Regulation of the Na+/H+ exchanger in fibroblasts overexpressing the Na+/Ca2+ exchanger

To assess the role of Ca²⁺in regulation of theNa⁺/H⁺exchanger (NHE1), we used CCL-39 fibroblasts overexpressing theNa⁺/Ca²⁺exchanger (NCX1). Expression of NCX1 markedly inhibited the transient cytoplasmic Ca²⁺ rise andlong-lasting cytoplasmic alkalinization (60-80% inhibition) induced by -thrombin. In contrast, coexpression of NCX1 did not inhibit this alkalinization in cells expressing the NHE1 mutant withthe calmodulin (CaM)-binding domain deleted (amino acids 637-656),suggesting that the effect of NCX1 transfection involves Ca²⁺-CaM binding. Expression ofNCX1 only slightly inhibited platelet-derived growth factor BB-inducedalkalinization and did not affect hyperosmolarity- or phorbol12-myristate 13-acetate-induced alkalinization. Downregulation ofprotein kinase C (PKC) inhibited thrombin-induced alkalinization partially in control cells and abolished it completely inNCX1-transfected cells, suggesting that the thrombin effect is mediatedexclusively via Ca²⁺ and PKC. Onthe other hand, deletion mutant study revealed that PKC-dependentregulation occurs through a small cytoplasmic segment (amino aids566-595). These data suggest that a mechanism involving directCa²⁺-CaM binding lasts for arelatively long period after agonist stimulation, despite apparentshort-lived Ca²⁺ mobilization, andfurther support our previous conclusion that Ca²⁺- and PKC-dependent mechanismsare mediated through distinct segments of the NHE1 cytoplasmic domain.

     

Amiloride and amiloride analogs inhibit Na+/K+-transporting ATPase and Na+-coupled alanine transport in rat hepatocytes

Amiloride, a commonly used inhibitor of Na+-H+ exchange, has been shown to exhibit a variety of nonspecific effects. Recently, the more potent amiloride analogs, 5-(N,N-dimethyl)amiloride hydrochloride (DMA) and 5-(N-ethyl-N-isopropyl)amiloride (EIA), have been used to control for the nonspecific effects of the parent compound. In the present study, we have explored the effects of these analogs on Na+/K+-transporting ATPase (Na+/K+-ATPase) and Na+-coupled alanine transport in primary rat hepatocyte cultures and rat liver plasma membranes, and we have compared the effects of these analogs with the effects of amiloride and ouabain. Amiloride, DMA, and EIA increased steady-state Na+ content and inhibited ouabain-sensitive 86Rb+ uptake in a reversible, concentration-dependent, ouabain-like manner, with estimated 50% inhibitory concentrations (IC50) of 3.0.10(-3) M, 5.2.10(-4) M, and 1.2.10(-4) M, respectively. Amiloride, DMA and EIA also inhibited ouabain-sensitive ATP hydrolysis in rat liver plasma membranes with similar potency (IC50 values of 2.2.10(-3) M, 2.2.10(-3) M, and 1.7.10(-4) M, respectively). In separate experiments, amiloride (5.10(-3) M), DMA (10(-3) M), and EIA (2.5.10(-4) M) decreased the uptake into hepatocytes of alanine by 20%, 61%, and 59%, respectively, and further studies with DMA (10(-3) M) demonstrated that this inhibition was largely due to a decrease in the Na+-dependent fraction of alanine uptake. These findings indicate that amiloride, DMA, and EIA inhibit hepatic Na+/K+-ATPase directly, reversibly, and with a relative rank order potency of EIA greater than DMA greater than amiloride. All three compounds also inhibit the hepatic uptake of alanine, and presumably could indirectly inhibit other Na+-coupled transport processes as well.     

Rapid Ca2+ extrusion via the Na+/Ca2+ exchanger of the human platelet

Summary This communication reports the kinetics of the Na⁺/ Ca²⁺ exchanger and of the plasma membrane (PM) Ca²⁺ pump of the intact human platelet. The kinetic properties of these two systems were deduced by studying the rate of Ca²⁺ extrusion and its Na⁺ dependence for concentrations of cytoplasmic free Ca²⁺ ([Ca²⁺]_cyt) in the 1–10-m range. The PM Ca²⁺ATPase was previously characterized (Johansson, J.S. Haynes, D.H. 1988. J. Membrane Biol. 104:147–163) for [Ca²⁺]_cyt] 1.5 m with the fluorescent Ca²⁺ indicator quin2 (K _d= 115 nm). That study determined that the PM Ca²⁺ pump in the basal state has a V _max = 0.098 mm/min, a K _m= 80 nm and a Hill coefficient = 1.7. The present study extends the measurable range of [Ca²⁺]_cyt with the intracellular Ca²⁺ probe, rhod2 (K _d= 500 nm), which has almost a fivefold lower affinity for Ca²⁺. An Appendix also describes the Mg²⁺ and pH dependence of the K _dand fluorescence characteristics of the commercially available dye, which is a mixture of two molecules. Rates of active Ca²⁺ extrusion were determined by two independent methods which gave good agreement: (i) by measuring Ca²⁺ extrusion into a Ca²⁺-free medium (above citation) or (ii) by the newly developed ionomycin short-circuit method, which determines the ionomycin concentration necessary to short circuit the PM Ca²⁺ extrusion systems. Absolute rates of extrusion were determined by knowledge of how many Ca²⁺ ions are moved by ionomycin per minute. The major findings are as follows: (i) The exchanger is saturable with respect to Ca²⁺ with a K _m= 0.97 ± 0.31 m and V_max = 1.0 ± 0.6 mm/ min. (ii) At high [Ca²⁺]_cyt, the exchanger works at a rate 10 times as large as the basal V _max of the PM Ca²⁺ extrusion pump. (iii) The exchanger can work in reverse after Na⁺ loading of the cytoplasm by monensin. (iv) The PM Ca²⁺ extrusion pump is activated by exposure to [Ca²⁺]_cyt 1.5 m for 20–50 sec. Activation raises the pump V _max to 1.6 ± 0.6 mm/min and the K _mto 0.55 ± 0.24 m. (v) The Ca²⁺ buffering capacity of the cytoplasm is 3.6 mm in the 0.1 to 3 m range of [Ca²⁺]_cyt. In summary, the results show that the human platelet can extrude Ca²⁺ very rapidly at high [Ca²⁺]_cyt. Both the Na⁺/Ca²⁺ exchanger and Ca²⁺ pump activation may prevent inappropriate platelet activation by marginal stimuli.Abbreviations cAMP cyclic adenosine 3,5-monophosphate - cGMP cyclic guanosine 3,5,-monophosphate - Ca-CAM calcium calmodulin; - DT dense tubules - B intrinsic cytoplasmic Ca²⁺ binding sites - R rhod2 or 5-(3,6-bis(dimethylamino)xanth-9-yl)-1-(2-amino-4-hy droxy lphenoxy)-2-(2-amino-5-methylphen- oxy)ethane-N,N,NN-tetraacetic acid - [Ca²⁺]_cyt cytoplasmic Ca²⁺ activity - quin2 2-[[2-bis[(carboxymethyl)amino]-5-methyl-phenoxy]methyl]-6-methoxy-8-[bis(carboxymethyl)amino]quinoline - V or V_extrusion true rate of Ca²⁺ extrusion - fura-2 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,NN-tetraacetic acid - AM acetoxymethyl ester - DMSO dimethylsulfoxide - CTC chlortetracycline - EGTA ethyleneglycol-bis(-aminoethyl ether) N,N,N,N- tetraacetic acid - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - NMDG N-methyl-d-glucamine - PIPES 1,4-piperazine-bis-(ethanesulfonic acid) - HPLC high performance liquid chromatography - _I fraction of high-affinity rhod2 complexed with Ca²⁺ - F the observed fluorescence - F_min the minimal fluorescence observed in the absence of Ca²⁺ - F_max the maximal fluorescence observed when the dye is saturated with Ca²⁺ - X₁ the fraction of high-affinity dye - K _d,1 dissociation constant of high-affinity dye - K _d,2 dissociation constant of the low-affinity dye - -d₁/dt rate of Ca²⁺ removal from the rhod2-Ca complex; - -dF/dt the slope representing the absolute rate of fluorescence decrease in a progress curve - F_max (F_max — F_min)_cyt difference between maximal and minimal fluorescence for cytoplasmic high affinity form of rhod2 - F₅₀ fluorescence of the high-affinity form ofrhod2for[Ca²⁺]_cyt=50 nM - [Ca²⁺]₀ external Ca²⁺concentration - K _p proportionality constant between the total number of Ca²⁺ ions moved and the change in high-affinity rhod2 complexation to Ca² - (d[Ca²⁺]_{cyt, T})/dt rate of Ca²⁺ influx obtained with maximal levels of ionomycin - k_leak rate constant for passive inward Ca²⁺ leakage - k_inno rate constant for ionomycin-mediated Ca²⁺ influx - T total - [rhod2]_cyt,T total intracellular rhod2 concentration - [quin2]_cyt,T total intracellular quin2 concentration - [B]_T total cytoplasmic buffering capacity - A[Ca²⁺]_cyt,T total number of Ca²⁺ ions moved into the cytoplasm - [rhod2-Ca]_{cyt, T} change in concentration of total intracellular high-affinity rhod2 complexed to Ca²⁺ - [B-Ca]_T change in concentration of total cytoplasmic binding sites complexed to Ca²⁺ - [quin2]_{cyt, T} change in concentration of total intracellular quinl complexed to Ca²⁺ - change in the degree of intracellular quin2 saturation - ₁ change in degree of saturation of cytoplasmic high-affinity rhod2 - ₁-/t rate of change in degree of saturation of cytoplasmic high affinityrhod2 - V_obs observed rate of Ca²⁺ removal from the rhod2-Ca complex - V_{8.3 m} the rate of Ca²⁺ removal from the high affinity rhod2-Ca complex at [Ca²⁺]_cyt = 8.3 m - /t rate of change in of the degree of quin2 saturation - [Ca²⁺]_cytT/_t initial linear rate of ionomycin-mediated Ca²⁺ influx - EC₅₀ effective concentration giving a half-maximal effect - [Na⁺]_cyt cytoplasmic Na⁺ activity - CAM calmodulin - ACN acetonitrile - TFA trifuloroacetic acid     

Na+/Na+ exchange and Na+/H+ antiport in rabbit erythrocytes: Two distinct transport systems

Summary Rabbit erythrocytes are well known for possessing highly active Na⁺/Na⁺ and Na⁺/H⁺ countertransport systems. Since these two transport systems share many similar properties, the possibility exists that they represent different transport modes of a single transport molecule. Therefore, we evaluated this hypothesis by measuring Na⁺ transport through these exchangers in acid-loaded cells. In addition, selective inhibitors of these transport systems such as ethylisopropyl-amiloride (EIPA) and N-ethylmaleimide (NEM) were used. Na⁺/Na⁺ exchange activity, determined as the Na _o ⁺ -dependent²²Na efflux or Na _i ⁺ -induced²²Na entry was completely abolished by NEM. This inhibitor, however, did not affect the H _i ⁺ -induced Na⁺ entry sensitive to amiloride (Na⁺/H⁺ exchange activity). Similarly, EIPA, a strong inhibitor of the Na⁺/H⁺ exchanger, did not inhibit Na⁺/Na^– countertransport, suggesting the independent nature of both transport systems. The possibility that the NEM-sensitive Na⁺/Na⁺ exchanger could be involved in Na⁺/H⁺ countertransport was suggested by studies in which the net Na⁺ transport sensitive to NEM was determined. As expected, net Na⁺ transport through this transport system was zero at different [Na⁺] _i /[Na⁺] _o ratios when intracellular pH was 7.2. However, at pH _i =6.1, net Na⁺ influx occurred when [Na⁺] _i was lower than 39mm. Valinomycin, which at low [K⁺] _o was lower than 39mm. Valinomycin, which at low [K⁺] _o clamps the membrane potential close to the K⁺ equilibrium potential, did not affect the net NEM-sensitive Na⁺ entry but markedly stimulated, the EIPA-and NEM-resistant Na⁺ uptake. This suggest that the net Na⁺ entry through the NEM-sensitive pathway at low pH _i , is mediated by an electroneutral process possibly involving Na⁺/H⁺ exchange. In contrast, the EIPA-sensitive Na⁺/H⁺ exchanger is not involved in Na⁺/Na⁺ countertransport, because Na⁺ transport through this mechanism is not affected by an increase in cell Na^– from 0.4 to 39mm. Altogether, these findings indicate that both transport systems: the Na⁺/Na⁺ and Na⁺/H⁺ exchangers, are mediated by distinct transport proteins.     

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.     

Amiloride and its analogs as tools in the study of ion transport

Amiloride inhibits most plasma membrane Na+ transport systems. We have reviewed the pharmacology of inhibition of these transporters by amiloride and its analogs. Thorough studies of the Na+ channel, the Na+/H+ exchanger, and the Na+/Ca2+ exchanger, clearly show that appropriate modification of the structure of amiloride will generate analogs with increased affinity and specificity for a particular transport system. Introduction of hydrophobic substituents on the terminal nitrogen of the guanidino moiety enhances activity against the Na+ channel; whereas addition of hydrophobic (or hydrophilic) groups on the 5-amino moiety enhances activity against the Na+/H+ exchanger. Activity against the Na+/Ca2+ exchanger and Ca2+ channel is increased with hydrophobic substituents at either of these sites. Appropriate modification of amiloride has produced analogs that are several hundred-fold more active than amiloride against specific transporters. The availability of radioactive and photoactive amiloride analogs, anti-amiloride antibodies, and analogs coupled to support matrices should prove useful in future studies of amiloride-sensitive transport systems. The use of amiloride and its analogs in the study of ion transport requires a knowledge of the pharmacology of inhibition of transport proteins, as well as effects on enzymes, receptors, and other cellular processes, such as DNA, RNA, and protein synthesis, and cellular metabolism. One must consider whether the effects seen on various cellular processes are direct or due to a cascade of events triggered by an effect on an ion transport system.     

The topology of the C-terminal sections of the NCX1 Na+/Ca2+ exchanger and the NCKX2 Na+/Ca2+-K+ exchanger

Mammalian Na⁺/Ca²⁺ (NCX) and Na⁺/Ca²⁺-K⁺ exchangers (NCKX) are polytopic membrane proteins that play critical roles in calcium homeostasis in many cells. Although hydropathy plots for NCX and NCKX are very similar, reported topological models for NCX1 and NCKX2 differ in the orientation of the three C-terminal transmembrane segments (TMS). NCX1 is thought to have 9 TMS and a re-entrant loop, whereas NCKX2 is thought to have 10 TMS. The current topological model of NCKX2 is very similar to the 10 membrane spanning helices seen in the recently reported crystal structure of NCX_MJ, a distantly related archaebacterial Na⁺/Ca²⁺ exchanger. Here we reinvestigate the orientation of the three C-terminal TMS of NCX1 and NCKX2 using mass-tagging experiments of substituted cysteine residues. Our results suggest that NCX1, NCKX2 and NCX_MJ all share the same 10 TMS topology.     

Contribution of the Na+/Ca2+ exchanger to rapid Ca2+ release in cardiomyocytes

Trigger Ca(2+) is considered to be the Ca(2+) current through the L-type Ca(2+) channel (LTCC) that causes release of Ca(2+) from the sarcoplasmic reticulum. However, cell contraction also occurs in the absence of the LTCC current (I(Ca)). In this article, we investigate the contribution of the Na(+)/Ca(2+) exchanger (NCX) to the trigger Ca(2+). Experimental data from rat cardiomyocytes using confocal microscopy indicating that inhibition of reverse mode Na(+)/Ca(2+) exchange delays the Ca(2+) transient by 3-4 ms served as a basis for the mathematical model. A detailed computational model of the dyadic cleft (fuzzy space) is presented where the diffusion of both Na(+) and Ca(2+) is taken into account. Ionic channels are included at discrete locations, making it possible to study the effect of channel position and colocalization. The simulations indicate that if a Na(+) channel is present in the fuzzy space, the NCX is able to bring enough Ca(2+) into the cell to affect the timing of release. However, this critically depends on channel placement and local diffusion properties. With fuzzy space diffusion in the order of four orders of magnitude lower than in water, triggering through LTCC alone was up to 5 ms slower than with the presence of a Na(+) channel and NCX.     

Proton-sensing Ca2+ binding domains regulate the cardiac Na+/Ca2+ exchanger

The cardiac Na(+)/Ca(2+) exchanger (NCX) regulates cellular [Ca(2+)](i) and plays a central role in health and disease, but its molecular regulation is poorly understood. Here we report on how protons affect this electrogenic transporter by modulating two critically important NCX C(2) regulatory domains, Ca(2+) binding domain-1 (CBD1) and CBD2. The NCX transport rate in intact cardiac ventricular myocytes was measured as a membrane current, I(NCX), whereas [H(+)](i) was varied using an ammonium chloride "rebound" method at constant extracellular pH 7.4. At pH(i) = 7.2 and [Ca(2+)](i) < 120 nM, I(NCX) was less than 4% that of its maximally Ca(2+)-activated value. I(NCX) increases steeply at [Ca(2+)](i) between 130-150 nM with a Hill coefficient (n(H)) of 8.0 ± 0.7 and K(0.5) = 310 ± 5 nM. At pH(i) = 6.87, the threshold of Ca(2+)-dependent activation of I(NCX) was shifted to much higher [Ca(2+)](i) (600-700 nM), and the relationship was similarly steep (n(H) = 8.0±0.8) with K(0.5) = 1042 ± 15 nM. The V(max) of Ca(2+)-dependent activation of I(NCX) was not significantly altered by low pH(i). The Ca(2+) affinities for CBD1 (0.39 ± 0.06 μM) and CBD2 (K(d) = 18.4 ± 6 μM) were exquisitely sensitive to [H(+)], decreasing 1.3-2.3-fold as pH(i) decreased from 7.2 to 6.9. This work reveals for the first time that NCX can be switched off by physiologically relevant intracellular acidification and that this depends on the competitive binding of protons to its C(2) regulatory domains CBD1 and CBD2.     

Na+/H+ exchanger inhibitor cariporide attenuates the mitochondrial Ca2+ overload and PTP opening

The Na(+)/H(+) exchanger (NHE) inhibitor cariporide has a cardioprotective effect in various animal models of myocardial ischemia-reperfusion. Recent studies have suggested that cariporide interacts with mitochondrial Ca(2+) overload and the mitochondrial permeability transition (MPT); however, the precise mechanisms remain unclear. Therefore, we examined whether cariporide affects mitochondrial Ca(2+) overload and MPT. Isolated adult rat ventricular myocytes were used to study the effects of cariporide on hypercontracture induced by ouabain or phenylarsine oxide (PAO). Mitochondrial Ca(2+) concentration ([Ca(2+)](m)) and the mitochondrial membrane potential (DeltaPsi(m)) were measured by loading myocytes with rhod-2 and JC-1, respectively. We also examined the effect of cariporide on the MPT using tetramethylrhodamine methyl ester (TMRM) and oxidative stress generated by laser illumination. Cariporide (1 microM) prevented ouabain-induced hypercontracture (from 40 +/- 2 to 24 +/- 2%, P < 0.05) and significantly attenuated ouabain-induced [Ca(2+)](m) overload (from 149 +/- 6 to 121 +/- 5% of the baseline value, P < 0.05) but did not affect DeltaPsi(m). These results indicate that cariporide attenuates the [Ca(2+)](m) overload without the accompanying depolarization of DeltaPsi(m). Moreover, cariporide increased the time taken to induce the MPT (from 79 +/- 11 to 137 +/- 20 s, P < 0.05) and also attenuated PAO-induced hypercontracture (from 59 +/- 3 to 50 +/- 4%, P < 0.05). Our data indicate that cariporide attenuates [Ca(2+)](m) overload and MPT. Thus these effects might potentially contribute to the mechanisms of cardioprotection afforded by NHE inhibitors.     

Regulation of the mitochondrial Na+/Ca2+ antiport by matrix pH

The effect of matrix pH (pHi) on the activity of the mitochondrial Na+/Ca2+ antiport has been studied using the fluorescence of SNARF-1 to monitor pHi and Na(+)-dependent efflux of accumulated Ca2+ to follow antiport activity. Heart mitochondria respiring in a KCl medium maintain a large delta pH (interior alkaline) and show optimal Na+/Ca2+ antiport only when the pH of the medium (pH0) is acid. Addition of nigericin to these mitochondria decreases delta pH and increases the membrane potential (delta psi). Nigericin strongly activates Na+/Ca2+ antiport at values of pH0 near 7.4 but inhibits antiport activity at acid pH0. When pHi is evaluated in these protocols, a sharp optimum in Na+/Ca2+ antiport activity is seen near pHi 7.6 in the presence or absence of nigericin. Activity falls off rapidly at more alkaline values of pHi. The effects of nigericin on Na+/Ca2+ antiport are duplicated by 20 mM acetate and by 3 mM phosphate. In each case the optimum rate of Na+/Ca2+ antiport is obtained at pHi 7.5 to 7.6 and changes in antiport activity do not correlate with changes in components of the driving force of the reaction (i.e., delta psi, delta pH, or the steady-state Na+ gradient). It is concluded that the Na+/Ca2+ antiport of heart mitochondria is very sensitive to matrix [H+] and that changes in pHi may contribute to the regulation of matrix Ca2+ levels.     

Ni2+ transport by the human Na+/Ca2+ exchanger expressed in Sf 9cells

The mechanism ofNi²⁺ block of theNa⁺/Ca²⁺exchanger was examined in Sf 9 cells expressing the human heartNa⁺/Ca²⁺exchanger (NCX1-NACA1). As predicted from the reported actions ofNi²⁺, its application reducedextracellular Na⁺-dependentchanges in intracellular Ca²⁺concentration (measured by fluo 3 fluorescence changes). However, contrary to expectation, the reduced fluorescence was accompanied bymeasured⁶³Ni²⁺entry. The⁶³Ni²⁺entry was observed in Sf 9 cells expressing theNa⁺/Ca²⁺exchanger but not in control cells. The established sequential transport mechanism of theNa⁺/Ca²⁺exchanger could be compatible with these results if one of the two iontranslocation steps is blocked byNi²⁺ and the other permitsNi²⁺ translocation. We concludethat, because Ni²⁺ entry wasinhibited by extracellular Ca²⁺and enhanced by extracellular Na⁺,the Ca²⁺ translocation step movedNi²⁺, whereas theNa⁺ translocation step wasinhibited by Ni²⁺. A model ispresented to discuss these findings.     

Cyclosporine A inhibits Ca2+-dependent stimulation of the Na+/H+ antiport in human T cells

The cyclic undecapeptide cyclosporine A (CsA) is a potent immunosuppressive agent that inhibits the initial activation of T lymphocytes. This agent appears to be most effective in blocking the action of mitogens such as concanavalin A and the calcium ionophore A23187, which cause an influx of Ca2+, but not those that may act by alternate mechanisms. These observations suggest that CsA may block a Ca2+-dependent step in T cell activation. We have shown that stimulation of the T3-T cell receptor complex-associated Ca2+ transporter activates the Na+/H+ antiport (Rosoff, P. M., and L. C. Cantley, 1985, J. Biol. Chem., 260: 14053-14059). The tumor-promoting phorbol esters, which are co-mitogenic for T cells, activate the exchanger by a separate pathway which is mediated by protein kinase C. Both the rise in intracellular Ca2+ and intracellular pH may be necessary for the successful triggering of cellular activation. In this report we show that CsA blocks the T3-T cell receptor-stimulated, Ca2+ influx-dependent activation of Na+/H+ exchange, but not the phorbol ester-mediated pathway in a transformed human T cell line. CsA inhibited mitogen-stimulation of interleukin-2 production in a separate cell line. CsA also inhibited vasopressin stimulation of the antiporter in normal rat kidney fibroblasts, but had no effect on serum or 12-O-tetradecanoyl phorbol 13-acetate stimulation. CsA did not affect serum or vasopressin or serum stimulation of normal rat kidney cell proliferation. CsA also had no effect on lipopolysaccharide or phorbol ester stimulation of Na+/H+ exchange activity or induction of differentiation in 70Z/3 pre-B lymphocytes in which these events are initiated by the protein kinase C pathway. These data suggest that mechanisms of activation of Na+/H+ exchange that involve an elevation in cytosolic Ca2+ are blocked by CsA but that C kinase-mediated regulation is unaffected. The importance of the Na+/H+ antiport in the regulation of growth and differentiation of T cells is discussed.     

Low density lipoproteins inhibit the Na+/H+ antiport in human platelets via activation of p38MAP kinase

Low density lipoproteins (LDL) inhibit the Na+/H+ antiport and thereby sensitize platelet towards agonist. However, mechanisms underlying the suppressing effect of LDL on Na+/H+ exchange are unclear. We here show that the lowering of intracellular pH and the suppression of the sodium propionate-induced Na+/H+ exchange in the presence of LDL are abolished by SKF86002, a selective inhibitor of p38MAP kinase (p38MAPK). The inhibitory effect of LDL on Na+/H+ exchange was mimicked by H2O2, which directly activates p38MAPK. Exposure of platelets to LDL or H2O2 led to phosphorylation of p38MAPK, its upstream regulator MAP kinase kinase 3/6 (MKK 3/6), and its downstream target heat shock protein 27 (HSP27), and this effect was abrogated in SKF86002-pretreated platelets. In addition, both LDL and H2O2 produced the SKF86002-sensitive phosphorylation of an oligopeptide encompassing p38MAPK phosphorylation sequence derived from NHE-1, a major Na+/H+ exchanger in platelets. We further show that the sensitizing effects of LDL on the thrombin-induced platelet activation, as reflected by aggregation and granule secretion, are abolished in cells pretreated with SKF86002. We conclude that activation of p38MAPK is required for the inhibitory effect of LDL on Na+/H+ antiport and thereby for LDL-dependent sensitization in human platelets.     

Inhibition of the Na+/Ca2+ antiport of heart mitochondria by diethylpyrocarbonate

Diethylpyrocarbonate inhibits Na⁺/Ca²⁺ antiport activity in isolated heart mitochondria. The inhibition is time-dependent with maximum activity developed after 5 min at 25°C. The reaction of diethylpyrocarbonate with the mitochondrial membrane is biphasic with 25–30 nmol mg^–1 reacting rapidly and an additional 30 nmol mg^–1 taken up slowly over a 30-min incubation. Inhibition of mitochondrial Na⁺/Ca²⁺ antiport by diethylpyrocarbonate decreases theV _max of the reaction, and the inhibition cannot be reversed by washing the mitochondria or addition of excess histidine. The inhibition occurs at levels of inhibitor that have little or no effect on Ca²⁺ uptake, Na⁺/H⁺ antiport, or succinate respiration. A portion of the Na⁺-dependent efflux of Ca²⁺ is insensitive to diethylpyrocarbonate and this component is abolished by diltiazem. The mechanism by which diethylpyrocarbonate inactivates Na⁺/Ca²⁺ antiport is still uncertain, but may involve the modification of an unprotonated histidine residue in the transporter.     

Ca2+ regulation in the Na+/Ca2+ exchanger involves two markedly different Ca2+ sensors

The plasma membrane Na+/Ca2+ exchanger (NCX) is almost certainly the major Ca2+ extrusion mechanism in cardiac myocytes. Binding of Na+ and Ca2+ ions to its large cytosolic loop regulates ion transport of the exchanger. We determined the solution structures of two Ca2+ binding domains (CBD1 and CBD2) that, together with an alpha-catenin-like domain (CLD), form the regulatory exchanger loop. CBD1 and CBD2 are very similar in the Ca2+ bound state and describe the Calx-beta motif. Strikingly, in the absence of Ca2+, the upper half of CBD1 unfolds while CBD2 maintains its structural integrity. Together with a 7-fold higher affinity for Ca2+, this suggests that CBD1 is the primary Ca2+ sensor. Specific point mutations in either domain largely allow the interchange of their functionality and uncover the mechanism underlying Ca2+ sensing in NCX.     

Regulation of mesangial cell Na+/Ca2+ exchanger isoforms

An isoform of the Na(+)/Ca(2+) exchanger (SDNCX1.10) was cloned from mesangial cells of Sprague-Dawley rat. Regulation of this isoform was compared to two other clones that were derived from the Dahl/Rapp salt sensitive (SNCX) and salt resistant rat (RNCX). All isoforms differ at the alternative splice site and at amino acid 218 for SNCX. PKC activates RNCX but not SNCX while SDNCX1.10 was also activated by PKC. Regulation of exchanger activities by intracellular calcium ([Ca(2+)](i)), pH, and kinases was assessed using Na-dependent (45)Ca(2+) uptake assays in OK-PTH cells expressing the vector, RNCX, SNCX, or SDNCX1.10. [Ca(2+)](i) was elevated from 50 to 125 nM (n = 4) with thapsigargin (40 nM) and reduced from 50 to 29 nM (n = 4) and 18 nM (n = 4) with 10 or 20 microM BAPTA, respectively. RNCX was active at all three [Ca(2+)](i) while SNCX and SDNCX1.10 were only active at lower [Ca(2+)](i). Varying extracellular pH (pH(e), without nigericin) or pH(e) and intracellular pH (pH(i), with 10 microM nigericin) from pH 7.4 to 6.2, 6.8, or 8.0 showed that SNCX activity was attenuated at both low and high pHs. SDNCX1.10 activity was attenuated only at pH 6.2 and 6.8 (with or without nigericin) while RNCX activity was attenuated at pH 6.2 (with or without nigericin) and pH 6.8 (with nigericin). Finally, only SDNCX1.10 activity was stimulated by 250 microM CPT-cAMP or 250 microM DB-cGMP treatment. Thus the differential regulation of [Ca(2+)](i) by these exchangers is dependent upon the pattern of cellular Na(+)/Ca(2+) exchanger isoform expression.     

Regulated release of Ca2+ from respiring mitochondria by Ca2+/2H+ antiport.

Simultaneous measurements of oxygen consumption and transmembrane transport of Ca2+, H+, and phosphate show that the efflux of Ca2+ from respiring tightly coupled rat liver mitochondria takes place by an electroneutral Ca2+/2H+ antiport process that is ruthenium red-insensitive and that is regulated by the oxidation-reduction state of the mitochondrial pyridine nucleotides. When mitochondrial pyridine nucleotides are kept in a reduced steady state, the efflux of Ca2+ is inhibited; when they are in an oxidized state, Ca2+ efflux is activated. These processes were demonstrated by allowing phosphate-depleted mitochondria respiring on succinate in the presence of rotenone to take up Ca2+ from the medium. Upon subsequent addition of ruthenium red to block Ca2+ transport via the electrophoretic influx pathway, and acetoacetate, to bring mitochondrial pyridine nucleotides into the oxidized state, Ca2+ efflux and H+ influx ensued. The observed H+ influx/Ca2+ efflux ratio was close to the value 2.0 predicted for the operation of an electrically neutral Ca2+/2H+ antiport process.     

Single alpha-domain constructs of the Na+/Ca2+ exchanger, NCLX, oligomerize to form a functional exchanger

Spliced isoforms of the Na+/Ca2+ exchanger, NCLX, truncated at the alpha-repeat region have been identified. The activity and functional organization of such proteins are, however, poorly understood. In the present work, we have studied Na+/Ca2+ exchange mediated by single alpha-repeat constructs (alpha1 and alpha2) of NCLX. Sodium-dependent calcium transport was fluorescently detected in both the reversal and forward modes; calcium-dependent outward currents were also recorded using a whole cell patch configuration in HEK293 cells heterologously expressing either the alpha1 or alpha2 single-domain proteins. In contrast, calcium transport and reversal currents were not detected when cells were transfected with a vector or with an alpha2 mutant (alpha2-S273T). Thus, our data indicate that the single alpha-domain constructs mediate electrogenic Na+/Ca2+ exchange. The alpha1 domain, but not the alpha2, exhibited partial sensitivity to the NCX inhibitor, KB-R7943, while Li+-dependent Ca2+ efflux was detected in cells expressing either the alpha1 or alpha2 construct. The functional organization of the single alpha-domain constructs was assessed using a dominant-negative approach. Coexpression of the alpha1 or alpha2 constructs with the nonfunctional alpha2-S273T mutant had a synergistic inhibitory effect on Na+/Ca2+ transport. Dose-dependence analysis of the inhibition of alpha2 construct activity by the alpha2-S273T mutant indicated that the functional unit is either a dimer or a trimer. Immunoprecipitation analysis indicated that the alpha2 construct indeed interacts with the alpha2-S273T mutant. Taken together, our data indicate that although single alpha1 or alpha2 domain constructs are independently capable of Na+/Ca2+ exchange, oligomerization is required for their activity. Such organization may give rise to transport activity with distinct kinetic parameters and physiological roles.