Na(+)-ionophore, monensin-induced rise in cytoplasmic free calcium depends on the presence of extracellular calcium in FRTL-5 rat thyroid cells

Calcium is an important regulator of cell function, and may be influenced by the intracellular sodium content. In the present study, the Na(+)-ionophore, monensin, was used to investigate the interrelationship between changes in intracellular Na+ concentration ([Na+]i) and elevation of cytosolic Ca2+ concentration ([Ca2+]i) in FRTL-5 thyroid cells. Cytoplasmic Ca2+ levels were measured using the fluorescent dye, indo-1. Monensin induced a dose-dependent increase in [Ca2+]i in FRTL-5 cells. Inhibitors of intracellular Ca2+ release, TMB-8 and ryanodine, were unable to prevent the monensin effect on [Ca2+]i. The alpha 1-receptor antagonist, prazosin, did not block the monensin-stimulated increase in [Ca2+]i. In the absence of extracellular calcium there was a marked diminution in the monensin effect on [Ca2+]i, yet calcium channel antagonists (nifedipine, diltiazem and verapamil) did not inhibit the response. Replacement of Na+ by choline chloride in the medium depressed the monensin-evoked rise in [Ca2+]i by up to 84%. Furthermore, addition of the Na(+)-channel agonist, veratridine, elicited an increase in [Ca2+]i, even though less dramatic than that caused by monensin. Ouabain increased the resting cytosolic Ca2+ concentration as well as the magnitude of the monensin effect on [Ca2+]i. The absence of any effect on the Na(+)-ionophore evoked increase in [Ca2+]i upon addition of tetrodotoxin (TTX) excluded a possible involvement of TTX-sensitive Na+ channels. These data show that the rise in [Ca2+]i induced by increasing [Na+]i is largely dependent on both external Na+ and Ca2+. Calcium entry appears not to involve voltage-dependent or alpha 1-receptor sensitive Ca2+ channels, but may result from activation of an Na(+)-Ca2+ exchange system.     

Transmembrane regulation of intracellular calcium by a plasma membrane sodium/calcium exchanger in mouse ova

Regulation of cytoplasmic free calcium concentration ([Ca2+)]i) is a key factor for maintenance of viability of cells, including oocytes. Indeed, during fertilization of an ovum, [Ca2+]i is known to undergo oscillations, but it is unknown how basal [Ca2+]i or calcium oscillations are regulated. In the present study we investigated the role of the plasma membrane in regulating [Ca2+]i of metaphase II-arrested mouse oocytes (ova). Ova were collected from B6C3F1 mice treated with eCG (10 IU) and hCG (5 IU), and intracellular calcium was determined by means of fura-2. Extracellular calcium flux across the zona pellucida was detected noninvasively by a calcium ion-selective, self-referencing microelectrode that was positioned by a computer-controlled micromanipulator. Under basal conditions ova exhibited a calcium net efflux of 20.6 +/- 5.2 fmol/cm2 per sec (n = 69). Treatment of ova with ethanol (7%) or thapsigargin (25 nM-2.5 microM) transiently increased intracellular calcium and stimulated calcium efflux that paralleled levels of [Ca2+]i. The presence of a Na+/Ca2+ exchanger was indicated by experiments employing both bepridil, an inhibitor of Na+/Ca2+ exchange, and sodium-depleted media. In the presence of bepridil, a net influx of calcium was revealed across the zona pellucida, which was reflected by an increase in the [Ca2+]i. In addition, replenishment of extracellular sodium to ova that had been incubated in sodium-depleted media induced a large calcium efflux, consistent with the actions of Na+/Ca2+ exchange. Sodium/calcium exchange in mouse ova may be an important mechanism that regulates [Ca2+]i.     

Cell Swelling, Blebbing, and Death Are Dependent on ATP Depletion and Independent of Calcium During Chemical Hypoxia in a Glial Cell Line (ROC-1)

The morphological and biochemical changes that occur during chemical hypoxic injury in a neural cell line were studied in the presence and absence of calcium. Oligodendroglial-glioma hybrid cells (ROC-1) were subjected to inhibitors of glycolytic and oxidative ATP synthesis (chemical hypoxia). Complete respiratory inhibition depleted [ATP] to less than 5% of control by 4 min. Blebs appeared on the cell surfaces and cells began to swell within a few minutes of ATP depletion. A 200% increase in cell volume and bleb coalescence preceded irreversible cell injury (lactate dehydrogenase release) which began at approximately 20 min with 50% cell death by 40 min. In energized cells an equivalent degree of osmotic swelling induced by ouabain inhibition of the Na+, K(+)-ATPase pump did not produce blebbing or cell death. Partial inhibition of respiration decreased [ATP] to approximately 10% of control by 40 min. Blebbing and swelling began at 40 min and bleb coalescence preceded plasma membrane disruption which began at approximately 55 min. ATP depletion, blebbing, swelling, and death followed similar time courses in the presence or absence of extracellular calcium ([Ca2+]e). Intracellular calcium ([Ca2+]i) was measured using fura-2. In calcium-containing medium metabolic inhibition caused a transient increase in resting [Ca2+]i (100 +/- 17 nM) followed by a low steady-state level preceding plasma membrane disruption. Following deenergization in calcium-free medium, [Ca2+]i remained below 60 nM throughout injury and death. These data suggest that decreased ATP initiates a sequence of events including bleb formation and cell swelling that lead to irreversible cell injury in the absence of large increases in [Ca2+]i.     

Generation and amplification of the cytosolic calcium signal during secretory responses to gonadotropin-releasing hormone

Gonadotropin-releasing hormone (GnRH) stimulates characteristic biphasic increases in cytosolic calcium concentration ([Ca2+]i) and in luteinizing hormone (LH) release in cultured gonadotrophs, with an early peak followed by a prolonged plateau in both responses. Analysis of [Ca2+]i by dual-wavelength fluorimetric assay and of LH release at 5-sec intervals in perifused pituitary cells revealed increases in both responses within a few seconds of exposure to GnRH. The maximum elevation of [Ca2+]i occurred within 20 sec, and the peak gonadotropin release in 35 sec; the total duration of the spike phase for both [Ca2+]i and LH release was 2.5 min. Under extracellular Ca2(+)-deficient conditions, the GnRH-induced peak in [Ca2+]i was reduced by about 20% and the plateau phase was abolished. Concomitantly, the magnitude of the acute phase of LH release was reduced by 40% and that of the second phase by about 90%. Recovery of the plateau phase of LH release occurred within 25 sec after addition of 1.25 mM Ca2+ to Ca2(+)-deficient medium. In a dose-dependent manner, the non-selective Ca2+ channel blockers Co2+ and Cd2+ reduced the Ca2+ current measured by whole-cell recording in pituitary gonadotrophs and abolished the extracellular Ca2(+)-dependent component of LH release. The selective calcium channel blocker, nifedipine, decreased the magnitude of the Ca2+ current and reduced the plateau phase of LH release by 50%; conversely, the dihydropyridine agonist methyl, 1,4,dihydro-2,6-dimethyl 3-nitro-4-(2-trifluorome) (Bay K 8644) consistently enhanced the amplitudes of both Ca2+ current and GnRH-induced LH release. These data reveal a close temporal correlation between changes in [Ca2+]i and LH release during GnRH action, with Ca2+ mobilization during the spike phase and Ca2+ influx through dihydropyridine-sensitive and insensitive sets of receptor-operated calcium channels during the spike and plateau phases. In addition, analysis of the magnitudes of the [Ca2+]i and LH responses to a wide range of GnRH concentrations in the presence and absence of extracellular Ca2+ is consistent with amplification of the [Ca2+]i signal in agonist-stimulated gonadotrops.     

The effects of cyanide on intracellular ionic exchange in ferret and rat ventricular myocardium

The effects of cyanide on Ca2+ exchange in isolated ventricular myocytes and on the intracellular concentrations of Ca2+, Na+ and H+ have been investigated to assess the contribution that mitochondria might play in cellular Ca2+ metabolism. Ionic levels were measured with ion-selective electrodes. KCN (2.5 mM) inhibited a component of Ca2+ exchange in myocytes that could be attributed to mitochondrial exchange, but was without effect on non-mitochondrial Ca2+ exchange. NaCN (2.5 mM) caused a transient reduction of [H+]i, [Na+]i and [Ca2+]i when applied to the superfusate bathing ventricular trabeculae or papillary muscles. The transient changes of [Na+]i were accentuated when the preparation was exposed to a solution which would be expected to increase the cellular calcium content. The reduction of [Na+]i which accompanies a reduction of the extracellular sodium concentration, [Na]o, was attenuated in the presence of NaCN, but the intracellular acidosis resulting from a reduction of [Na]o was unaffected by NaCN. A small, but significant, rise of [Ca2+]i accompanied a reduction of [Na]o but only when NaCN was present in the superfusate. It is concluded that cyanide ions have a reasonably specific action on cardiac cellular ionic metabolism. Its primary action is to prevent mitochondrial Ca2+ sequestration. It is postulated that a Na+/H+ exchange, possibly at the sarcolemma, could account for some of the changes to sarcoplasmic ionic levels observed. In a solution of low [Na]o, it is concluded that mitochondria could sequester at least 30% of the calcium accumulated by the cell even though the sarcoplasmic [Ca2+] does not exceed 0.3 microM.     

Acetylcholine-evoked increase in the cytoplasmic Ca2+ concentration and Ca2+ extrusion measured simultaneously in single mouse pancreatic acinar cells.

The intracellular free Ca2+ concentration ([free Ca2+]i) was measured simultaneously with the Ca2+ extrusion from single isolated mouse pancreatic acinar cells placed in a microdroplet of extracellular solution using the fluorescent probes fura-2 and fluo-3. The extracellular solution had a low total calcium concentration (15-35 microM), and acetylcholine (ACh), applied by microionophoresis, therefore only evoked a transient elevation of [free Ca2+]i lasting about 2-5 min. The initial sharp rise in [free Ca2+]i from about 100 nM toward 0.5-1 microM was followed within seconds by an increase in the total calcium concentration in the microdroplet solution ([Ca]o). The rate of this rise of [Ca]o was dependent on the [free Ca2+]i elevation, and as [free Ca2+]i gradually decreased Ca2+ extrusion declined with the same time course. Ca2+ extrusion following ACh stimulation was not influenced by removal of all Na+ in the microdroplet solution indicating that the Ca2+ extrusion is not mediated by Na(+)-Ca2+ exchange but by the Ca2+ pump. The amount of Ca2+ extruded during the ACh-evoked transient rise in [free Ca2+]i corresponded to a decrease in the total intracellular Ca concentration of about 0.7 mM which is close to previously reported values (0.5-1 mM) for the total concentration of mobilizable calcium in these cells. Our results therefore demonstrate directly the ability of the Ca2+ pump to rapidly remove the large amount of Ca2+ released from the intracellular pools during receptor activation.     

Intracellular-free magnesium in the smooth muscle of guinea pig taenia caeci: a concomitant analysis for magnesium and pH upon sodium removal

This study is concerned with the regulation of intracellular-free Mg2+ concentration ([Mg2+]i) in the smooth muscle of guinea pig taenia caeci. To assess an interaction of Ca2+ on the Na(+)-dependent Mg(2+)- extrusion mechanism (Na(+)-Mg2+ exchange), effects of Na+ removal (N- methyl-D-glucamine substitution) were examined in Ca(2+)-containing solutions. As changes in pHi in Na(+)-free solutions perturb estimation of [Mg2+]i using the single chemical shift only of the beta-ATP peak in 31P NMR (nuclear magnetic resonance) spectra, [Mg2+]i and pHi were concomitantly estimated from the chemical shifts of the gamma- and beta- peaks. When extracellular Na+ was substituted with N-methyl-D- glucamine, [Mg2+]i was reversibly increased. This increase in [Mg2+]i was eliminated in Mg(2+)-free solutions and enhanced in excess Mg2+ solutions. ATP content fluctuated little during removal and readmission of Na+, indicating that [Mg2+]i changes were not induced by Mg2+ release from ATP, and that Mg(2+)-extruding system would not be inhibited by fuel restriction. A slow acidification in Na(+)-free solutions and transient alkalosis by a readmission of Na+ were observed regardless of the extracellular Mg2+ concentration. When the extracellular Ca2+ concentration was increased from normal (2.4 mM) to 12 mM, only a marginal increase in [Mg2+]i was caused by Na+ removal, whereas a similar slow acidosis was observed, indicating that extracellular Ca2+ inhibits Mg2+ entry, and that the increase in [Mg2+]i is negligible through competition between Mg2+ and Ca2+ in intracellular sites. These results imply that Na(+)-Mg2+ exchange is the main mechanism to maintain low [Mg2+]i even under physiological conditions.     

The regulation of the intracellular sodium ion concentration in cultured chick embryo heart cells.

Using digital imaging microscopy with the fluorescent indicator sodium-binding benzofuran isophtalate, we examined the cytosolic Na+ concentration ([Na+]i) in individual chick embryo heart cells. Inhibition of the Na(+)-H+ exchanger using Na(+)-free (Li+ substituted) medium and inhibition of the Na(+)-efflux through the Na(+)-Ca2+ exchanger using Ca(2+)-free medium didn't change the [Na+]i. The opening of voltage-dependent Na+ channels with veratridine (150 micrograms/ml) and inhibition of the Na(+)-K(+)-Cl(-)-cotransporter with bumetanide (10 microM) led to an increase in [Na+]i by 107% and 86%, respectively, suggesting that the Na+ channels and the Na(+)-K(+)-Cl- cotransporter predominantly regulate the [Na+]i in cultured chick embryo heart cells.     

Occurrence of Na(+)-Ca2+ exchange in the ciliate Euplotes crassus and its role in Ca2+ homeostasis.

Ciliates possess diverse Ca2+ homeostasis systems, but little is known about the occurrence of a Na(+)-Ca2+ exchanger. We studied Na(+)-Ca2+ exchange in the ciliate Euplotes crassus by digital imaging. Cells were loaded with fura-2/AM or SBF1/AM for fluorescence measurements of cytosolic Ca2+ and Na+ respectively. Ouabain pre-treatment and Na+o substitution in fura-2/AM-loaded cells elicited a bepridil-sensitive [Ca2+]i rise followed by partial recovery, indicating the occurrence of Na(+)-Ca2+ exchanger working in reverse mode. In experiments on prolonged effects, ouabain, Na+o substitution, and bepridil all caused Ca2+o-dependent [Ca2+]i increase, showing a role for Na(+)-Ca2+ exchange in Ca2+ homeostasis. In addition, by comparing the effect of orthovanadate (affecting not only Ca2+ ATPase, but also Na(+)-K+ ATPase and, hence, Na(+)-Ca2+ exchange) to that of bepridil on [Ca2+]i, it was shown that Na(+)-Ca2+ exchange contributes to Ca2+ homeostasis. In electrophysiological experiments, no membrane potential variation was observed after bepridil treatment suggesting compensatory mechanisms for ion effects on cell membrane voltage, which also agrees with membrane potential stability after ouabain treatment. In conclusion, data indicate the presence of a Na(+)-Ca2+ exchanger in the plasma membrane of E. crassus, which is essential for Ca2+ homeostasis, but could also promote Ca2+ entry under specific conditions.     

Effect of vasopressin on Na+ kinetics in cultured rat vascular smooth muscle cells

The effect of arginine vasopressin (AVP) on Na+ kinetics was examined in cultured rat vascular smooth muscle cells (VSMC) and rat renal papillary collecting tubule cells (RPCT) by the direct measurement of intracellular sodium concentration [(Na+]i) using fluorescence dye; SBFI. AVP increased [Na+]i in a dose-dependent manner at a concentration of 10(-9) M or higher in rat VSMC but did not affect [Na+]i in rat RPCT. The calcium (Ca2+)-free solution completely blocked the increasing effect of AVP on [Na+]i in rat VSMC. A Ca2+ ionophore, ionomycin (1-2 x 10(-6) M) increased [Na+]i both in rat VSMC and RPCT. The Ca2(+)-free solution abolished the ionomycin-increased [Na+]i both in rat VSMC and RPCT. These results therefore indicate that after binding the V1 receptor AVP increases [Na+]i mediated through an increase in cellular Ca2+ uptake in VSMC.     

The effect of isoflurane during reoxygenation on the sarcoplasmic reticulum and cellular injury in isolated ventricular myocytes

In contrast to pretreatment with isoflurane its benefit when applied during reperfusion in rat hearts was only modest. As cellular injury during reoxygenation is greatly determined by sarcoplasmic reticulum (SR) calcium [Ca2+] handling we investigated the effect of isoflurane after simulated ischemia in rat ventricular myocytes. Hypoxic metabolic inhibition was induced by exposure to an acidic medium (pH: 6.3) containing deoxyglucose. Ambient pO2 was reduced to <15 mm Hg. After 30 min, cells were reoxygenated for 30 min with a glucose containing medium (pH: 7.4) in air (Air) or in the presence of isoflurane (Iso), or two SR blockers, i.e. either 3 microM ryanodine (Rya) or 10 microM of cyclopiazonic acid (CPA). During inhibition, diastolic cytosolic calcium ([Ca2+]i) increased and systolic cell shortening decreased. [Ca2+]i further increased in all groups towards the end of reoxygenation. However, [Ca2+]i in the Iso and the Rya group climbed twice as high as in the Air and the CPA group (P < 0.05). Hypercontracture occurred in 23% and 18% in the Iso and the Rya and in 10% and 9% in the Air and the CPA group, respectively (P < 0.05). Cell relengthening and shortening was impaired in Iso, Rya, and CPA treated cells (P < 0.05 vs. Air). Isoflurane given solely during reoxygenation appears to augment cellular injury. Its action seems to be blockade of SR Ca2+ release and Ca2+ efflux. SR Ca2+ overload induces spontaneous Ca2+ oscillations that cause hypercontracture. However, [Ca2+]i does not independently govern cellular systolic and diastolic dysfunction.     

Roles of Na(+)-Ca2+ exchange and of mitochondria in the regulation of presynaptic Ca2+ and spontaneous glutamate release

The release of neurotransmitter from presynaptic terminals depends on an increase in the intracellular Ca2+ concentration ([Ca2+]i). In addition to the opening of presynaptic Ca2+ channels during excitation, other Ca2+ transport systems may be involved in changes in [Ca2+]i. We have studied the regulation of [Ca2+]i in nerve terminals of hippocampal cells in culture by the Na(+)-Ca2+ exchanger and by mitochondria. In addition, we have measured changes in the frequency of spontaneous excitatory postsynaptic currents (sEPSC) before and after the inhibition of the exchanger and of mitochondrial metabolism. We found rather heterogeneous [Ca2+]i responses of individual presynaptic terminals after inhibition of Na(+)-Ca2+ exchange. The increase in [Ca2+]i became more uniform and much larger after additional treatment of the cells with mitochondrial inhibitors. Correspondingly, sEPSC frequencies changed very little when only Na(+)-Ca2+ exchange was inhibited, but increased dramatically after additional inhibition of mitochondria. Our results provide evidence for prominent roles of Na(+)-Ca2+ exchange and mitochondria in presynaptic Ca2+ regulation and spontaneous glutamate release.     

Trypanosoma cruzi: mechanisms of intracellular calcium homeostasis.

Regulation of intracellular Ca2+ homeostasis was characterized in epimastigote forms of Trypanosoma cruzi using the fluorescence probe Fura-2. Despite an increase in extracellular Ca2+, [Ca2+]o, from 0 to 2 mM, cytosolic Ca2+, [Ca2+]i, increased only from 85 +/- 9 to 185 +/- 21 nM, indicating the presence of highly efficient mechanisms for maintaining [Ca2+]i. Exposure to monovalent Na+ (monensin)-, K+ (valinomycin, nigericin)-, and divalent Ca2+ (ionomycin)-specific ionophores, uncouplers of mitochondrial respiration (oligomycin), inhibitors of Na+/K(+)-ATPase (ouabain), and Ca(2+)-sensitive ATPase (orthovanadate) in 0 or 1 mM [Ca2+]o resulted in perturbations of [Ca2+]i, the patterns of which suggested both sequestration and extrusion mechanisms. Following equilibration in 1 mM [Ca2+]o, incubation with orthovanadate markedly increased [Ca2+]i, results which are compatible with an active uptake of [Ca2+]i by endoplasmic reticulum. In contrast, equilibration in 0 or 1 mM [Ca2+]o did not influence the relatively smaller increase in [Ca2+]i following incubation with oligomycin, suggesting a minor role for the mitochondrial compartment. In cells previously equilibrated in 1 mM [Ca2+]o, exposure to monensin or ouabain, conditions known to decrease the [Na+]o/[Na+]i gradient, upon which the Na+/Ca2+ exchange pathways are dependent, markedly increased [Ca2+]i. In a complementary manner, decreasing the extracellular Na+ gradient with Li+ increased [Ca2+]i in a dose-dependent manner. Finally, the calcium channel blockers verapamil and isradipine inhibited the uptake of Ca2+ by greater than 50%, whereas diltiazem, nifedipine, and nicardipine were ineffective. The results suggest that epimastigote forms of T. cruzi maintain [Ca2+]i by uptake, sequestration, and extrusion mechanisms, with properties common to eukaryotic organisms.     

Presence of Na+/Ca2+ exchange activity and its role in regulation of intracellular calcium concentration in bovine adrenal chromaffin cells.

The presence of a Na+/Ca2+ exchanger in bovine adrenal chromaffin cells was demonstrated by measuring the efflux of 45Ca2+ which had been preloaded into cells by a brief depolarization. The efflux of 45Ca2+ was dependent on extracellular Na+ (Na+o); 45Ca2+ efflux was significantly decreased by replacing Na+o with N-methylglucamine (NMG), or Li+. Replacement of Na+o by NMG increased the resting intracellular Ca2+ concentration ([Ca2+]i) of freshly isolated chromaffin cells. This could be reversed by adding Na+, suggesting that Na+/Ca2+ exchanger activity was involved in maintaining [Ca2+]i at its resting level. The initial rate of Na(+)-dependent [Ca2+]i recovery after Ca2+ loading by depolarization was dependent on the level of [Ca2+]i. There was an apparent linear relationship between the activity of the Na+/Ca2+ exchanger and [Ca2+]i both in the presence and absence of Na+o. When cells were treated with other stimuli, including 10 microM DMPP or 40 mM caffeine, the ability of the stimulated cells to decrease [Ca2+]i was significantly reduced upon replacing Na+o with NMG. Our data show that the Na+/Ca2+ exchanger is one of the major pathways for regulating [Ca2+]i in chromaffin cells in both resting and stimulated states.     

Cytoplasmic calcium transients due to single action potentials and voltage-clamp depolarizations in mouse pancreatic B-cells.

Changes in the cytoplasmic free calcium concentration ([Ca2+]i) in pancreatic B-cells play an important role in the regulation of insulin secretion. We have recorded [Ca2+]i transients evoked by single action potentials and voltage-clamp Ca2+ currents in isolated B-cells by the combination of dual wavelength emission spectrofluorimetry and the patch-clamp technique. A 500-1000 ms depolarization of the B-cell from -70 to -10 mV evoked a transient rise in [Ca2+]i from a resting value of approximately 100 nM to a peak concentration of 550 nM. Similar [Ca2+]i changes were associated with individual action potentials. The depolarization-induced [Ca2+]i transients were abolished by application of nifedipine, a blocker of L-type Ca2+ channels, indicating their dependence on influx of extracellular Ca2+. Following the voltage-clamp step, [Ca2+]i decayed with a time constant of approximately 2.5 s and summation of [Ca2+]i occurred whenever depolarizations were applied with an interval of less than 2 s. The importance of the Na(+)-Ca2+ exchange for B-cell [Ca2+]i maintenance was evidenced by the demonstration that basal [Ca2+]i rose to 200 nM and the magnitude of the depolarization-evoked [Ca2+]i transients was markedly increased after omission of extracellular Na+. However, the rate by which [Ca2+]i returned to basal was not affected, suggesting the existence of additional [Ca2+]i buffering processes.     

Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchange and regulation of cytoplasmic concentration of calcium in rat cerebellar neurons treated with glutamate

In the present work, the forward and/or reversed Na+/Ca2+ exchange in cerebellar granular cells was suppressed by substitution of Na+o by Li+ before, during, and after exposure to glutamate for varied time and also using the inhibitor KB-R7943 of the reversed exchange. After glutamate challenge for 1 min, Na+o/Li+ substitution did not influence the recovery of low [Ca2+]i in a calcium-free medium. A 1-h incubation with 100 microM glutamate induced in the neurons a biphasic and irreversible [Ca2+]i rise (delayed calcium deregulation (DCD)), enhancement of [Na+]i, and decrease in the mitochondrial potential. If Na+o had been substituted by Li+ before the application of glutamate, i.e. the exchange reversal was suppressed during the exposure to glutamate, the number of cells with DCD was nearly fourfold lowered. However, addition of the Na+/K+-ATPase inhibitor ouabain (0.5 mM) not preventing the exchange reversal also decreased DCD in the presence of glutamate. Both exposures decreased the glutamate-caused loss of intracellular ATP. Glucose deprivation partially abolished protective effects of the Na+o/Li+ substitution and ouabain. KB-R7943 (10 microM) increased 7.4-fold the number of cells with the [Ca2+]i decreased to the basal level after the exposure to glutamate. Thus, reversal of the Na+/Ca2+ exchange reinforced the glutamate-caused perturbations of calcium homeostasis in the neurons and slowed the recovery of the decreased [Ca2+]i in the post-glutamate period. However, for development of DCD, in addition to the exchange reversal, other factors are required, in particular a decrease in the intracellular concentration of ATP.     

Factors controlling the intracellular concentration of calcium and the spontaneous activity of the ureter

The role of the electrogenic Na(+)-Ca(2+)-exchange mechanism in regulating the spike activity of the ureter was studied. The ureter cells were shown to be capable of generating action potentials (AP) in sodium-free Krebs solution. The time during which the spikes are generated is in exponential dependence on the concentration of calcium ions in the medium, [Ca2+]o within 2.5 to 15 mmol/l. Simultaneously with the generation of the spikes, accumulation of calcium in the muscles is observed, proportional to the increase of [Ca2+]o. The addition of as little as 20 mmol/l Na+ or Li+ ions into the solution restores the prolonged electrical activity of the ureter. Under these conditions, the decrease of intracellular Ca2+ within 5 min was more than two times larger as compared with that in sodium-free medium. Upon substituting Ba2+ ions for Ca2+ ions in Krebs solution AP are generated within an interval which was the longer the higher the Ba2+ concentration in the medium. Li+ ions can replace Na+ ions in maintaining AP and in extruding calcium from the cell. It is supposed that the generation of the stable spike activity of the ureter depends on the functioning of Na(+)-Ca(2+)-exchange mechanism.     

Interrelationship between insulin-like growth factor I-induced activation of the Na+/H(+)-antiporter and intracellular Ca(2+)-mobilization in thyroid cells.

Insulin-like growth factor I (IGF-I) increased cytoplamic pH (pHi) and cytoplasmic Ca2+ [( Ca2+]i) in cultured porcine thyroid cells. Inhibition of the Na+/H(+)-antiporter by dimethylamiloride or a reduction of external Na(+)-concentrations attenuates the increases in pHi and [Ca2+]i. The [Ca2+]i response to IGF-I is a pHi-dependent process. IGF-I activates Na+/H(+)-antiporter and alkalinizes thyroid cells. The resulting increase in pHi facilitates the [Ca2+]i response by adjusting the pHi closer to the pHi-optimum of the intracellular Ca(2+)-mobilizing system. One of the biological functions of IGF-I-induced activation of the Na+/H(+)-antiporter is to shift the pHi to an optimal value for the [Ca2+]i response.     

Cytosolic Free Calcium and Gene Expression During Chemical Hypoxia

Understanding the cellular response to hypoxia may help elucidate the role of altered oxidation in neuronal death or abnormal cell function. In PC12 cells, 30 min of chemical hypoxia (i.e., KCN) reduced ATP concentrations by 92%, but diminished viability by only 10%. Ten minutes of hypoxia increased cytosolic free calcium ([Ca2+]i) 2.5-fold above control, but after 30 min of hypoxia, [Ca2+]i was slightly below that of nonhypoxic cells. Short periods of hypoxia also exaggerated the K(+)-induced elevation of [Ca2+]i, but by 30 min these ATP-depleted cells reestablished a calcium gradient that was equal to nonhypoxic, K(+)-depolarized cells. Thus, 30 min of severe ATP depletion left [Ca2+]i and viability relatively unaffected. Nerve growth factor caused slight, but significant, improvements in ATP and viability of hypoxic cells, but had no effect on [Ca2+]i. Although [Ca2+]i was equivalent in control and hypoxic cells after 30 or 60 min, hypoxia abolished the K(+)-stimulated elevation of [Ca2+]i. The nerve growth factor induction of c-fos, an indicator of the genomic response, was diminished by approximately 80%. Thus, hypoxic PC12 cells with greatly reduced ATP stores maintained normal [Ca2+]i, but their ability to respond to external stimulation was impaired. Further, the reduced oxidation that occurs in the brain in a variety of pathological conditions may interfere with the cellular response to stimulation and growth factors.     

Axial stretch enhances sarcoplasmic reticulum Ca2+ leak and cellular Ca2+ reuptake in guinea pig ventricular myocytes: experiments and models

Cardiac cellular calcium (Ca2+) handling is the well-investigated mediator of excitation-contraction coupling, the process that translates cardiac electrical activation into mechanical events. The reverse--effects of mechanical stimulation on cardiomyocyte Ca2+ handling--are much less well understood, in particular during the inter-beat period, called 'diastole'. We have investigated the effects of diastolic length changes, applied axially using a pair of carbon fibres attached to opposite ends of Guinea pig isolated ventricular myocytes, on the availability of Ca2+ in the main cellular stores (the sarcoplasmic reticulum; SR), by studying the rest-decay of SR Ca2+ content [Ca2+]SR, and the reloading of the SR after prior depletion of Ca2+ from the cell. Cells were loaded with Fura-2 AM (an indicator of the cytosolic 'free' Ca2+ concentration, [Ca2+]i), and pre-conditioned by field-stimulation (2 Hz) at 37 degrees C, while [Ca2+]i transients and sarcomere length (SL) were recorded simultaneously. After reaching a steady state in the behaviour of observed parameters, stimulation was interrupted for between 5 and 60s, while cells were either held at resting length, or stretched (controlled to cause a 10% increase in SL, to aid inter-individual comparison). Thereafter, each cell was returned to its original resting length, followed by swift administration of 10mM of caffeine (in Na+/Ca2+-free solution), which causes the release of Ca2+ from the SR (caffeine), but largely prevents extrusion of Ca2+ from the cytosol to the cell exterior (Na+/Ca2+-free solution). By comparing the [Ca2+]i in cells exposed/not exposed to diastolic stretch of different duration, we assessed the rest-decay dynamics of [Ca2+]SR. To assess SR reloading after initial Ca2+ depletion, the same stretch protocol was implemented after prior emptying of the cell by application of 10mM of caffeine in normal Tyrode solution (which causes Ca2+ to be released from the SR and extruded from the cell via the Na+/Ca2+ exchanger; NCX). Axial stretch enhanced the rate of both rest-decay and reloading of [Ca2+]SR. Application of 40 microM streptomycin, a blocker of stretch-activated ion channels, did not affect the stretch-induced increase in SR reloading. This behaviour was reproduced in a computer simulation study, using a modified version of the 2006 Iribe-Kohl-Noble model of single cardiac myocyte Ca2+ handling, suggesting that stretch increases both Ca2+ leak from the SR and Ca2+ influx via the sarcolemma. This may have important implications for the mobilisation of Ca2+ in stretched cells, and could contribute to the regional 'matching' of individual cardiomyocyte contractility to dynamic, and regionally varying, changes in mechanical loads, such as diastolic pre-load, of cardiac tissue.