Sodium-dependent calcium efflux from adrenal chromaffin cells following exocytosis. Possible role of secretory vesicle membranes.

Although cytosolic Ca2+ transients are known to influence the magnitude and duration of hormone and neurotransmitter release, the processes regulating the decay of such transients after cell stimulation are not well understood. Na(+)-dependent Ca2+ efflux across the secretory vesicle membrane, following its incorporation into the plasma membrane, may play a significant role in Ca2+ efflux after stimulation of secretion. We have measured an enhanced 45Ca2+ efflux from cultured bovine adrenal chromaffin cells following cell stimulation with depolarizing medium (75 mM K+) or nicotine (10 microM). Such stimulation also causes Ca2+ uptake via voltage-gated Ca2+ channels and secretion of catecholamines. Na+ replacement with any of several substitutes (N-methyl-glucamine, Li+, choline, or sucrose) during cell stimulation inhibited the enhanced 45Ca2+ efflux, indicating and Na(+)-dependent Ca2+ efflux process. Na+ deprivation did not inhibit 45Ca2+ uptake or catecholamine secretion evoked by elevated K+. Suppression of exocytotic incorporation of secretory vesicle membranes into the plasma membrane with hypertonic medium (620 mOsm) or by lowering temperature to 12 degrees C inhibited K(+)-stimulated 45Ca2+ efflux in Na(+)-containing medium but did not inhibit the stimulated 45Ca2+ uptake. Enhancement of exocytotic secretion with pertussis toxin resulted in an enhanced 45Ca2+ efflux without affecting calcium uptake. The combined results suggest that Na(+)-dependent Ca2+ efflux across secretory vesicle membranes, following their incorporation into the plasma membrane during exocytosis, plays a significant role in regulating calcium efflux and the decay of cytosolic Ca2+ in adrenal chromaffin cells and possibly in related secretory cells.     

Inhibition by taurine of Na(+)-Ca2+ exchange in sarcolemmal membrane vesicles from bovine and guinea pig hearts

1. Taurine, but not GABA, beta-alanine and glycine, inhibited Na(+)-dependent Ca2+ uptake in bovine cardiac sarcolemmal membrane vesicles in a dose-dependent manner. 2. The inhibition of Na(+)-dependent Ca2+ uptake was noncompetitive with respect to Ca2+ concentration. 3. The inhibitory effect of taurine on the exchange was also observed in cardiac sarcolemmal vesicles prepared from guinea pig, but not from rat. 4. Taurine did not affect Na(+)-dependent Ca2+ efflux nor ATP-dependent Ca2+ uptake in the bovine cardiac membranes.     

Parathyroid hormone increases sodium/calcium exchange activity in renal cells and the blunting of the response in aging

Na+-dependent Ca2+ efflux was demonstrated in cells isolated from the rat renal cortex, suggestive of the presence of a Na+/Ca2+ exchange carrier in the cells. Parathyroid hormone, when incubated with the cells in vitro, increased Na+-dependent Ca2+ efflux about 60%. The effect of the hormone was specific for biologically active parathyroid hormone analogs and could be mimicked by cyclic nucleotides and forskolin. The effects of parathyroid hormone concentration on Ca2+ efflux and cyclic AMP formation were similar. These findings would be consistent with the view that the cyclic nucleotide might act as the intracellular messenger to increase Na+/Ca2+ exchange activity. Cells isolated from parathyroidectomized rats had decreased Na+-dependent Ca2+ efflux. When these cells were treated in vitro with parathyroid hormone, Na+-dependent Ca2+ efflux was enhanced to the same rate as found with cells from sham-operated animals. Parathyroid hormone-sensitive Na+/Ca2+ exchange activity was markedly blunted in cells from senescent (24 months) rats. Basal Na+-dependent Ca2+ efflux and Na+-independent Ca2+ efflux were not altered in the aged animal. Parathyroid-stimulated adenylate cyclase was also decreased in aging. In contrast, forskolin-stimulated Na+-dependent Ca2+ efflux and adenylate cyclase did not change with senescence. These findings would be compatible with a mechanism of desensitization that occurred at the level of the receptor or hormone-receptor coupling to adenylate cyclase. These results may be of physiological significance in understanding calcium homeostasis and the imbalances in mineral metabolism associated with old age.     

Effects of variation in the structure of spermine on the association with DNA and the induction of DNA conformational changes.

Manganese shares the uniport mechanism of mitochondrial calcium influx, accumulates in mitochondria and is cleared only very slowly from brain. Using dual-label isotope techniques, we have investigated both Mn2+ and Ca2+ mitochondrial efflux kinetics. We report that (1) there is no significant Na(+)-dependent Mn2+ efflux from brain mitochondria; (2) Mn2+ inhibits both Na(+)-dependent and Na(+)-independent Ca2+ efflux in brain, in a mode that appears to be primarily competitive and with apparent Ki values of 5.1 and 7.9 nmol/mg respectively; and (3) Ca2+ does not appear to inhibit Mn2+ efflux from brain mitochondria. Findings (1) and (2) suggest the possibility of mitochondrial accumulation of both Mn2+ and Ca2+ in Mn2(+)-intoxicated brain.     

Alcohol stimulates Na+/Ca2+ exchange in brain mitochondria

Ethanol, at low concentrations, specifically stimulates the Na(+)-dependent Ca2(+)-efflux in brain mitochondria. In addition, at higher concentrations, ethanol inhibits the Na(+)-independent Ca2(+)-efflux. The electrogenic Ca(+)-uptake system is not affected by ethanol. The specific stimulation of Na+/Ca2+ exchange reaches a maximum of 60% stimulation, with half-maximal stimulation at 130 mM ethanol. The inhibition of the Na(+)-independent efflux is proportional to the ethanol concentration, becoming significant only above 200 mM, with 50% inhibition at 0.5 M. The inhibition of the Na(+)-independent efflux is, in large part, due to an inhibition of the activation of the Cyclosporin-sensitive pore. Long-term ethanol-feeding had no effect on the Ca2+ transport systems and their sensitivity to acute ethanol treatment. It is suggested that the stimulation of the Na(+)-dependent Ca2(+)-efflux, which is the dominant Ca2+ efflux pathway in brain mitochondria, contributes to the intoxicating effects of ethanol.     

A sodium zinc exchange mechanism is mediating extrusion of zinc in mammalian cells

Zinc influx, driven by a steep inward electrochemical gradient, plays a fundamental role in zinc signaling and in pathophysiologies linked to intracellular accumulation of toxic zinc. Yet, the cellular transport mechanisms that actively generate or maintain the transmembrane gradients are not well understood. We monitored Na+-dependent Zn2+ transport in HEK293 cells and cortical neurons, using fluorescent imaging. Treatment of the HEK293 cells with CaPO4 precipitates induced Na+-dependent Zn2+ extrusion, against a 500-fold transmembrane zinc gradient, or zinc influx upon reversal of Na+ gradient, thus indicating that Na+/Zn2+ exchange is catalyzing active Zn2+ transport. Depletion of intracellular ATP did not inhibit the Na+-dependent Zn2+ extrusion, consistent with a mechanism involving a secondary active transporter. Inhibitors of the Na+/Ca2+ exchanger failed to inhibit Na+-dependent Zn2+ efflux. In addition, zinc transport was unchanged in HEK293 cells heterologously expressing functional cardiac or neuronal Na+/Ca2+ exchangers, thus indicating that the Na+/Zn2+ exchange activity is not mediated by the Na+/Ca2+ exchanger. Sodium-dependent zinc exchange, facilitating the removal of intracellular zinc, was also monitored in neurons. To our knowledge, the Na+/Zn2+ exchanger described here is the first example of a mammalian transport mechanism capable of Na+-dependent active extrusion of zinc. Such mechanism is likely to play an important role, not only in generating the transmembrane zinc gradients, but also in protecting cells from the potentially toxic effects of permeation of this ion.     

Calcium mobilization by cadmium or decreasing extracellular Na+ or pH in coronary endothelial cells

Replacing extracellular Na+ with choline transiently increased cytoplasmic free Ca2+ ([Ca2+]i) more than 5-fold in coronary endothelial cells. Removing external Na+ stimulated 45Ca2+ efflux approximately 4-fold and influx approximately 1.7-fold. The stimulation of efflux was independent of extracellular Ca2+ and the osmotic Na+ substitute. The release of stored Ca2+, rather than Ca2+ influx via Na(+)-Ca2+ exchange, probably causes the increase in [Ca2+]i and 45Ca2+ efflux. Cadmium or decreasing external, not intracellular, pH transiently increased [Ca2+]i. Cd2+ and some other divalent metals also stimulated 45Ca2+ efflux. The potency order of the metals that stimulated efflux was Cd2+ greater than CO2+ greater than Ni2+ greater than Fe2+ greater than Mn2+. Incubating the cells with Zn2+ prior to assaying efflux in the absence of Zn2+ strongly inhibited the stimulation of 45Ca2+ efflux by Cd2+, pH 6, and the removal of external Na+ without affecting the stimulation of efflux by ATP. These findings support the hypothesis that certain trace metals or decreasing external Na+ or pH trigger the release of stored Ca2+ by stimulating a cell surface "receptor."     

The interrelations between the transport of sodium and calcium in mitochondria of various mammalian tissues.

Addition of ruthenium red to mitochondria isolated from brain, adrenal cortex, parotid gland and skeletal muscle inhibits further uptake of Ca2+ by these mitochondria but induces little or no net Ca2+ efflux; the further addition of Na+, however, induces rapid efflux of Ca2+. The velocity of the Na+-induced efflux of Ca2+ from these mitochondria exhibits a sigmoidal dependence on the [Na+]. Addition of Na+ to mitochondria exhibiting the most active Na+-dependent efflux of Ca2+ (brain and adrenal cortex) also releases Ca2+ in the absence of ruthenium red and, under these conditions, the mitochondria become uncoupled. It is concluded that the efflux of Ca2+ from these mitochondria occurs via a Na+-dependent pathway, possibly a Na+-Ca2+ antiporter, that is distinct from the ruthenium-red-sensitive carrier that catalyses energy-linked Ca2+-influx. The possible role of the Na+-dependent efflux process in the distribution of Ca2+ between the mitochondria and the cytosol is discussed. In contrast, mitochondria from liver, kidney, lung, uterus muscle and ileum muscle exhibit no Na+-dependent efflux of Ca2+.     

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.     

Changes in the nature of calcium transport systems on the porcine sperm plasma membrane during epididymal maturation.

Comparative studies of 45Ca(2+)-transport across the plasma membrane were performed using porcine caput, corpus and cauda epididymal sperm. The Ca(2+)-uptake is dependent on the presence of the substrates for respiration and is sensitive to verapamil. The Ca(2+)-efflux is mediated by both Na(+)-dependent and -independent systems. In the immature sperm in caput epididymis, Na(+)-independent efflux is predominant, but it is gradually replaced by Na(+)-dependent efflux during the epididymal transit. The net activity of Ca2+ accumulation into sperm increases with the epididymal maturation.     

Inhibition of sodium-calcium exchange by ceramide and sphingosine

Na(+)/Ca(2+) exchange activity in Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger was inhibited by the short chain ceramide analogs N-acetylsphingosine and N-hexanoylsphingosine (5-15 micrometer). The sphingolipids reduced exchange-mediated Ba(2+) influx by 50-70% and also inhibited the Ca(2+) efflux mode of exchange activity. The biologically inactive ceramide analog N-acetylsphinganine had only modest effects on exchange activity. Cells expressing the Delta(241-680) and Delta(680-685) deletion mutants of the Na(+)/Ca(2+) exchanger were not inhibited by ceramide; these mutants show defects in both Na(+)-dependent and Ca(2+)-dependent regulatory behavior. Another mutant, which was defective only in Na(+)-dependent regulation, was as sensitive to ceramide inhibition as the wild-type exchanger. Inhibition of exchange activity by ceramide was time-dependent and was accelerated by depletion of internal Ca(2+) stores. Sphingosine (2.5 micrometer) also inhibited the Ca(2+) influx and efflux modes of exchange activity in cells expressing the wild-type exchanger; sphingosine did not affect Ba(2+) influx in the Delta(241-680) mutant. The effects of the exogenous sphingolipids were reproduced by blocking cellular ceramide utilization pathways, suggesting that exchange activity is inhibited by increased levels of endogenous ceramide and/or sphingosine. We propose that sphingolipids impair Ca(2+)-dependent activation of the exchanger and that in cardiac myocytes, this process serves as a feedback mechanism that links exchange activity to the diastolic concentration of cytosolic Ca(2+).     

Effects of extracellular ATP on ion transport systems and [Ca2+]i in rat parotid acinar cells. Comparison with the muscarinic agonist carbachol

The effects of extracellular ATP on ion fluxes and the intracellular free Ca2+ concentration ([Ca2+]i) were examined using a suspension of rat parotid acinar cells and were contrasted with the effects of the muscarinic agonist carbachol. Although ATP and carbachol both rapidly increased [Ca2+]i about threefold above the resting level (200-250 nM), the effect of ATP was due primarily to an influx of Ca2+ across the plasma membrane, while the initial response to carbachol was due to a release of Ca2+ from intracellular stores. Within 10 s, ATP (1 mM) and carbachol (20 microM) reduced the cellular Cl- content by 39-50% and cell volume by 15-25%. Both stimuli reduced the cytosolic K+ content by 57-65%, but there were marked differences in the rate and pattern of net K+ movement as well as the effects of K+ channel inhibitors on the effluxes initiated by the two stimuli. The maximum rate of the ATP-stimulated K+ efflux (approximately 2,200 nmol K+/mg protein per min) was about two-thirds that of the carbachol-initiated efflux rate, and was reduced by approximately 30% (vs. 60% for the carbachol-stimulated K+ efflux) by TEA (tetraethylammonium), an inhibitor of the large conductance (BK) K+ channel. Charybdotoxin, another K+ channel blocker, was markedly more effective than TEA on the effects of both agonists, and reduced the rate of K+ efflux initiated by both ATP and carbachol by approximately 80%. The removal of extracellular Ca2+ reduced the ATP- and the carbachol-stimulated rates of K+ efflux by 55 and 17%, respectively. The rate of K+ efflux initiated by either agonist was reduced by 78-95% in cells that were loaded with BAPTA to slow the elevation of [Ca2+]i. These results indicated that ATP and carbachol stimulated the efflux of K+ through multiple types of K(+)-permeable channels, and demonstrated that the relative proportion of efflux through the different pathways was different for the two stimuli. ATP and carbachol also stimulated the rapid entry of Na+ into the parotid cell, and elevated the intracellular Na+ content to 4.4 and 2.6 times the normal level, respectively. The rate of Na+ entry through Na(+)-K(+)-2Cl- cotransport and Na(+)-H+ exchange was similar whether stimulated by ATP, carbachol, or ionomycin, and uptake through these two carrier-mediated transporters accounted for 50% of the ATP-promoted Na+ influx. The remainder may be due to a nonselective cation channel and an ATP-gated cation channel that is also permeable to Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intracellular pH regulation in cultured embryonic chick heart cells. Na(+)-dependent Cl-/HCO3- exchange

The contribution of Cl-/HCO3- exchange to intracellular pH (pHi) regulation in cultured chick heart cells was evaluated using ion-selective microelectrodes to monitor pHi, Na+ (aiNa), and Cl- (aiCl) activity. In (HCO3- + CO2)-buffered solution steady-state pHi was 7.12. Removing (HCO3- + CO2) buffer caused a SITS (0.1 mM)-sensitive alkalinization and countergradient increase in aiCl along with a transient DIDS-sensitive countergradient decrease in aiNa. SITS had no effect on the rate of pHi recovery from alkalinization. When (HCO3- + CO2) was reintroduced the cells rapidly acidified, aiNa increased, aiCl decreased, and pHi recovered. The decrease in aiCl and the pHi recovery were SITS sensitive. Cells exposed to 10 mM NH4Cl became transiently alkaline concomitant with an increase in aiCl and a decrease in aiNa. The intracellular acidification induced by NH4Cl removal was accompanied by a decrease in aiCl and an increase in aiNa that led to the recovery of pHi. In the presence of (HCO3- + CO2), addition of either amiloride (1 mM) or DIDS (1 mM) partially reduced pHi recovery, whereas application of amiloride plus DIDS completely inhibited the pHi recovery and the decrease in aiCl. Therefore, after an acid load pHi recovery is HCO3o- and Nao- dependent and DIDS sensitive (but not Ca2+o dependent). Furthermore, SITS inhibition of Na(+)-dependent Cl-/HCO3- exchange caused an increase in aiCl and a decrease in the 36Cl efflux rate constant and pHi. In (HCO3- + CO2)-free solution, amiloride completely blocked the pHi recovery from acidification that was induced by removal of NH4Cl. Thus, both Na+/H+ and Na(+)-dependent Cl-/HCO3- exchange are involved in pHi regulation from acidification. When the cells became alkaline upon removal of (HCO3- + CO2), a SITS-sensitive increase in pHi and aiCl was accompanied by a decrease of aiNa, suggesting that the HCO3- efflux, which can attenuate initial alkalinization, is via a Na(+)-dependent Cl-/HCO3- exchange. However, the mechanism involved in pHi regulation from alkalinization is yet to be established. In conclusion, in cultured chick heart cells the Na(+)-dependent Cl-/HCO3- exchange regulates pHi response to acidification and is involved in the steady-state maintenance of pHi.     

Functional differences of Na+/Ca2+ exchanger expression in Ca2+ transport system of smooth muscle of guinea pig stomach

The plasma membrane ATP-dependent Ca2+ pump and the Na+/Ca2+ exchanger (NCX) are the major means of Ca2+ extrusion in smooth muscle. However, little is known regarding distribution and function of the NCX in guinea pig gastric smooth muscle. The expression pattern and distribution of NCX isoforms suggest a role as a regulator of Ca2+ transport in cells. Na+ pump inhibition and the consequent to removal of K+ caused gradual contraction in fundus. In contrast, the response was significantly less in antrum. Western blotting analysis revealed that NCX1 and NCX2 are the predominant NCX isoforms expressed in stomach, the former was expressed strongly in antrum, whereas the latter displayed greater expression in fundus. Isolated plasma membrane fractions derived from gastric fundus smooth muscle were also investigated to clarify the relationship between NCX protein expression and function. Na+-dependent Ca2+ uptake increased directly with Ca2+ concentration. Ca2+ uptake in Na+-loaded vesicles was markedly elevated in comparison with K+-loaded vesicles. Additionally, Ca2+ uptake by the Na+- or K+-loaded vesicles was substantially higher in the presence of A23187 than in its absence. The result can be explained based on the assumption that Na+ gradients facilitate downhill movement of Ca2+. Na+-dependent Ca2+ uptake was abolished by the monovalent cationic ionophore, monensin. NaCl enhanced Ca2+ efflux from vesicles, and this efflux was significantly inhibited by gramicidin. Results documented evidence that NCX2 isoform functionally contributes to Ca2+ extrusion and maintenance of contraction-relaxation cycle in gastric fundus smooth muscle.     

Ionic regulation of the cardiac sodium-calcium exchanger

The Na(+)-Ca(2+) exchanger (NCX) links transmembrane movements of Ca(2+) ions to the reciprocal movement of Na(+) ions. It normally functions primarily as a Ca(2+) efflux mechanism in excitable tissues such as the heart, but it can also mediate Ca(2+) influx under certain conditions. Na(+) and Ca(2+) ions exert complex regulatory effects on NCX activity. Ca(2+) binds to two regulatory sites in the exchanger's central hydrophilic domain, and this interaction is normally essential for activation of exchange activity. High cytosolic Na(+) concentrations, however, can induce a constitutive activity that by-passes the need for allosteric Ca(2+) activation. Constitutive NCX activity can also be induced by high levels of phopshotidylinositol-4,5-bisphosphate (PIP?) and by mutations affecting the regulatory calcium binding domains. In addition to promoting constitutive activity, high cytosolic Na(+) concentrations also induce an inactivated state of the exchanger (Na(+)-dependent inactivation) that becomes dominant when cytosolic pH and PIP? levels fall. Na(+)-dependent inactivation may provide a means of protecting cells from Ca(2+) overload due to NCX-mediated Ca(2+) influx during ischemia.     

Identification of a peptide inhibitor of the cardiac sarcolemmal Na(+)-Ca2+ exchanger

The deduced amino acid sequence of the cardiac sarcolemmal Na(+)-Ca2+ exchanger has a region which could represent a calmodulin binding site. As calmodulin binding regions of proteins often have an autoinhibitory role, a synthetic peptide with this sequence was tested for functional effects on Na(+)-Ca2+ exchange activity. The peptide inhibits the Na(+)-dependent Ca2+ uptake (KI approximately 1.5 microM) and the Nao(+)-dependent Ca2+ efflux of sarcolemmal vesicles in a noncompetitive manner with respect to both Na+ and Ca2+. The peptide is also a potent inhibitor (KI approximately 0.1 microM) of the Na(+)-Ca2+ exchange current of excised sarcolemmal patches. The binding site for the peptide on the exchanger is on the cytoplasmic surface of the membrane. The exchanger inhibitory peptide binds calmodulin with a moderately high affinity. From the characteristics of the inhibition of the exchange of sarcolemmal vesicles, we deduce that only inside-out sarcolemmal vesicles participate in the usual Na(+)-Ca2+ exchange assay. This contrasts with the common assumption that both inside-out and right-side-out vesicles exhibit exchange activity.     

Decreasing extracellular Na+ concentration triggers inositol polyphosphate production and Ca2+ mobilization

Removing extracellular Na+ (Na+o) evoked a large increase in cytosolic free Ca2+ concentration ([Ca2+]i in human skin fibroblasts. Decreasing [Na+]o from 120 to 14 mM caused the half-maximal peak increase in [Ca2+]i. Removing Na+o strongly stimulated 45Ca2+ efflux and decreased total cell Ca2+ by about 40%. Bradykinin caused changes in [Ca2+]i, total Ca2+, and 45Ca2+ fluxes similar to those evoked by removing Na+o. Prior stimulation of the cells with bradykinin prevented Na+o removal from increasing [Ca2+]i and vice versa. Na+o removal rapidly increased [3H]inositol polyphosphate production. Loading the cells with Na+ had no effect on the increase in 45Ca2+ efflux produced by Na+o removal. Therefore, decreasing [Na+]o probably stimulates a "receptor(s)" which is sensitive to extracellular, not intracellular, Na+. Removing Na+o also mobilized intracellular Ca2+ in smooth muscle and endothelial cells cultured from human umbilical and dog coronary arteries, respectively.     

Stimulatory effect of enkephalins on calcium efflux from bovine adrenal chromaffin cells in culture

The effects of leucine- and methionine-enkephalin, opiate peptides, on Ca2+ efflux from cultured bovine adrenal chromaffin cells were examined. These enkephalins stimulated the efflux of 45Ca2+ from cells in a concentration-dependent manner (10(-8) M-10(-6) M). Leucine-enkephalin did not increase the intracellular free Ca2+ level, 45Ca2+ uptake, catecholamine secretion, cAMP level or cGMP level. The peptide-stimulated 45Ca2+ efflux was not inhibited by incubation in Ca2+-free medium, but was inhibited by incubation in Na+-free medium. These results indicate that enkephalins stimulate extracellular Na+-dependent 45Ca2+ efflux from cultured bovine adrenal chromaffin cells, probably by stimulating membrane Na+/Ca2+ exchange.     

Calcium export by sodium-calcium exchange in bovine chromaffin cells

Calcium efflux from bovine chromaffin cells in tissue culture has been examined after loading them with small amounts of Ca2+ by brief depolarization in media containing 20 mumol/l to 1 mmol/l Ca2+ and 45Ca2+ in trace amounts. In the presence of normal external Na+ and Ca2+ concentrations cells depolarized in media containing up to 200 mumol/l Ca2+ exported nearly 100% of their accumulated Ca2+ loads within 10 min and 20% within the first 5 s. In the absence of external Na+ and Ca2+ the proportion of a small (i.e., depolarization in 20 mumol/l calcium) Ca2+ load exported at any time point in the range to 10 min was approximately two thirds of the total efflux measured in their presence indicating that under these conditions the external Na+/Ca(2+)-dependent and Na+/Ca(2+)-independent mechanisms both contribute significantly to the export of calcium. At higher cellular loads of calcium (i.e., depolarization in 200 mumol/l to 1 mmol/l calcium) the Na+/Ca(2+)-dependent mechanism exported a progressively greater proportion of the accumulated Ca2+. Both sodium and calcium alone promoted a component of Ca2+ efflux; Ca2+ (i.e. calcium-calcium exchange) was as effective as Na+ (i.e. sodium-calcium exchange). The Km for Na+ stimulation of Ca(2+)-efflux (KNa) was approximately 65 mM. Increased external Mg2+ (from 1.2 to 10 mmol/l) increased the apparent KNa to 90 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of Li+ on Na-Ca exchange in cardiac sarcolemmal vesicles

The effects of Li+ on Na-Ca exchange in bovine cardiac sarcolemmal vesicles were examined. The initial rate of Na(+)-dependent Ca2+ uptake and efflux was inhibited by Li+ in a dose dependent manner. The initial rate of Na(+)-dependent Ca2+ uptake was inhibited 49.8 +/- 2.9% (S.E.) (n = 6) in the presence of Li+ compared to activity in external K+ or choline+. Kinetic analysis indicated that Li+ increased the Km for Ca2+ (96.3 microM) compared to K+ and choline+ (25.5 and 22.9 microM respectively) while Vmax (1.4, 1.2 and 1.1 nmol Ca2+/mg protein/sec respectively) remained unchanged. Li+ did not alter the experimentally derived stoichiometry of the exchange reaction of 3 Na+ for 1 Ca2+.