Modulation of intracellular Na+ and Ca2+ induced by the cardiac Na+-Ca2+ exchanger in guinea pig ventricular myocytes

The Na+-Ca2+ exchanger current was measured in single guinea pig ventricular myocytes, using the whole-cell voltage-clamp technique, and intracellular free calcium concentration ([Ca2+](i)) was monitored simultaneously with the fluorescent probe Indo-1 applied intracellularly through a perfused patch pipette. In external solutions, which have levels of Ca2+ (approximately 66 microM Ca2+) thought low enough to inhibit exchanger turnover, the removal of external Na+ (by replacement with Li+) induced both an outward shift of the holding current and an increase in [Ca2+](i), even though the recording pipette contained 30 mM bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), sufficient to completely block phasic contractions. The effects of Na+ removal were blocked either by the extracellular application of 2 mM Ni2+ or by chelating extracellular Ca2+ with 1 mM EGTA. In the presence of 10 microM Ryanodine, the effects of external Na+ substitution with Li(+) on both membrane current and [Ca2+](i) were attenuated markedly in amplitude and at a much slower time course. Reversal potentials were estimated by using ramp pulses and by defining exchange currents as the Ni2+-sensitive components. The experimental values of the reversal potential and [Ca2+](i) were used to calculate cytosolic Na+ ([Na+](i)) by assuming an exchanger stoichiometry of 3Na+ : 1Ca2+. These calculations suggested that in the nominal absence of external Ca2+ ( approximately 66 microM under our experimental conditions), the exchanger operates at -40 mV as though approximately 40 mM Na+ had accumulated in the vicinity of the intracellular binding sites. We conclude that under the conditions of low extracellular Ca2+ and high intracellular Ca2+ buffering, the Na+-Ca2+ exchanger can still generate sufficient Ca2+ influx on the removal of external Na+ to markedly increase cytosolic free Ca2+.     

Intracellular calibration of the fluorescent Mg2+ indicator furaptra in rat ventricular myocytes

Single ventricular myocytes enzymatically isolated from rat hearts were loaded with the Mg2+ indicator furaptra, and the relationship between the fluorescence ratio signal (R) and the intracellular free concentration of Mg2+ ([Mg2+]i) was studied in situ at 25 degrees C. After the application of ionophores (ionomycin, monensin, nigericin and valinomycin), an immediate change in furaptra R was noted, followed by a slow change in R that reached a steady level in 2-4 h. The direction of the early change in R that accompanied rigor contraction was independent of the extracellular Mg2+ concentration ([Mg2+]o), and was consistent with the breakdown of ATP and release of bound Mg2+. The intracellular calibration curve was constructed from the steady levels of R obtained at various [Mg2+]o between 0 and 47 mM. The dissociation constant of intracellular furaptra was estimated to be 5.3 mM, which was 44% higher than that determined in salt solutions (3.7 mM). The basal [Mg2+]i of rat ventricular myocytes calculated with the intracellular curve averaged 0.91 mM.     

Transport of magnesium by two isoforms of the Na+-Ca2+ exchanger expressed in CCL39 fibroblasts

Cytoplasmic concentrations of Ca2+ ([Ca2+]i) and Mg2+ ([Mg2+]i) were measured with fluorescent indicators in CCL39 cells, a cell line established from Chinese hamster lung fibroblasts, transfected with complementary deoxyribonucleic acid (cDNA) of the Na+-Ca2+ exchanger isolated either from canine heart (NCX1) or from rat brain (NCX3). Raising extracellular [Mg2+] to 10 mM increased Mg2+ influx and the resultant change in [Mg2+]i (delta[Mg2+]i) was monitored with furaptra under Ca2+-free conditions. In control (vector-transfected) cells, delta[Mg2+]i at 45 min was similar with or without extracellular Na+ (130 mM or 0 mM) and when [Na+]i was raised by 1 mM ouabain treatment. delta[Mg2+]i in NCX1-transfected cells was attenuated significantly in the presence of 130 mM Na+, but became comparable to (or slightly larger than) that in control cells on either removal of extracellular Na+ or treatment with 1 mM ouabain. Cells expressing NCX3 showed an intermediate dependence of delta[Mg2+]i on Na+, probably reflecting a lower degree of expression of the exchanger protein. Extracellular Na+-dependent changes in [Ca2+]i (measured with fura-2 in the presence of extracellular Ca2+ and 10 microM ionomycin, a Ca2+ ionophore) were minimal in control cells, marked in the NCX1-transfected cells and intermediate in the NCX3-transfected cells. These results suggest that the Na+-Ca2+ exchanger (either NCX1 or NCX3) can transport Mg2+ and may play a role in the extrusion of magnesium from cells.     

Myoplasmic Mg2+ concentration in Xenopus muscle fibres at rest, during fatigue and during metabolic blockade.

Intracellular free Mg2+ concentration ([Mg2+]i) was measured in isolated single fibres of Xenopus muscle using the fluorescent Mg2+ indicator furaptra. In resting muscle the [Mg2+]i was 1.7 mM in a Mg(2+)-free Ringer solution. There was no significant change in [Mg2+]i over 2 h in Mg(2+)-free Ringer solution. Elevating extracellular [Mg2+] to 40 mM for 5 min caused a small rise (0.13 mM) in [Mg2+]i. There was no detectable rise in [Mg2+]i after 5 min in Na(+)-free Ringer solution. These results suggest that the membrane is relatively impermeable to Mg2+ and that there was no detectable Na(+)-Mg2+ exchange over 5 min. When muscle fibres were fatigued by repeated tetani continued until force declined to about 40% of control, [Mg2+]i showed characteristic changes. During the early period of fatigue when force first showed a small decline and then became almost stable, [Mg2+]i was unchanged; during the final period of fatigue when force declined more rapidly, [Mg2+]i increased by 0.8 mM. Recovery of [Mg2+]i took about 30 min. Recovery of force was complex: tetanic force first declined (post-contractile depression) and then slowly recovered to control. Since the minimum force occurred at about the time when [Mg2+]i had recovered, it seems unlikely that post-contractile depression is caused by elevated [Mg2+]i. Rigor, produced by inhibiting oxidative phosphorylation and glycolysis, was associated with a larger increase (1.6 mM) in [Mg2+]i than fatigue. The rise in [Mg2+]i during fatigue and metabolic blockade could be explained as release of Mg2+ normally bound to ATP. A model of the metabolic changes and the resulting increase in [Mg2+]i explains our results reasonably well.     

The Na+-activated K+ channel contributes to K+ efflux in Na+-loaded guinea-pig but not rat ventricular myocytes

Activation of the Na+-activated K+ channels (KNa channels) has been suggested to contribute to the ischaemia-induced accumulation of extracellular K+ (K+e) in the mammalian myocardium. Recent evidence shows that these channels are not present in rat ventricular myocytes [9]. We have therefore investigated the effect of raised intracellular Na+ activity (aiNa) on intracellular K+ activity (aiK) in guinea-pig myocytes, which possess the channels, and on rat ventricular myocytes which do not. The Na+-activated K+ current was activated by an increase in aiNa induced by removing extracellular Ca2+ and Mg2+ and inhibiting the Na-pump. The aiNa increased and the aiK decreased in both guinea-pig and rat myocytes superfused with Ca2+- and Mg2+-free Tyrode. The new steady-state increase in aiNa and decline in aiK were similar in both species. Inhibition of the Na-pump resulted in an additional increase in aiNa and decrease in aiK in both species. However, both the increase in aiNa and decrease in aiK were greater in guinea-pig myocytes and the decline in aiK in guinea-pig myocytes followed the development of a large Na+-activated K+ current. When Li+ replaced Na+ in the superfusate the Na+-activated K+ current did not develop and the fall in aiK was reduced. In Na+-loaded rat myocytes, which do not have a Na+-activated K+ current, the decline in aiK was reduced and blocked by 2 mM Mg2+ suggesting that a Mg2+-sensitive non-specific cation channel may be involved in the K+ efflux from rat myocytes [12]. These data suggest that KNa channels are a major route for K+ efflux from Na+-loaded guinea-pig myocytes.     

Effects of the calcium ionophore A23187 on pancreatic acinar cell membrane potentials and amylase release.

1. The effects of the Ca2+-ionophore A23187 and the non-metabolizable cholinergic agonist bethanechol on acinar cell membrane potentials and amylase release from the superfused mouse pancreas were studied. 2. In the presence of extracellular Ca2+ (2.56 mM), A23187 (10(-5)M) and bethanechol (3 X 10(-5)M) caused an equal increase in the release of amylase. Both stimulants depolarized theacinar cells, A23187 by 6-0 mV and bethanechol by 12-3 mV. 3. When Ca2+ and Mg2+ were removed from the superfusate, the ability of A23187 to increase the rate of amylase release was virtually abolished, while the effect of bethanechol remained unaltered. Similarly, in the absence of these divalent cations, A23187 did not cause depolarization of the acinar cells, while depolarization in response to bethanechol was largely normal. Consequently it is unlikely that cholinergic agonists initiate secretion by activating a Ca2+-ionophore-like mechanism in the cell membrane. 4. When the concentration of Ca2+ in the medium was raised to 10 mM was the only extracellular divalent cation present, the depolarization in response to A23187 was increased to 11-8 mV. When Mg2+ in a concentration of 10 mM was the only extracellular divalent cation, the depolarization was only 2-1 mV. 5. The Ca2+ dependent, A23187-induced depolarization was abolished in the absence of Na+ (Tris substitution). Addition of Na+ to the superfusate caused an immediate depolarization. 6. It is concluded that the Ca2+ dependent depolarization of pancreatic acinar cells induced by A23187 is not directly due to an increased divalent cation conductance. Our findings are consistent with the view that the depolarization is due to an increased influx of Na+ resulting from a Ca2+ mediated increase in Na+ permeability.     

Ca2+ signalling and membrane current activated by cADPr in starfish oocytes

Cyclic ADP-ribose (cADPr) is a second messenger that regulates intracellular free [Ca2+] ([Ca2+](i)) in a variety of cell types, including immature oocytes from the starfish Astropecten auranciacus. In this study, we employed confocal laser scanning microscopy and voltage clamp techniques to investigate the source of the cADPr-elicited Ca2+ wave originating from the cortical Ca2+ patches we have described previously. The Ca2+ swing was accompanied by a membrane current with a reversal potential of approximately +20 mV. Decreasing external Na+ almost abolished the current without affecting the Ca2+ response. Removal of extracellular Ca2+ altered neither the Ca2+ transient nor the ionic current, nor did the holding potential exert any effect on the Ca2+ wave. Both the Ca2+ response and the membrane current were abolished when BAPTA, ruthenium red or 8-NH(2)-cADPr were preinjected into the oocytes, while perfusion with ADPr did not elicit any [Ca2+](i) increase or ionic current. However, elevating [Ca2+](i) by uncaging Ca2+ from nitrophenyl- (NP-EGTA) or by photoliberating inositol 1,4,5-trisphosphate (InsP(3)) induced an ionic current with biophysical properties similar to that elicited by cADPr. These results suggest that cADPr activates a Ca2+ wave by releasing Ca2+ from intracellular ryanodine receptors and that the rise in [Ca2+](i) triggers a non-selective monovalent cation current that does not seem to contribute to the global Ca2+ elevation.     

Caffeine-induced depolarization in amphibian skeletal muscle fibres: role of Na+/Ca2+ exchange and K+ release.

Caffeine (4 mM) produces a depolarization of about 10 mV in frog muscle fibres (Leptodactylus ocellatus). The aim of this work was to study the mechanisms of this effect. An approximately threefold rise in membrane resistance [Cl--free (SO(4)2-) medium] substantially increased, and both Na+-free medium and Ni2+ (5 mM) reduced, the caffeine-induced depolarization. In voltage-clamped (-60 mV) short fibres from lumbricalis muscle of the toad (Buffo arenarum), caffeine generated an inward current of 4.13 +/- 0.48 microA cm(-2). This caffeine-induced current was reduced by 60% in Na+-free medium, 44% in the presence of 5 mM amiloride and 48% by 5 mM Ni2+, suggesting that the activation of the Na+-Ca2+ exchanger in its forward mode may play a role in the observed electrical effects of the drug. Caffeine also produced a marked release of K+. Net K+ efflux increased from 3.5 +/- 0.2 (control) to 22.1 +/- 2.3 pmol s(-1) cm(-2) (caffeine). It is shown that in the presence of the drug, [K+] in the lumen of the T tubules may well increase to levels which could produce, in part, both the observed depolarization and the caffeine-induced current under voltage clamp conditions. The caffeine-induced K+ efflux was not reduced by 5 mM Ni2+. At a holding potential of 30 mV the caffeine-induced current was reversed (outward) and roughly halved by 5 mM Ni2+. The Ni2+-sensitive fraction of the caffeine-induced current, assumed to represent the Na+-Ca2+ exchanger current, had an estimated reversal potential close to 12 mV ([Na+]o = 115 mM; [Ca2+]o = 1 mM). In conclusion, the depolarizing effect of caffeine described here would be produced by two mechanisms: (a) an inward current generated by the activation of the Na+-Ca2+ exchanger in its forward mode, and (b) the rise of the external [K+] in restricted spaces like the T tubules.     

Membrane permeability during low potassium depolarization in sheep cardiac Purkinje fibers.

Exposure of sheep Purkinje fibers to low [K]o leads to marked depolarization to a stable potential of about -40 mV. This level is equivalent to the plateau of the Purkinje fiber action potential. The low [K]o depolarization could be prevented by removal of [Na]o and was modified by tetrodotoxin. The membrane potential in the depolarized state was unresponsive to changes in [Cl]o or [Ca]o and it was poorly responsive to changes in [K]o between 0 and 2 mM. Repolarization was induced by decrease in [Na]o with a slope response of 30 mV/10-fold change in [Na]o. Average internal K activity (aK) in the resting state with a [K]o of 5 mM was 121.4 mM for a membrane potential of -80 mV. During low K depolarization aK was 119.7 mM with a membrane potential of -34 mV. The depolarization was therefore due to a change in membrane permeability, with little change in aK. Upon restoration of [K]o the fiber repolarized to values transiently more negative than the prior resting potential. These transient potentials were more negative than the K equilibrium potential (VK), if it is calculated assuming a uniform [K]o. The hyperpolarization was reduced by ouabain [10(-6)] or by low [Ca]o.     

Block of recombinant KCNQ1/KCNE1 K+ channels (IKs) by intracellular Na+ and its implications on action potential repolarization

I(Ks), the slow component of delayed rectifier K+ current, plays an important role for the repolarization of ventricular action potential. We investigated the block of I(Ks) by intracellular Na+ ([Na+](i)), using a heterologous expression system (KCNQ1/KCNE1 expressed in COS7 cells), since this well-known blocking action on various K+ channels has not been fully or quantitatively characterized in I(Ks) current. The Na+ block of I(Ks) was concentration- and voltage-dependent and was described by a conventional binding-site model (Woodhull AM: J Gen Physiol 61: 687-708, 1973). In physiological ionic conditions, the blocking action was operating noticeably with Delta ("electrical" distance of the block site) approximately 0.6 and K(d)(0) (apparent dissociation constant at 0 mV) approximately 300 mM. Because K(d)(0) was a function of intra- and extracellular K+ concentrations, changes in ionic environments not only of [Na+](i), but also of [K+](o), affected the amplitude of I(Ks) through the modulation of the Na+ block. Based on these experimental data, we analyzed the effects of Na+ block on action potentials by a computer simulation study, using the Luo-Rudy model. In a physiological ionic environment, the Na+ block of I(Ks) contributed little to modifying action potentials. However, when action potential duration (APD) was marginally prolonged because of decreased I(Ks), as observed in M cells under the conditions of bradycardia and low [K+](o), the Na+ block of I(Ks) may contribute to arrhythmogenesis through the facilitation of early afterdepolarizations (EADs).     

The ionic mechanism of the excitatory action of glutamate upon the membranes of motoneurones of the frog

Simultaneous intra- and extracellular recordings with K+, Na+, Ca2+, and Cl- sensitive microelectrodes were performed in motoneurones of the spinal cord of the frog during depolarizations mediated by glutamate (GLUT) and by experimentally increased extracellular K+. Depolarization resulting from increased K+ activity (alpha K+) in the bathing solution evoked a decrease of intracellular Na+ activity (alpha Na+i); a transient increase of alpha Na+i accompanied by a decrease of alpha Na+e was observed during the depolarization induced by GLUT. Both modes of depolarization led to an increase of alpha Cl-i and a concomitant decrease of alpha Cl-e. An experimental increase of alpha K+e led to a threshold dependent increase of alpha Ca2+i by at least one order of magnitude and to an equally threshold dependent strong decrease of alpha Ca2+e. The threshold of these changes of alpha Ca2+ was at a membrane potential of -25 mV. During a depolarization of half the amplitude induced by GLUT a comparable increase of alpha Ca2+i and a smaller decrease of alpha Ca2+e were observed. The GLUT mediated changes of alpha Ca2+ were not threshold dependent and occurred synchronously with the onset of depolarization. A transient decrease of alpha K+i and a parallel strong increase of alpha K+e occurred during the GLUT induced depolarization. Depolarization evoked by an experimental increase of alpha K+e led to an increase of alpha K+i. The observed changes in the ionic composition of the intra- and extracellular fluids indicate that GLUT evokes an increase in membrane permeability to Na+ and Ca2+ and a subsequent influx of these ions into motoneurones, while the inward shift of Cl- and the outward shift of K+ are presumably passive. A voltage dependent Ca2+ influx is triggered at -25 mV membrane potential.     

Changes in glutamate-evoked currents of frog motoneurones by extracellular Mg2+

Responses of frog motoneurones to glutamate were studied using a single electrode voltage clamp method. At resting membrane potential glutamate evoked biphasic effects: firstly a transient outward current with reversal potential between -90 and -100 mV. Secondly an inward current with reversal potential near 0 mV and non-linearly related to the holding potential. Mg2+ (1 mM) had no effect on the outward current but reduced the inward current and its slope conductance at holding potentials more negative than -60 mV. These data indicate that the operation of the inward current activated by glutamate was partly but not solely controlled by extracellular Mg2+.     

Properties of the inwardly rectifying K+ conductance in the toad retinal pigment epithelium.

An inwardly rectifying K+ current was analysed in isolated toad retinal pigment epithelial (RPE) cells using the perforated-patch clamp technique. The zero-current potential (Vo) of RPE cells averaged -71 mV when the extracellular K+ concentration ([K+]o) was 2 mM. Increasing [K+]o from 0.5 to 5 mM shifted V0 by +43 mV, indicating a relative K+ conductance (TK) of 0.74. At [K+]o greater than 5 mM, TK decreased to 0.53. Currents were larger in response to hyperpolarizing voltage pulses than depolarizing pulses, indicating an inwardly rectifying conductance. Currents were time independent except in response to voltage pulses to potentials positive to 0 mV, where the outward current decayed with an exponential time course. Both the inwardly rectifying current and the transient outward current were eliminated by the addition of 0.5 mM Ba2+, 5 mM Cs+ or 2 mM Rb+ to the extracellular solution. The current blocked by these ions reversed near the K+ equilibrium potential (EK) over a wide range of [K+]o, indicating a highly selective K+ channel. The current-voltage relationship of the isolated K+ current exhibited mild inward rectification at voltages negative to -20 mV and a negative slope conductance at voltages positive to -20 mV. The Cs(+)- and Ba(2+)-induced blocks of the K+ current were concentration dependent but voltage independent. The apparent dissociation constants were 0.8 mM for Cs+ and 40 microM for Ba2+. The K+ conductance decreased when extracellular Na+ was removed. Increasing [K+]o decreased the K+ chord conductance (gK) at negative membrane potentials. In the physiological voltage range, increasing [K+]o from 2 to 5 mM caused gK to decrease by approximately 25%. We conclude that the inwardly rectifying K+ conductance represents the resting K+ conductance of the toad RPE apical membrane. The unusual properties of this conductance may enhance the ability of the RPE to buffer [K+]o changes that take place in the subretinal space at the transition between dark and light.     

Reduced Mg2+ blockade of synaptically activated N-methyl-D-aspartate receptor-channels in CA1 pyramidal neurons in kainic acid-lesioned rat hippocampus.

Unilateral kainic acid lesion in the hippocampus caused a long-term change in the balance between excitatory and inhibitory drive onto CA1 pyramidal cells, making these cells hyperexcitable several weeks post-lesion. In this study, we have shown an enhanced N-methyl-D-aspartate receptor-mediated component in the excitatory synaptic transmission together with a reduced GABA(A) receptor-mediated inhibition in CA1 pyramidal cells one-week post kainic acid lesion. In these cells, pharmacologically isolated N-methyl-D-aspartate receptor-mediated whole-cell excitatory postsynaptic currents were significantly larger at negative holding potentials, and the voltage-dependence of N-methyl-D-aspartate receptor channels was shifted in the hyperpolarizing direction. The plot of relative conductance (g/gMax) shifted significantly (P<0.01) to more negative holding potentials by 19 mV (-28+/-4 mV in control slices and -47+/-4 mV in kainic acid slices) at the half maximal conductance point (g/gMax =0.5). This shift gives a larger N-methyl-D-aspartate receptor-mediated component in the excitatory synaptic transmission at resting membrane potentials (around -60 mV). The shifted voltage dependence is highly sensitive to extracellular Mg2+ ions. Moderate increases in [Mg2+]o from 1 mM to 2.6 mM more than compensated for the negative shift and effectively suppressed the population epileptiform bursting activity. Fitting the voltage dependence to an ionic block model revealed a higher dissociation constant of N-methyl-D-aspartate receptor channels for Mg2+ in kainic acid-lesioned slices (52 mM at 0 mV; 330 microM at -60 mV) than in control slices (7.7 mM at 0 mV; 93 microM at -60 mV). While a simple single site model adequately fitted the control data for [Mg2+]o at 1 mM and 2.6 mM, no consistent model of this form was found for the kainic acid-lesioned slices. These results revealed changed properties of N-methyl-D-aspartate receptor channels in the kainic acid-lesioned model of epilepsy. The reduced Mg2+ blockade of N-methyl-D-aspartate receptor channels contributed significantly to the epileptiform bursting activity.     

No correlation between membrane potential and increased cytosolic free Ca2+ concentration, 86Rb+ influx or subsequent [3H]-thymidine incorporation in neoplastic human B cells stimulated with antibodies to surface immunoglobulin

We have followed the changes in the membrane potential of neoplastic B cells after addition of antibodies to cell specific surface immunoglobulin heavy chains (anti-Ig). Two methods were used, both based on the distribution of lipophilic fluorescent indicators: the anionic bis(1,3-diethylthiobarbiturate)-trimethine oxonol (fluorescence measurement made on whole cell suspensions) and the cationic 3,3-dihexyloxacarbocyanine iodide (diOC6-(3] (using flow cytofluorimetric analysis). By the latter method, the resting membrane potential was calculated to range from -40 to -63 mV. The stimulatory effect of anti-Ig was investigated on two B cell populations. In one, which may be stimulated in this way to [3H]-thymidine incorporation, the membrane potential was apparently unchanged, at least during the first hour of stimulation. In the other population, anti-Ig induced a rapid depolarization (oxonol method), but these cells did not, on the other hand, respond with [3H]-thymidine incorporation. The nature of this depolarization was further investigated. Both Na+ and K+ permeabilities appeared increased, while Ca2+ influx did not contribute to the change in membrane potential. Nor did depolarization itself change free cytosolic Ca+ concentration, [Ca2+]i, as estimated by the quin-2 method. Furthermore, anti-Ig stimulation increased [Ca2+]i in these cells, even when depolarization was abolished by lowering the extracellular Na+ concentration. Thus, in cells where depolarization could be demonstrated, it was linked neither to early changes in [Ca2+]i nor to late [3H]-thymidine incorporation. In contrast, such effects of anti-Ig stimulation were observed in other cells without any significant early change in the membrane potential.     

Contribution of Na+/Ca2+ exchanger to the regulation of myogenic tone in isolated rat small arteries.

The contribution of the Na+/Ca2+ exchanger to the myogenic vascular tone was examined in rat isolated skeletal muscle small arteries (ASK) with pronounced myogenic tone and mesenteric small arteries (AMS) with little myogenic tone. Myogenic tone was assessed by the vascular inner diameter at transmural pressures of 40 and 100 mmHg. To depress the Na+/Ca2+ exchanger, the extracellular Na+ concentration ([Na+]o) was lowered from 143 to 1.2 mM by substituting choline-Cl for NaCl. The ASK developed significant myogenic tone and constricted further in low [Na+]o. Nifedipine (1 microM) reduced both myogenic tone and low [Na+]o-induced contraction. Because the membrane potential of ASK was not changed by low [Na+]o (-35 +/- 2 mV at 143 mM [Na+]o, -37 +/- 3 mV at 1.2 mM [Na+]o), depolarization-induced Ca2+ influx was not a cause of the low [Na+]o-induced contraction. The AMS did not develop significant myogenic tone. Although low [Na+]o also constricted AMS, the magnitude of constriction was significantly weaker than that in ASK (17 +/- 4 vs. 47 +/- 6%, P < 0.01, at 58 mM Na+). With Bay K 8644, AMS developed myogenic tone, and low [Na+]o-induced constriction was significantly increased. In conclusion, Na+/Ca2+ exchanger may play an important role in regulating myogenic tone, likely via mediating Ca2+-extrusion.     

Relationship between global cytosolic Ca2+ concentration and Ca2+-activated K+ current in rabbit cerebral arterial myocyte.

In smooth muscle cells, the sarcoplasmic reticulum (SR) has been identified as the primary storage site for intracellular Ca2+. The peripheral SR is in close proximity with plasma membrane to make a narrow subsarcolemmal space. In this study, we investigated the regulation of subsarcolemmal [Ca2+] ([Ca2+]sl) and global cytosolic [Ca2+] ([Ca2+]c) of rabbit arterial smooth muscle using whole cell patch clamp technique and microspectrofluorimetry. The Ca2+-activated K+ current (IK(Ca)) and the ratio of fura-2 fluorescence (R340/380) were considered to reflect the [Ca2+]sl and [Ca2+]c, respectively. At a holding potential of 0 mV, extracellular application of 10 mM caffeine, a well known Ca2+-releasing agent, induced transient increase of IK(Ca) and R340/380 (IK(Ca)-transient and R340/380-transient, respectively). The increase and decay of IK(Ca) transient was faster than R340/380-transient. By repetitive application of caffeine, when the refilling state of SR was supposed to be lower than the control condition, IK(Ca)-transient and R340/380 transient were suppressed to different levels; e.g. the second application 20 sec after the first could induce smaller IK(Ca) transient than R340/380-transient. Dissociation of IK(Ca)-transient and R340/380-transient was removed by sufficient (>3 min) washout of caffeine. Recovery from the dissociation was also dependent upon the membrane potential; faster recovery was observed at negative (-40 mV) holding potential than at depolarized (0 mV) condition. Dissociation of IK(Ca) from [Ca2+]c was also partially prevented by perfusion with Na+-free (replaced by NMDG+) extracellular solution. These results suggest that, 1) there is prominent spatial inhomogeneity of [Ca2+] in cerebral arterial myocyte, 2) [Ca2+]Sl is preferentially affected by the interference from nearby plasmalemmal Ca2+ regulation mechanism which is partly dependent upon extracellular Na+.     

Inactivation of outward Na(+)-Ca2+ exchange current in guinea-pig ventricular myocytes.

1. Outward Na(+)-Ca2+ exchange currents were measured in freshly dissociated guinea-pig myocytes to probe in intact cells the functional status of exchanger inactivation reactions, described previously in giant excised cardiac membranes patches. 2. When the cytoplasmic (pipette) solution contained 40 mM Na+ and 0.1 microM free Ca2+ (50 mM EGTA), the outward exchange current activated by extracellular Ca2+ decayed with time (time constant, 13.1 +/- 2.6 s; n = 6), and an inward current transient was observed upon removal of extracellular Ca2+. Both the current decay and the subsequent inward current transient were remarkably diminished with a saturating (100 mM) pipette Na+ concentration. 3. With 100 mM cytoplasmic Na+ and 140 mM extracellular Na+, a significant fraction of the exchanger population is predicted to be in an inactive state. Intracellular application of 2 mg ml-1 chymotrypsin and 5 microM sodium tetradecylsulphate, both of which decrease Na(+)-dependent inactivation in giant membrane patches, increased the outward exchange current by about 160-170%, suggesting that about 60-70% of exchangers might be inactivated. 4. With 100 mM cytoplasmic Na+ and no extracellular Na+ (replaced with 140 mM Li+), application of extracellular Ca+ was predicted to reorient exchanger binding sites from the extracellular side to the cytoplasmic side and thereby favour inactivation. During such protocols, the outward exchange current decayed by 60-80% when activated by extracellular Ca2+. The current decayed similarly when extracellular Ca2+ and Na+ were applied together, whereby current magnitudes were about 3-fold smaller. 5. The decay of outward exchange current usually followed a biexponential time course (5.8 +/- 3.5 and 27.3 +/- 16.3 s, means +/- S.D., n = 11). Intracellular application of 0.5-2 mg ml-1 trypsin attenuated the fast component more than the slow component, suggesting that the fast component reflects an inactivation process. 6. Current-voltage (I-V) relations of the outward exchange current became less steep during the inactivation protocols, but this flattening could not be correlated with inactivation. 7. Replacement of extracellular Li+ with N-methyl-D-glucamine (NMG), tetraethylammonium (TEA), sucrose or Cs+ resulted in a flattening of I-V relations and a decrease of the outward exchange current amplitude by approximately 3-fold, but the kinetics and extent of inactivation were not remarkably changed. Thus, the mechanism of inactivation appears to be independent of the mechanism(s) of activation by extracellular monovalent cations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Functional Na+/K+ pump in rat dorsal root ganglia neurons.

Steady-state Na+/K+ pump current (Ip) in isolated adult rat dorsal root ganglia neurons was studied to determine if the plasma membrane Na+/K+ pump activity is uniform across the population of dorsal root ganglia neurons. Cells were voltage-clamped at -40 mV and holding current (Ih) was recorded using whole-cell patch-clamp techniques under conditions that stimulate the Na+/K+ pump (60 mM intracellular Na+ and 5.4 mM extracellular K+). Ip was defined as the 1 mM ouabain-sensitive fraction of Ih. Data suggest the existence of three subpopulations of dorsal root ganglia neurons having mean steady-state Ip densities of 1.6+/-0.1, 3.8+/-0.2 and 7.5+/-0.4 pA/pF. Neurons with small Ip had an average soma perimeter of 95+/-3 microm, while neurons with medium and large Ip density had significantly larger soma sizes (131+/-8 and 141+/-7 microm, respectively). Neurons with a large Ip density had a significantly lower specific membrane resistance (Rm; mean 4.0+/-0.3 kohm x cm2) than neurons with medium or small Ip density (19+/-6 and 31+/-6 kohm x cm2, respectively). Regardless of these differences, in all groups of neurons Ip had a low sensitivity to ouabain (Ip half inhibition by ouabain was observed at 80-110 microM). These data suggest that the Na+/K+ pump site density and/or its activity is not uniform throughout the dorsal root ganglia neuron population; however, this non-uniformity does not appear to relate to the functional expression of the different alpha isoforms of the Na+/K+ pump. The major functional Na+/K+ pump in the dorsal root ganglia neuron plasma membrane appeared to be the low ouabain affinity (alpha1) isoform.     

Contractile dysfunctions in ATP-dependent K+ channel-deficient mouse muscle during fatigue involve excessive depolarization and Ca2+ influx through L-type Ca2+ channels

Muscles deficient in ATP-dependent potassium (KATP) channels develop contractile dysfunctions during fatigue that may explain their apparently faster rate of fatigue compared with wild-type muscles. The objectives of this study were to determine: (1) whether the contractile dysfunctions, namely unstimulated force and depressed force recovery, result from excessive membrane depolarization and Ca2+ influx through L-type Ca2+ channels; and (2) whether reducing the magnitude of these two contractile dysfunctions reduces the rate of fatigue in KATP channel-deficient muscles. To reduce Ca2+ influx, we lowered the extracellular Ca2+ concentration ([Ca2+]o) from 2.4 to 0.6 mM or added 1 microM verapamil, an L-type Ca2+ channel blocker. Flexor digitorum brevis (FDB) muscles deficient in KATP channels were obtained by exposing wild-type muscles to 10 microM glibenclamide or by using FDB from Kir6.2-/- mice. Fatigue was elicited with one contraction per second for 3 min at 37 degrees C. In wild-type FDB, lowered [Ca2+]o or verapamil did not affect the decrease in peak tetanic force and unstimulated force during fatigue and force recovery following fatigue. In KATP channel-deficient FDB, lowered [Ca2+]o or verapamil slowed down the decrease in peak tetanic force recovery, reduced unstimulated force and improved force recovery. In Kir6.2-/- FDB, the rate of fatigue became slower than in wild-type FDB in the presence of verapamil. The cell membrane depolarized from -83 to -57 mV in normal wild-type FDB. The depolarizations in some glibenclamide-exposed fibres were similar to those of normal FDB, while in other fibres the cell membrane depolarized to -31 mV in 80 s, which was also the time when these fibres supercontracted. It is concluded that: (1) KATP channels are crucial in preventing excessive membrane depolarization and Ca2+ influx through L-type Ca2+ channels; and (2) they contribute to the decrease in force during fatigue.