Cerebral microvascular nNOS responds to lowered oxygen tension through a bumetanide-sensitive cotransporter and sodium-calcium exchanger

Na(+) cotransporters have a substantial role in neuronal damage during brain hypoxia. We proposed these cotransporters have beneficial roles in oxygen-sensing mechanisms that increase periarteriolar nitric oxide (NO) concentration ([NO]) during mild to moderate oxygen deprivation. Our prior studies have shown that cerebral neuronal NO synthase (nNOS) is essential for [NO] responses to decreased oxygen tension and that endothelial NO synthase (eNOS) is of little consequence. In this study, we explored the mechanisms of three specific cotransporters known to play a role in the hypoxic state: KB-R7943 for blockade of the Na(+)/Ca(2+) exchanger, bumetanide for the Na(+)-K(+)-2Cl(-) cotransporter, and amiloride for Na(+)/H(+) cotransporters. In vivo measurements of arteriolar diameter and [NO] at normal and locally reduced oxygen tension in the rat parietal cortex provided the functional analysis. As previously found for intestinal arterioles, bumetanide-sensitive cotransporters are primarily responsible for sensing reduced oxygen because the increased [NO] and dilation were suppressed. The Na(+)/Ca(2+) exchanger facilitated increased NO formation because blockade also suppressed [NO] and dilatory responses to decreased oxygen. Amiloride-sensitive Na(+)/H(+) cotransporters did not significantly contribute to the microvascular regulation. To confirm that nNOS rather than eNOS was primarily responsible for NO generation, eNOS was suppressed with the fusion protein cavtratin for the caveolae domain of eNOS. Although the resting [NO] decreased and arterioles constricted as eNOS was suppressed, most of the increased NO and dilatory response to oxygen were preserved because nNOS was functional. Therefore, nNOS activation secondary to Na(+)-K(+)-2Cl(-) cotransporter and Na(+)/Ca(2+) exchanger functions are key to cerebral vascular oxygen responses.     

Modification of sarcolemmal Na+-K+-ATPase and Na+/Ca2+ exchanger expression in heart failure by blockade of renin-angiotensin system

The activities of both sarcolemmal (SL) Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchanger, which maintain the intracellular cation homeostasis, have been shown to be depressed in heart failure due to myocardial infarction (MI). Because the renin-angiotensin system (RAS) is activated in heart failure, this study tested the hypothesis that attenuation of cardiac SL changes in congestive heart failure (CHF) by angiotensin-converting enzyme (ACE) inhibitors is associated with prevention of alterations in gene expression for SL Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchanger. CHF in rats due to MI was induced by occluding the coronary artery, and 3 wk later the animals were treated with an ACE inhibitor, imidapril (1 mg.kg(-1).day(-1)), for 4 wk. Heart dysfunction and cardiac hypertrophy in the infarcted animals were associated with depressed SL Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchange activities. Protein content and mRNA levels for Na(+)/Ca(2+) exchanger as well as Na(+)-K(+)-ATPase alpha(1)-, alpha(2)- and beta(1)-isoforms were depressed, whereas those for alpha(3)-isoform were increased in the failing heart. These changes in SL activities, protein content, and gene expression were attenuated by treating the infarcted animals with imidapril. The beneficial effects of imidapril treatment on heart function and cardiac hypertrophy as well as SL Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchange activities in the infarcted animals were simulated by enalapril, an ACE inhibitor, and losartan, an angiotensin receptor antagonist. These results suggest that blockade of RAS in CHF improves SL Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchange activities in the failing heart by preventing changes in gene expression for SL proteins.     

Ionic mechanisms of cardiac cell swelling induced by blocking Na+/K+ pump as revealed by experiments and simulation

Although the Na(+)/K(+) pump is one of the key mechanisms responsible for maintaining cell volume, we have observed experimentally that cell volume remained almost constant during 90 min exposure of guinea pig ventricular myocytes to ouabain. Simulation of this finding using a comprehensive cardiac cell model (Kyoto model incorporating Cl(-) and water fluxes) predicted roles for the plasma membrane Ca(2+)-ATPase (PMCA) and Na(+)/Ca(2+) exchanger, in addition to low membrane permeabilities for Na(+) and Cl(-), in maintaining cell volume. PMCA might help maintain the [Ca(2+)] gradient across the membrane though compromised, and thereby promote reverse Na(+)/Ca(2+) exchange stimulated by the increased [Na(+)](i) as well as the membrane depolarization. Na(+) extrusion via Na(+)/Ca(2+) exchange delayed cell swelling during Na(+)/K(+) pump block. Supporting these model predictions, we observed ventricular cell swelling after blocking Na(+)/Ca(2+) exchange with KB-R7943 or SEA0400 in the presence of ouabain. When Cl(-) conductance via the cystic fibrosis transmembrane conductance regulator (CFTR) was activated with isoproterenol during the ouabain treatment, cells showed an initial shrinkage to 94.2 +/- 0.5%, followed by a marked swelling 52.0 +/- 4.9 min after drug application. Concomitantly with the onset of swelling, a rapid jump of membrane potential was observed. These experimental observations could be reproduced well by the model simulations. Namely, the Cl(-) efflux via CFTR accompanied by a concomitant cation efflux caused the initial volume decrease. Then, the gradual membrane depolarization induced by the Na(+)/K(+) pump block activated the window current of the L-type Ca(2+) current, which increased [Ca(2+)](i). Finally, the activation of Ca(2+)-dependent cation conductance induced the jump of membrane potential, and the rapid accumulation of intracellular Na(+) accompanied by the Cl(-) influx via CFTR, resulting in the cell swelling. The pivotal role of L-type Ca(2+) channels predicted in the simulation was demonstrated in experiments, where blocking Ca(2+) channels resulted in a much delayed cell swelling.     

Sarcolemmal cation channels and exchangers modify the increase in intracellular calcium in cardiomyocytes on inhibiting Na+-K+-ATPase

Although inhibition of the sarcolemmal (SL) Na(+)-K(+)-ATPase is known to cause an increase in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) by stimulating the SL Na(+)/Ca(2+) exchanger (NCX), the involvement of other SL sites in inducing this increase in [Ca(2+)](i) is not fully understood. Isolated rat cardiomyocytes were treated with or without different agents that modify Ca(2+) movements by affecting various SL sites and were then exposed to ouabain. Ouabain was observed to increase the basal levels of both [Ca(2+)](i) and intracellular Na(+) concentration ([Na(+)](i)) as well as to augment the KCl-induced increases in both [Ca(2+)](i) and [Na(+)](i) in a concentration-dependent manner. The ouabain-induced changes in [Na(+)](i) and [Ca(2+)](i) were attenuated by treatment with inhibitors of SL Na(+)/H(+) exchanger and SL Na(+) channels. Both the ouabain-induced increase in basal [Ca(2+)](i) and augmentation of the KCl response were markedly decreased when cardiomyocytes were exposed to 0-10 mM Na(+). Inhibitors of SL NCX depressed but decreasing extracellular Na(+) from 105-35 mM augmented the ouabain-induced increase in basal [Ca(2+)](i) and the KCl response. Not only was the increase in [Ca(2+)](i) by ouabain dependent on the extracellular Ca(2+) concentration, but it was also attenuated by inhibitors of SL L-type Ca(2+) channels and store-operated Ca(2+) channels (SOC). Unlike the SL L-type Ca(2+)-channel blocker, the blockers of SL Na(+) channel and SL SOC, when used in combination with SL NCX inhibitor, showed additive effects in reducing the ouabain-induced increase in basal [Ca(2+)](i). These results support the view that in addition to SL NCX, SL L-type Ca(2+) channels and SL SOC may be involved in raising [Ca(2+)](i) on inhibition of the SL Na(+)-K(+)-ATPase by ouabain. Furthermore, both SL Na(+)/H(+) exchanger and Na(+) channels play a critical role in the ouabain-induced Ca(2+) increase in cardiomyocytes.     

Modification of intracellular calcium concentration in cardiomyocytes by inhibition of sarcolemmal Na+/H+ exchanger

Although the Na(+)/H(+) exchanger (NHE) is considered to be involved in regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) through the Na(+)/Ca(2+) exchanger, the exact mechanisms of its participation in Ca(2+) handling by cardiomyocytes are not fully understood. Isolated rat cardiomyocytes were treated with or without agents that are known to modify Ca(2+) movements in cardiomyocytes and exposed to an NHE inhibitor, 5-(N-methyl-N-isobutyl)amiloride (MIA). [Ca(2+)](i) in cardiomyocytes was measured spectrofluorometrically with fura 2-AM in the absence or presence of KCl, a depolarizing agent. MIA increased basal [Ca(2+)](i) and augmented the KCl-induced increase in [Ca(2+)](i) in a concentration-dependent manner. The MIA-induced increase in basal [Ca(2+)](i) was unaffected by extracellular Ca(2+), antagonists of the sarcolemmal (SL) L-type Ca(2+) channel, and inhibitors of the SL Na(+)/Ca(2+) exchanger, SL Ca(2+) pump ATPase and mitochondrial Ca(2+) uptake. However, the MIA-induced increase in basal [Ca(2+)](i) was attenuated by inhibitors of SL Na(+)-K(+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+) transport. On the other hand, the MIA-mediated augmentation of the KCl response was dependent on extracellular Ca(2+) concentration and attenuated by agents that inhibit SL L-type Ca(2+) channels, the SL Na(+)/Ca(2+) exchanger, SL Na(+)-K(+)-ATPase, and SR Ca(2+) release channels and the SR Ca(2+) pump. However, the effect of MIA on the KCl-induced increase in [Ca(2+)](i) remained unaffected by treatment with inhibitors of SL Ca(2+) pump ATPase and mitochondrial Ca(2+) uptake. MIA and a decrease in extracellular pH lowered intracellular pH and increased basal [Ca(2+)](i), whereas a decrease in extracellular pH, in contrast to MIA, depressed the KCl-induced increase in [Ca(2+)](i) in cardiomyocytes. These results suggest that NHE may be involved in regulation of [Ca(2+)](i) and that MIA-induced increases in basal [Ca(2+)](i), as well as augmentation of the KCl-induced increase in [Ca(2+)](i), in cardiomyocytes are regulated differentially.     

Calcium signalling and regulation in olfactory neurons.

The odorant-induced Ca(2+) increase inside the cilia of vertebrate olfactory sensory neurons controls both excitation and adaptation. The increase in the internal concentration of Ca(2+) in the cilia has recently been visualized directly and has been attributed to Ca(2+) entry through cAMP-gated channels. These recent results have made it possible to further characterize Ca(2+)'s activities in olfactory neurons. Ca(2+) exerts its excitatory role by directly activating Cl(-) channels. Given the unusually high concentration of ciliary Cl(-), Ca(2+)'s activation of Cl(-) channels causes an efflux of Cl(-) from the cilia, contributing high-gain and low-noise amplification to the olfactory neuron depolarization. Moreover, in combination with calmodulin, Ca(2+) mediates odorant adaptation by desensitizing cAMP-gated channels. The restoration of the Ca(2+) concentration to basal levels occurs via a Na(+)/Ca(2+) exchanger, which extrudes Ca(2+) from the olfactory cilia.     

Reversed Na+/Ca2+ exchange contributes to Ca2+ influx and respiratory burst in microglia

Phagocytosis and the ensuing NADPH-mediated respiratory burst are important aspects of microglial activation that require calcium ion (Ca(2+)) influx. However, the specific Ca(2+) entry pathway(s) that regulates this mechanism remains unclear, with the best candidates being surface membrane Ca(2+)-permeable ion channels or Na(+)/Ca(2+) exchangers. In order to address this issue, we used quantitative real-time RT-PCR to assess mRNA expression of the Na(+)/Ca(2+) exchangers, Slc8a1-3/NCX1-3, before and after phagocytosis by rat microglia. All three Na(+)/Ca(2+) exchangers were expressed, with mRNA levels of NCX1 > NCX3 > NCX2, and were unaltered during the one hour phagocytosis period. We then carried out a biophysical characterization of Na(+)/Ca(2+) exchanger activity in these cells. To investigate conditions under which Na(+)/Ca(2+) exchange was functional, we used a combination of perforated patch-clamp analysis, fluorescence imaging of a Ca(2+) indicator (Fura-2) and a Na(+) indicator (SBFI), and manipulations of membrane potential and intracellular and extracellular ions. Then, we used a pharmacological toolbox to compare the contribution of Na(+)/Ca(2+) exchange with candidate Ca(2+)-permeable channels, to the NADPH-mediated respiratory burst that was triggered by phagocytosis. We find that inhibiting the reversed mode of the Na(+)/Ca(2+) exchanger with KB-R7943, dose dependently reduced the phagocytosis-stimulated respiratory burst; whereas, blockers of store-operated Ca(2+) channels or L-type voltage-gated Ca(2+) channels had no effect. These results provide evidence that Na(+)/Ca(2+) exchangers are potential therapeutic targets for reducing the bystander damage that often results from microglia activation in the damaged CNS.     

Oscillatory transepithelial H(+) flux regulates a rhythmic behavior in C. elegans

In C. elegans, rhythmic defecation is timed by oscillatory Ca(2+) signaling in the intestine [1-5]. Here, by using fluorescent biosensors in live, unrestrained worms, we show that intestinal pH also oscillates during defecation and that transepithelial proton movement is essential for defecation signaling. The intestinal cytoplasm is acidified by proton influx from the lumen during defecation. Acidification is predicted to trigger Na(+)/H(+) exchange activity and subsequent proton efflux. The Na(+)/H(+) exchanger NHX-7 (PBO-4) extrudes protons across the basolateral membrane and is necessary for both acute acidification of the pseudocoelom and for strong contractions of the posterior body wall muscles during defecation. This suggests that secreted protons transmit a signal between the intestine and muscle. NHX-2 is a second Na(+)/H(+) exchanger whose distribution is limited to the apical membranes facing the intestinal lumen. RNA interference of nhx-2 reduces the basal pH of the intestinal cells, reduces the rate of proton movement between the lumen and the cytoplasm during defecation, and extends the defecation period. Thus, the cell may integrate both pH and calcium signals to regulate defecation timing. Overall, these results establish the defecation cycle as a model system for studying transepithelial proton flux in tissues that maintain systemic acid-base balance.     

Na(+)/Ca(2+) exchange facilitates Ca(2+)-dependent activation of endothelial nitric-oxide synthase.

Recent evidence suggests the expression of a Na(+)/Ca(2+) exchanger (NCX) in vascular endothelial cells. To elucidate the functional role of endothelial NCX, we studied Ca(2+) signaling and Ca(2+)-dependent activation of endothelial nitric-oxide synthase (eNOS) at normal, physiological Na(+) gradients and after loading of endothelial cells with Na(+) ions using the ionophore monensin. Monensin-induced Na(+) loading markedly reduced Ca(2+) entry and, thus, steady-state levels of intracellular free Ca(2+) ([Ca(2+)](i)) in thapsigargin-stimulated endothelial cells due to membrane depolarization. Despite this reduction of overall [Ca(2+)](i), Ca(2+)-dependent activation of eNOS was facilitated as indicated by a pronounced leftward shift of the Ca(2+) concentration response curve in monensin-treated cells. This facilitation of Ca(2+)-dependent activation of eNOS was strictly dependent on the presence of Na(+) ions during treatment of the cells with monensin. Na(+)-induced facilitation of eNOS activation was not due to a direct effect of Na(+) ions on the Ca(2+) sensitivity of the enzyme. Moreover, the effect of Na(+) was not related to Na(+) entry-induced membrane depolarization or suppression of Ca(2+) entry, since neither elevation of extracellular K(+) nor the Ca(2+) entry blocker 1-(beta-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenethyl)-1H-imidazol e hydrochloride (SK&F 96365) mimicked the effects of Na(+) loading. The effects of monensin were completely blocked by 3', 4'-dichlorobenzamil, a potent and selective inhibitor of NCX, whereas the structural analog amiloride, which barely affects Na(+)/Ca(2+) exchange, was ineffective. Consistent with a pivotal role of Na(+)/Ca(2+) exchange in Ca(2+)-dependent activation of eNOS, an NCX protein was detected in caveolin-rich membrane fractions containing both eNOS and caveolin-1. These results demonstrate for the first time a crucial role of cellular Na(+) gradients in regulation of eNOS activity and suggest that a tight functional interaction between endothelial NCX and eNOS may take place in caveolae.     

Distribution of proteins implicated in excitation-contraction coupling in rat ventricular myocytes

We have examined the distribution of ryanodine receptors, L-type Ca(2+) channels, calsequestrin, Na(+)/Ca(2+) exchangers, and voltage-gated Na(+) channels in adult rat ventricular myocytes. Enzymatically dissociated cells were fixed and dual-labeled with specific antibodies using standard immunocytochemistry protocols. Images were deconvolved to reverse the optical distortion produced by wide-field microscopes equipped with high numerical aperture objectives. Every image showed a well-ordered array of fluorescent spots, indicating that all of the proteins examined were distributed in discrete clusters throughout the cell. Mathematical analysis of the images revealed that dyads contained only ryanodine receptors, L-type Ca(2+) channels, and calsequestrin, and excluded Na(+)/Ca(2+) exchangers and voltage-gated Na(+) channels. The Na(+)/Ca(2+) exchanger and voltage-gated Na(+) channels were distributed largely within the t-tubules, on both transverse and axial elements, but were not co-localized. The t-tubule can therefore be subdivided into at least three structural domains; one of coupling (dyads), one containing the Na(+)/Ca(2+) exchanger, and one containing voltage-gated Na(+) channels. We conclude that if either the slip mode conductance of the Na(+) channel or the reverse mode of the Na(+)/Ca(2+) exchanger are to contribute to the contractile force, the fuzzy space must extend outside of the dyad.     

Electrogenic Na(+)/Ca(2+) exchange. A novel amplification step in squid olfactory transduction

Olfactory receptor neurons (ORNs) from the squid, Lolliguncula brevis, respond to the odors l-glutamate or dopamine with increases in internal Ca(2+) concentrations ([Ca(2+)](i)). To directly asses the effects of increasing [Ca(2+)](i) in perforated-patched squid ORNs, we applied 10 mM caffeine to release Ca(2+) from internal stores. We observed an inward current response to caffeine. Monovalent cation replacement of Na(+) from the external bath solution completely and selectively inhibited the caffeine-induced response, and ruled out the possibility of a Ca(2+)-dependent nonselective cation current. The strict dependence on internal Ca(2+) and external Na(+) indicated that the inward current was due to an electrogenic Na(+)/Ca(2+) exchanger. Block of the caffeine-induced current by an inhibitor of Na(+)/Ca(2+) exchange (50-100 microM 2',4'-dichlorobenzamil) and reversibility of the exchanger current, further confirmed its presence. We tested whether Na(+)/Ca(2+) exchange contributed to odor responses by applying the aquatic odor l-glutamate in the presence and absence of 2', 4'-dichlorobenzamil. We found that electrogenic Na(+)/Ca(2+) exchange was responsible for approximately 26% of the total current associated with glutamate-induced odor responses. Although Na(+)/Ca(2+) exchangers are known to be present in ORNs from numerous species, this is the first work to demonstrate amplifying contributions of the exchanger current to odor transduction.     

Targeted disruption of Na+/Ca2+ exchanger gene leads to cardiomyocyte apoptosis and defects in heartbeat

Ca(2+), which enters cardiac myocytes through voltage-dependent Ca(2+) channels during excitation, is extruded from myocytes primarily by the Na(+)/Ca(2+) exchanger (NCX1) during relaxation. The increase in intracellular Ca(2+) concentration in myocytes by digitalis treatment and after ischemia/reperfusion is also thought to result from the reverse mode of the Na(+)/Ca(2+) exchange mechanism. However, the precise roles of the NCX1 are still unclear because of the lack of its specific inhibitors. We generated Ncx1-deficient mice by gene targeting to determine the in vivo function of the exchanger. Homozygous Ncx1-deficient mice died between embryonic days 9 and 10. Their hearts did not beat, and cardiac myocytes showed apoptosis. No forward mode or reverse mode of the Na(+)/Ca(2+) exchange activity was detected in null mutant hearts. The Na(+)-dependent Ca(2+) exchange activity as well as protein content of NCX1 were decreased by approximately 50% in the heart, kidney, aorta, and smooth muscle cells of the heterozygous mice, and tension development of the aortic ring in Na(+)-free solution was markedly impaired in heterozygous mice. These findings suggest that NCX1 is required for heartbeats and survival of cardiac myocytes in embryos and plays critical roles in Na(+)-dependent Ca(2+) handling in the heart and aorta.     

Constitutive overexpression of phosphomimetic phospholemman S68E mutant results in arrhythmias, early mortality, and heart failure: potential involvement of Na+/Ca2+ exchanger

Expression and activity of cardiac Na(+)/Ca(2+) exchanger (NCX1) are altered in many disease states. We engineered mice in which the phosphomimetic phospholemman S68E mutant (inhibits NCX1 but not Na(+)-K(+)-ATPase) was constitutively overexpressed in a cardiac-specific manner (conS68E). At 4-6 wk, conS68E mice exhibited severe bradycardia, ventricular arrhythmias, increased left ventricular (LV) mass, decreased cardiac output (CO), and ～50% mortality compared with wild-type (WT) littermates. Protein levels of NCX1, calsequestrin, ryanodine receptor, and α(1)- and α(2)-subunits of Na(+)-K(+)-ATPase were similar, but sarco(endo)plasmic reticulum Ca(2+)-ATPase was lower, whereas L-type Ca(2+) channels were higher in conS68E hearts. Resting membrane potential and action potential amplitude were similar, but action potential duration was dramatically prolonged in conS68E myocytes. Diastolic intracellular Ca(2+) ([Ca(2+)](i)) was higher, [Ca(2+)](i) transient and maximal contraction amplitudes were lower, and half-time of [Ca(2+)](i) transient decline was longer in conS68E myocytes. Intracellular Na(+) reached maximum within 3 min after isoproterenol addition, followed by decline in WT but not in conS68E myocytes. Na(+)/Ca(2+) exchange, L-type Ca(2+), Na(+)-K(+)-ATPase, and depolarization-activated K(+) currents were decreased in conS68E myocytes. At 22 wk, bradycardia and increased LV mass persisted in conS68E survivors. Despite comparable baseline CO, conS68E survivors at 22 wk exhibited decreased chronotropic, inotropic, and lusitropic responses to isoproterenol. We conclude that constitutive overexpression of S68E mutant was detrimental, both in terms of depressed cardiac function and increased arrhythmogenesis.     

alpha-Adrenoceptor stimulation-mediated negative inotropism and enhanced Na(+)/Ca(2+) exchange in mouse ventricle

Mechanisms underlying the negative inotropic response to alpha-adrenoceptor stimulation in adult mouse ventricular myocardium were studied. In isolated ventricular tissue, phenylephrine (PE), in the presence of propranolol, decreased contractile force by approximately 40% of basal value. The negative inotropic response was similarly observed under low extracellular Ca(2+) concentration ([Ca(2+)](o)) conditions but was significantly smaller under high-[Ca(2+)](o) conditions and was not observed under low-[Na(+)](o) conditions. The negative inotropic response was not affected by nicardipine, ryanodine, ouabain, or dimethylamiloride (DMA), inhibitors of L-type Ca(2+) channel, Ca(2+) release channel, Na(+)-K(+) pump, or Na(+)/H(+) exchanger, respectively. KB-R7943, an inhibitor of Na(+)/Ca(2+) exchanger, suppressed the negative inotropic response mediated by PE. PE reduced the magnitude of postrest contractions. PE caused a decrease in duration of the late plateau phase of action potential and a slight increase in resting membrane potential; time courses of these effects were similar to that of the negative inotropic effect. In whole cell voltage-clamped myocytes, PE increased the L-type Ca(2+) and Na(+)/Ca(2+) exchanger currents but had no effect on the inwardly rectifying K(+), transient outward K(+), or Na(+)-K(+)-pump currents. These results suggest that the sustained negative inotropic response to alpha-adrenoceptor stimulation of adult mouse ventricular myocardium is mediated by enhancement of Ca(2+) efflux through the Na(+)/Ca(2+) exchanger.     

Regulation of in vivo cardiac contractility by phospholemman: role of Na+/Ca2+ exchange

Phospholemman (PLM), when phosphorylated at serine 68, relieves its inhibition on Na(+)-K(+)-ATPase but inhibits Na(+)/Ca(2+) exchanger 1 (NCX1) in cardiac myocytes. Under stress when catecholamine levels are high, enhanced Na(+)-K(+)-ATPase activity by phosphorylated PLM attenuates intracellular Na(+) concentration ([Na(+)](i)) overload. To evaluate the effects of PLM on NCX1 on in vivo cardiac contractility, we injected recombinant adeno-associated virus (serotype 9) expressing either the phosphomimetic PLM S68E mutant or green fluorescent protein (GFP) directly into left ventricles (LVs) of PLM-knockout (KO) mice. Five weeks after virus injection, ～40% of isolated LV myocytes exhibited GFP fluorescence. Expression of S68E mutant was confirmed with PLM antibody. There were no differences in protein levels of α(1)- and α(2)-subunits of Na(+)-K(+)-ATPase, NCX1, and sarco(endo)plasmic reticulum Ca(2+)-ATPase between KO-GFP and KO-S68E LV homogenates. Compared with KO-GFP myocytes, Na(+)/Ca(2+) exchange current was suppressed, but resting [Na(+)](i), Na(+)-K(+)-ATPase current, and action potential amplitudes were similar in KO-S68E myocytes. Resting membrane potential was slightly lower and action potential duration at 90% repolarization (APD(90)) was shortened in KO-S68E myocytes. Isoproterenol (Iso; 1 μM) increased APD(90) in both groups of myocytes. After Iso, [Na(+)](i) increased monotonically in paced (2 Hz) KO-GFP but reached a plateau in KO-S68E myocytes. Both systolic and diastolic [Ca(2+)](i) were higher in Iso-stimulated KO-S68E myocytes paced at 2 Hz. Echocardiography demonstrated similar resting heart rate, ejection fraction, and LV mass between KO-GFP and KO-S68E mice. In vivo closed-chest catheterization demonstrated enhanced contractility in KO-S68E compared with KO-GFP hearts stimulated with Iso. We conclude that under catecholamine stress when [Na(+)](i) is high, PLM minimizes [Na(+)](i) overload by relieving its inhibition of Na(+)-K(+)-ATPase and preserves inotropy by simultaneously inhibiting Na(+)/Ca(2+) exchanger.     

The co-presence of Na+/Ca2+-K+ exchanger and Na+/Ca2+ exchanger in bovine adrenal chromaffin cells

We have previously shown that there is high Na(+)/Ca(2+) exchange (NCX) activity in bovine adrenal chromaffin cells. In this study, by monitoring the [Ca(2+)](i) change in single cells and in a population of chromaffin cells, when the reverse mode of exchanger activity has been initiated, we have shown that the NCX activity is enhanced by K(+). The K(+)-enhanced activity accounted for a significant proportion of the Na(+)-dependent Ca(2+) uptake activity in the chromaffin cells. The results support the hypothesis that both NCX and Na(+)/Ca(2+)-K(+) exchanger (NCKX) are co-present in chromaffin cells. The expression of NCKX in chromaffin cells was further confirmed using PCR and northern blotting. In addition to the plasma membrane, the exchanger activity, measured by Na(+)-dependent (45)Ca(2+) uptake, was also present in membrane isolated from the chromaffin granules enriched fraction and the mitochondria enriched fraction. The results support that both NCX and NCKX are present in bovine chromaffin cells and that the regulation of [Ca(2+)](i) is probably more efficient with the participation of NCKX.     

Inhibition of sodium-calcium exchange by ceramide and sphingosine

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

Frequency-dependent regulation of cardiac Na(+)/Ca(2+) exchanger

The activity of the cardiac Na(+)/Ca(2+) exchanger (NCX1.1) undergoes continuous modulation during the contraction-relaxation cycle because of the accompanying changes in the electrochemical gradients for Na(+) and Ca(2+). In addition, NCX1.1 activity is also modulated via secondary, ionic regulatory mechanisms mediated by Na(+) and Ca(2+). In an effort to evaluate how ionic regulation influences exchange activity under pulsatile conditions, we studied the behavior of the cloned NCX1.1 during frequency-controlled changes in intracellular Na(+) and Ca(+) (Na(i)(+) and Ca(i)(2+)). Na(+)/Ca(2+) exchange activity was measured by the giant excised patch-clamp technique with conditions chosen to maximize the extent of Na(+)- and Ca(2+)-dependent ionic regulation so that the effects of variables such as pulse frequency and duration could be optimally discerned. We demonstrate that increasing the frequency or duration of solution pulses leads to a progressive decline in pure outward, but not pure inward, Na(+)/Ca(2+) exchange current. However, when the exchanger is permitted to alternate between inward and outward transport modes, both current modes exhibit substantial levels of inactivation. Changes in regulatory Ca(2+), or exposure of patches to limited proteolysis by alpha-chymotrypsin, reveal that this "coupling" is due to Na(+)-dependent inactivation originating from the outward current mode. Under physiological ionic conditions, however, evidence for modulation of exchange currents by Na(i)(+)-dependent inactivation was not apparent. The current approach provides a novel means for assessment of Na(+)/Ca(2+) exchange ionic regulation that may ultimately prove useful in understanding its role under physiological and pathophysiological conditions.