Effects of sulfhydryl reagents on Na+-Ca2+ exchange in rat brain microsomal membranes

Effects of six thiol reagents with different physico-chemical properties were tested on the Na+-dependent 45Ca2+ transport into the rat brain microsomal membrane vesicles. The mercurials p-chlormercuribenzoate and Mersalyl effectively inhibited 45Ca2+ uptake with IC50 values in the order of 10(-4) mol X l-1 in the medium. N-ethylmaleimide and its more lipophilic analog N-(4-(2-benzoxazolyl)phenyl)maleimide were much less effective at the same concentrations. 2,2'-dithiodipyridine markedly reduced 45Ca2+ uptake already at concentrations below 10(-4) mol X l-1, whereas 5,5'-dithiobis-2-nitrobenzoate in a concentration range 10(-6)-10(-3) mol X l-1 was a weak inhibitor. Inhibitory effects of the most potent inhibitors p-chlormercuribenzoate and 2,2'-dithiodipyridine were readily reversed by 1 mmol X l-1 dithiothreitol. The results suggest that free SH groups of membrane polypeptides are involved in the functioning of the Na+-Ca2+ exchanger in the nerve tissue cell membranes.     

Inhibition of Na+-Ca2+ exchange by calcium antagonists in rat brain microsomal membranes

Na+-Ca2+ exchange rates were studied in native and/or pronase pretreated rat brain microsomal membranes in the presence of calcium channel antagonists verapamil, nimodipine and nifedipine. In native membranes all the substances used inhibited Na+-Ca2+ exchange. A relatively stronger inhibition was observed in membranes pretreated with pronase. The values of Ki for nimodipine and nifedipine did not change and it fell to about one half for verapamil. Lineweaver-Burk's plots have revealed that the verapamil inhibition in native membranes as well as in pronase pretreated ones was of a non-competitive type; Km for calcium oscillated around 15 mumol.l-1. It is suggested that the inhibition strength depends on the access of inhibitors to the membrane binding sites as well as on the solubility of inhibitors in membrane lipids.     

Na(+)-Ca2+ exchange in the rat brain during ontogeny

A rise of Na(+)-Ca2+ exchange during ontogenic development was found in the rat brain which parallels brain maturation. Nerve endings are the main structure which contributes to the rise of the exchange activity.     

Na+-Ca2+ exchange activity in rabbit lymphocyte plasma membranes

Plasma membranes of rabbit thymus lymphocytes accumulated Ca2+ when a Na+ gradient (intravesicular greater than extravesicular) was formed across the membranes. Dissipation of the Na+ gradient by the addition of Na+ to the external medium decreased Ca2+ uptake. Ca2+ preloaded into the lymphocytes was extruded when Na+ was added to the external medium. The Ca2+ uptake decreased at acidic pH but increased at alkaline pH (above 8) and the activity was saturable for Ca2+ (apparent Km for Ca2+ was 61 microM and apparent Vmax was 11.5 nmol/mg protein per min). Na+-dependent uptake of Ca2+ was inhibited by tetracaine and verapamil, and partially inhibited by La3+. The uptake was not influenced by orthovanadate.     

The modulation of rat brain Na(+)-Ca2+ exchange by K+

The involvement of potassium ions in the Na(+)-Ca2+ exchange process was studied in rat brain synaptic plasma membrane (SPM) vesicles. Addition of equimolar [K+] to the intravesicular and the extravesicular medium led to a stimulation of the Na+ gradient-dependent Ca2+ influx; this stimulation was noticeable already at 0.5 mM and reached its maximum at 2 mM K+. The magnitude of the K+ stimulation was between 1.3-2.5-fold in different SPM preparations. K+ ions also stimulated the Na(+)-dependent Ca2+ efflux. K+ stimulation of Na(+)-Ca2+ exchange is of considerable specificity, since it is not mimicked by either Li+ or H+. The following lines of evidence suggest that K+ modulation of Na(+)-Ca2+ exchange involves the catalytic moiety of the transporter itself and not an unrelated K+ channel which modulates the membrane potential. 1) K+ stimulation of the transport process was conserved following reconstitution of the transporter into phospholipid-rich liposomes, an experimental condition which presumably separates the native membrane proteins among different vesicular structures. 2) K+ stimulation of Na+ gradient-dependent Ca2+ influx persists also when the build up of negative inside membrane potential is prevented by addition of carbonyl cyanide p-trifluoromethoxy phenylhydrazone which renders the membrane highly permeable to protons both in the native and the reconstituted preparation. 3) K+ stimulation of Na+ gradient-dependent Ca2+ influx is obtained also when tetraethylammonium chloride, 2,3-diaminopyridine and Cs+ are added to the Ca2+ uptake medium. Reconstituted SPM vesicles take up 86Rb+ in response to activation of Na+ gradient-dependent Ca2+ influx. The ratio of Ca2+ taken up by SPM vesicles in a Na+ gradient-dependent manner to the corresponding amounts of Rb+ taken up varies between 8-5 in different SPM preparations. If the stoichiometry of the process is 1 Rb+/1 Ca2+, then Rb+ cotransport is mediated by 10-20% of the transporters present in the preparation.     

Na+-Ca2+ exchange in plasma membranes of crayfish striated muscle

Na+-Ca2+ exchange rates and some physico-chemical properties of the exchanger were studied in crayfish striated muscle membranes enriched in plasma membranes prepared by differential centrifugation of muscle microsomal fraction on discontinuous sucrose density gradient. The lightest subfraction with the highest Na+, K+-ATPase and Mg2+-ATPase activities also showed the highest Na+-Ca2+ exchange rates. A number of physico-chemical characteristics of the Na+-Ca2+ exchanger found in the present experiments were similar to those reported for excitable membranes of mammals, except for the temperature optimum (20 degrees C for the crayfish).     

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.     

Tricyclic antidepressants inhibit voltage-dependent calcium channels and Na(+)-Ca2+ exchange in rat brain cortex synaptosomes

We have used a resting (5 mM K+) or depolarizing (60 mM K+) choline-based medium, and a nondepolarizing sodium-based or choline-based medium, to characterize the inhibitory potential of tricyclic antidepressants against the voltage-dependent calcium channels or the Na(+)-Ca2+ exchange process, respectively, in synaptosomes from rat brain cortex. Imipramine, desipramine, amitriptyline, and clomipramine inhibited net K(+)-induced 45Ca uptake with similar IC50 values (26-31 microM), and this uptake was also inhibited by diltiazem with an IC50 of 36 microM; these results indicate an inhibition of voltage-dependent calcium channels by tricyclic antidepressants. The net uptake of 45Ca induced by Na(+)-Ca2+ exchange was also inhibited by the four tricyclic antidepressants tested, but not by diltiazem; imipramine (IC50 = 94 microM) was a more potent inhibitor of this process than desipramine (IC50 = 151 microM), and the IC50 values of amitriptyline (107 microM) and clomipramine (97 microM) were similar to that of imipramine. Some degree (approximately 25%) of brain calcium channel blockade could be present at the steady-state concentrations of tricyclic antidepressants expected to occur therapeutic use of these compounds to treat depression or panic disorder.     

Na(+)-Ca2+ exchange in locust striated muscles

High Na+ + Ca2+ exchange rates comparable with those reported for crayfish striated muscle, rat heart and rat brain, were observed in locust striated muscle homogenates and membrane preparations. The Na(+)-Ca2+ exchange followed the 1st order kinetics with a Km value of 18 mumol.l-1 for Ca, the pH optimum was at 8, the temperature optimum at 30 degrees C, and the exchange was inhibited in the presence of sodium in the incubation medium, with a KiNa of approx. 25 mmol.l-1. The present results suggest a high Na(+)-Ca2+ exchange in locust striated muscles which operate on the calcium electrogenesis principle.     

Purification of the cardiac Na+-Ca2+ exchange protein

We have used fractionation procedures to enrich solubilized cardiac sarcolemma in the Na+-Ca2+ exchange protein. Sarcolemma is extracted with an alkaline medium to remove peripheral proteins and is then solubilized with decylmaltoside. Next, the exchanger is applied to DEAE-Sepharose and eluted with high salt. The DEAE fraction is applied to WGA-agarose, and a small fraction of protein, enriched in the exchanger, can be eluted by changing the detergent to Triton X-100. This fraction is reconstituted into asolectin proteoliposomes for measurement of Na+-Ca2+ exchange activity and gel electrophoresis. The purified fraction has a Na+-Ca2+ exchange activity of 600 nmol Ca2+/mg of protein per s at 10 microM Ca2+ and a purification factor of about 30 as compared with control reconstituted sarcolemmal vesicles. Ca2+-Ca2+ exchange and Na+-Ca2+ exchange activities were both present in the same final reconstituted vesicles indicating that the same protein is responsible for both transport activities. SDS-PAGE reveals two prominent protein bands at 70 and 120 kDa. After mild chymotrypsin treatment (1 microgram/ml), there is no loss of exchange activity, but the 120 kDa band disappears and the 70 kDa band becomes more dense. This suggests that the 70 kDa band is due to an active proteolytic fragment of the 120 kDa protein. Under non-reducing gel conditions, only a single protein band is seen with an apparent molecular weight of 160 kDa. Antibodies to the purified exchanger preparation are able to immunoprecipitate exchange activity and confirm that the 70 kDa protein derives from the 120 kDa protein. We propose that both the 70 and 120 kDa proteins are associated with the Na+-Ca2+ exchanger.     

Na+-Ca2+ exchange activity in rat skeletal myotubes: effect of lithium ions

The whole-cell patch-clamp technique coupled with intracellular [Ca2+] measurements was used to investigate the sodium-calcium exchange mechanism in rat skeletal muscle cells in primary culture. Replacing external Na+ ions with Li+ or N-methyl-D-glucamine (NMDG+) ions generated outward currents which were correlated with significant increases of free cytosolic-calcium concentration. These results strongly argue for a functional Na+-Ca2+ exchange mechanism working in its reverse mode. Moreover, the outward currents were sensitive to the new compound KB-R7943 (10 microM), which has been shown to be a potent inhibitor of the sodium-calcium exchanger. Outward Na+-Ca2+ exchange current densities were reduced in the presence of external Li+ as compared to those measured in the presence of NMDG+. After replacing internal sodium by lithium ions, rapid changes of external lithium concentrations generated sarcolemmal currents which were accompanied by subsequent variations of intracellular calcium activity. The currents were dependent on extracellular Li+ with a half-maximal activation at 67 mM and a Hill coefficient of 2.9. This work shows that the Na+-Ca2+ exchanger is able to significantly influence the myoplasmic calcium concentration of cultured rat myotubes. On the other hand, our results suggest that Li+ ions may substitute Na+ ions to catalyse an electrogenic Li+/Ca2+ counter transport.     

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.     

Up-regulation of Na(+)-Ca2+ exchange activity by interferon-gamma in cultured rat microglia

The Na(+)-Ca2+ exchanger (NCX) plays a role in regulating intracellular Ca2+ concentration, but little is known about NCX in microglia. We examined mRNA expression of NCX isoforms in cultured rat microglia and the effect of interferon-gamma (IFN-gamma) on NCX activity. RT-PCR showed that all of the known NCX isoforms, NCX1-3, are expressed in cultured microglia. Ouabain and monensin increased 45Ca2+ uptake and intracellular Ca2+ concentration in microglia, suggesting the presence of NCX activity in the reverse mode. Treatment with IFN-gamma (100 U/mL) caused a biphasic increase in NCX activity. The transient increase in NCX activity by IFN-gamma for 1 h was blocked by the protein kinase C (PKC) inhibitors, staurosporine and GF109203X, and the tyrosine kinase inhibitor, herbimycin A. The delayed increase in NCX activity by IFN-gamma for 24 h was blocked by the protein synthesis inhibitor cycloheximide and actinomycin D. Treatment with IFN-gamma for 24 h increased NCX mRNA and protein levels. The increase in NCX activity and NCX protein by IFN-gamma for 24 h was blocked by staurosporine, GF109203X, herbimycin A and the extracellular signal-regulated kinase inhibitor, PD98059. These findings suggest that NCX is up-regulated by IFN-gamma in a biphasic manner in microglia. Moreover, PKC and tyrosine kinase are involved in the up-regulation of NCX and the extracellular signal-regulated protein kinase is also involved in the delayed increase in NCX activity.     

Stimulation of Na+-Ca2+ exchange in heart sarcolemma by insulin

Insulin was found to stimulate Na+-dependent Ca2+ uptake in dog heart sarcolemma in a concentration dependent manner (0.001 to 1 milliunits/ml). Maximal stimulation (160 to 170%) was seen at 0.1 to 1 milliunits/ml of insulin. Unlike Na+-dependent Ca2+ uptake, ATP-dependent Ca2+ uptake was unaltered by 1 microunit/ml of insulin. However, high concentrations of insulin (0.01 to 1 milliunits/ml) significantly increased the ATP-dependent Ca2+ uptake activity of heart sarcolemma; maximal increase (60%) was observed at 1 milliunit/ml of insulin. The Na+ K+-ATPase activity did not change upon incubating sarcolemma with insulin. The membrane preparation exhibited specific insulin binding characteristics. The Scatchard plot analysis of the data indicated two binding sites for insulin; the association constants for the high and low affinity sites were 2 X 10(9) M-1 and 4.4 X 10(8) M-1, respectively. These results support the view regarding the presence of insulin receptors in the heart cell membrane and indicate a dramatic effect of insulin on the sarcolemmal Ca2+ transport systems.     

Effects of diabetes and hypertension on myocardial Na+-Ca2+ exchange

Abnormalities in cardiac function have been extensively documented in experimental and clinical diabetes. These aberrations are well known to be exaggerated when hypertension and diabetes co-exist. The objective of the present study was to examine whether alterations in the activity of the myocardial Na+-Ca2+ exchanger (NCX) can account for the deleterious effects of diabetes and (or) hypertension on the heart. To this aim, the following experimental groups were studied: (i) control; (ii) diabetic; (iii) hypertensive; and (iv) hypertensive-diabetic. Wistar rats served as the control group (C) while Wistar rats injected with streptozotocin (STZ, 55 mg/kg) served as the diabetic (D) group. Spontaneously hypertensive (SH) rats were used as the hypertensive group (H) while SH rats injected with STZ served as the hypertensive-diabetic (HD) group. Sarcolemma was isolated from the ventricles of the C, D, H, and HD groups and NCX activity was examined using rapid quenching techniques to study initial rates over a [Ca2+]o range of 10-160 microM. The Vmax of NCX was lower in the D group when compared with the C group (D, 2.96 +/- 0.26 vs. C, 4.0 +/- 0.46 nmol x mgprot(-1) x s(-1), P < 0.05), however combined diabetes and hypertension (HD) did not affect the Vmax of NCX activity (HD, 3.84 +/- 0.88 vs. H, 3.59 +/- 0.24 nmol x mgprot(-1) x s(-1), P > 0.05). However, analysis of the Km values for Ca2+ indicated that both the D and HD groups exhibited a significantly lower Km when compared with their respective control groups (D, 42 +/- 4 vs. C, 56 +/- 4 microM, P < 0.05; HD, 33 +/- 7 vs. H, 51 +/- 8 microM, P < 0.05). Immunoblotting using polyclonal antibodies (against canine cardiac NCX) exhibited the typical banding of 160, 120, and 70 kDa. The 120 kDa band is believed to represent the native exchanger with its post-translational modifications. Examination of the blots revealed a lower intensity of the 120 kDa band in the D group when compared with the C group, however, no significant difference in the HD group was observed. We speculate that the lower Vmax in the D group may be due to a reduced concentration of exchanger protein in the membrane. The absence of this defect in the HD group may be a result of compensatory mechanisms to the overall hemodynamic overload, however, this remains to be determined. The increased affinity for Ca2+ in both the D and HD groups (determined by the lower Km values) is an interesting finding and may be due to changes in sarcolemmal lipid bilayer composition secondary to diabetes-induced hyperlipidemia.     

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.