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1.
Three distinctmammalianNa+/Ca2+exchangers have been cloned: NCX1, NCX2, and NCX3. We have undertaken adetailed functional comparison of these three exchangers. Eachexchanger was stably expressed at high levels in the plasma membranesof BHK cells. Na+/Ca2+exchange activity was assessed using three different complementary techniques: Na+ gradient-dependent45Ca2+uptake into intact cells, Na+gradient-dependent45Ca2+uptake into membrane vesicles isolated from the transfected cells, andexchange currents measured using giant patches of excised cellmembrane. Apparent affinities for the transported ionsNa+ andCa2+ were markedly similar for thethree exchangers at both membrane surfaces. Likewise, generally similarresponses to changes in pH, chymotrypsin treatment, and application ofvarious inhibitors were obtained. Depletion of cellular ATP inhibitedNCX1 and NCX2 but did not affect the activity of NCX3. Exchangeactivities of NCX1 and NCX3 were modestly increased by agents thatactivate protein kinases A and C. All exchangers were regulated byintracellular Ca2+. NCX1-inducedexchange currents were especially large in excised patches and, likethe native myocardial exchanger, were stimulated by ATP. Results may beinfluenced by our choice of expression system and specific splicevariants, but, overall, the three exchangers appear to have verysimilar properties.

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2.
The Na+/Ca2+ exchanger is the major Ca2+ extrusion mechanism in cardiac myocytes. The activity of the cardiac Na+/Ca2+ exchanger is dynamically regulated by intracellular Ca2+. Previous studies indicate that Ca2+ binding to a high-affinity Ca2+-binding domain (CBD1) in the large intracellular loop is involved in regulation. We generated transgenic zebrafish with cardiac-specific expression of CBD1 linked to yellow and cyan fluorescent protein. Ca2+ binding to CBD1 induces conformational changes, as detected by fluorescence resonance energy transfer. With this transgenic fish model, we were able to monitor conformational changes of the Ca2+ regulatory domain of Na+/Ca2+ exchanger in intact hearts. Treatment with the positive inotropic agents ouabain and isoproterenol increased both Ca2+ transients and Ca2+-induced changes in fluorescence resonance energy transfer. The results indicate that Ca2+ regulation of the Na+/Ca2+ exchanger domain CBD1 changes with inotropic state. The transgenic fish models will be useful to further characterize the regulatory properties of the Na+/Ca2+ exchanger in vivo. Ca2+-binding domain; sodium/calcium exchange; zebrafish; fluorescence resonance energy transfer  相似文献   

3.
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 (INCX) and the Ca2+ transient. We found that the integral of INCX, 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 (VNCX) 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 INCX 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  相似文献   

4.
La3+ uptake was measured in fura 2-loaded Chinese hamster ovary cells expressing the bovine cardiac Na+/Ca2+ exchanger (NCX1.1). La3+ was taken up by the cells after an initial lag phase of 50-60 s and achieved a steady state within 5-6 min. Neonatal cardiac myocytes accumulated La3+ in a similar manner. La3+ uptake was due to the activity of the exchanger, because no uptake was seen in nontransfected cells or in transfected cells that had been treated with gramicidin to remove cytosolic Na+. The low rate of La3+ uptake during the lag period resulted from insufficient cytosolic Ca2+ to activate the exchanger at its regulatory sites, as shown by the following observations. La3+ uptake occurred without a lag period in cells expressing a mutant of NCX1.1 that does not exhibit regulatory activation by cytosolic Ca2+. The rate of La3+ uptake by wild-type cells was increased, and the lag phase was reduced or eliminated, when the cytosolic Ca2+ concentration was increased before initiating La3+ uptake. La3+ could substitute for Ca2+ at very low concentrations to activate exchange activity. Thus preloading cells expressing NCX1.1 with a small quantity of La3+ increased the rate of exchange-mediated Ca2+ influx by 20-fold; in contrast, cytosolic La3+ partially inhibited Ca2+ uptake by the regulation-deficient mutant. With an estimated KD of 30 pM for the binding of La3+ to fura 2, we conclude that cytosolic La3+ activates exchange activity at picomolar concentrations. We speculatively suggest that endogenous trace metals might activate exchange activity under physiological conditions. fura 2; NCX1.1; myocyte  相似文献   

5.
In cardiac-specific Na+-Ca2+ exchanger (NCX) knockout (KO) mice, the ventricular action potential (AP) is shortened. The shortening of the AP, as well as a decrease of the L-type Ca2+ current (ICa), provides a critical mechanism for the maintenance of Ca2+ homeostasis and contractility in the absence of NCX (Pott C, Philipson KD, Goldhaber JI. Excitation-contraction coupling in Na+-Ca2+ exchanger knockout mice: reduced transsarcolemmal Ca2+ flux. Circ Res 97: 1288–1295, 2005). To investigate the mechanism that underlies the accelerated AP repolarization, we recorded the transient outward current (Ito) in patch-clamped myocytes isolated from wild-type (WT) and NCX KO mice. Peak Ito was increased by 78% and decay kinetics were slowed in KO vs. WT. Consistent with increased Ito, ECGs from KO mice exhibited shortened QT intervals. Expression of the Ito-generating K+ channel subunit Kv4.2 and the K+ channel interacting protein was increased in KO. We used a computer model of the murine AP (Bondarenko VE, Szigeti GP, Bett GC, Kim SJ, and Rasmusson RL. Computer model of action potential of mouse ventricular myocytes. Am J Physiol Heart Circ Physiol 287: 1378–1403, 2004) to determine the relative contributions of increased Ito, reduced ICa, and reduced NCX current (INCX) on the shape and kinetics of the AP. Reduction of ICa and elimination of INCX had relatively small effects on the duration of the AP in the computer model. In contrast, AP repolarization was substantially accelerated when Ito was increased in the computer model. Thus, the increase in Ito, and not the reduction of ICa or INCX, is likely to be the major mechanism of AP shortening in KO myocytes. The upregulation of Ito may comprise an important regulatory mechanism to limit Ca2+ influx via a reduction of AP duration, thus preventing Ca2+ overload in situations of reduced myocyte Ca2+ extrusion capacity. genetically altered mice; cardiac myocytes; short QT interval; transient outward current  相似文献   

6.
Physiologicalfunctions of the intracellular regulatory domains of theNa+/Ca2+ exchanger NCX1 were studied byexamining Ca2+ handling in CCL39 cells expressing alow-affinity Ca2+ regulatory site mutant (D447V/D498I), anexchanger inhibitory peptide (XIP) region mutant displaying noNa+ inactivation (XIP-4YW), or a mutant lacking most of thecentral cytoplasmic loop (246-672). We found that D447V/D498Iwas unable to efficiently extrude Ca2+ from the cytoplasm,particularly during a small rise in intracellular Ca2+concentration induced by the physiological agonist -thrombin orthapsigargin. The same mutant took up Ca2+ much lessefficiently than the wild-type NCX1 in Na+-free medium whentransfectants were not loaded with Na+, although itappeared to take up Ca2+ normally in transfectantspreloaded with Na+. XIP-4YW and, to a lesser extent,246-672, but not NCX1 and D447V/D498I, markedly accelerated theloss of viability of Na+-loaded transfectants. Furthermore,XIP-4YW was not activated by phorbol ester, whereas XIP-4YW andD447V/D498I were resistant to inhibition by ATP depletion. The resultssuggest that these regulatory domains play important roles in thephysiological and pathological Ca2+ handling by NCX1, aswell as in the regulation of NCX1 by protein kinase C or ATP depletion.

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7.
High concentrations of cytosolic Na+ ions induce the time-dependent formation of an inactive state of the Na+/Ca2+ exchanger (NCX), a process known as Na+-dependent inactivation. NCX activity was measured as Ca2+ uptake in fura 2-loaded Chinese hamster ovary (CHO) cells expressing the wild-type (WT) NCX or mutants that are hypersensitive (F223E) or resistant (K229Q) to Na+-dependent inactivation. As expected, 1) Na+-dependent inactivation was promoted by high cytosolic Na+ concentration, 2) the F223E mutant was more susceptible than the WT exchanger to inactivation, whereas the K229Q mutant was resistant, and 3) inactivation was enhanced by cytosolic acidification. However, in contrast to expectations from excised patch studies, 1) the WT exchanger was resistant to Na+-dependent inactivation unless cytosolic pH was reduced, 2) reducing cellular phosphatidylinositol-4,5-bisphosphate levels did not induce Na+-dependent inactivation in the WT exchanger, 3) Na+-dependent inactivation did not increase the half-maximal cytosolic Ca2+ concentration for allosteric Ca2+ activation, 4) Na+-dependent inactivation was not reversed by high cytosolic Ca2+ concentrations, and 5) Na+-dependent inactivation was partially, but transiently, reversed by an increase in extracellular Ca2+ concentration. Thus Na+-dependent inactivation of NCX expressed in CHO cells differs in several respects from the inactivation process measured in excised patches. The refractoriness of the WT exchanger to Na+-dependent inactivation suggests that this type of inactivation is unlikely to be a strong regulator of exchange activity under physiological conditions but would probably act to inhibit NCX-mediated Ca2+ influx during ischemia. ischemia; cytosolic calcium concentration; cytosolic sodium concentration; cellular phosphatidylinositol-4,5-bisphosphate  相似文献   

8.
A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMC) is an important stimulus for cell contraction, migration, and proliferation. Depletion of intracellular Ca2+ stores opens store-operated Ca2+ channels (SOC) and causes Ca2+ entry. Transient receptor potential (TRP) cation channels that are permeable to Na+ and Ca2+ are believed to form functional SOC. Because sarcolemmal Na+/Ca2+ exchanger has also been implicated in regulating [Ca2+]cyt, this study was designed to test the hypothesis that the Na+/Ca2+ exchanger (NCX) in cultured human PASMC is functionally involved in regulating [Ca2+]cyt by contributing to store depletion-mediated Ca2+ entry. RT-PCR and Western blot analyses revealed mRNA and protein expression for NCX1 and NCKX3 in cultured human PASMC. Removal of extracellular Na+, which switches the Na+/Ca2+ exchanger from the forward (Ca2+ exit) to reverse (Ca2+ entry) mode, significantly increased [Ca2+]cyt, whereas inhibition of the Na+/Ca2+ exchanger with KB-R7943 (10 µM) markedly attenuated the increase in [Ca2+]cyt via the reverse mode of Na+/Ca2+ exchange. Store depletion also induced a rise in [Ca2+]cyt via the reverse mode of Na+/Ca2+ exchange. Removal of extracellular Na+ or inhibition of the Na+/Ca2+ exchanger with KB-R7943 attenuated the store depletion-mediated Ca2+ entry. Furthermore, treatment of human PASMC with KB-R7943 also inhibited cell proliferation in the presence of serum and growth factors. These results suggest that NCX is functionally expressed in cultured human PASMC, that Ca2+ entry via the reverse mode of Na+/Ca2+ exchange contributes to store depletion-mediated increase in [Ca2+]cyt, and that blockade of the Na+/Ca2+ exchanger in its reverse mode may serve as a potential therapeutic approach for treatment of pulmonary hypertension. sodium-calcium exchange; calcium homeostasis; vascular smooth muscle  相似文献   

9.
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.  相似文献   

10.
Na+/Ca2+ exchangers are low affinity, high capacity transporters that rapidly transport calcium at the plasma membrane, mitochondrion, endoplasmic (and sarcoplasmic) reticulum, and the nucleus. Na+/Ca2+ exchangers are widely expressed in diverse cell types where they contribute homeostatic balance to calcium levels. In animals, Na+/Ca2+ exchangers are divided into three groups based upon stoichiometry: Na+/Ca2+ exchangers (NCX), Na+/Ca2+/K+ exchangers (NCKX), and Ca2+/Cation exchangers (CCX). In mammals there are three NCX genes, five NCKX genes and one CCX (NCLX) gene. The genome of the nematode Caenorhabditis elegans contains ten Na+/Ca2+ exchanger genes: three NCX; five CCX; and two NCKX genes. Here we set out to characterize structural and taxonomic specializations within the family of Na+/Ca2+ exchangers across the phylum Nematoda. In this analysis we identify Na+/Ca2+ exchanger genes from twelve species of nematodes and reconstruct their phylogenetic and evolutionary relationships. The most notable feature of the resulting phylogenies was the heterogeneous evolution observed within exchanger subtypes. Specifically, in the case of the CCX exchangers we did not detect members of this class in three Clade III nematodes. Within the Caenorhabditis and Pristionchus lineages we identify between three and five CCX representatives, whereas in other Clade V and also Clade IV nematode taxa we only observed a single CCX gene in each species, and in the Clade III nematode taxa that we sampled we identify NCX and NCKX encoding genes but no evidence of CCX representatives using our mining approach. We also provided re-annotation for predicted CCX gene structures from Heterorhabditis bacteriophora and Caenorhabditis japonica by RT-PCR and sequencing. Together, these findings reveal a complex picture of Na+/Ca2+ transporters in nematodes that suggest an incongruent evolutionary history of proteins that provide central control of calcium dynamics.  相似文献   

11.
A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMC) is a trigger for pulmonary vasoconstriction and a stimulus for PASMC proliferation and migration. Multiple mechanisms are involved in regulating [Ca2+]cyt in human PASMC. The resting [Ca2+]cyt and Ca2+ entry are both increased in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH), which is believed to be a critical mechanism for sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in these patients. Here we report that protein expression of NCX1, an NCX family member of Na+/Ca2+ exchanger proteins is upregulated in PASMC from IPAH patients compared with PASMC from normal subjects and patients with other cardiopulmonary diseases. The Na+/Ca2+ exchanger operates in a forward (Ca2+ exit) and reverse (Ca2+ entry) mode. By activating the reverse mode of Na+/Ca2+ exchange, removal of extracellular Na+ caused a rapid increase in [Ca2+]cyt, which was significantly enhanced in IPAH PASMC compared with normal PASMC. Furthermore, passive depletion of intracellular Ca2+ stores using cyclopiazonic acid (10 µM) not only caused a rise in [Ca2+]cyt due to Ca2+ influx through store-operated Ca2+ channels but also mediated a rise in [Ca2+]cyt via the reverse mode of Na+/Ca2+ exchange. The upregulated NCX1 in IPAH PASMC led to an enhanced Ca2+ entry via the reverse mode of Na+/Ca2+ exchange, but did not accelerate Ca2+ extrusion via the forward mode of Na+/Ca2+ exchange. These observations indicate that the upregulated NCX1 and enhanced Ca2+ entry via the reverse mode of Na+/Ca2+ exchange are an additional mechanism responsible for the elevated [Ca2+]cyt in PASMC from IPAH patients. transient receptor potential channel; reverse and forward mode; proliferation  相似文献   

12.
The cardiac sarcolemmal Na+-Ca2+ exchanger is modulated by intrinsic regulatory mechanisms. A large intracellular loop of the exchanger participates in the regulatory responses. We have proposed (Li, Z., D.A. Nicoll, A. Collins, D.W. Hilgemann, A.G. Filoteo, J.T. Penniston, J.N. Weiss, J.M. Tomich, and K.D. Philipson. 1991. J. Biol. Chem. 266:1014–1020) that a segment of the large intracellular loop, the endogenous XIP region, has an autoregulatory role in exchanger function. We now test this hypothesis by mutational analysis of the XIP region. Nine XIP-region mutants were expressed in Xenopus oocytes and all displayed altered regulatory properties. The major alteration was in a regulatory mechanism known as Na+-dependent inactivation. This inactivation is manifested as a partial decay in outward Na+-Ca2+ exchange current after application of Na+ to the intracellular surface of a giant excised patch. Two mutant phenotypes were observed. In group 1 mutants, inactivation was markedly accelerated; in group 2 mutants, inactivation was completely eliminated. All mutants had normal Na+ affinities. Regulation of the exchanger by nontransported, intracellular Ca2+ was also modified by the XIP-region mutations. Binding of Ca2+ to the intracellular loop activates exchange activity and also decreases Na+-dependent inactivation. XIP-region mutants were all still regulated by Ca2+. However, the apparent affinity of the group 1 mutants for regulatory Ca2+ was decreased. The responses of all mutant exchangers to Ca2+ application or removal were markedly accelerated. Na+-dependent inactivation and regulation by Ca2+ are interrelated and are not completely independent processes. We conclude that the endogenous XIP region is primarily involved in movement of the exchanger into and out of the Na+-induced inactivated state, but that the XIP region is also involved in regulation by Ca2+.  相似文献   

13.
A precise temporal and spatial control of intracellular Ca2+ concentration is essential for a coordinated contraction of the heart. Following contraction, cardiac cells need to rapidly remove intracellular Ca2+ to allow for relaxation. This task is performed by two transporters: the plasma membrane Na+-Ca2+ exchanger (NCX) and the sarcoplasmic reticulum (SR) Ca2+‐ATPase (SERCA). NCX extrudes Ca2+ from the cell, balancing the Ca2+entering the cytoplasm during systole through L-type Ca2+ channels. In parallel, following SR Ca2+ release, SERCA activity replenishes the SR, reuptaking Ca2+ from the cytoplasm.The activity of the mammalian exchanger is fine-tuned by numerous ionic allosteric regulatory mechanisms. Micromolar concentrations of cytoplasmic Ca2+ potentiate NCX activity, while an increase in intracellular Na+ levels inhibits NCX via a mechanism known as Na+-dependent inactivation. Protons are also powerful inhibitors of NCX activity. By regulating NCX activity, Ca2+, Na+ and H+ couple cell metabolism to Ca2+ homeostasis and therefore cardiac contractility. This review summarizes the recent progress towards the understanding of the molecular mechanisms underlying the ionic regulation of the cardiac NCX with special emphasis on pH modulation and its physiological impact on the heart.  相似文献   

14.
We investigated the roles and relationships of plasma membrane Ca2+-ATPase (PMCA), sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2, and Na+/Ca2+ exchanger (NCX) in bladder smooth muscle contractility in Pmca-ablated mice: Pmca4-null mutant (Pmca4–/–) and heterozygous Pmca1 and homozygous Pmca4 double gene-targeted (Pmca1+/–Pmca4–/–) mice. Gene manipulation did not alter the amounts of PMCA1, SERCA2, and NCX. To study the role of each Ca2+ transport system, contraction of circular ring preparations was elicited with KCl (80 mM) plus atropine, and then the muscle was relaxed with Ca2+-free physiological salt solution containing EGTA. We measured the contributions of Ca2+ clearance components by inhibiting SERCA2 (with 10 µM cyclopiazonic acid) and/or NCX (by replacing NaCl with N-methyl-D-glucamine/HCl plus 10 µM KB-R7943). Contraction half-time (time to 50% of maximum tension) was prolonged in the gene-targeted muscles but marginally shortened when SERCA2 or NCX was inhibited. The inhibition of NCX significantly inhibited this prolongation, suggesting that NCX activity might be augmented to compensate for PMCA4 function in the gene-targeted muscles under nonstimulated conditions. Inhibition of SERCA2 and NCX as well as gene targeting all prolonged the relaxation half-time. The contribution of PMCA to relaxation was calculated to be 25–30%, with that of SERCA2 being 20% and that of NCX being 70%. PMCA and SERCA2 appeared to function additively, but the function of NCX might overlap with those of other components. In summary, gene manipulation of PMCA indicates that PMCA, in addition to SERCA2 and NCX, plays a significant role in both excitation-contraction coupling and the Ca2+ extrusion-relaxation relationship, i.e., Ca2+ homeostasis, of bladder smooth muscle. ATP2B; sarco(endo)plasmic reticulum Ca2+-ATPase 2; Na+/Ca2+ exchanger; homeostasis  相似文献   

15.
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.  相似文献   

16.
Store-operated Ca2+ entry (SOCE), which is Ca2+ entry triggered by the depletion of intracellular Ca2+ stores, has been observed in many cell types, but only recently has it been suggested to occur in cardiomyocytes. In the present study, we have demonstrated SOCE-dependent sarcoplasmic reticulum (SR) Ca2+ loading (loadSR) that was not altered by inhibition of L-type Ca2+ channels, reverse mode Na+/Ca2+ exchange (NCX), or nonselective cation channels. In contrast, lowering the extracellular [Ca2+] to 0 mM or adding either 0.5 mM Zn2+ or the putative store-operated channel (SOC) inhibitor SKF-96365 (100 µM) inhibited loadSR at rest. Interestingly, inhibition of forward mode NCX with 30 µM KB-R7943 stimulated SOCE significantly and resulted in enhanced loadSR. In addition, manipulation of the extracellular and intracellular Na+ concentrations further demonstrated the modulatory role of NCX in SOCE-mediated SR Ca2+ loading. Although there is little knowledge of SOCE in cardiomyocytes, the present results suggest that this mechanism, together with NCX, may play an important role in SR Ca2+ homeostasis. The data reported herein also imply the presence of microdomains unique to the neonatal cardiomyocyte. These findings may be of particular importance during open heart surgery in neonates, in which uncontrolled SOCE could lead to SR Ca2+ overload and arrhythmogenesis. cardiac ontogeny; cardiac excitation-contraction coupling; calcium homeostasis  相似文献   

17.
We hypothesized that activation of heat shock protein 70 (HSP70) by preconditioning, which is known to confer delayed cardioprotection, attenuates the impaired handling of Ca2+ at multiple sites. To test the hypothesis, we determined how the ryanodine receptor (RyR), sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), and Na+/Ca2+ exchanger (NCX) handled Ca2+ in rat ventricular myocytes preconditioned with a -opioid receptor agonist, U50488H (UP), followed by blockade of HSP70 with a selective antisense oligonucleotide and subsequently subjected to simulated ischemia. We determined the following: 1) the Ca2+ transients induced by electrical stimulation and caffeine, which provide the overall picture of Ca2+ homeostasis; 2) expression of RyR, SERCA, and NCX; and 3) Ca2+ fluxes via NCX by the use of 45Ca2+ in the rat ventricular myocyte. We found that UP increased the activity of RyR, SERCA, and NCX and the expression of RyR and SERCA. These effects led to increases in the release of Ca2+ from the sarcoplasmic reticulum via RyR and in the removal of Ca2+ from the cytoplasm by reuptake of Ca2+ to the SR via SERCA and by extrusion of Ca2+ out of the cell via NCX. UP also reduced mitochondrial Ca2+ accumulation. All of the effects of UP were either abolished or significantly attenuated by blockade of HSP70 synthesis with a selective antisense oligonucleotide. The results are evidence that activation of HSP70 by preconditioning improves the ischemia-impaired Ca2+ homeostasis at multiple sites in the heart, which may be responsible, at least partly, for attenuated Ca2+ overload, improved recovery in contractile function, and cardioprotection. intracellular Ca2+, -opioid receptor; Na+/Ca2+ exchanger; ryanodine receptor; sarco(endo)plasmic reticulum Ca2+-ATPase  相似文献   

18.
We compared the properties of three mammalianNa+/Ca2+exchanger isoforms, NCX1, NCX2, and NCX3, by analyzing the effects of Ni2+ and other cations as well asthe recently identified inhibitor isothiourea derivatives onintracellular Na+-dependent45Ca2+uptake into CCL-39 (Dede) fibroblasts stably expressingeach isoform. All these NCX isoforms had similar affinities for the extracellular transport substratesCa2+ andNa+.Ni2+ inhibited45Ca2+uptake by competing with Ca2+ forthe external transport site, with 10-fold less affinity in NCX3 than inNCX1 or NCX2. Ni2+ andCo2+ were most efficient in suchdiscrimination of NCX isoforms, although their inhibitory potencieswere less than those of La3+ andCd2+. The monovalent cationLi+ stimulated45Ca2+uptake rate by all NCX isoforms similarly with low affinity, althoughthe extent of stimulation was somewhat smaller in NCX1. On the otherhand, the isothiourea derivative KB-R7943 was threefold more inhibitoryto NCX3 than to NCX1 or NCX2. Thus distinct differences in the kineticand pharmacological properties were detected between NCX3 and the othertwo isoforms.

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19.
The Na+-Ca2+ exchanger (NCX) links transmembrane movements of Ca2+ ions to the reciprocal movement of Na+ ions. It normally functions primarily as a Ca2+ efflux mechanism in excitable tissues such as the heart, but it can also mediate Ca2+ influx under certain conditions. Na+ and Ca2+ ions exert complex regulatory effects on NCX activity. Ca2+ binds to two regulatory sites in the exchanger's central hydrophilic domain, and this interaction is normally essential for activation of exchange activity. High cytosolic Na+ concentrations, however, can induce a constitutive activity that by-passes the need for allosteric Ca2+ activation. Constitutive NCX activity can also be induced by high levels of phopshotidylinositol-4,5-bisphosphate (PIP2) and by mutations affecting the regulatory calcium binding domains. In addition to promoting constitutive activity, high cytosolic Na+ concentrations also induce an inactivated state of the exchanger (Na+-dependent inactivation) that becomes dominant when cytosolic pH and PIP2 levels fall. Na+-dependent inactivation may provide a means of protecting cells from Ca2+ overload due to NCX-mediated Ca2+ influx during ischemia.  相似文献   

20.
The cardiacNa+/Ca2+ exchanger (NCX) in troutexhibits profoundly lower temperature sensitivity in comparison to themammalian NCX. In this study, we attempt to characterize the regions of the NCX molecule that are responsible for its temperature sensitivity. Chimeric NCX molecules were constructed using wild-type trout andcanine NCX cDNA and expressed in Xenopus oocytes.NCX-mediated currents were measured at 7, 14, and 30°C using thegiant excised-patch technique. By using this approach, the differentialtemperature dependence of NCX was found to reside within theNH2-terminal region of the molecule. Specifically, we foundthat ~75% of the Na+/Ca2+ exchangedifferential energy of activation is attributable to sequencedifferences in the region that include the first four transmembranesegments, and the remainder is attributable to transmembrane segmentfive and the exchanger inhibitory peptide site.

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