Modulation of Na+-Ca2+ exchange and Ca2+ permeability in cardiac sarcolemmal vesicles by doxylstearic acids

We examine the effects of 5-, 12- and 16-doxylstearic acids on the Na+-Ca2+ exchange and passive Ca2+ permeability of cardiac sarcolemmal vesicles. Stearic acid is a weak stimulator of Na+-Ca2+ exchange. A doxyl moiety potentiates stimulation with the order of increasing potency being 5-, 12- and then 16-doxylstearic acid. Stearic acid has little effect on vesicle Ca2+ permeability but again the doxylstearates are more effective. The sequence of potency is reversed, however, from that for increasing Na+-Ca2+ exchange. 5-Doxylstearic acid most markedly exchanges passive Ca2+ flux followed by the 12-, and then 16-doxylstearic acids. Methyl esters of the doxylstearates have no effect on either Na+-Ca2+ exchange or Ca2+ permeability. We model the results as follows. For a fatty acid to stimulate Na+-Ca2+ exchange activity, an anionic charge is required to interact with the exchanger protein at the membrane surface. Stimulation is potentiated by a perturbation (such as provided by a doxyl group) within the lipid bilayer. The perturbation is most effective at a location towards the center of the bilayer. To increase passive Ca2+ permeability an anionic charge is again essential. Disorder within the bilayer is also important, but now the most important site is near the membrane surface. Results of experiments with linolenic and gamma-linolenic acid and previous studies with other fatty acids also support this model.     

Alterations by saponins of passive Ca2+ permeability and Na+-Ca2+ exchange activity of canine cardiac sarcolemmal vesicles

Saponins can both permeabilize cell plasma membranes and cause positive inotropic effects in isolated cardiac muscles. Different saponins vary in their relative abilities to cause each effect suggesting that different mechanisms of action may be involved. To investigate this possibility, we have compared the effects of seven different saponins on the passive Ca2+ permeability and Na+-Ca2+ exchange activity of isolated canine cardiac sarcolemmal membranes. Saponins having hemolytic activity reversibly increased the passive efflux of Ca2+ from sarcolemmal vesicles preloaded with 45Ca2+ with the following order of potency: echinoside-A greater than echinoside-B greater than holothurin-A greater than holothurin-B greater than sakuraso-saponin. Ginsenoside-Rd and desacyl-jego-saponin, which lack hemolytic activity, had no significant effect on this variable. The saponins also stimulated Na+-Ca2+ exchange activity measured as Na+-dependent Ca2+ uptake by sarcolemmal vesicles. Ginsenoside-Rd and desacyl-jego-seponin, which did not affect passive Ca2+ permeability, stimulated the uptake, while in contrast, echinoside-A and -B only slightly increased or decreased this latter variable. Thus, the abilities of these compounds to enhance Na+-Ca2+ exchange activity seem to be inversely related to their abilities to increase the Ca2+ permeability. Effects by the echinosides on Na+-Ca2+ exchange may be masked by the loss of Ca2+ from the vesicles due to the increased permeability. These results suggest that the saponins interact with membrane constituent(s) that can influence the passive Ca2+ permeability and the Na+-Ca2+ exchange activity of cardiac sarcolemmal membranes.     

In dialyzed squid axons Ca2+i activates Ca2+o-Na+i and Na+o-Na+i exchanges in the absence of Ca chelating agents

We used internally dialyzed squid axons to explore whether the reported activatory effect of Ca2+i on the partial reactions of the Na+-Ca2+ exchange (essential activator) is secondary to the presence of Ca2+ chelating agents in the internal medium. The effect of Ca2+i pulses on both the reverse (Ca2+o-dependent Na+ efflux) and Na+-Na+ exchange (Na+o-dependent Na+ efflux) modes of the Na+-Ca2+ exchange was studied in axons dialyzed without EGTA. For these experiments a substantial inhibition of the Ca2+ buffer capacity of the axoplasm was achieved by the use of Ruthenium red (10-20 microM), cyanide (1 mM) and vanadate (1 mM) in the dialysis solution. Our results indicate that the Ca2+i requirement of the reverse and Na+-Na+ exchange can not be explained by a direct inhibition of the Na+-Ca2+ exchanger by EGTA. In fact, both modes of operation of the exchanger can be activated by internal Ca2+ ions in the complete absence of Ca2+ chelating agents thus indicating that the 'catalytic' effect of Ca2+i on the Na+-Ca2+ exchanger is a real phenomenon.     

Symmetry properties of the Na+-Ca2+ exchange mechanism in cardiac sarcolemmal vesicles

The Na+-Ca2+ exchange mechanism in cardiac sarcolemmal vesicles can catalyze the exchange of Ca2+ on either side of the sarcolemmal membrane for Na+ on the opposing side. Little is known regarding the relative affinities of Na+ and Ca2+ for exchanger binding sites on the intra- and extracellular membrane surfaces. We have previously reported (Philipson, K.D. and Nishimoto, A.Y. (1982) J. Biol. Chem. 257, 5111-5117) a method for measuring the Na+-Ca2+ exchange of only the inside-out vesicles in a mixed population of sarcolemmal vesicles (predominantly right-side-out). We concluded that the apparent Km(Ca2+) for Na+i-dependent Ca2+ uptake was similar for inside-out and right-side-out vesicles. In the present study, we examine in detail Na+o-dependent Ca2+ efflux from both the inside-out and the total population of vesicles. To load vesicles with Ca2+ prior to measurement of Ca2+ efflux, four methods are used: 1, Na+-Ca2+ exchange; 2, passive Ca2+ diffusion; 3, ATP-dependent Ca2+ uptake; 4, exchange of Ca2+ for Na+ which has been actively transported into vesicles by the Na+ pump. The first two methods load all sarcolemmal vesicles with Ca2+, while the latter two methods selectively load inside-out vesicles with Ca2+. We are able to conclude that the dependence of Ca2+ efflux on the external Na+ concentration is similar in inside-out and right-side-out vesicles. Thus the apparent Km(Na+) values (approximately equal to 30 mM) of the Na+-Ca2+ exchanger are similar on the two surfaces of the sarcolemmal membrane. In other experiments, external Na+ inhibited the Na+i-dependent Ca2+ uptake of the total population of vesicles much more potently than that of the inside-out vesicles. Apparently Na+ can compete for the Ca2+ binding site more effectively on the external surface of right-side-out than on the external surface of inside-out vesicles. Thus, although affinities for Na+ or Ca2+ (in the absence of the other ion) appear symmetrical, the interactions between Na+ and Ca2+ at the two sides of the exchanger are not the same. The Na+-Ca2+ exchanger is not a completely symmetrical transport protein.     

Effects of fatty acids on Na+-Ca2+ exchange and Ca2+ permeability of cardiac sarcolemmal vesicles

We have previously reported that anionic phospholipids (Philipson, K.D., and Nishimoto, A.Y. (1984) J. Biol. Chem. 259, 16-19) and other anionic amphiphiles (Philipson, K.D. (1984) J. Biol. Chem. 259, 13999-14002) stimulate Na+-Ca2+ exchange in cardiac sarcolemmal vesicles. To further these studies, we have now investigated the effects of a variety of fatty acids on both Na+-Ca2+ exchange and passive Ca2+ permeability. Na+-Ca2+ exchange was stimulated by fatty acids by up to 150%. Unsaturated fatty acids were more potent than saturated fatty acids, and the stimulation was primarily due to a decrease in the apparent KM (Ca2+). There was a positive correlation between the ability of a fatty acid to stimulate Na+-Ca2+ exchange and to increase passive Ca2+ permeability. The methyl esters of fatty acids had no effects on either exchange or permeability indicating the importance of anionic charge. We conclude that the combination of local lipid disorder and anionic charge regulate Na+-Ca2+ exchange. Perturbations of the bilayer hydrophobic region and increased negative surface charge are both required for fatty acids to increase passive Ca2+ flux. Na+-Ca2+ exchange is stimulated when the ratio of membrane free fatty acid to phospholipid is about 5%. This level of fatty acid is achieved during 1 h of myocardial ischemia (Chien, K. R., Han, A., Sen, A., Buja, L. M., and Willerson, J. T. (1984) Circ. Res. 54, 313-322), indicating that ischemia could induce altered sarcolemmal Ca2+ transport due to fatty acid accumulation.     

Effect of amiloride on diaphragmatic contractility: evidence of a role for Na(+)-Ca2+ exchange

Extracellular Ca2+ has been shown to be important for the normal function of the diaphragm. In this study we have examined the potential importance of Na(+)-Ca2+ exchange as a mechanism for Ca2+ influx during the contractile process by studying the effect of inhibition or stimulation of Na(+)-Ca2+ exchange. Blockade of Na(+)-Ca2+ exchange with amiloride attenuated the twitch response, altered the force-frequency response curve, and enhanced the development of fatigue. The effect of amiloride could be partially reversed by increasing the extracellular Ca2+ concentration. The ability of amiloride to decrease force was associated with decreased Ca2+ uptake by the diaphragm. Enhancing intracellular Na(+)-extracellular Ca2+ exchange by inhibiting the Na(+)-K+ pump significantly decreased the rate of the development of muscle fatigue (89%). The maximal inhibition of diaphragmatic force produced by the amiloride analogue benzamil, which possesses 10-fold greater selectivity for Na(+)-Ca2+ exchange, was not significantly different from that produced by amiloride (76.2 +/- 1.1%), with a concentration that decreased maximum force by 50% equal to 46 microM compared with 460 microM for amiloride. Both agents slowed the maximal rate of relaxation up to 90%. Benzamil elevated resting tension during continuous stimulation of the diaphragm at 0.15 Hz. The results suggest that Na(+)-Ca2+ exchange may have a role in the normal function of the diaphragm.     

Role of phosphatidylinositol in cardiac sarcolemmal membrane sodium-calcium exchange

The purpose of this investigation was to study the effects of a distinct type of phospholipase C on sarcolemmal Na+-Ca2+ exchange. With this phospholipase C (Staphylococcus aureus), treatment of cardiac sarcolemmal vesicles resulted in a specific hydrolysis of membrane phosphatidylinositol. This hydrolysis of phosphatidylinositol also released two proteins (110 and 36 kDa) from the sarcolemmal membrane. Phospholipase C pretreatment of the sarcolemma resulted in an unexpected stimulation of Na+-Ca2+ exchange. The Vmax of Na+-Ca2+ exchange was increased but the Km for Ca2+ was not altered. This stimulation was specific to the Na+-Ca2+ exchange pathway. ATP-dependent Ca2+ uptake was depressed after phospholipase C treatment, but passive membrane permeability to Ca2+ was unaffected. Sarcolemmal Na+,K+-ATPase activity was not altered, whereas passive Ca2+ binding was modestly decreased after phospholipase C pretreatment. The stimulation of Na+-Ca2+ exchange after phosphatidylinositol hydrolysis was greater in inside-out vesicles than in a total population of vesicles of mixed orientation. This finding suggests that the cardiac sarcolemmal Na+-Ca2+ exchanger is functionally asymmetrical. The results also suggest that membrane phosphatidylinositol is inhibitory to the Na+-Ca2+ exchanger or, alternatively, this phospholipid may anchor an endogenous inhibitory protein in the sarcolemmal membrane. The observation that a transsarcolemmal Ca2+ flux pathway may be stimulated solely by phosphatidylinositol hydrolysis independently of phosphoinositide metabolic products like inositol triphosphate is novel.     

Dependence of Na+-Ca2+ exchange and Ca2+-Ca2+ exchange on monovalent cations

Two mechanisms of passive Ca2+ transport, Na+-Ca2+ exchange and Ca2+-Ca2+ exchange, were studied using highly-purified dog heart sarcolemmal vesicles. About 80% of the Ca2+ accumulated by Na+-Ca2+ exchange or Ca2+-Ca2+ exchange could be released as free Ca2+, while up to 20% was probably bound. Na+-Ca2+ exchange was simultaneous, coupled countertransport of Na+ and Ca2+. The movement of anions during Na+-Ca2+ exchange did not limit the initial rate of Na+-Ca2+ exchange. Na+-Ca2+ exchange was electrogenic, with a reversal potential of about -105 mV. The apparent flux ratio of Na+-Ca2+ exchange was 4 Na+:1 Ca2+. Coupled cation countertransport by the Na+-Ca2+ exchange mechanism required a monovalent cation gradient with the following sequence of ion activation: Na+ much greater than Li+ greater than Cs+ greater than K+ greater than Rb+. In contrast to Na+-Ca2+ exchange, Ca2+-Ca2+ exchange did not require a monovalent cation gradient, but required the presence of Ca2+ plus a monovalent cation on both sides of the vesicle membrane. The sequence of ion activation of Ca2+-Ca2+ exchange was: K+ much greater than Rb+ greater than Na+ greater than Li+ greater than Cs+. Na+ inhibited Ca2+-Ca2+ exchange when Ca2+-Ca2+ exchange was supported by another monovalent cation. Both Na+-Ca2+ exchange and Ca2+-Ca2+ exchange were inhibited, but with different sensitivities, by external MgCl2, quinidine, or verapamil.     

Na+-Ca2+ exchange and Ca2+ efflux in transfected Chinese hamster ovary cells

The objective of this study was to assess the contribution of Na+-Ca2+ exchange activity to Ca2+ efflux at various cytosolic Ca2+ concentrations ([Ca2+]i) in transfected Chinese hamster cells expressing the bovine cardiac Na+-Ca2+ exchanger. Ionomycin was added to fura-2 loaded cells and the resulting [Ca2+]i transient was monitored in Ca2+-free media with or without extracellular Na+. The presence of Na+ reduced both the amplitude and duration of the [Ca2+]i transient. Na+ had similar effects when the peak of the [Ca2+]i transient was buffered to 100 nM by cytosolic EGTA, or when Ca2+ was slowly released from internal stores with thapsigargin. Ca2+ efflux following ionomycin addition was directly measured with extracellular fura-2 and followed a biphasic time course (t(1/2) approximately = 10 s and 90s). The proportion of total efflux owing to the rapid phase was increased by Na+ and reduced by EGTA-loading. Na+ accelerated the initial rate of Ca2+ efflux by 65% in unloaded cells but only by 16% in EGTA-loaded cells. In both cases, the stimulation by Na+ was less than expected, given the pronounced effects of Na+ on the [Ca2+]i transient. We conclude that the exchanger contributes importantly to Ca2+ efflux activity at all [Ca2+]i values above 40 nM. We also suggest that Ca2+ efflux pathways may involve non-cytosolic or local routes of Ca2+ traffic.     

Physiological characterization of the Na(+)/Ca(2+) exchanger (NCX) in hepatopancreatic and antennal gland basolateral membrane vesicles isolated from the freshwater crayfish Procambarus clarkii

The purpose of this study was to physiologically characterize the basolateral Na(+)/Ca(2+) exchanger (NCX) in basolateral membrane vesicles (BLMVs) of hepatopancreas and antennal gland of intermolt crayfish. Conditions were optimized to measure Na(+)-dependent Ca(2+) uptake and retention in the BLMV including use of intravesicular (IV) oxalate and measuring initial uptake rates at 20 s. Na(+)-dependent Ca(2+) uptake rate into BLMV was temperature insensitive. Na(+)-dependent Ca(2+) uptake rate was dependent upon free Ca(2+) with saturable Michaelis-Menten kinetics determined as follows: hepatopancreas, maximal uptake rate (J(max))=2.45 nmol/mg per min, concentration at which carrier operates at half-maximal uptake rate (K(m))=0.69 microM Ca(2+); antennal gland, J(max)=13.2 nmol/mg per min, K(m)=0.59 microM Ca(2+). The two vesicle populations exhibited different sensitivity to putative NCX inhibitors. Benzamil had no effect on Na(+)-dependent Ca(2+) uptake rate in hepatopancreas; in antennal gland it was inhibitory at concentrations up to 30 microM and was stimulatory at higher concentrations. Conversely the inhibitor quinacrine was inhibitory at 10 microM in hepatopancreas and was stimulatory at 1000 microM; meanwhile it was ineffective in antennal gland BLMV. Short circuiting the BLMV had no effect on Na(+)-dependent Ca(2+) uptake rate suggesting that the process may be electroneutral. Compared with another prominent basolateral transporter in hepatopancreas the plasma membrane Ca(2+) ATPase (PMCA), the NCX has 70-fold greater J(max) (at comparable temperature) and a lower affinity. In antennal gland the NCX has 40-fold greater J(max) and a lower affinity. In hepatopancreas and antennal gland BLMV NCX appears to determine the rate of basolateral Ca(2+) efflux in intermolt.     

Intestinal calcium transport in spontaneously hypertensive rats (SHR) and their genetically matched WKY rats

Calcium transport across the basolateral membranes of the enterocyte represents the active step in calcium translocation. This step occurs by two mechanisms, an ATP-dependent pump and a Ca2+/Na+ exchange process. These studies were designed to investigate these two processes in jejunal basolateral membrane vesicles (BLMV) of the spontaneously hypertensive rats (SHR) and their genetically matched controls, Wistar-Kyoto (WKY) rats. The ATP-dependent calcium uptake was stimulated several-fold compared with no ATP condition in both SHR and WKY, but no differences were noted between rate of calcium uptake in SHR and WKY. Kinetics of ATP-dependent calcium uptake at concentrations between 0.01 and 1.0 microM revealed a Vmax of 0.67 +/- 0.03 nmol/mg protein/20 sec and a Km of 0.2 +/- 0.03 microM in SHR and Vmax of 0.69 +/- 0.12 and a Km of 0.32 +/- 0.14 microM in WKY rats. Ca2+/Na+ exchange in jejunal BLMV of SHR and WKY was investigated in two ways. First, sodium was added to the incubation medium (cis-Na+). Second, Ca2+ efflux from BLMV was studied in the presence of extravesicular Na+ (trans-Na+). Both studies suggest a decreased exchange of calcium and Na+. Kinetic parameters of Na(+)-dependent Ca2+ uptake at concentrations between 0.01 and 1.0 microM exhibited Vmax of 0.05 +/- 0.01 nanmol/mg protein/5 sec and a Km of 0.21 +/- 0.13 microM in SHR and Vmax of 0.11 +/- 0.02 nanmol/mg protein/5 sec and a Km of 0.09 +/- 0.05 in WKY, respectively. These results confirm that the intestinal BLMV of SHR and WKY rats have two mechanisms for calcium extrusion, an ATP-dependent Ca2+ transport process and a Na+/Ca2+ exchange process. The ATP-dependent process appears to be functional in SHR; however, the Ca2+/Na+ exchange mechanism appears to have a marked decrease in its maximal capacity. These findings suggest that calcium extrusion via Ca2+/Na+ is impaired in the SHR, which may lead to an increase in intracellular calcium concentration. These findings may have relevance to the development of hypertension.     

Na+-Ca2+ exchange in squid optic nerve membrane vesicles is activated by internal calcium

The role of intracellular Ca2+ as essential activator of the Na+-Ca2+ exchange carrier was explored in membrane vesicles containing 67% right-side-out and 10% inside-out vesicles, isolated from squid optic nerves. Vesicles containing 100 microM free calcium exhibited a 2-fold increase in the initial rate of Na+i-dependent Ca2+ uptake as compared with vesicles where intravesicular calcium was chelated by 2 mM EGTA or 10 mM HEDTA. The activatory effect exerted by intravesicular Ca2+ on the reverse mode of Na+-Ca2+ exchange (i.e. Na+i-Ca2+o exchange) is saturated at about 100 microM Ca2+i and displays an apparent K 1/2 of 12 microM. Intravesicular Ca2+ produced activation of Na+i-Ca2+i exchange activity rather than an increase in Ca2+ uptake due to Ca2+-Ca2+ exchange. The presence of Ca2+i was essential for the Na+i-dependent Na+ influx, a partial reaction of the Na+-Ca2+ exchanger. In fact, the Na+ influx levels in vesicles loaded with 2 mM EGTA were close to those expected from diffusional leak while in vesicles containing Ca2+i an additional Na+-Na+ exchange was measured. The results suggest that in nerve membrane vesicles Ca2+ at the inner aspect of the membrane acts as an activator of the Na+-Ca2+ exchange system.     

ATP-dependent calcium pump and Na+-Ca2+ exchange in plasma membrane vesicles from squid optic nerve

Purified plasma membrane vesicles from the optic nerve of the squid Sepiotheutis sepioidea accumulate calcium in the presence of Mg2+ and ATP. Addition of the Ca2+ ionophore A23187 to vesicles which have reached a steady state of calcium-active uptake induces complete discharge of the accumulated cation. Kinetic analysis of the data indicates that the apparent Km for free Ca2+ and ATP are 0.2 muM and 21 muM, respectively. The average Vmax is 1 nmol Ca2+/min per mg protein at 25 degrees C. This active transport is inhibited by orthovanadate in the micromolar range. An Na+-Ca2+ exchange mechanism is also present in the squid optic nerve membrane. When an outwardly directed Na+ gradient is imposed on the vesicles, they accumulate calcium in the absence of Mg2+ and/or ATP. This ability to accumulate Ca2+ is absolutely dependent on the Na+ gradient: replacement of Na+ by K+, or passive dissipation of the Na+ gradient, abolishes transport activity. The apparent Km for Ca2+ of the Na+-Ca2+ exchange is more than 10-fold higher than that of the ATP-driven pump (app. Km=7.5 muM). While the apparent Km for Na+ is 74 mM, the Vmax of the exchanger is 27 nmol Ca2+/min per mg protein at 25 degrees C. These characteristics are comparable to those displayed by the uncoupled Ca pump and Na+-Ca2+ exchange previously described in dialyzed squid axons.     

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.     

Mechanism of sodium independent calcium efflux from rat liver mitochondria

On the basis of primarily two types of observations, it has been suggested that the Na+-independent Ca2+ efflux mechanism of rat liver mitochondria is a passive Ca2+-2H+ exchanger. First, when a pulse of acid is added to a suspension of mitochondria loaded with Ca2+, a pulse of intramitochondrial Ca2+ is often released, even in the presence of the inhibitor of mitochondrial Ca2+ influx, ruthenium red. Second, at a pH near 7, the stoichiometry of Ca2+ released to H+ taken up by Ca2+-loaded mitochondria, following treatment with ruthenium red, has been observed to be 1:2. This evidence for a Ca2+-2H+ exchanger is reexamined here by studying the release of Ca2+ upon acidification of the medium by addition of buffer, the dependence of liver mitochondrial Ca2+ efflux on external medium pH and intramitochondrial pH, and the Ca2+-Ca2+ exchange properties of the Ca2+ efflux mechanism. These studies show no pulse of mitochondrial Ca2+ efflux when pH is abruptly lowered by addition of buffer. The stoichiometry between Ca2+ and H+ fluxes is found to be highly pH dependent. The reported 1:2 stoichiometry between Ca2+ efflux and H+ influx is only observed at one pH. Furthermore, the rate of Ca2+ efflux from mitochondria is found to increase only very slightly at most as suspension pH is decreased. The rate of Ca2+ efflux is not found to increase with increasing intramitochondrial pH. Finally, no Ca2+-Ca2+ isotope exchange can be demonstrated over the Na+-independent efflux mechanism (i.e., in the presence of ruthenium red). It is concluded that these data do not support the hypothesis that the Na+-independent Ca2+ efflux mechanism is a passive Ca2+-2H+ exchanger.     

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.     

A role for Na+/Ca2+ exchange in the generation of superoxide radicals by human neutrophils

A Na+/Ca2+ exchange mechanism has been recently described in human neutrophils that constitutes the principal pathway for Ca2+ influx into resting cells. The potential role of this system in regulating the respiratory burst in response to activation by the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine was explored. In the presence of 1 mM Ca2+, a variety of di- and trivalent cations suppressed the generation of O(-2) radicals in a series of decreasing efficacy: La3+ approximately Zn2+ much greater than Sr2+ approximately Cd2+ greater than Ba2+ greater than Co2+ greater than Ni2+ approximately Mg2+. This sequence is similar to their rank order of activity in inhibiting 45Ca2+ influx via Na+/Ca2+ counter-transport. Benzamil, phenamil, and 2',4'-dichlorobenzamil, analogues of amiloride which selectively block Na+/Ca2+ exchange in neutrophils, likewise suppressed the release of O(-2) with apparent Ki values of approximately 30 microM. The effect of the cations was competitive with Ca2+, while the interaction between the benzamil derivatives and Ca2+ appeared to be noncompetitive in nature. Both the divalent cations and benzamil also inhibited the rise in cytoplasmic Ca2+ as monitored by fura-2 fluorescence: these agents reduced peak cytosolic Ca2+ levels after N-formyl-methionyl-leucyl-phenylalanine stimulation to values seen in the absence of extracellular Ca2+. These results are compatible with the hypothesis that the influx of Ca2+ via Na+/Ca2+ exchange contributes to the transient elevation in intracellular free Ca2+. The polyvalent cations block the entry of critical Ca2+ ions by competing with Ca2+ for binding to the translocation site on the exchange carrier, while benzamil acts by lowering the maximal transport rate. These studies emphasize that Na+/Ca2+ exchange through its effects on cytoplasmic Ca2+ plays a major regulatory role in activation of the respiratory burst in chemotactic factor-stimulated neutrophils.     

Inhibition of Na+-dependent Ca2+ efflux from heart mitochondria by amiloride analogues

The Na+-induced release of accumulated Ca2+ from heart mitochondria is inhibited by amiloride, benzamil and several other amiloride analogues. These drugs do not affect uptake or release of Ca2+ mediated by the ruthenium red-sensitive uniporter and their effects, like those of diltiazem and other Ca2+-antagonists, appear to be localized principally at the Na+/Ca2+ antiporter of the mitochondrion. Benzamil inhibits Na+/Ca2+ antiport non-competitively with respect to [Na+] with a Ki of 167 microM. In the presence of 1.5 mM Pi the Ki for benzamil inhibition of this reaction is decreased to 87 microM.     

Regulation of the plasma membrane Ca2+ pump by cyclic nucleotides in cultured vascular smooth muscle cells

We investigated the mechanisms of Ca2+ extrusion from cultured rat aortic smooth muscle cells while monitoring changes in the cytosolic Ca2+ concentration ([Ca2+]i) using fura 2 fluorescence. 45Ca2+ efflux from these cells consisted of two major mechanisms; one was dependent on the extracellular sodium concentration (Na+o) and the other was independent of Na+o. Na+o-dependent efflux increased monotonically with increasing [Ca2+]i between 0.1 and 1.0 microM, whereas Na+o-independent efflux reached a plateau at 0.6-1 microM [Ca2+]i with a half-maximum obtained at about 0.16 microM. At [Ca2+]i below 1 microM, the latter was significantly greater than the former. Unlike the Na+o-dependent mechanism, Na+o-independent 45Ca2+ efflux was inhibited almost entirely by extracellularly added La3+ or a combination of high extracellular pH (pH 8.8) and 20 mM Mg2+. It was also inhibited, although not completely, by compound 48/80, a calmodulin antagonist, and vanadate. These results strongly suggest that Na+o-dependent and Na+o-independent 45Ca2+ effluxes occur via the Na+/Ca2+ exchanger and the ATP-dependent Ca2+ pump, respectively. Sodium nitroprusside and atrial natriuretic factor, which are agents that stimulate intracellular production of cGMP, and 8-BrcGMP significantly accelerated the Na+o-independent 45Ca2+ efflux especially at low [Ca2+]i. Forskolin, dibutyryl cAMP, and 8-Br-cAMP, however, showed no stimulation. These results suggest that the plasma membrane Ca2+ pump is regulated by cGMP but not by cAMP in intact vascular smooth muscle cells.     

Light-dependent Na(+)-Ca2+ exchange in retinal rod discs

Experiments are described demonstrating that Na(+)-Ca2+ exchange of retinal rod disc membrane is highly sensitive to light. The Na(+)-Ca2+ exchanger was shown to possess two types of binding sites with different affinities for calcium. The low affinity binding sites (KCaD = 5.8 mumol/l) are light-insensitive. After bleaching, KD of the high affinity Ca2(+)-binding sites an Ki for Na+ changed from 0.2 to 0.3 mumol/l and from 3.2 to 0.7 nmol/l, respectively. Light inhibits the steady-state Ca2+ uptake by a factor of 1.5. Photocontrol of the Na(+)-Ca2+ exchanger affinity is observed at the physiological level of rhodopsin bleaching.