Role of Na+/H+ exchange in thrombin- and arachidonic acid-induced Ca2+ influx in platelets

Platelet activation is accompanied by an increase of cytosolic free Ca2+ concentration, [Ca2+]i, (due to both extracellular Ca2+ influx and Ca2+ movements from the dense tubular system) and an Na+ influx associated with H+ extrusion. The latter event is attributable to the activation of Na+/H+ exchange, which requires Na+ in the extracellular medium and is inhibited by amiloride and its analogs. The present study was carried out to determine whether a link exists between Ca2+ transients (measured by the quin2 method and the 45CaCl2 technique) and Na+/H+ exchange activation (studied with the pH-sensitive intracellular probe, 6-carboxyfluorescein) during platelet stimulation. Washed human platelets, stimulated with thrombin and arachidonic acid, showed: (1) a large and rapid [Ca2+]i rise, mostly due to a Ca2+ influx through the plasma membrane; (2) a marked intracellular alkalinization. Both phenomena were markedly inhibited in the absence of extracellular Na+ or in the presence of an amiloride analog (EIPA). Monensin, a cation exchanger which elicits Na+ influx and alkalinization, and NH4Cl, which induces alkalinization only, were able to evoke an increase in [Ca2+]i, mostly as an influx from the extracellular medium. Our results suggest that Ca2+ influx induced by thrombin and arachidonic acid in human platelets is strictly dependent on Na+/H+-exchange activation.     

Importance of Ca2+ influx by Na+/Ca2+ exchange under normal and sodium-loaded conditions in mammalian ventricles

Na⁺/Ca²⁺ exchange (NCX) is a major Ca²⁺ extrusion system in cardiac myocytes, but can also mediate Ca²⁺ influx and trigger sarcoplasmic reticulum Ca²⁺ release. Under conditions such as digitalis toxicity or ischemia/reperfusion, increased [Na⁺]_i may lead to a rise in [Ca²⁺]_i through NCX, causing Ca²⁺ overload and triggered arrhythmias. Here we used an agent which selectively blocks Ca²⁺ influx by NCX, KB-R7943 (KBR), and assessed twitch contractions and Ca²⁺ transients in rat and guinea pig ventricular myocytes loaded with indo-1. KBR (5 M) did not alter control steady-state twitch contractions or Ca²⁺ transients at 0.5 Hz in rat, but significantly decreased them in guinea pig myocytes. When cells were Na⁺-loaded by perfusion of strophanthidin (50 M), the addition of KBR reduced diastolic [Ca²⁺]_i and abolished spontaneous Ca²⁺ oscillations. In guinea pig papillary muscles exposed to substrate-free hypoxic medium for 60 min, KBR (10 M applied 10 min before and during reoxygenation) reduced both the incidence and duration of reoxygenation-induced arrhythmias. KBR also enhanced the recovery of developed tension after reoxygenation. It is concluded that (1) the importance of Ca²⁺ influx via NCX for normal excitation-contraction coupling is species-dependent, and (2) Ca²⁺ influx via NCX may be critical in causing myocardial Ca²⁺ overload and triggered activities induced by cardiac glycoside or reoxygenation.     

Na+/Ca2+ exchange in coated microvesicles.

Coated microvesicles isolated from bovine neurohypophyses could be loaded with Ca2+ in two different ways, either by incubation in the presence of ATP or by imposition of an outwardly directed Na+ gradient. Na+, but not K+, was able to release Ca2+ accumulated by the coated microvesicles. These results suggest the existence of an ATP-dependent Ca2+-transport system as well as of a Na+/Ca2+ carrier in the membrane of coated microvesicles similar to that present in the membranes of secretory vesicles from the neurohypophysis. A kinetic analysis of transport indicates that the apparent Km for free Ca2+ of the ATP-dependent uptake was 0.8 microM. The average Vmax. was 2 nmol of Ca2+/5 min per mg of protein. The total capacity of microvesicles for Ca2+ uptake was 3.7 nmol/mg of protein. Both nifedipine (10 microM) and NH4Cl (50 mM) inhibited Ca2+ uptake. The ATPase activity in purified coated-microvesicles fractions from brain and neurohypophysis was characterized. Micromolar concentrations of Ca2+ in the presence of millimolar concentrations of Mg2+ did not change enzyme activity. Ionophores increasing the proton permeability across membranes activated the ATPase activity in preparations of coated microvesicles from brain as well as from the neurohypophysis. Thus the enzyme exhibits properties of a proton-transporting ATPase. This enzyme seems to be linked to the ion accumulation by coated microvesicles, although the precise coupling of the proton transport to Ca2+ and Na+ fluxes remains to be determined.     

In depolarized and glucose-deprived neurons,Na+ influx reverses plasmalemmal K+-dependent and K+-independent Na+/Ca2+ exchangers and contributes to NMDA excitotoxicity

Cerebellar granule cells (CGCs) express K+-dependent (NCKX) and K+-independent (NCX) plasmalemmal Na+/Ca2+ exchangers which, under plasma membrane-depolarizing conditions and high cytosolic [Na+], may reverse and mediate potentially toxic Ca2+ influx. To examine this possibility, we inhibited NCX or NCKX with KB-R7943 or K+-free medium, respectively, and studied how gramicidin affects cytosolic [Ca2+] and 45Ca2+ accumulation. Gramicidin forms pores permeable to alkali cations but not Ca2+. Therefore, gramicidin-induced Ca2+ influx is indirect; it results from fluxes of monovalent cations. In the presence of Na+, but not Li+ or Cs+, gramicidin induced Ca2+ influx that was inhibited by simultaneous application of KB-R7943 and K+-free medium. The data indicate that gramicidin-induced Na+ influx reverses NCX and NCKX. To test the role of NCX and/or NCKX in excitotoxicity, we studied how NMDA affects the viability of glucose-deprived and depolarized CGCs. To assure depolarization of the plasma membrane, we inhibited Na+,K+-ATPase with ouabain. Although inhibition of NCX or NCKX reversal failed to significantly limit 45Ca2+ accumulation and excitotoxicity, simultaneously inhibiting NCX and NCKX reversal was neuroprotective and significantly decreased NMDA-induced 45Ca2+ accumulation. Our data suggest that NMDA-induced Na+ influx reverses NCX and NCKX and leads to the death of depolarized and glucose-deprived neurons.     

Alcohol stimulates Na+/Ca2+ exchange in brain mitochondria

Ethanol, at low concentrations, specifically stimulates the Na(+)-dependent Ca2(+)-efflux in brain mitochondria. In addition, at higher concentrations, ethanol inhibits the Na(+)-independent Ca2(+)-efflux. The electrogenic Ca(+)-uptake system is not affected by ethanol. The specific stimulation of Na+/Ca2+ exchange reaches a maximum of 60% stimulation, with half-maximal stimulation at 130 mM ethanol. The inhibition of the Na(+)-independent efflux is proportional to the ethanol concentration, becoming significant only above 200 mM, with 50% inhibition at 0.5 M. The inhibition of the Na(+)-independent efflux is, in large part, due to an inhibition of the activation of the Cyclosporin-sensitive pore. Long-term ethanol-feeding had no effect on the Ca2+ transport systems and their sensitivity to acute ethanol treatment. It is suggested that the stimulation of the Na(+)-dependent Ca2(+)-efflux, which is the dominant Ca2+ efflux pathway in brain mitochondria, contributes to the intoxicating effects of ethanol.     

Development and application of Na+/Ca2+ exchange inhibitors

The Na+/Ca2+ exchanger (NCX) is an ion transporter that exchanges Na+ and Ca2+ in either Ca2+ efflux or Ca2+ influx mode, depending on the ion gradients across the plasma membrane and the membrane potential. In heart, smooth muscle cells, neurons, and nephron cells, the NCX is thought to play an important role in the regulation of intracellular Ca2+ concentration. Recently, a novel selective inhibitor (KB-R7943 and SEA0400) of the Ca2+ influx mode of the NCX has been developed. NCX inhibitor is expected to be a pharmaceutical agent that offers effective protection against ischemia/reperfusion injury in several organs such as heart and kidney. Here, we summarize pharmacological profiles of KB-R7943 and SEA0400, the molecular mechanism of its action, and its future prospect as a novel pharmaceutical agent.     

A constitutive, transient receptor potential-like Ca2+ influx pathway in presynaptic nerve endings independent of voltage-gated Ca2+ channels and Na+/Ca2+ exchange

Calcium levels in the presynaptic nerve terminal are altered by several pathways, including voltage-gated Ca(2+) channels, the Na(+)/Ca(2+) exchanger, Ca(2+)-ATPase, and the mitochondria. The influx pathway for homeostatic control of [Ca(2+)](i) in the nerve terminal has been unclear. One approach to detecting the pathway that maintains internal Ca(2+) is to test for activation of Ca(2+) influx following Ca(2+) depletion. Here, we demonstrate that a constitutive influx pathway for Ca(2+) exists in presynaptic terminals to maintain internal Ca(2+) independent of voltage-gated Ca(2+) channels and Na(+)/Ca(2+) exchange, as measured in intact isolated nerve endings from mouse cortex and in intact varicosities in a neuronal cell line using fluorescence spectroscopy and confocal imaging. The Mg(2+) and lanthanide sensitivity of the influx pathway, in addition to its pharmacological and short hairpin RNA sensitivity, and the results of immunostaining for transient receptor potential (TRP) channels indicate the involvement of TRPC channels, possibly TRPC5 and TRPC1. This constitutive Ca(2+) influx pathway likely serves to maintain synaptic function under widely varying levels of synaptic activity.     

Genetic manipulation of cardiac Na+/Ca2+ exchange expression

The Na+/Ca2+ exchanger (NCX) is the primary Ca2+ extrusion mechanism in cardiomyocytes. To further investigate the role of NCX in excitation-contraction coupling and Ca2+ homeostasis, we created murine models with altered expression levels of NCX. Homozygous overexpression of NCX resulted in mild cardiac hypertrophy. Decline of the Ca2+ transient and relaxation of contraction were increased and the reverse mode of NCX was augmented. Overexpression also led to a higher susceptibility to ischemia-reperfusion injury and to a greater ability of NCX to trigger Ca2+-induced Ca2+ release. Furthermore, an increase in peak L-type Ca2+ current was observed suggesting a direct influence of NCX on L-type Ca2+ current. Whereas global knockout of NCX led to prenatal death, a recently generated cardiac-specific NCX knockout mouse was viable with surprisingly normal contractile properties. Expression levels of other Ca2+-handling proteins were not altered. Ca2+ influx in these animals is limited by a decrease of peak L-type Ca2+ current. An alternative Ca2+ efflux mechanism, presumably the plasma membrane Ca2+-ATPase, is sufficient to maintain Ca2+-homeostasis in the NCX knockout mice.     

Na+/Ca2+ exchange activity in neonatal rabbit ventricular myocytes

Much less is known about the contributions of the Na⁺/Ca²⁺ exchanger (NCX) and sarcoplasmic reticulum (SR) Ca²⁺ pump to cell relaxation in neonatal compared with adult mammalian ventricular myocytes. Based on both biochemical and molecular studies, there is evidence of a much higher density of NCX at birth that subsequently decreases during the next 2 wk of development. It has been hypothesized, therefore, that NCX plays a relatively more important role for cytosolic Ca²⁺ decline in neonates as well as, perhaps, a role in excitation-contraction coupling in reverse mode. We isolated neonatal ventricular myocytes from rabbits in four different age groups: 3, 6, 10, and 20 days of age. Using an amphotericin-perforated patch-clamp technique in fluo-3-loaded myocytes, we measured the caffeine-induced inward NCX current (I_NCX) and the Ca²⁺ transient. We found that the integral of I_NCX, an indicator of SR Ca²⁺ content, was greatest in myocytes from younger age groups when normalized by cell surface area and that it decreased with age. The velocity of Ca²⁺ extrusion by NCX (V_NCX) was linear with [Ca²⁺] and did not indicate saturation kinetics until [Ca²⁺] reached 1–3 µM for each age group. There was a significantly greater time delay between the peaks of I_NCX and the Ca²⁺ transient in myocytes from the youngest age groups. This observation could be related to structural differences in the subsarcolemmal microdomains as a function of age. ontogeny of cardiac excitation-contraction coupling; sodium/calcium exchanger; cytosolic calcium concentration; subsarcolemmal calcium concentration; sarcoplasmic reticulum calcium content     

Calcineurin inhibits Na+/Ca2+ exchange in phenylephrine-treated hypertrophic cardiomyocytes

The cardiac Na(+)/Ca(2+) exchanger (NCX1) is the predominant mechanism for the extrusion of Ca(2+) from beating cardiomyocytes. The role of protein phosphorylation in the regulation of NCX1 function in normal and diseased hearts remains unclear. In our search for proteins that interact with NCX1 using a yeast two-hybrid screen, we found that the C terminus of calcineurin Abeta, containing the autoinhibitory domain, binds to the beta1 repeat of the central cytoplasmic loop of NCX1 that presumably constitutes part of the allosteric Ca(2+) regulatory site. The association of NCX1 with calcineurin was significantly increased in the BIO14.6 cardiomyopathic hamster heart compared with that in the normal control. In hypertrophic neonatal rat cardiomyocytes subjected to chronic phenylephrine treatment, we observed a marked depression of NCX activity measured as the rate of Na(+)(i)-dependent (45)Ca(2+) uptake or the rate of Na(+)(o)-dependent (45)Ca(2+) efflux. Depressed NCX activity was partially and independently reversed by the acute inhibition of calcineurin and protein kinase C activities with little effect on myocyte hypertrophic phenotypes. Studies of NCX1 deletion mutants expressed in CCL39 cells were consistent with the view that the beta1 repeat is required for the action of endogenous calcineurin and that the large cytoplasmic loop may be required to maintain the interaction of the enzyme with its substrate. Our data suggest that NCX1 is a novel regulatory target for calcineurin and that depressed NCX activity might contribute to the etiology of in vivo cardiac hypertrophy and dysfunction occurring under conditions in which both calcineurin and protein kinase C are chronically activated.     

Na+/Ca2+ Exchange and Cellular Ca2+ Homeostasis

The Na⁺/Ca²⁺ exchange system is the primary Ca²⁺ efflux mechanism in cardiac myocytes, and plays an important role in controlling the force of cardiac contraction. The exchanger protein contains 11 transmembrane segments plus a large hydrophilic domain between the 5th and 6th transmembrane segments; the transmembrane regions are reponsible for mediating ion translocation while the hydrophilic domain is responsible for regulation of activity. Exchange activity is regulated in vitro by interconversions between an active state and either of two inactive states. High concentrations of cytosolic Na⁺ or the absence of cytosolic Ca²⁺ promote the formation of the inactive states; phosphatidylinositol-(4,5)bisphosphate (or other negatively charged phospholipids) and cytosolic Ca²⁺ counteract the inactivation process. The importance of these mechanisms in regulating exchange activity under normal physiological conditions is uncertain. Exchanger function is also dependent upon cytoskeletal interactions, and the exchanger's location with respect to intracellular Ca²⁺-sequestering organelles. An understanding of the exchanger's function in normal cell physiology will require more detailed information on the proximity of the exchanger and other Ca²⁺-transporting proteins, their interactions with the cytoskeleton, and local concentrations of anionic phospholipids and transported ions.     

Physiological roles of Na+/Ca2+ exchange in Limulus ventral photoreceptors

In previous work we have presented evidence for electrogenic Na+/Ca2+ exchange in Limulus ventral photoreceptors (1989. J. Gen. Physiol. 93:473-492). This article assesses the contributions to photoreceptor physiology from Na+/Ca2+ exchange. Four separate physiological processes were considered: maintenance of resting sensitivity, light-induced excitation, light adaptation, and dark adaptation. (a) Resting sensitivity: reduction of [Na+]o caused a [Ca2+]o-dependent reduction in light sensitivity and a speeding of the time courses of the responses to individual test flashes; this effect was dependent on the final value to which [Na+]o was reduced. The desensitization caused by Na+ reduction was dependent on the initial sensitivity of the photoreceptor; in fully dark-adapted conditions no desensitization was observed; in light-adapted conditions, extensive desensitization was observed. (b) Excitation: Na+ reduction in fully dark-adapted conditions caused a Ca2+o-dependent depolarizing phase in the receptor potential that persisted beyond the stimulus duration and was evoked by a bright adapting flash. (c) Light adaptation: the degree of desensitization induced by a bright adapting flash was Na+o dependent, being larger with lower [Na+]o. Na+ reduction enhanced light adaptation only at intensities brighter than 4 x 10(-6) W/cm2. In addition to being Na+o dependent, light adaptation was Ca2+o dependent, being greater at higher [Ca2+]o. (d) Dark adaptation: the recovery of light sensitivity after adapting illumination was Na+o dependent. Dark adaptation after bright illumination in voltage-clamped and in unclamped conditions was faster in normal-Na+ saline than in reduced Na+ saline. The final sensitivity to which photoreceptors recovered was lower in reduced-Na+ saline when bright adapting illumination was used. The results suggest the involvement of Na+/Ca2+ exchange in each of these physiological processes. Na+/Ca2+ exchange may contribute to these processes by counteracting normal elevations in [Ca2+]i.     

ATP stimulation of Na+/Ca2+ exchange in cardiac sarcolemmal vesicles

In cardiacsarcolemmal vesicles, MgATP stimulatesNa⁺/Ca²⁺exchange with the following characteristics:1) increases 10-fold the apparentaffinity for cytosolic Ca²⁺;2) a Michaelis constant for ATP of~500 µM; 3) requires micromolar vanadate while millimolar concentrations are inhibitory;4) not observed in the presence of20 µM eosin alone but reinstated when vanadate is added;5) mimicked by adenosine5'-O-(3-thiotriphosphate), without the need for vanadate, but not by ,-methyleneadenosine 5'-triphosphate; and 6) notaffected by unspecific protein alkaline phosphatase but abolished by aphosphatidylinositol-specific phospholipase C (PI-PLC). The PI-PLCeffect is counteracted by phosphatidylinositol. In addition, in theabsence of ATP,L--phosphatidylinositol4,5-bisphosphate (PIP₂) was ableto stimulate the exchanger activity in vesicles pretreated with PI-PLC.This MgATP stimulation is not related to phosphorylation of thecarrier, whereas phosphorylation appeared in the phosphoinositides,mainly PIP₂, thatcoimmunoprecipitate with the exchanger. Vesicles incubated with MgATPand no Ca²⁺ show a markedsynthesis ofL--phosphatidylinositol4-monophosphate (PIP) with little production ofPIP₂; in the presence of 1 µM Ca²⁺, the net synthesis of PIP issmaller, whereas that of PIP₂increases ninefold. These results indicate thatPIP₂ is involved in the MgATPstimulation of the cardiacNa⁺/Ca²⁺exchanger through a fast phosphorylation chain: aCa²⁺-independent PIP formationfollowed by a Ca²⁺-dependentsynthesis of PIP₂.

     

Evidence for electrogenic Na+/Ca2+ exchange in Limulus ventral photoreceptors

The Ca2+ indicator photoprotein, aequorin, was used to estimate and monitor intracellular Ca2+ levels in Limulus ventral photoreceptors during procedures designed to affect Na+/Ca2+ exchange. Dark levels of [Ca2+]i were estimated at 0.66 +/- 0.09 microM. Removal of extracellular Na+ caused [Ca2+]i to rise transiently from an estimated 0.5-0.6 microM in a typical cell to approximately 21 microM; [Ca2+]i approached a plateau level in 0-Na+ saline of approximately 5.5 microM; restoration of normal [Na+]o lowered [Ca2+]i to baseline with a time course of 1 log10 unit per 9 s. The apparent rate of Nao+-dependent [Ca2+]i decline decreased with decreasing [Ca2+]i. Reintroduction of Ca2+ to 0-Na+, 0-Ca2+ saline in a typical cell caused a transient rise in [Ca2+]i from an estimated 0.36 microM (or lower) to approximately 16.5 microM. This was followed by a decline in [Ca2+]i approaching a plateau of approximately 5 microM; subsequent removal of Cao2+ caused [Ca2+]i to decline slowly (1 log unit in approximately 110 s). Intracellular injection of Na+ in the absence of extracellular Na+ caused a transient rise in [Ca2+]i in the presence of normal [Ca2+]o; in 0-Ca2+ saline, however, no such rise in [Ca2+]i was detected. Under constant voltage clamp (-80 mV) inward currents were measured after the addition of Nao+ to 0-Na+ 0-Ca2+ saline and outward currents were measured after the addition of Cao2+ to 0-Na+ 0-Ca2+ saline. The results suggest the presence of an electrogenic Na+/Ca2+ exchange process in the plasma membrane of Limulus ventral photoreceptors that can operate in forward (Nao+-dependent Ca2+ extrusion) or reverse (Nai+-dependent Ca2+ influx) directions.     

Phospholemman modulates Na+/Ca2+ exchange in adult rat cardiac myocytes

Previous studies have shown that overexpression of phospholemman (PLM) affected contractile function and Ca(2+) homeostasis in adult rat myocytes. We tested the hypothesis that PLM modulated Na(+)/Ca(2+) exchanger (NCX1) activity. PLM was overexpressed in adult rat myocytes by adenovirus-mediated gene transfer. After 72 h, the half-time of relaxation from caffeine-induced contracture, an estimate of forward NCX1 activity, was prolonged 1.8-fold (P < 0.003) in myocytes overexpressing PLM compared with control myocytes overexpressing green fluorescent protein alone. Reverse NCX1 current (3 Na(+) out:1 Ca(2+) in) was significantly (P < 0.0001) lower in PLM myocytes, especially at more positive voltages. Immunofluorescence demonstrated colocalization of PLM and NCX1 to the plasma membrane and t-tubules. Resting membrane potential, action potential amplitude and duration, myocyte size, and NCX1 and calsequestrin protein levels were not affected by PLM overexpression. At 5 mM extracellular [Ca(2+)] ([Ca(2+)](o)), the depressed contraction amplitudes in PLM myocytes were increased towards normal by cooverexpression with NCX1. At 0.6 mM [Ca(2+)](o), the supranormal contraction amplitudes in PLM myocytes were reduced by cooverexpression with NCX1. We conclude that PLM modulated myocyte contractility partly by inhibiting Na(+)/Ca(2+) exchange.     

Direct evidence of Na+/Ca2+ exchange in squid rhabdomeric membranes

Na⁺/Ca²⁺exchange has been investigated in squid(Loligopealei) rhabdomeric membranes.Ca²⁺-containing vesicles have beenprepared from purified rhabdomeric membranes by extrusion throughpolycarbonate filters of 1-µm pore size. After removal of externalCa²⁺, up to 90% of the entrappedCa²⁺ could be specificallyreleased by the addition of Na⁺;this finding indicates that most of the vesicles containedNa⁺/Ca²⁺exchanger. The Na⁺-inducedCa²⁺ efflux had a half-maximumvalue (K_1/2) of~44 mM and a Hill coefficient of ~1.7. The maximalNa⁺-inducedCa²⁺ efflux was ~0.6 nmolCa²⁺ · s¹ · mgprotein¹. SimilarNa⁺-inducedCa²⁺ effluxes were measured ifK⁺ was replaced withLi⁺ orCs⁺. Vesicles loaded withCa²⁺ byNa⁺/Ca²⁺exchange also released this Ca²⁺byNa⁺/Ca²⁺exchange, suggesting thatNa⁺/Ca²⁺exchange operated in both forward and reverse modes. Limited proteolysis by trypsin resulted in a rate ofCa²⁺ efflux enhanced byapproximately fivefold when efflux was activated with 95 mM NaCl. For vesicles subjected to limited proteolysis by trypsin,Na⁺/Ca²⁺exchange was characterized by aK_1/2 of ~25 mMand a Hill coefficient of 1.6. For these vesicles, the maximalNa⁺-inducedCa²⁺ efflux was about twice asgreat as in control vesicles. We conclude thatNa⁺/Ca²⁺exchange proteins localized in rhabdomeric membranes mediate Ca²⁺ extrusion in squid photoreceptors.