the intracellular magnesium concentration and the membrane potential. Inward currents were not altered by the concentrations of magnesium used (between 0 and 22.5 mmol/l). In Mg free solutions a linear current-voltage relation can also be measured in the range of outward Na currents. At +60 mV (symmetrical Na solutions, single channel conductance 24 pS) a half maximal block of cardiac Na channels by intracellular magnesium was found at 2.1 mmol/l. From the analysis of single channel current-voltage relationships the concentration and voltage-dependent block by intracellular magnesium of cardiac sodium channels could be described as binding of Mg at one site with a Kd value of 5.1 mmol/l at 0 mV. The site is located at an electrical distance of 0.18 from the inside.

Animals↗
PubMed1990

Properties of a potassium-selective ion channel in human melanoma cells.

Currents through ion channels were measured from cells of a human melanin-producing melanoma cell line (IRG 1) with the patch clamp technique. In these cells the most frequently observed channel is a potassium channel. The channel activates slowly at depolarizing voltage steps but does not inactivate. Single channel potassium currents can be measured in cell-attached patches at the resting potential of melanoma cells. The channel has a conductance of approximately 10 pS. As measured from the reversal potentials of single channel currents, the permeability ratio for sodium and potassium, PNa/PK, is between 0.03 and 0.04. Open probability is increased at positive potentials. Mean open times are prolonged at voltage steps to more positive potentials. Closed time histograms are fitted by two exponentials. The slow shut time is decreased at positive potentials. In whole cell measurements, cell conductance measured between -20 and + 70 mV was reduced by 10 mM tetraethylammonium chloride from 6.4 +/- 1.2 nS (n = 4) to 0.8 +/- 0.3 nS (n = 3). Application of isoproterenol decreases the probability of the channel being open without any change in the single channel conductance. A possible role of the 10 pS potassium channel in the growth of melanoma cells is discussed.

Cyclic AMP↗
PubMed1990

Permeation properties of a non-selective cation channel in human vascular endothelial cells.

Endothelial cells obtained from human umbilical chord have been studied by the patch clamp method. An ion channel is described that is activated by microM concentrations of histamine and shows a slow run-down in cell-attached patches. After excision, channel activity quickly runs down to zero open probability. In symmetrical potassium concentrations (140 mM K in the bath and the pipette), the single channel conductance is 28 +/- 2 pS and the reversal potential is 0.3 +/- 0.8 mV (mean +/- SEM, n = 4). With 140 mM Na in the pipette, the conductance is 26 +/- 2 pS. A reversal potential of -1.5 +/- 0.9 mV (n = 7) was measured. With 60 mM Ca and 70 mM Na in the pipette, 140 mM K in the bath, the reversal potential was -11 +/- 3 mV, the single channel conductance in 16 +/- 3 pS (n = 5). The single channel conductance in 110 mM Ca (pipette) and 140 mM K (bath) is 8 +/- 2 pS and the reversal potential is -18 +/- 6 mV (n = 3). From analysis of the reversal potentials, a permeation ratio of K:Na:Ca = 1:0.9:0.2 was calculated. This ligand-gated non-selective cation channel in human endothelial cells is Ca permeable and could induce a sustained agonist mediated Ca influx.

Calcium↗
PubMed1990

Effects of thrombin on single calcium channels in frog ventricular cells.

Single calcium channel (Ca channel) currents were measured using the patch-clamp technique in isolated ventricular myocytes of the frog (Rana esculenta). Sodium was used as the charge carrier. After formation of cell-attached patches, the proteolytic enzyme thrombin was added to the bath solution, where it increased the amplitude of the averaged currents more than twofold, by decreasing the number of empty sweeps and reducing the time constant of the slow exponential term of the shut-time histogram. Single channel conductance was not changed by thrombin. If the activation kinetics of the Ca channels are described by the commonly used C1-C2-O model, where C1 and C2 indicate closed states 1 and 2 respectively and O denotes the open state, thrombin increases the open-state probability in the non-empty sweeps by increasing the rate constant (k1) for the transition from C1 to C2. It is shown that thrombin acts via an H-7 blockable pathway.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Free energy of ATP-hydrolysis fails to affect ATP-dependent potassium channels in isolated mouse ventricular cells.

The single channel recording technique has been used to study the adenosine-5'-triphosphate (ATP)-dependent K+ channel in isolated mouse ventricular cells. The aim of this work was to determine if the activity of the K+ channel depends on the free energy of ATP-hydrolytic reaction (phosphorylation potential) in addition to the well-studied direct block by ATP. When the phosphorylation potential was changed from 60 to 50, to 40 and back to 60 kJ/mol at a constant ATP-concentration of 10(-4) mol/l, the ATP-channel activity showed a run down that is best described by a linear function. Changing the ATP-concentration of the bath solution from 0.01 to 1 and back to 0.01 mmol/l by a factor of 10 at a constant phosphorylation potential of 50 kJ/mol, resulted in a run down of the mean average current by a reduction of the open state probability in a concentration dependent manner. A dissociation constant of about 0.14 mmol/l could be estimated. The amplitude of the single channel current was not affected by ATP-concentrations in contrast to changes in phosphorylation potential. A significant increase in the single channel current accompanying a decrease in the phosphorylation potential at constant ATP concentrations was observed. This effect might be due to a decrease of free Mg2(+)-concentration by an increase of the ADP concentration in solutions with lower phosphorylation potentials. An allosteric regulation of the ATP-dependent K+ channel dependent on the free ATP concentration together with the Mg2(+)-ADP concentration seems to be a more likely explanation than regulation by phosphorylation/dephosphorylation secondary to a breakdown of ATP.

Action Potentials↗
PubMed1990

Modulation of the T-type cardiac Ca channel by changes in proton concentration.

Single-channel measurements and whole-cell experiments with the two suction electrode, voltage clamp technique were used to investigate the effects of external and internal proton concentrations on T-type Ca channels in heart muscle cells of the guinea pig. As in the L-type Ca channel, an increase in the external proton concentration decreases T-type currents, while external alkalinization enlarges the currents. In contrast to the L-type Ca channel, however, a change in the internal proton concentration does not modulate T-type Ca currents. The T-type Ca channel is much more sensitive to variations in pHo than the L-type Ca channel. By the combination of single-channel and whole-cell experiments we can conclude that the observed changes in macroscopic currents are due to (a) changes in the single-channel conductance and in the probability of the T-type Ca channel being open, and (b) the titration of the negative surface charges in the neighborhood of the T-type Ca channel with shifts of both the activation and inactivation processes of the channel. The pHo-induced changes in the maximal conductance (gmax) of the T-type Ca channel show an apparent pKa in the range of 7.1-7.5, while the titration of the negative surface charges near the channel shows an apparent pKa of 7.1 with a concomitant surface potential of -24.6 mV at 5.4 mM [Ca]o. These pKa values, less acid than the pKa values found for the pHo-induced, L-type Ca channel modulation, might imply a physiological importance of this novel type of channel modulation.

Action Potentials↗
PubMed1990

Ion channels in human endothelial cells.

Ion channels were studied in human endothelial cells from umbilical cord by the patch clamp technique in the cell attached mode. Four different types of ion channels were recorded: i) potassium channel current that rectifies at positive potentials in symmetrical potassium solutions (inward rectifier); ii) low-conductance non-selective cation channel with a permeability ratio K:Na:Ca = 1:0.9:0.2; iii) high-conductance cation-selective channel that is about 100 times more permeable for calcium than for sodium or potassium; iv) high-conductance potassium channel with a permeability ratio K:Na = 1:0.05. The extrapolated reversal potential of the inwardly rectifying current was near to the potassium equilibrium potential. The slope conductance decreased from 27 pS in isotonic KCl solution to 7 pS with 5.4 mmol/l KCl and 140 mmol/l NaCl in the pipette but 140 mmol/l KCl in the bath. The low-conductance non-selective cation channel showed a single-channel conductance of 26 pS with 140 mmol/l Na outside, 28 pS with 140 mmol/l K outside, and rectified in inward direction in the presence of Ca (60 mmol/l Ca, 70 mmol/l Na, 2.7 mmol/l K in the pipette) at negative potentials. The current could be observed with either chloride or aspartate as anion. The high-conductance non-selective channel did not discriminate between Na and K. The single-channel conductance was about 50 pS. The extrapolated reversal potential was more positive than +40 mV (140 K or 140 Na with 5 Ca outside). Both the 26 and 50 pS channel showed a run-down, and they rapidly disappeared in excised patches. The high-conductance potassium channel with a single-channel conductance of 170 pS was observed only rarely. It reversed near the expected potassium equilibrium potential. The 26 pS channel could be stimulated with histamine and thrombin from outside in the cell-attached mode. Both the 26 pS as well as the 50 pS channel can mediate calcium flux into the endothelial cell.

Cells, Cultured↗
PubMed1990

Changes of calcium channel inactivation during run-down.

Run down of calcium channel currents carried by Ba2+ was studied in enzymatically isolated ventricular cells from guinea-pig hearts using the suction pipette method. The decay of the Ba currents elicited by 100 ms voltage steps from -60 to 0 mV could be best described biexponentially. The peak current amplitude declined monotonously during dialysis of cell interior, whereas the inactivation showed a biphasic behaviour. After start of intracellular perfusion, the time constants of the fast and slow exponential component decreased up to the 6th and 10th minute, respectively. Thereafter the inactivation decelerated. These changes of the inactivation behaviour could be described by a model of current dependent inactivation. It was assumed, that in the first phase the decay is accelerated due to predominating of washout of phosphorylating agents and in the second phase the reduced conductance of the cell membrane for Ba2+ due to progressive irreversible closure of Ca channels causes slowing down of inactivation.

Animals↗
PubMed1990

Caffeine and the myoplasmic calcium removal mechanisms in cut frog skeletal muscle fibres.

Antipyrylazo III myoplasmic calcium transients were recorded in cut skeletal muscle fibres of the frog (Rana esculenta), using the double vaseline-gap voltage-clamp system. Intracellular calcium removal mechanisms were analysed, using a slightly modified model taken from the literature. Parameter values reported here are generally consistent with those obtained by the original model. Caffeine (0.5 mmol.l-1) moderately enhanced the overall myoplasmic calcium removal. In particular, the rate constant of the non-saturable uptake increased by 51% on the average, but there was a considerable fiber-to-fiber variation. The kinetic features of the binding sites representing the saturable uptake did not change significantly while the concentration of the available sites decreased by 36%. It is concluded that the caffeine-induced changes of the calcium removal components can be explained by supposing an increased resting myoplasmatic Ca2+ concentration in the presence of the drug.

Animals↗