A new topological model of the cardiac sarcolemmal Na+-Ca2+ exchanger

The current topological model of the Na+-Ca2+ exchanger consists of 11 transmembrane segments with extracellular loops a, c, e, g, i, and k and cytoplasmic loops b, d, f, h, and j. Cytoplasmic loop f, which plays a role in regulating the exchanger, is large and separates the first five from the last six transmembrane segments. We have tested this topological model by mutating residues near putative transmembrane segments to cysteine and then examining the effects of intracellular and extracellular applications of sulfhydryl-modifying reagents on exchanger activity. To aid in our topological studies, we also constructed a cysteineless Na+-Ca2+ exchanger. This mutant is fully functional in Na+ gradient-dependent 45Ca2+ uptake measurements and displays wild-type regulatory properties. It is concluded that the 15 endogenous cysteine residues are not essential for either activity or regulation of the exchanger. Our data support the current model by placing loops c and e at the extracellular surface and loops d, j, and l at the intracellular surface. However, the data also support placing Ser-788 of loop h at the extracellular surface and Gly-837 of loop i at the intracellular surface. To account for these data, we propose a revision of the model that places transmembrane segment 6 in cytoplasmic loop f. Additionally, we propose that putative transmembrane segment 9 does not span the membrane, but may form a "P-loop"-like structure.     

Interaction of the Na(+)-Ca2+ exchanger with small molecules on cell Ca2+ signaling

BACKGROUND: Evaluation of outcome after CPR in severe hypothermic patients. DESIGN: Perspective study from October 1995 to April 1996. SETTING: First aid team of Italian Red Cross, Busto Arsizio (Varese), Italy. METHODS: A population of 22 patients in cardiac arrest in which CPR was performed immediately after rescue team's arrival is studied. ECG, core temperature, SpO2 and MAP were monitored whereas vital parameters were present during Basic Life Support. Outcome after CPR was evaluated with GOS scale. RESULTS: It has been observed that severe hypothermia and time of cardiac arrest impact on the clinical outcome after CPR. The high mortality rate after CPR with BLS standard is worsened by a core temperature < or = 33 degrees C. CONCLUSIONS: Severe hypothermia seems to have a dangerous effect upon outcome after cardiopulmonary resuscitation; heating systems for body temperature could prevent this situation improving CPR results.     

A novel molecular determinant for cAMP-dependent regulation of the frog heart Na+-Ca2+ exchanger

Na+-Ca2+ exchanger is one of the major sarcolemmal Ca2+ transporters of cardiac myocytes. In frog ventricular myocytes the exchanger is regulated by isoproterenol via a beta-adrenoreceptor/adenylate-cyclase/cAMPdependent signaling pathway providing a molecular mechanism for the relaxant effect of the hormone. Here, we report on the presence of a novel exon of 27-base pair insertion, which generates a nucleotide binding motif (P-loop) in the frog cardiac Na+-Ca2+ exchanger. To examine the functional role of this motif, we constructed a full-length frog heart Na+-Ca2+ exchanger cDNA (fNCX1a) containing this exon. The functional expression of fNCX1a in oocytes showed characteristic voltage dependence, divalent (Ni2+, Cd2+) inhibition, and sensitivity to cAMP in a manner similar to that of native exchanger in frog myocytes. In oocytes expressing the dog heart NCX1 or the frog mutant (DeltafNCX1a) lacking the 9-amino acid exon, cAMP failed to regulate Na+-dependent Ca2+ uptake. We suggest that this motif is responsible for the observed cAMP-dependent functional differences between the frog and the mammalian hearts.     

Rapid charge translocation by the cardiac Na(+)-Ca2+ exchanger after a Ca2+ concentration jump

The kinetics of Na(+)-Ca2+ exchange current after a cytoplasmic Ca2+ concentration jump (achieved by photolysis of DM-nitrophen) was measured in excised giant membrane patches from guinea pig or rat heart. Increasing the cytoplasmic Ca2+ concentration from 0.5 microM in the presence of 100 mM extracellular Na+ elicits an inward current that rises with a time constant tau 1 < 50 microseconds and decays to a plateau with a time constant tau 2 = 0.65 +/- 0.18 ms (n = 101) at 21 degrees C. These current signals are suppressed by Ni2+ and dichlorobenzamil. No stationary current, but a transient inward current that rises with tau 1 < 50 microseconds and decays with tau 2 = 0.28 +/- 0.06 ms (n = 53, T = 21 degrees C) is observed if the Ca2+ concentration jump is performed under conditions that promote Ca(2+)-Ca2+ exchange (i.e., no extracellular Na+, 5 mM extracellular Ca2+). The transient and stationary inward current is not observed in the absence of extracellular Ca2+ and Na+. The application of alpha-chymotrypsin reveals the influence of the cytoplasmic regulatory Ca2+ binding site on Ca(2+)-Ca2+ and forward Na(+)-Ca2+ exchange and shows that this site regulates both the transient and stationary current. The temperature dependence of the stationary current exhibits an activation energy of 70 kj/mol for temperatures between 21 degrees C and 38 degrees C, and 138 kj/mol between 10 degrees C and 21 degrees C. For the decay time constant an activation energy of 70 kj/mol is observed in the Na(+)-Ca2+ and the Ca(2+)-Ca2+ exchange mode between 13 degrees C and 35 degrees C. The data indicate that partial reactions of the Na(+)-Ca2+ exchanger associated with Ca2+ binding and translocation are very fast at 35 degrees C, with relaxation time constants of about 6700 s-1 in the forward Na(+)-Ca2+ exchange and about 12,500 s-1 in the Ca(2+)-Ca2+ exchange mode and that net negative charge is moved during Ca2+ translocation. According to model calculations, the turnover number, however, has to be at least 2-4 times smaller than the decay rate of the transient current, and Na+ inward translocation appears to be slower than Ca2+ outward movement.     

Signal transduction mechanism for the stimulation of the sarcolemmal Na(+)-Ca2+ exchanger by insulin

The changes of opioid peptide reactivity in seizure activity have been well studied in animals. Increased enkephalin and dynorphin immunoreactivity in the hippocampi of animals are interpreted as the result of seizure induced mossy fibre sprouting. We studied the hippocampi of six patients with a history of long-standing grand mal seizures and six age-matched control patients with no history of epilepsy or neurologic disease, using frozen sections which were immunostained with antibodies against Leu-enkephalin and Met-enkephalin. The staining intensity in the CA3, CA4 and internal molecular layer of the dentate fascia in each case was quantified using optical densitometry image analysis. The CA3 and CA4 of the epileptic hippocampi showed highly significant increase in Leu-enkephalin-like immunoreactivity compared to the controls (P < 0.005) while the inner molecular layer showed only significant increase (P < 0.05). Met-Enkephalin-like immunoreactivity was only significantly increased in CA4 of the epileptic hippocampi (P < 0.05).     

Structure-function analysis of CALX1.1, a Na+-Ca2+ exchanger from Drosophila. Mutagenesis of ionic regulatory sites

Cytoplasmic Na+ and Ca2+ regulate the activity of Na+-Ca2+ exchange proteins, in addition to serving as the transported ions, and protein regions involved in these processes have been identified for the canine cardiac Na+-Ca2+ exchanger, NCX1.1. Although protein regions associated with Na+i- and Ca2+i-dependent regulation are highly conserved among cloned Na+-Ca2+ exchangers, it is unknown whether or not the structure-function relationships characteristic of NCX1.1 apply to any other exchangers. Therefore, we studied structure-function relationships in a Na+-Ca2+ exchanger from Drosophila, CALX1.1, which is unique among characterized members of this family of proteins in that microM levels of Ca2+i inhibit exchange current. Wild-type and mutant CALX1.1 exchangers were expressed in Xenopus oocytes and characterized electrophysiologically using the giant excised patch technique. Mutations within the putative regulatory Ca2+i binding site of CALX1. 1, like corresponding alterations in NCX1.1, led to reduced ability (i.e. D516V and D550I) or inability (i.e. G555P) of Ca2+i to inhibit Na+-Ca2+ exchange activity. Similarly, mutations within the putative XIP region of CALX1.1, as in NCX1.1, led to two distinct phenotypes: acceleration (i.e. K306Q) and elimination (i.e. Delta310-313) of Na+i-dependent inactivation. These results indicate that the respective regulatory roles of the Ca2+i binding site and XIP region are conserved between CALX1.1 and NCX1.1, despite opposite responses to Ca2+i. We extended these findings using chimeric constructs of CALX1.1 and NCX1.1 to determine whether or not functional interconversion of Ca2+i regulatory phenotypes was feasible. With one chimera (i.e. CALX:NCX:CALX), substitution of a 193-amino acid segment, from the large intracellular loop of NCX1.1, for the corresponding 177-amino acid segment of CALX1.1 led to an exchanger that was stimulated by Ca2+i. This result indicates that the regulatory Ca2+i binding site of NCX1.1 retains function in a CALX1. 1 parent transporter and that the substituted segment contains some of the amino acid sequence(s) required for transduction of the Ca2+i binding signal.     

Na+-Ca2+ exchange is affected by membrane potential in cardiac sarcolemmal vesicles

The effect of membrane potential on the Na+-Ca2+ exchange activity of isolated sarcolemmal vesicles from dog ventricles is examined. Na+-Ca2+ exchange is monitored as Nai+-dependent Ca2+ uptake as described by Reeves and Sutko ((1979) Proc. Natl. Acad. Sci. U. S. A. 76,590-594). Membrane potential is controlled by varying internal and external K+ in the presence of valinomycin. Inside-positive potentials stimulate Nai+-dependent Ca2+ influx. This stimulation is independent of Ca2+ concentration. The results indicate that Na+-Ca2+ exchange by itself can generate a substantial potential (approximately -60mV) in the sarcolemmal vesicles. The data are consistent with an electrogenic Na+-Ca2+ exchange mechanism in which three or more Na+ are exchanged for one Ca2+. This electrogenic exchange may have important implications in the control of myocardial tension development.     

Parameters of lymphocyte Na+-Ca2+ regulation and blood pressure: the gender effect

Alterations in cellular Ca2+ and Na+ regulation play a role in the pathogenesis of essential hypertension. Using peripheral lymphocytes from 68 normal persons, we observed the following relationships for major cellular Ca2+ regulatory parameters. Among men and women, Na+-Ca2+ exchanger activity was positively correlated with the resting cytosolic free Ca2+ ([Ca2+]c) (r=0.43, P=0.0003), and the resting [Ca2+]c was positively correlated with cytosolic Na+ ([Na+]c) (r=0.50, P=0.0001). For men only, store-operated Ca2+ entry was positively correlated with Na+-Ca2+ exchanger activity (r=0.63, P=0.0001). In addition, systolic and diastolic blood pressures were positively correlated with [Na+]c in men (r=0.53, P=0.001, and r=0. 41, P=0.017, respectively) but not in women (r=0.30, P=0.088, and r=0.24, P=0.17, respectively). Some of the relationships between cellular and blood pressure parameters were confounded by serum triglycerides. These observations indicate a gender effect on cellular Ca2+-Na+ regulation and its relationship with blood pressure.     

Relationship between Na+-Ca2+-exchanger protein levels and diastolic function of failing human myocardium

BACKGROUND: In the failing human heart, sarcoplasmic reticulum (SR) calcium handling is impaired, and therefore, calcium elimination and diastolic function may depend on the expression of sarcolemmal Na+-Ca2+ exchanger. METHODS AND RESULTS: Force-frequency relations were studied in ventricular muscle strip preparations from failing human hearts (n=29). Protein levels of Na+-Ca2+ exchanger and SR Ca2+-ATPase were measured in the same hearts. Hearts were divided into 3 groups by discriminant analysis according to the behavior of diastolic function when stimulation rate of muscle strips was increased from 30 to 180 min-1. At 180 compared with 30 min-1, diastolic force was increased by 160%, maximum rate of force decline was decreased by 46%, and relaxation time was unchanged in group III. In contrast, in group I, diastolic force and maximum rate of force decline did not change, and relaxation time decreased by 20%. Na+-Ca2+ exchanger was 66% higher in group I than in group III. Na+-Ca2+ exchanger was inversely correlated with the frequency-dependent rise of diastolic force when stimulation rate was increased (r=-0.74; P<0.001). Compared with nonfailing human hearts (n=6), SR Ca2+-ATPase was decreased and Na+-Ca2+ exchanger unchanged in group III, whereas Na+-Ca2+ exchanger was increased and SR Ca2+-ATPase unchanged in group I. Results with group II hearts were between those of group I and group III hearts. CONCLUSIONS: By discriminating failing human hearts according to their diastolic function, we identified different phenotypes. Disturbed diastolic function occurs in hearts with decreased SR Ca2+-ATPase and unchanged Na+-Ca2+ exchanger, whereas increased expression of the Na+-Ca2+ exchanger is associated with preserved diastolic function.     

Subcellular redistribution is involved in acute regulation of the brush border Na+/H+ exchanger isoform 3 in human colon adenocarcinoma cell line Caco-2. Protein kinase C-mediated inhibition of the exchanger

Na+/H+ exchanger isoform 3 (NHE3), an epithelial brush border isoform of the Na+/H+ exchanger gene family, plays an important role in reabsorption of Na+ in the small intestine, the colon, and the kidney. In several cell types, phorbol 12-myristate 13-acetate (PMA) acutely inhibits NHE3 activity by changes in Vmax, but the mechanism of this inhibition is unknown. We investigated the role of subcellular redistribution of NHE3 in the PMA-induced inhibition of endogenous brush border NHE3 in a model human colon adenocarcinoma cell line, Caco-2. Subcellular localization of NHE3 was examined by confocal morphometric analysis complemented with cell surface biotinylation and compared with NHE3 activity evaluated by fluorometric measurement of intracellular pH. PMA inhibited NHE3 activity by 28% (p < 0.01), which was associated with a decrease of the ratio of the brush border/subapical cytoplasmic compartment of NHE3 from approximately 4.3 to approximately 2.4. This translocation resulted in 10-15% of the total cell NHE3 being shifted from the brush border pool to the cytoplasmic pool. These effects were mediated by protein kinase C, since they were blocked by the protein kinase C inhibitor H7. We conclude that inhibition of NHE3 by protein kinase C in Caco-2 cells involves redistribution of the exchanger from brush border into a subapical cytoplasmic compartment, and that this mechanism contributes approximately 50% to the overall protein kinase C-induced inhibition of the exchanger.     

Na+/Ca2+ exchanger modulates kainate-triggered Ca2+ signaling in Bergmann glial cells in situ

The role of sodium-calcium exchanger in calcium homeostasis in Bergmann glial cells in situ was investigated by monitoring cytoplasmic calcium ([Ca2+]i) and sodium ([Na+]i) concentrations. The [Ca2+]i and [Na+]i transients were measured either separately by using fluorescent indicators fura-2 and SBFI, respectively, or simultaneously using the indicators fluo-3 and SBFI. Since the removal of extracellular Na+ induced a relatively small (approximately 50 nM) elevation of [Ca2+]i, the Na+/Ca2+ exchanger seems to play a minor role in regulation of resting [Ca2+]i. In contrast, kainate-triggered [Ca2+]i increase was significantly suppressed by lowering of the extracellular Na+ concentration ([Na+]o). In addition, manipulations with [Na+]o dramatically affected the recovery of the kainate-induced [Ca2+]i transients. Simultaneous recordings of [Ca2+]i and [Na+]i revealed that kainate-evoked [Ca2+]i transients were accompanied with an increase in [Na+]i. Moreover, kainate induced significantly larger [Ca2+]i and smaller [Na+]i transients under current-clamp conditions as compared to those recorded when the membrane voltage was clamped at -70 mV. The above results demonstrate that the Na(+)-Ca2+ exchanger is operative in Bergmann glial cells in situ and is able to modulate dynamically the amplitude and kinetics of [Ca2+]i signals associated with an activation of ionotropic glutamate receptors.     

Topology of a functionally important region of the cardiac Na+/Ca2+ exchanger

The cardiac Na+/Ca2+ exchanger, NCX1, has been modeled to consist of 11 transmembrane segments and a large cytoplasmic loop (loop f). Cysteine mutagenesis and sulfhydryl modification experiments demonstrate that the loop connecting transmembrane segments 1 and 2 (loop b) is located on the cytoplasmic side of the membrane, as previously modeled. A mutation in loop b, asparagine 101 to cysteine (N101C), renders the exchanger insensitive to regulation by cytoplasmic Na+ and Ca2+. Nearby mutations at residue threonine 103 (T103C or T103V) increase the apparent affinity of the exchanger for cytoplasmic Na+ and also produce a significant Li+ transport capacity. The evidence suggests that the region at the interface of cytoplasmic loop b and transmembrane segment 2 is important in Na+ transport and also in secondary regulation. Thus, this region may form part of the link between the ion translocation pathway formed by the transmembrane segments and regulatory sites that have previously been localized to loop f.     

Identification of a mitochondrial Na+/H+ exchanger

The electroneutral exchange of protons for Na+ and K+ across the mitochondrial inner membrane contributes to organellar volume and Ca2+ homeostasis. The molecular nature of these transporters remains unknown. In this report, we characterize a novel gene (YDR456w; renamed NHA2) in Saccharomyces cerevisiae whose deduced protein sequence is homologous to members of the mammalian Na+/H+ exchanger gene family. Fluorescence microscopy showed that a Nha2-green fluorescent protein chimera colocalizes with 4',6-diamidino-2-phenylindole staining of mitochondrial DNA. To assess the function of Nha2, we deleted the NHA2 gene by homologous disruption and found that benzamil-inhibitable, acid-activated 22Na+ uptake into mitochondria was abolished in the mutant strain. It also showed retarded growth on nonfermentable carbon sources and severely reduced survival during the stationary phase of the cell cycle compared with the parental strain, consistent with a defect in aerobic metabolism. Sequence comparisons revealed that Nha2 has highest identity to a putative Na+/H+ exchanger homologue (KIAA0267; renamed NHE6) in humans. Northern blot analysis demonstrated that NHE6 is ubiquitously expressed but is most abundant in mitochondrion-rich tissues such as brain, skeletal muscle, and heart. Fluorescence microscopy showed that a NHE6-green fluorescent protein chimera also accumulates in mitochondria of transfected HeLa cells. These data indicate that NHA2 and NHE6 encode homologous Na+/H+ exchangers and suggest they may be important for mitochondrial function in lower and higher eukaryotes, respectively.     

Relation of exocytotic release of gamma-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the gamma-aminobutyric acid (GABA) carrier, NNC-711, (1-[(2-diphenylmethylene)amino]oxyethyl)- 1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride, blocks the Ca(2+)-independent release of [3H]GABA from rat brain synaptosomes induced by 50 mM K+ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca(2+)-dependent release of [3H]GABA in the total absence of carrier-mediated release. Reversal of the Na+/Ca2+ exchanger was used to increase the intracellular free Ca2+ concentration ([Ca2+]i) to test whether an increase in [Ca2+]i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca2+]i may rise to values above 400 nM, as a result of Na+/Ca2+ exchange, without inducing release of [3H]GABA, but subsequent K+ depolarization immediately induced [3H]GABA release. Thus, a rise of only a few nanomolar Ca2+ in the cytoplasm induced by 50 mM K+ depolarization, after loading the synaptosomes with Ca2+ by Na+/Ca2+ exchange, induced exocytotic [3H]GABA release, whereas the rise in cytoplasmic [Ca2+] caused by reversal of the Na+/Ca2+ exchanger was insufficient to induce exocytosis, although the value for [Ca2+]i attained was higher than that required for exocytosis induced by K+ depolarization. The voltage-dependent Ca2+ entry due to K+ depolarization, after maximal Ca2+ loading of the synaptosomes by Na+/Ca2+ exchange, and the consequent [3H]GABA release could be blocked by 50 microM verapamil. Although preloading the synaptosomes with Ca2+ by Na+/Ca2+ exchange did not cause [3H]GABA release under any conditions studied, the rise in cytoplasmic [Ca2+] due to Na+/Ca2+ exchange increased the sensitivity to external Ca2+ of the exocytotic release of [3H]GABA induced by subsequent K+ depolarization. Thus, our results show that the vesicular release of [3H]GABA is rather insensitive to bulk cytoplasmic [Ca2+] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca2+ entry through Ca2+ channels near the active zones.     

Both Na+-K+ ATPase and Na +-H+ exchanger are immediately active upon post-ischemic reperfusion in isolated rat hearts

Lipases contained in commercial samples of lipase extracts from Rhizopus niveus (RNL) and Candida rugosa (CRL) have been selectively adsorbed on hydrophobic supports at very low ionic strength. Under these conditions, adsorption of other proteins (including some esterases) is almost negligible. More interestingly, these lipases could be separated in several active fractions as a function of a different rate or a different intensity of adsorption on supports activated with different hydrophobic groups (butyl-, phenyl- and octyl-agarose). Thus, although RNL seemed to be a homogeneous sample by SDS-PAGE, it could be separated, via sequential adsorption on the different supports, into three different fractions with very different thermal stability and substrate specificity. For example, one fraction hydrolyzed more rapidly ethyl acetate than ethyl butyrate, while another hydrolyzed the acetate ester 7-fold slower than the butyrate. Similar results were obtained with samples of CRL. Again, we could obtain three different fractions showing very different properties. For example, enantioselectivity for the hydrolysis of (R,S) 2-hydroxy-4-phenylbutanoic acid ethyl ester ranged from 1.2 to 12 for different CRL fractions. It seems that very slight structural differences may promote a quite different interfacial adsorption of lipases on hydrophobic supports as well as a quite different catalytic behavior. In this way, this new 'interfacial affinity chromatography' seems to be very suitable for an easy separation of such slightly different lipase forms.     

Immunohistochemical localization of Na+/Ca2+ exchanger in human retina and retinal pigment epithelium

OBJECTIVE: Validation a self-administered form used by patients to record their food intake and compare the recorded data with the observed intake. DESIGN: Data were obtained from an unselected cross-sectional group of hospitalized patients. SUBJECTS: Forty-five adult men and women volunteered to participate. Five of these dropped out. METHODS: Observed intake at breakfast, lunch and dinner was obtained by recording the servings of food before they were served to the patients and subtracting weighed leftovers. At meal times the patients recorded food items eaten in fractions of amount served to the nearest 25%. SETTING: Inpatients from five different wards at Rikshospitalet, Oslo. RESULTS: There was a significant under-reporting of the number of foods served (P < 0.005) resulting in a significant underestimation of energy 231 kJ (P < 0.02). There was good agreement between the patients and the observers for the portions of most foods (Kappa 0.44-0.92, P < 0.00001). The differences in amount had little influence on the difference in total energy. The difference in number of foods correlated with the difference in energy (r = 0.68, P < 0.001) and with the difference in protein (r = 0.50, P < 0.01). Patients with an underestimation of energy above 20% had forgotten seven or more food items. CONCLUSIONS: For most patients, the self-administered form adapted to the hospital menu appears to have acceptable validity, but for some patients it was unacceptable, mainly owing to food items being omitted and not because of incorrect estimate of amounts of food.     

Na(+)-Ca2+ exchange currents in cortical neurons: concomitant forward and reverse operation and effect of glutamate

Na(+)-Ca2+ exchanger-associated membrane currents were studied in cultured murine neocortical neurons, using whole-cell recording combined with intracellular perfusion. A net inward current specifically associated with forward (Na+(o)-Ca2+(i)) exchange was evoked at -40 mV by switching external 140 mM Li+ to 140 mM Na+. The voltage dependence of this current was consistent with that predicted for 3Na+:1Ca2+ exchange. As expected, the current depended on internal Ca2+, and could be blocked by intracellular application of the exchanger inhibitory peptide, XIP. Raising internal Na+ from 3 to 20 mM or switching the external solution from 140 mM Li+ to 30 mM Na+ activated outward currents, consistent with reverse (Na+(i)-Ca2+(o)) exchange. An external Ca2(+)-sensitive current was also identified as associated with reverse Na(+)-Ca2+ exchange based on its internal Na+ dependence and sensitivity to XIP. Combined application of external Na+ and Ca2+ in the absence of internal Na+ triggered a 3.3-fold larger inward current than the current activated in the presence of 3 mM internal Na+, raising the intriguing possibility that Na(+)-Ca2+ exchangers might concurrently operate in both the forward and the reverse direction, perhaps in different subcellular locations. With this idea in mind, we examined the effect of excitotoxic glutamate receptor activation on exchanger operation. After 3-5 min of exposure to 100-200 microM glutamate, the forward exchanger current was significantly increased even when external Na+ was reduced to 100 mM, and the external Ca2(+)-activated reverse exchanger current was eliminated.     

Catecholamine secretion from bovine adrenal chromaffin cells: the role of the Na+/Ca2+ exchanger and the intracellular Ca2+ pool

The role of the Na+/Ca2+ exchanger and intracellular nonmitochondrial Ca2+ pool in the regulation of cytosolic free calcium concentration ([Ca2+]i) during catecholamine secretion was investigated. Catecholamine secretion and [Ca2+]i were simultaneously monitored in a single chromaffin cell. After high-K+ stimulation, control cells and cells in which the Na+/Ca2+ exchange activity was inhibited showed similar rates of [Ca2+]i elevation. However, the recovery of [Ca2+]i to resting levels was slower in the inhibited cells. Inhibition of the exchanger increased the total catecholamine secretion by prolonging the secretion. Inhibition of the Ca2+ pump of the intracellular Ca2+ pool with thapsigargin caused a significant delay in the recovery of [Ca2+]i and greatly enhanced the secretory events. These data suggest that both the Na+/Ca2+ exchanger and the thapsigargin-sensitive Ca2+ pool are important in the regulation of [Ca2+]i and, by modulating the time course of secretion, are important in determining the extent of secretion.