Evidence for Na(+)-Ca2+ exchange and Ca(2+)-induced Ca2+ release in a cultured vascular smooth muscle cell line from the rat.

Measurements of intracellular calcium (Cai2+) and sodium (Nai+) have been made in single smooth muscle cells from the rat aortic cell line (A10) using the Ca(2+)- and Na(+)-sensitive dyes Fura-2 and SBFI (sodium-binding benzofuran isophthalate). The effects of manipulation of intracellular and extracellular Na+ on Cai2+ have been investigated. Reversal of the Na+ gradient in control cells does not result in any measurable increase in Cai2+ or change in the rate of recovery of the cells from agonist stimulation, suggesting that there is little functional Na(+)-Ca2+ exchange. In ouabain-pre-treated cells however, the recovery from agonist stimulation is significantly slowed, suggesting that in the presence of an elevated intracellular Na+ concentration there is an alteration in the Ca(2+)-handling mechanisms. Reversal of the Na+ gradient in ouabain-pre-treated cells results in a transient increase in Cai2+ followed by a slow secondary rise. The transient component of this rise is absent on a second activation of the cell or by prior mobilization of the intracellular stores of Ca2+ by agonist. Data presented in this paper suggest the possibility that the transient component is due to a Ca(2+)-induced Ca(2+)-release mechanism triggered by an initial influx of Ca2+. The mechanism underlying this influx is not known but may involve the Na(+)-Ca2+ exchanger operating in reverse. The possible modulation of the Na(+)-Ca2+ exchanger and Ca(2+)-induced Ca2+ release by internal Na+ is discussed.     

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+.     

Ryanodine receptors and the mediation of Ca2+-dependent anion secretion across rat colon

The properties of the Na(+)-Ca(2+) exchanger in isolated crypts from rat colon were studied using the Fura-2 imaging technique. The transport mode of the exchanger was reversed by replacing extracellular Na(+) by the impermeable cation, N-methyl-D-glucamine (NMDG(+)), so that the transporter mediated a Ca(2+) influx into the cells. Depletion of intracellular Ca(2+) stores by inhibitors of sarcoplasmatic endoplasmatic calcium ATPases (SERCA), i.e., cyclopiazonic acid (10(-5) mol l(-1)) or thapsigargin (10(-6) mol l(-1)), reduced the increase in [Ca(2+)](i) evoked by superfusion with NMDG(+), suggesting a cross-talk between the exchanger and the Ca(2+) stores. However, measurement of Ca(2+) influx with the Mn(2+) quench technique revealed that the activity of the exchanger was independent of the filling state of the stores. Instead, the obvious inhibition of the [Ca(2+)](i)response by SERCA blockers was due to a reduction of Ca(2+)-induced Ca(2+) release after inhibition of store-refilling. The functional presence of ryanodine receptors was demonstrated by the increase in [Ca(2+)](i)evoked by ryanodine (10(-7) to 3x10(-4) mol l(-1)) in a concentration-dependent manner. This effect was mimicked by cADP ribose (10(-5) mol l(-1)) in crypts permeabilized with saponin (10 mg l(-1)). Ruthenium red (5x10(-5) mol l(-1)) or high concentrations of ryanodine (3x10(-4) mol l(-1)) inhibited this response. In Ussing chamber experiments ruthenium red (5x10(-4) mol l(-1)) or a high concentration of ryanodine (10(-3) mol l(-1)) inhibited the increase in short-circuit current evoked by the cholinergic agonist, carbachol (5x10(-5) mol l(-1)). Consequently, Ca(2+)-induced Ca(2+) release may act as kind of amplifier during Ca(2+)-dependent Cl(-) secretion in order to maintain a long-lasting increase in the intracellular Ca(2+) concentration.     

Electrogenic Ca2+ entry in the rat colonic epithelium

Capacitative Ca2+ entry in isolated rat colonic crypts was induced by dialysing the cells in the whole-cell patch-clamp mode with a pipette solution having a high Ca(2+)-buffering capacity. Under these conditions crypt cell resting potential was lower than normal. Flufe-namate, La3+ and Gd3+, blockers of non-selective cation channels, hyperpolarized the crypt cells and decreased membrane current. This current exhibited a cation selectivity of Na+>Ca2+. In contrast to Na+, Ca(2+) inhibited the current at concentrations exceeding 1 mmol/l. Indirect evidence suggests that the non-selective cation conductance is activated after stimulation of muscarinic receptors. Carbachol, a cholinergic agonist, evoked a transient hyperpolarization and an increase in membrane outwards current. The half-time of the decay of the carbachol response was shortened strongly in the presence of La3+. Fura-2 experiments with isolated crypts confirmed that La3+ inhibited the carbachol-induced increase in intracellular Ca2+. In parallel Ussing chamber experiments, La3+ suppressed the induction of Cl- secretion by carbachol. These results demonstrate that a non-selective cation conductance activated by store depletion may be involved in the regulation of electrolyte transport by agonists of the Ca2+ signalling pathway.     

Simulation of spike-burst generation and Ca(2+) oscillation in pancreatic beta-cells.

Based on the experimental evidence that Na(+)-Ca(2+) exchange participates in the regulation of intracellular Ca(2+) concentration in pancreatic beta-cells, we construct a mathematical model for the cyclic spike-bursts and oscillations of intracellular Ca(2+) concentration. In our model, an increase in ATP concentration by the stimulation of glucose metabolism leads to the closure of ATP-sensitive K(+) channels (K(ATP) channels) and gradual depolarization to the threshold of voltage-gated Ca(2+) channels. Spikes are generated by the alternate activation of voltage-gated Ca(2+) and K(+) channels, causing Ca(2+) entry. The accumulated Ca(2+) ions are extruded by Na(+)-Ca(2+) exchange and Ca(2+) active transport. An increase in Na(+) influx through Na(+)-Ca(2+) exchangers results in a rise in intracellular Na(+) concentration and the activation of Na(+)-K(+) active transport. The consumption of ATP during the process of Ca(2+) extrusion leads to the opening of K(ATP) channels and repolarization. The present model could reproduce the main experimental features of the spike-burst activity and Ca(2+) oscillations following changes in the extracellular glucose concentration. As the rate of ATP production increases, the spike-burst pattern changes from bursts with long silent phases to continuous spiking. Changes in the pattern of electrical activity produced by the alteration of extracellular Na(+) and K(+) concentrations and the addition of ouabain could be reproduced in the present model.     

Monovalent cation permeability and Ca2+ block of the store-operated Ca2+ current I CRAC in rat basophilic leukemia cells

Like voltage-operated Ca(2+) channels, store-operated CRAC channels become permeable to monovalent cations in the absence of external divalent cations. Using the whole-cell patch-clamp technique, we have characterized the permeation and selectivity properties of store-operated channels in the rat basophilic leukemia (RBL-1) cell line. Store depletion by dialysis with InsP(3) and 10 mM EGTA resulted in the rapid development of large inward currents in Na(+)- and Li(+)-based divalent-free solutions. Cs(+) permeated the channels poorly (P(Cs)/ P(Na)=0.01). Trimethylamine (TMA(+)), tetramethylammonium (TeMA(+)), tetraethylammonium (TEA(+)), N-methyl- D-glucamine (NMDG(+)) and TRIS(+) were not measurably permeant. NH(4)(+) was conducted well. We estimated the minimum pore diameter under divalent-free conditions to be between 0.32 nm and 0.55 nm. When cells were dialysed with buffered Ca(2+) solution and I(CRAC) activated by application of thapsigargin, P(Cs)/ P(Na) was still low (0.08). Outward currents through CRAC channels were carried by intracellular Na(+), K(+) and, to a much lesser extent, by Cs(+). Currents were unaffected by dialysis with Mg(2+)-free solution. The Na(+) current was inhibited by external Ca(2+) (half-maximal blocking concentration of 10 microM). This Ca(2+)-dependent block could be alleviated by hyperpolarization. The monovalent Na(+) current was voltage dependent, increasing as the holding potential depolarized above 0 mV. Our results suggest that CRAC channels in RBL-1 cells have a smaller pore diameter than voltage-operated Ca(2+) channels, discriminate between Group I cations, and differ markedly in their selectivity from CRAC channels reported in lymphocytes.     

The effect of [Na+]i and [Na+]o on Ca2+ mobilization in the rat aortic smooth muscle cell line A7r5.

Measurements of intracellular calcium (Ca2+i) and sodium (Na+i) have been made in single smooth muscle cells in confluent cultures of the A7r5 cell line using the Ca(2+)- and Na(+)-sensitive dyes Fura-2 and sodium-binding benzofuran isophthalate (SBFI). Reversal of the Na+ gradient in control cells results in a small increase in Ca2+i and slows the rate of recovery in Ca2+i following agonist stimulation. This suggests that a Na(+)-Ca2+ exchange mechanism may be functioning in these cells. In ouabain-pretreated cells, Na+i is elevated and the recovery from agonist stimulation is significantly slowed. This suggests that the elevation of Na+i alters Ca2+ homeostasis. Reversal of the Na+ gradient in ouabain-pretreated cells results in a transient increase in Ca2+i which was larger than in control cells. This response is reduced during a second or third exposure to zero Na+o.NA+i, in Na(+)-loaded cells, falls in the absence of external Na+. This fall is slowed in the absence of external Ca2+ supporting the idea that the Na+ loss is via Na(+)-Ca2+ exchange. The possible modulation of the Na(+)-Ca2+ exchanger and Ca2+ mobilization by internal Na+ is discussed.     

Ion selectivity of stretch-activated cation currents in mouse ventricular myocytes

Stretch-activated non-selective cation currents ( I(SAC)) constitute a mechanism that can induce cardiac arrhythmias. We studied I(SAC) in mouse ventricular myocytes by stretching part of the cell surface between the patch-pipette and a motor-driven glass stylus. In non-clamped cells, local stretch depolarised and induced after-depolarisations and extrasystoles. In voltage-clamped cells (K(+) currents suppressed) I(SAC) activated by local stretch had a nearly linear voltage dependence and reversed polarity between -12 and 0 mV. Conductance G(SAC) increased with the extent of local stretch. I(SAC) was not a Cl(-) current (insensitivity to replacement of Cl(-) by aspartate(-)). I(SAC) was not a Ca(2+)-activated current (insensitivity to 5 mM intracellular BAPTA). G(SAC) was blocked by 5 micro M GdCl(3) or by 75 mM extracellular (e.c.) CaCl(2). Removal of e.c. CaCl(2) increased G(SAC) 2.5-fold, as if G(SAC) were sensitive to Ca(2+) and Gd(3+). Replacement of 150 mM e.c. Na(+) by 150 mM Cs(+), Li(+), tetraethylammonium (TEA(+)) or N-methyl d-glucosamine (NMDG(+)) yielded currents that suggested for the conductance a selectivity G(Cs)> G(Na)> G(Li)> G(TEA)> G(NMDG). I(SAC) was suppressed by cytochalasin D, as if an intact F-actin cytoskeleton were necessary for activation of I(SAC).     

Na+-Ca2+ exchange induces low Na+contracture in frog skeletal muscle fibers after partial inhibition of sarcoplasmic reticulum Ca2+-ATPase

Contractile responses due to reduction in external sodium concentration ([Na+]o) were investigated in twitch skeletal muscle fibers of frog semitendinosus. Experiments were conducted after partial inhibition of sarcoplasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid (CPA). In the absence of CPA, Na+ withdrawal failed to produce any change in resting tension. In the presence of CPA (2-10 microM), [Na+]o reduction induced a transient contracture without a significant change in the resting membrane potential. The amplitude of the contracture displayed a step dependence on [Na+]o, was increased by K(+)-free medium and was prevented in Ca(2+)-free medium. This contracture was inhibited by various blockers of the Na(+)-Ca2+ exchange but was little affected by inhibitors of sarcolemmal Ca(2+)-ATPase or mitochondria. When sarcoplasmic reticulum function was impaired, low-Na+ solutions caused no contracture. These results provide evidence that skeletal muscle fibers possess a functional Na(+)-Ca2+ exchange which can mediate sufficient Ca2+ entry to activate contraction by triggering Ca2+ release from sarcoplasmic reticulum when the sodium electrochemical gradient is reduced, and sarcoplasmic reticulum Ca(2+)-ATPase is partially inhibited. This indicates that when the sarcoplasmic reticulum Ca(2+)-ATPase is working (no CPA), Ca2+ fluxes produced by the exchanger are buffered by the sarcoplasmic reticulum. Thus the Na(+)-Ca2+ exchange may be one of the factors determining sarcoplasmic reticulum Ca2+ content and thence the magnitude of the release of Ca2+ from the sarcoplasmic reticulum.     

Na(+)-Ca(2+) exchanger controls the gain of the Ca(2+) amplifier in the dendrites of amacrine cells

We have previously shown that disabling forward-mode Na(+)-Ca(2+) exchange in amacrine cells greatly prolongs the depolarization-induced release of transmitter. To investigate the mechanism for this, we imaged [Ca(2+)](i) in segments of dendrites during depolarization. Removal of [Na(+)](o) produced no immediate effect on resting [Ca(2+)](i) but did prolong [Ca(2+)](i) transients induced by brief depolarization in both voltage-clamped and unclamped cells. In some cells, depolarization gave rise to stable patterns of higher and lower [Ca(2+)] over micrometer-length scales that collapsed once [Na(+)](o) was restored. Prolongation of [Ca(2+)](i) transients by removal of [Na(+)](o) is not due to reverse mode operation of Na(+)-Ca(2+) exchange but is instead a consequence of Ca(2+) release from endoplasmic reticulum (ER) stores over which Na(+)-Ca(2+) exchange normally exercises control. Even in normal [Na(+)](o), hotspots for [Ca(2+)] could be seen following depolarization, that are attributable to local Ca(2+)-induced Ca(2+) release. Hotspots were seen to be labile, probably reflecting the state of local stores or their Ca(2+) release channels. When ER stores were emptied of Ca(2+) by thapsigargin, [Ca(2+)] transients in dendrites were greatly reduced and unaffected by the removal of [Na(+)](o) implying that even when Na(+)-Ca(2+) exchange is working normally, the majority of the [Ca(2+)](i) increase by depolarization is due to internal release rather than influx across the plasma membrane. Na(+)-Ca(2+) exchange has an important role in controlling [Ca(2+)] dynamics in amacrine cell dendrites chiefly by moderating the positive feedback of the Ca(2+) amplifier.     

Ionic mechanisms of regulatory volume increase (RVI) in the human hepatoma cell-line HepG2

We studied the effects of hypertonic stress on ion transport and cell volume regulation (regulatory volume increase; RVI) in the human tumor cell-line HepG2. Ion conductances were monitored in intracellular current-clamp measurements with rapid ion-substitutions and in whole-cell patch-clamp recordings; intracellular pH buffering capacity and activation of Na(+)/H(+) antiport were determined fluorometrically; the rates of Na(+)-K(+)-2Cl(-) symport and Na(+)/K(+)-ATPase were quantified on the basis of time-dependent and furosemide- or ouabain-sensitive (86)Rb(+) uptake, respectively; changes in cell volume were recorded by means of confocal laser-scanning microscopy. It was found that hypertonic conditions led to the activation of a cation conductance that was inhibited by Gd(3+), flufenamate as well as amiloride, but not by benzamil or ethyl-isopropyl-amiloride (EIPA). Most likely, this cation conductance was non-selective for Na(+) over K(+). Hypertonic stress did not change K(+) conductance, whereas possible changes in Cl(-) conductance remain ambiguous. The contribution of Na(+)/H(+)antiport to the RVI process appeared to be minor. Under hypertonic conditions an approximately 3.5-fold stimulation of Na(+)-K(+)-2Cl(-)symport was observed but this transporter did not significantly contribute to the overall RVI process. Hypertonic stress did not increase the activity of Na(+)/K(+)-ATPase, which even under isotonic conditions appeared to be working at its limit. It is concluded that the main mechanism in the RVI of HepG2 cells is the activation of a novel non-selective cation conductance. In contrast, there is little if any contribution of K(+) conductance, Na(+)/H(+) antiport, Na(+)-K(+)-2Cl(-) symport, and Na(+)/K(+)-ATPase to this process.     

Exposure of N-formyl-L-methionyl-L-leucyl-L-phenylalanine-activated human neutrophils to the Pseudomonas aeruginosa-derived pigment 1-hydroxyphenazine is associated with impaired calcium efflux and potentiation of primary granule enzyme release.

The effects of pathologically relevant concentrations (0.38 to 12.5 microM) of the proinflammatory, Pseudomonas aeruginosa-derived pigment 1-hydroxyphenazine (1-hp) on Ca2+ metabolism and intracellular cyclic AMP (cAMP) in N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP; 1 microM)-activated human neutrophils, as well as on the release of myeloperoxidase (MPO) and elastase from these cells, have been investigated in vitro. Ca2+ fluxes were measured by the combination of a fura-2/AM-based spectrofluorimetric method and radiometric procedures, which together enable distinction between net efflux and influx of the cation, while radioimmunoassay and colorimetric methods were used to measure cAMP and granule enzymes, respectively. Coincubation of neutrophils with 1-hp did not affect intracellular cAMP levels or the FMLP-activated release of Ca2+ from intracellular stores but did retard the subsequent decline in the chemoattractant-induced increase in the concentration of cytosolic free Ca2+. These effects of 1-hp on the clearance of Ca2+ from the cytosol of activated neutrophils were associated with decreased efflux of the cation from the cells and increased release of MPO and elastase, while the delayed store-operated influx of the cation into the cells was unaffected by the pigment. The plasma membrane Ca2+-ATPase rather than a Na+-Ca2+ exchanger appeared to be the primary target of 1-hp. These observations suggest that the proinflammatory interactions of 1-hp with activated human neutrophils are a consequence of interference with the efflux of cytosolic Ca2+ from these cells.     

The activity of the Na+/Ca2+ exchanger largely modulates the Ca2+i signal induced by hypo-osmotic stress in rat cerebellar astrocytes. The effect of osmolarity on exchange activity

We recently demonstrated that rat cerebellar Type-1 astrocytes express a very active Na(+)/Ca(2+) exchanger highly colocalized with ryanodine receptors (RyRs), which in turn play a key role in glutamate-induced Ca(2+) signaling through a calcium-induced calcium release (CICR) mechanism. In this work we have explored whether the Na(+)/Ca(2+) exchanger has any role in the Ca(2+)(i) signal induced by hypo-osmotic stress in these cells, using microspectrofluorometric measurements with Fura-2, pharmacological tools, and confocal microscopy image analysis. We present evidence for the first time that the increase in [Ca(2+)](i) in rat cerebellar Type-1 astrocytes, resulting from moderate hypotonic shock, is mediated by Ca(2+) release from ryanodine-operated Ca(2+)(i) stores, and that the magnitude of the intracellular Ca(2+) signal induced by hypotonicity in the short term (up to 240 s) is small and controlled by the activity of the Na(+)/Ca(2+) exchanger operating in its extrusion mode. With longer times in the hypotonic medium, intracellular Ca(2+) store depletion leads to Ca(2+) entry through store-operated Ca(2+) channels. We found it interesting that the activity of the Na(+)/Ca(2+) exchanger measured during this reverse mode operation (Ca(2+) entry in exchange for internal Na(+)) was found to be greatly increased in hypotonic solutions and decreased in hypertonic ones. The buffering of the [Ca(2+)](i) rise induced by hypo-osmotic stress may prevent excessive increases in [Ca(2+)](i), which otherwise might impair the normal function of this glial cell.     

ATP and nitric oxide modulate a Ca(2+)-activated non-selective cation current in macrovascular endothelial cells

We have studied the properties of a non-selective cation current (NSC(Ca)) in macrovascular endothelial cells derived from human umbilical vein (EA cells) that is activated by an increase of intracellular Ca(2+) concentration, [Ca(2+)](i). Current-voltage relationships are linear and the kinetics of the current is time-independent. Current-[Ca(2+)](i) relationships were fitted to a Ca(2+) binding site model with a concentration for half-maximal activation of 417 +/- 76 nM, a Hill coefficient of 2.3 +/- 0.8 and a maximum current of -23.9 +/- 2.7 pA/pF at -50 mV. The Ca(2+)-activated channel is more permeable to Na(+) than for Cs(+) ( P(Cs)/ P(Na)=0.58, n=7), but virtually impermeable to Ca(2+). Current activation was transient if ATP was omitted from the pipette solution. The maximal currents at 300 and 500 nM [Ca(2+)](i) were smaller than in the absence of ATP, but were not significantly different at 2 microM. The intracellular Ca(2+) concentration for half-maximal activation of the Ca(2+)-activated current was shifted to 811 +/- 12 nM in the absence of ATP. Substitution of ATP by the non-hydrolysable ATP analogue adenylylimidodiphosphate (AMP-PNP) did not affect current activation. Sodium nitroprusside (SNP) decreased NSC(Ca) in a concentration-dependent manner. The nitric oxide (NO) donors S-nitroso- N-acetylpenicillamine (SNAP) and 3-morpholinosydnonimine (SIN-1) also inhibited NSC(Ca). In contrast, nitro- L-arginine (NLA), which inhibits all NO-synthases, potentiated NSC(Ca), whereas superoxide dismutase (SOD), which inhibits the breakdown of NO, inhibited NSC(Ca). It is concluded that the Ca(2+)-activated non-selective action channel in EA cells is modulated by the metabolic state of the cell and by NO.     

A store-operated Ca(2+) influx pathway in the bag cell neurons of Aplysia

Although store-operated Ca(2+) influx has been well-studied in nonneuronal cells, an understanding of its nature in neurons remains poor. In the bag cell neurons of Aplysia californica, prior work has suggested that a Ca(2+) entry pathway can be activated by Ca(2+) store depletion. Using fura-based imaging of intracellular Ca(2+) in cultured bag cell neurons, we now characterize this pathway as store-operated Ca(2+) influx. In the absence of extracellular Ca(2+), the endoplasmic reticulum Ca(2+)-ATPase inhibitors, cyclopiazonic acid (CPA) or thapsigargin, depleted intracellular stores and elevated intracellular free Ca(2+). With the subsequent addition of extracellular Ca(2+), a prominent Ca(2+) influx was observed. The ryanodine receptor agonist, chloroethylphenol (CEP), also increased intracellular Ca(2+) but did not initiate store-operated Ca(2+) influx, despite overlap between CEP- and CPA-sensitive stores. Bafilomycin A, a vesicular H(+)-ATPase inhibitor, liberated intracellular Ca(2+) from acidic stores and attenuated subsequent Ca(2+) influx, presumably by replenishing CPA-depleted stores. Store-operated Ca(2+) influx was partially blocked by low concentrations of La(3+) or BTP2, and strongly inhibited by either 1-[b-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365) or a high concentration of Ni(2+). Regarding IP(3) receptor blockers, 2-aminoethyldiphenyl borate, but not xestospongin C, prevented store-operated Ca(2+) influx. However, jasplakinolide, an actin stabilizer reported to inhibit this pathway in smooth muscle cell lines, was ineffective. The bag cell neurons initiate reproductive behavior through a prolonged afterdischarge associated with intracellular Ca(2+) release and neuropeptide secretion. Store-operated Ca(2+) influx may serve to replenish stores depleted during the afterdischarge or participate in the release of peptide that triggers behavior.     

Hypoxia delays the intracellular Ca2+ clearance by Na+-Ca2+ exchanger in human adult cardiac myocytes.

Transient myocardial ischemia during cardiac surgery causes a loss of energy sources, contractile depression, and accumulation of metabolites and H+ ion resulting in intracellular acidosis. The reperfusion following ischemic cardioplegia recovers intracellular pH, activates Na+-H+ exchange and Na+-Ca2+ exchange transports and consequently produces Ca2+ overload, which yields cell death. Among the various Ca2+ entry pathways, the Na+-Ca2+ exchanger is known to play one of the major roles during the ischemia/reperfusion of cardioplegia. Consequently, information on the changes in intracellular Ca2+ activities of human cardiac myocytes via the Na+-Ca2+ exchanger is imperative despite previous measurements of Ca2+ current of human single myocytes. In this study, human single myocytes were isolated from the cardiac tissues obtained during open-heart surgery and intracellular Ca2+ activity was measured with cellular imaging techniques employing fluorescent dyes. We report that the Na+-Ca2+ exchanger of adult cardiac myocytes is more susceptible to hypoxic insult than that of young patients.     

N-Methyl-D-aspartate receptor activation inhibits protein synthesis in cortical neurons independently of its ionic permeability properties

Transient cerebral ischemia, which is accompanied by a sustained release of glutamate, strongly depresses protein synthesis. We have previously demonstrated in cortical neurons that a glutamate-induced increase in intracellular Ca(2+) is likely responsible for the blockade of the elongation step of protein synthesis. In this study, we provide evidence indicating that NMDA mobilizes a thapsigargin-sensitive pool of intracellular Ca(2+). Exposure of cortical neurons to NMDA, in the absence of external Ca(2+), produced a transient rise in intracellular Ca(2+) that was suppressed by pretreatment with thapsigargin. This rise in intracellular Ca(2+) did not result from an influx of Na(+) via reversal of the mitochondrial Na(+)/Ca(2+) exchanger since it persisted in a Na(+)-free medium or in the presence of CGP 37157, an inhibitor of the exchanger. Moreover, the NMDA-induced increase in intracellular Ca(2+) required the presence of D-serine, was blocked by D(-)-2-amino-5-phosphonopentanoic acid, but was not reduced in the presence of external Mg(2+). This unexpected non-ionotropic effect of NMDA was associated with an inhibition of protein synthesis that was also insensitive to the absence of external Ca(2+) or Na(+), or presence of Mg(2+). NMDA treatment resulted in an increase in the phosphorylation of eEF-2 in the absence or presence of external Ca(2+). The initiation step of protein synthesis was not blocked by NMDA since the phosphorylation of initiation factor eIF-2alpha subunit was not altered by NMDA treatment. In conclusion, we provide evidence indicating that NMDA can inhibit protein synthesis in cortical neurons through a process that involves the mobilization of intracellular Ca(2+) stores via a mechanism that is not linked to the ionic properties of NMDA receptors.     

Relaxation in ferret ventricular myocytes: unusual interplay among calcium transport systems.

Transport systems responsible for removing Ca2+ from the myoplasm during relaxation in isolated ferret ventricular myocytes were studied using caffeine-induced contractures. Internal calcium concentration ([Ca2+]i) was measured with the fluorescent calcium indicator indo-1, and the results were compared with our recent detailed characterizations in rabbit and rat myocytes. Relaxation and [Ca2+]i decline during a twitch in ferret myocytes were fast and similar to that in rat myocytes (i.e. half-time, t 1/2 approximately 100-160 ms). During a caffeine-induced contracture (SR Ca2+ accumulation prevented), relaxation was still relatively fast (t 1/2 = 0.57 s) and similar to relaxation in rabbit supported mainly by a strong Na(+)-Ca2+ exchange. When both the SR Ca2+ uptake and Na(+)-Ca2+ exchange are blocked (by caffeine and 0 Na+, 0 Ca2+ solution) relaxation in the ferret myocyte is remarkably fast (approximately 5-fold) compared with rabbit and rat myocytes. The decline of the Cai2+ transient was also fast under these conditions. These values were similar to those in rat under conditions where relaxation is due primarily to Na(+)-Ca2+ exchange. Additional inhibition of either the sarcolemmal Ca(2+)-ATPase or mitochondrial Ca2+ uptake caused only modest slowing of the relaxation of caffeine-induced contracture in 0 Na+, 0 Ca2+ (t 1/2 increased to approximately 3 s). In rabbit myocytes the relaxation t 1/2 is slowed to 20-30 s by these procedures. Even when the systems responsible for slow relaxation in rabbit ventricular myocytes are inhibited (i.e. sarcolemmal Ca(2+)-ATPase and mitochondrial Ca2+ uptake) along with the SR Ca(2+)-ATPase and Na(+)-Ca2+ exchange, relaxation and [Ca2+]i decline in ferret myocytes remain rapid compared with rabbit myocytes. Ca2+ taken up by mitochondria in rabbit myocytes during a caffeine contracture in 0 Na+, 0 Ca2+ solution gradually returns to the SR after caffeine removal, but this component appears to be much smaller in ferret myocytes under the same conditions. We tested for possible residual Ca2+ transport by each of the four systems which suffice to explain Ca2+ removal from the cytoplasm in rabbit (SR Ca(2+)-ATPase, Na(+)-Ca2+ exchange, sarcolemmal Ca(2+)-ATPase and mitochondrial Ca2+ uptake). We conclude that there is an additional calcium transport system at work in ferret myocytes. For this additional system, our results are most compatible with a trans-sarcolemmal Ca2+ transport, but neither a cation exchanger nor a Ca(2+)-ATPase with characteristics like that in other cardiac cells. This additional system appears able to transport Ca2+ nearly as fast as the Na(+)-Ca2+ exchange in rat ventricular myocytes.     

Ion Currents in Smooth Muscle Cells from Human Small Bronchioles: Presence of an Inward Rectifier K+ Current and Three Types of Large Conductance K+ Channel

Bronchoconstriction of small bronchioles plays a major role in the increase in airway resistance following agonist challenge. There is evidence that the airway smooth muscle (ASM) of small bronchioles differs functionally from that in larger airways. Little is known however about the electrophysiology of small bronchioles. Ion currents were therefore studied in airway smooth muscle cells freshly dissociated from human intralobular bronchioles, with a diameter between 0.3 and 1.0 mm. As previously reported for human large airways, the major outward current in these cells was due to activity of large conductance K+ (BK) channels, with a relatively minor component due to a voltage-gated delayed rectifier current (IDR), which was only observed in 30 % of cells. Three distinct types of iberiotoxin- and TEA-sensitive large conductance K+ channel contributed to large conductance K+ current (IBK). These included a highly voltage- and Ca2+-sensitive 200 pS channel previously reported in human large airways, and two smaller channels of 150 and 100 pS previously seen only in human fetal or cultured ASM. In contrast to large airways, ASM cells from bronchioles also demonstrated a voltage-gated inward rectifier current (IIR). IIR was activated by hyperpolarisation below the K+ equilibrium potential and could be blocked by submillimolar concentrations of Cs+ or Ba2+, and partially by physiological concentrations of Na+. Corresponding single channels with a conductance of 17 pS could also be recorded in the cell-attached configuration. A small voltage-independent current was also observed which was resistant to classic K+ and Cl- channel blockers but which could be abolished by replacement of Na+ with the impermeant cation N-methyl-D-glucamine (NMDG+). Corresponding non-selective single channels of 20 pS could be seen in inside-out mode. These results demonstrate that ASM from small bronchioles differs in terms of ion currents and channels from ASM derived from large airways, with possible implications for function.     

The sodium-calcium exchanger is a mechanosensitive transporter

This report describes the influence of fluid flow and osmotically induced volume changes on Na(+)-Ca(2+) exchange (NCX) activity in transfected CHO cells. Exchange activity was measured as Na(+)-dependent Ca(2+) or Ba(2+) fluxes using the fluorescent probe fura-2. When exchange activity was initiated by superfusing Ba(2+)-containing solutions over the cells for a 20 s interval, a high rate of Ba(2+) uptake was observed while the solution was being applied but the rate of Ba(2+) uptake declined > 10-fold when the solution flow ceased. Ba(2+) efflux in exchange for extracellular Na(+) or Ca(2+) (Ba(2+)-Ca(2+) exchange) was similarly biphasic. During NCX-mediated Ca(2+) uptake, a rapid increase in cytosolic [Ca(2+)] to a peak value occurred, followed by a decline in [Ca(2+)](i) to a lower steady-state value after solution flow ceased. When NCX activity was initiated by an alternate procedure that minimized the duration of solution flow, the rapid phase of Ba(2+) influx was greatly reduced in magnitude and Ca(2+) uptake became nearly monophasic. Solution superfusion did not produce any obvious changes in cell shape or volume. NCX-mediated Ba(2+) and Ca(2+) influx were also sensitive to osmotically induced changes in cell volume. NCX activity was stimulated in hypotonic media and inhibited in hypertonic media; the osmotically induced changes in activity occurred within seconds and were rapidly reversible. We conclude that NCX activity is modulated by both solution flow and osmotically induced volume changes.