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.     

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.     

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.     

Evidence for Mg2, ATP-dependent accumulation of Ca2+ by the intracellular non-mitochondrial calcium store in uterine smooth muscle cells

Using carbachol contracture as the experimental model for testing the properties of the intracellular calcium store in intact tissue and 45Ca2+ accumulation in the chemically skinned by digitonin smooth muscle cells isolated from oestrogen-dominated rat uterus the evidence for the presence of Mg2+, ATP-dependent Ca2+ pump in the non-mitochondrial store has been found which is supposed to play a key role in the process of refilling' of the store on the cytoplasmic level. The experiments performed on intact muscle showed that the functional activity of the carbachol-releasable Ca2+ store is critically dependent on Ca2+ entry. It is found that Ca2+ entry via voltage operated Ca2+ channels or on the Na(+)-Ca2+ exchange was needed to refill the store in this tissue. However, when Ca2+ extrusion systems located in the plasma membrane were inhibited by La3+, the store retained its ability to discharge and reaccumulate Ca2+ released on the regular basis suggesting the presence of the energy-dependent Ca2+ accumulating system in the store. The process of the store refilling was totally inhibited by cyclopiazonic acid. Chemically skinned uterine smooth muscle cells demonstrated the presence of Mg2+, ATP-dependent accumulation of Ca2+ in the non-mitochondrial (ruthenium red insensitive) intracellular store(s) potentiated by Ca(2+)-precipitating anions (potassium oxalate and phosphate), in a time- and concentration dependent way which was inhibited by Ca(2+)-ionophore A 23187 (5 microM) and cyclopiazonic acid with Ki = 0.4 microM. It is suggested that in the uterine smooth muscle of the oestrogen-dominated rats, nonmitochondrial receptor-operated intracellular calcium store is represented by endoplasmic reticulum.     

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.     

A novel antagonist, No. 7943, of the Na+/Ca2+ exchange current in guinea-pig cardiac ventricular cells

1. The effects of No. 7943 on the Na+/Ca2+ exchange current and on other membrane currents were investigated in single cardiac ventricular cells of guinea-pig with the whole-cell voltage-clamp technique. 2. No. 7943 at 0.1-10 microM suppressed the outward Na+/Ca2+ exchange current in a concentration-dependent manner. The suppression was reversible and the IC50 value was approximately 0.32 microM. 3. No. 7943 at 5-50 microM suppressed also the inward Na+/Ca2+ exchange current in a concentration-dependent manner but with a higher IC50 value of approximately 17 microM. 4. In a concentration-response curve, No. 7943 raised the K(m)Ca2+ value, but did not affect the Imax value, indicating that No. 7943 is a competitive antagonist with external Ca2+ for the outward Na+/ Ca2+ exchange current. 5. The voltage-gated Na+ current, Ca2+ current and the inward rectifier K+ current were also inhibited by No. 7943 with IC50S of approximately 14, 8 and 7 microM, respectively. 6. In contrast to No. 7943, 3', 4'-dichlorobenzamil (DCB) at 3-30 microM suppressed the inward Na+/Ca2+ exchange current with IC50 of 17 microM, but did not affect the outward exchange current at these concentrations. 7. We conclude that No. 7943 inhibits the outward Na+/Ca2+ exchange current more potently than any other currents as a competitive inhibitor with external Ca2+. This effect is in contrast to DCB which preferentially inhibits the inward rather than the outward Na+/Ca2+ exchange current.     

Heat shock proteins come of age: primitive functions acquire new roles in an adaptive world

We measured [Ca2+]i and [Na+]i in isolated transgenic (TG) mouse myocytes overexpressing the Na+-Ca2+ exchanger and in wild-type (WT) myocytes. In TG myocytes, the peak systolic level and amplitude of electrically stimulated (ES) [Ca2+]i transients (0.25 Hz) were not significantly different from those in WT myocytes, but the time to peak [Ca2+]i was significantly prolonged. The decline of ES [Ca2+]i transients was significantly accelerated in TG myocytes. The decline of a long-duration (4-s) caffeine-induced [Ca2+]i transient was markedly faster in TG myocytes, and [Na+]i was identical in TG and WT myocytes, indicating that the overexpressed Na+-Ca2+ exchanger is functionally active. The decline of a short-duration (100-ms) caffeine-induced [Ca2+]i transient in 0 Na+/0 Ca2+ solution did not differ between the two groups, suggesting that the sarcoplasmic reticulum (SR) Ca2+-ATPase function is not altered by overexpression of the Na+-Ca2+ exchanger. There was no difference in L-type Ca2+ current density in WT and TG myocytes. However, the sensitivity of ES [Ca2+]i transients to nifedipine was reduced in TG myocytes. This maintenance of [Ca2+]i transients in nifedipine was inhibited by Ni2+ and required SR Ca2+ content, consistent with enhanced Ca2+ influx by reverse Na+-Ca2+ exchange, and the resulting Ca2+-induced Ca2+ release from SR. The rate of rise of [Ca2+]i transients in nifedipine in TG myocytes was much slower than when both the L-type Ca2+ current and the Na+-Ca2+ exchange current function together. In TG myocytes, action potential amplitude and action potential duration at 50% repolarization were reduced, and action potential duration at 90% repolarization was increased, relative to WT myocytes. These data suggest that under these conditions, overexpression of the Na+-Ca2+ exchanger in TG myocytes accelerates the decline of [Ca2+]i during relaxation, indicating enhanced forward Na+-Ca2+ exchanger function. Increased Ca2+ influx also appears to occur, consistent with enhanced reverse function. These findings provide support for the physiological importance of both these modes of Na+-Ca2+ exchange.     

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.     

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.     

An effect of voltage on binding of Na+ at the cytoplasmic surface of the Na(+)-K+ pump

This work utilizes proteoliposomes reconstituted with renal Na(+)-K(+)-ATPase to study effects of electrical potential (40-80 mV) on activation of pump-mediated fluxes of Na+ or Rb+ (K+) ions and on inhibitory effects of Rb+ ions or organic cations. The latter include guanidinium derivatives that are competitive Na(+)-like antagonists (David, P., Mayan, H., Cohen, H., Tal, D. M., and Karlish, S.J.D. (1992) J. Biol. Chem. 267, 1141-1149). Cytoplasmic side-positive diffusion potentials significantly decreased the K0.5 of Na+ at the cytoplasmic surface for activation of ATP-dependent Na(+)-K+ exchange but did not affect the inhibitory potency of Rb+ (K+) or any Na(+)-like antagonist. Diffusion potentials did not affect activation of Rb(+)-Rb+ exchange by Rb+ ions at the cytoplasmic surface and had only a minor effect on Rb+ activation at the extracellular surface. Previously, we proposed that the cation binding domain consists of two negatively charged sites, to which two K+ or two Na+ ions bind, and one neutral site for the third Na+ (Glynn, I. M., and Karlish, S.J.D. (1990) Annu. Rev. Biochem. 59, 171-205). The present experiments suggest that binding of a Na+ ion in the neutral site at the cytoplasmic surface is sensitive to voltage. By contrast, binding of Rb+ ions at the extracellular surface of renal pumps appears to be only weakly or insignificantly affected by voltage. Inferences on the identity of the charge-carrying steps, based on experiments using proteoliposomes, are discussed in relation to recent evidence that dissociation of Na+ or association of K+ ions, at the extracellular surface, represent the major charge-carrying steps.     

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.     

Membrane potential regulates inositol 1,4,5-trisphosphate-controlled cytoplasmic Ca2+ oscillations in pituitary gonadotrophs

The influence of membrane potential (Vm) on cytoplasmic calcium ([Ca2+]i) oscillations during the sustained extracellular Ca(2+)-dependent phase of the Ca2+ signaling response to gonadotropin-releasing hormone (GnRH) was analyzed in cultured pituitary gonadotrophs. In agonist- and inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3)-stimulated cells, sustained [Ca2+]i oscillations were extinguished by hyperpolarization after 3-15 min despite the availability of Ca2+ in the extracellular medium. Single depolarizing pulses transiently restored the amplitude of the sustained spiking in a dihydropyridine- and extracellular Ca(2+)-sensitive manner. The responses to depolarization showed a marked dependence on Vm that was correlated with the steady-state inward Ca2+ current. In addition, repetitive application of brief depolarizing pulses modulated the frequency of agonist- and Ins(1,4,5)P3-controlled spiking; depolarization pulses at frequencies lower than the intrinsic rate of episodic Ca2+ release triggered large transients between the autonomous spikes, whereas higher frequencies of depolarizing pulses overcame the original Ca2+ spiking frequency. These extrinsically driven and extracellular Ca(2+)-dependent oscillations were sensitive to the Ca(2+)-ATPase blocker, thapsigargin, but not to ryanodine. On the other hand, spontaneous firing and application of depolarizing pulses to nonstimulated cells failed to induce thapsigargin-sensitive oscillations. These findings demonstrate that the pattern of Ca2+ signaling in gonadotrophs does not depend exclusively on the Ins(1,4,5)P3 concentration, but also on the excitable status of the cell. Such modulation of the Ins(1,4,5)P3-controlled Ca2+ signaling system by changes in Vm could provide a mechanism for the integration of multiple inputs that utilize diverse signal transduction pathways.     

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.     

Effects of extracellular calcium on electrical bursting and intracellular and luminal calcium oscillations in insulin secreting pancreatic beta-cells

The extracellular calcium concentration has interesting effects on bursting of pancreatic beta-cells. The mechanism underlying the extracellular Ca2+ effect is not well understood. By incorporating a low-threshold transient inward current to the store-operated bursting model of Chay, this paper elucidates the role of the extracellular Ca2+ concentration in influencing electrical activity, intracellular Ca2+ concentration, and the luminal Ca2+ concentration in the intracellular Ca2+ store. The possibility that this inward current is a carbachol-sensitive and TTX-insensitive Na+ current discovered by others is discussed. In addition, this paper explains how these three variables respond when various pharmacological agents are applied to the store-operated model.     

Ischemia/reperfusion-induced arrhythmias in anaesthetized rats: a role of Na+ and Ca2+ influx

We hypothesized that by limiting the Na+ and Ca2+ loading by a blocker/inhibitor of the Na+ channel (lidocaine), Na+ overload (R56865: N-[1-[4-(4-fluorophenoxy)butyl]-4-piperidinyl]-N-methyl-2-benzothiazo lamine), Ca2+ channel (verapamil), Na+ -H+ exchange (ethylisobutyl amiloride) or of Na+ -Ca2+ exchange (No. 7943: 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate), it should be possible to reduce ischemia/reperfusion-induced arrhythmias. To test this hypothesis, we used anaesthetized rats subjected to 5 min of coronary artery occlusion followed by 10 min of reperfusion to study antiarrhythmic effects of above compounds on reperfusion-induced ventricular premature beats, ventricular tachycardia, and reversible and irreversible ventricular fibrillation. Compound or saline was administered as an intravenous bolus injection at 5 min before ischemia. Pretreatment with lidocaine (5 mg/kg), verapamil (0.63 mg/kg), R56865 (0.63 mg/kg) or ethylisobutyl amiloride (1.25 mg/kg) significantly reduced or abolished all types of ventricular arrhythmias. However, pretreatment with verapamil was associated with second or third degree heart block in 3 out of 12 animals. Pretreatment with No. 7943 did not significantly influence the ischemia/reperfusion-induced ventricular arrhythmias. The present results suggest that both intracellular Na+ -and Ca2+ -loading play important roles in reperfusion-induced ventricular arrhythmias and the inhibition of Na+ -Ca2+ exchange to limit Ca2+ loading probably does not play any important role in ischemia/reperfusion-induced arrhythmias in anaesthetized rats.     

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.     

Subcellular distribution of ankyrin in developing rabbit heart--relationship to the Na+-Ca2+ exchanger

Ankyrins are a multigene family of proteins that function as adapters between the cytoskeleton and trans-membrane proteins, such as ion channels. Previous studies have shown the linkage between ankyrin and ionic transport proteins such as Na+-K+ ATPase, voltage-dependent Na+ channels and Ca2+ channels. In the present study, we have investigated the subcellular distribution of ankyrin and its relationship to the Na+-Ca2+ exchange protein in immature and adult rabbit ventricular myocytes. Isolated single cardiomyocytes from neonatal, juvenile and adult rabbit hearts were examined by immunofluorescence labeling techniques, using antibodies against ankyrin and the Na+-Ca2+ exchanger. We found that in neonatal rabbit cardiac myocytes, ankyrin labeling was mainly present at the Z disk, whereas the Na+-Ca2+ exchanger was only present on the peripheral sarcolemma. At 2 weeks of age, ankyrin labeling was still predominantly observed at the level of the Z disks as well as in the partially developed T-tubules. In the adult cells, however, ankyrin and the Na+-Ca2+ exchanger seem to be co-localized within T-tubules and at the costamere region of the peripheral sarcolemma. Immunogold labeling studies at the higher resolution electron microscopic level using cyrosection tissues of rabbit heart at different ages confirm these findings. These results indicate that the distribution pattern of ankyrin and the Na+-Ca2+ exchanger changes with development in rabbit ventricular myocytes.     

Warm cells revealed by microfluorimetry of Ca2+ in cultured dorsal root ganglion neurons

Warm cells were identified by Fura-PE3-based microfluorimetry of Ca2+ in cultured dorsal root ganglion (DRG) neurons. In response to a physiologically relevant stimulus temperature (43 degrees C), a subpopulation of small DRG neurons from new born rats increased the intracellular Ca2+ concentration ([Ca2+]i). Seven percent of the cells responded to the warm stimulus. The stimulus evoked elevation in [Ca2+]i from 52.5 +/- 9.5 nM (mean +/- S.D., n = 18) to 171.0 +/- 15.6 nM in cells between 15 and 25 microns in diameter. The depletion of extracellular Ca2+ diminished the Ca2+ elevation. The Na(+)-free condition also diminished the response. We concluded that the heat stimulation opens nonselective cation channels in putative warm cells from DRG neurons.     

Kinetics of Ca2+ release evoked by photolysis of caged InsP3 in rat submandibular cells

Quantitative time-resolved measurements of cytosolic Ca2+ release by photolysis of caged InsP3 have been made in single rat submandibular cells using patch clamp whole-cell recording to measure the Ca2+-activated Cl- and K+ currents. Photolytic release of InsP3 from caged InsP3 at 100 Joules caused transient inward (V(H) = 60 mV) and outward (V(H) = 0 mV) currents, which were nearly symmetric in their time course. The inward current was reduced when pipette Cl- concentration was decreased, and the outward current was suppressed by K+ channel blockers, indicating that they were carried by Cl- and K+, respectively. Intracellular pre-loading of the InsP3 receptor antagonist heparin or the Ca2+ chelator EGTA clearly prevented both inward and outward currents, indicating that activation of Ca2+-dependent Cl- and K+ currents underlies the inward and the outward currents. At low flash intensities, InsP3 caused Ca2+ release which normally activated the K+ and Cl- currents in a mono-transient manner. At higher intensities, however, InsP3 induced an additional delayed outward K+ current (I[K,(delay)]). I[K(delay)] was independent of the initial K+ current, independent of extracellular Ca2+, inhibited by TEA, and gradually prolongated by repeated flashes. The photolytic release of Ca2+ from caged Ca2+ did not mimic the I[K(delay)]. It is suggested that Ca2+ releases from the InsP3-sensitive pools in an InsP3 concentration-dependent manner. Low concentrations of InsP3 induce the transient Ca2+-dependent Cl- and K+ currents, which reflects the local Ca2+ release, whereas high concentrations of InsP3 induce a delayed Ca2+-dependent K+ current, which may reflect the Ca2+ wave propagation.     

Effect of H+ extracellular and intracellular buffering capacity on the activity Ca2+/H+ exchange in the lymphocyte plasma membrane

Formation of delta pH is registered when studying Ca2+ passive transport through lymphocytes' plasma membrane (PM). The pHi values strongly depended on pH0. Changes of pH0 lead to unidirectional changes of pHi and affect Ca2+ concentration in cytoplasm of the intact cells. The presence of Ca(2+)-channels antagonists does not affect this phenomenon. Ca2+/H+ exchange is supposed to exist in PM. It is also of great interest that cytoplasmic Ca2+ and H+ activities are some equal in physiological range. Besides, H(+)-buffering as well as Ca(2+)-buffering systems are present in the cell and have their maximal capacity about 7.2 in the intact cells. The spectrofluorimetric study of internal lymphocytes' H(+)-buffering capacity with titration technique using weak base, acid or other buffer addition has demonstrated maximal value of 9.0-1.1 mM depending on the substance to be added.