Journal articles on the topic 'Ca2+ exchanger'
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1
Hryshko, L. V., S. Matsuoka, D. A. Nicoll, J. N. Weiss, E. M. Schwarz, S. Benzer, and K. D. Philipson. "Anomalous regulation of the Drosophila Na(+)-Ca2+ exchanger by Ca2+." Journal of General Physiology 108, no. 1 (July 1, 1996): 67–74. http://dx.doi.org/10.1085/jgp.108.1.67.
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The Na(+)-Ca2+ exchanger from Drosophila was expressed in Xenopus and characterized electrophysiologically using the giant excised patch technique. This protein, termed Calx, shares 49% amino acid identity to the canine cardiac Na(+)-Ca2+ exchanger, NCX1. Calx exhibits properties similar to previously characterized Na(+)-Ca2+ exchangers including intracellular Na+ affinities, current-voltage relationships, and sensitivity to the peptide inhibitor, XIP. However, the Drosophila Na(+)-Ca2+ exchanger shows a completely opposite response to cytoplasmic Ca2+. Previously cloned Na(+)-Ca2+ exchangers (NCX1 and NCX2) are stimulated by cytoplasmic Ca2+ in the micromolar range (0.1-10 microM). This stimulation of exchange current is mediated by occupancy of a regulatory Ca2+ binding site separate from the Ca2+ transport site. In contrast, Calx is inhibited by cytoplasmic Ca2+ over this same concentration range. The inhibition of exchange current is evident for both forward and reverse modes of transport. The characteristics of the inhibition are consistent with the binding of Ca2+ at a regulatory site distinct from the transport site. These data provide a rational basis for subsequent structure-function studies targeting the intracellular Ca2+ regulatory mechanism.
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Matsuoka, S., D. A. Nicoll, L. V. Hryshko, D. O. Levitsky, J. N. Weiss, and K. D. Philipson. "Regulation of the cardiac Na(+)-Ca2+ exchanger by Ca2+. Mutational analysis of the Ca(2+)-binding domain." Journal of General Physiology 105, no. 3 (March 1, 1995): 403–20. http://dx.doi.org/10.1085/jgp.105.3.403.
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The sarcolemmal Na(+)-Ca2+ exchanger is regulated by intracellular Ca2+ at a high affinity Ca2+ binding site separate from the Ca2+ transport site. Previous data have suggested that the Ca2+ regulatory site is located on the large intracellular loop of the Na(+)-Ca2+ exchange protein, and we have identified a high-affinity 45Ca2+ binding domain on this loop (Levitsky, D. O., D. A. Nicoll, and K. D. Philipson. 1994. Journal of Biological Chemistry. 269:22847-22852). We now use electrophysiological and mutational analyses to further define the Ca2+ regulatory site. Wild-type and mutant exchangers were expressed in Xenopus oocytes, and the exchange current was measured using the inside-out giant membrane patch technique. Ca2+ regulation was measured as the stimulation of reverse Na(+)-Ca2+ exchange (intracellular Na+ exchanging for extracellular Ca2+) by intracellular Ca2+. Single-site mutations within two acidic clusters of the Ca2+ binding domain lowered the apparent Ca2+ affinity at the regulatory site from 0.4 to 1.1-1.8 microM. Mutations had parallel effects on the affinity of the exchanger loop for 45Ca2+ binding (Levitsky et al., 1994) and for functional Ca2+ regulation. We conclude that we have identified the functionally important Ca2+ binding domain. All mutant exchangers with decreased apparent affinities at the regulatory Ca2+ binding site also have a complex pattern of altered kinetic properties. The outward current of the wild-type Na(+)-Ca2+ exchanger declines with a half time (th) of 10.8 +/- 3.2 s upon Ca2+ removal, whereas the exchange currents of several mutants decline with th values of 0.7-4.3 s. Likewise, Ca2+ regulation mutants respond more rapidly to Ca2+ application. Study of Ca2+ regulation has previously been possible only with the exchanger operating in the reverse mode as the regulatory Ca2+ and the transported Ca2+ are then on opposite sides of the membrane. The use of exchange mutants with low affinity for Ca2+ at regulatory sites also allows demonstration of secondary Ca2+ regulation with the exchanger in the forward or Ca2+ efflux mode. In addition, we find that the affinity of wild-type and mutant Na(+)-Ca2+ exchangers for intracellular Na+ decreases at low regulatory Ca2+. This suggests that Ca2+ regulation modifies transport properties and does not only control the fraction of exchangers in an active state.
3
Ruknudin, A., C. Valdivia, P. Kofuji, W. J. Lederer, and D. H. Schulze. "Na+/Ca2+ exchanger in Drosophila: cloning, expression, and transport differences." American Journal of Physiology-Cell Physiology 273, no. 1 (July 1, 1997): C257—C265. http://dx.doi.org/10.1152/ajpcell.1997.273.1.c257.
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cDNAs for the Na+/Ca2+ exchanger from Drosophila melanogaster (Dmel/Nck) have been cloned by homology screening using the human heart Na+/Ca2+ exchanger cDNA. The overall deduced protein structure for Dmel/Nck is similar to that of mammalian Na+/Ca2+ exchanger genes NCX1 and NCX2, having six hydrophobic regions in the amino terminus separated from six at the carboxy-terminal end by a large intracellular loop. Sequence comparison of the Drosophila exchanger cDNAs with NCX1 and NCX2 Na+/Ca2+ exchangers are approximately 46% identical at the deduced amino acid level. Consensus phosphorylation sites for both protein kinase C and protein kinase A are present on the intracellular loop region of the Dmel/Nck. Alternative splicing for the Dmel/Nck gene is suggested in the same intracellular loop region as demonstrated for NCX1. Functionally, the Drosophila Na+/ Ca2+ exchanger expressed in oocytes differs from expressed mammalian NCX1 with regard to Ca2+ transport in Ca2+/ Ca2+ exchange and the effect of monovalent-dependent Ca2+/ Ca2+ exchange. The Dmel/Nck gene maps to chromosome 3 (93A-B) using in situ hybridization to polytene chromosomes, the same position as the Na(+)-K(+)-ATPase, a related transporter. We conclude that, although extracellular Na+ concentration-dependent Ca2+ transport is subserved by both human and Drosophila Na+/Ca2+ exchangers, there are clear and important differences in the transporters, which should be useful in deducing how the Na+/Ca2+ exchanger protein function depends on its structure.
4
Schoenmakers, T. J., and G. Flik. "Sodium-extruding and calcium-extruding sodium/calcium exchangers display similar calcium affinities." Journal of Experimental Biology 168, no. 1 (July 1, 1992): 151–59. http://dx.doi.org/10.1242/jeb.168.1.151.
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Na+/Ca2+ exchange activities in purely inside-out and mixed inside-out and right-side-out fish enterocyte basolateral plasma membrane vesicle preparations display equal affinities for Ca2+, showing that only the intracellular Ca2+ transport site of the Na+/Ca2+ exchanger is detected in experiments on vesicle preparations with mixed orientation. Therefore, Ca2+ pump and Na+/Ca2+ exchange activity may be compared directly without correction for vesicle orientation. The Na+/Ca2+ exchange activity in fish enterocyte vesicles is compared to the activity found in dog erythrocyte vesicles. The calcium-extruding exchanger in fish basolateral plasma membranes shows values of Km and V(max) for calcium similar to those found for the sodium-extruding exchanger in dog erythrocyte membranes, indicating that differences in electrochemical gradients underlie the difference in cellular function of the two exchangers.
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Omelchenko, Alexander, Christopher Dyck, Mark Hnatowich, John Buchko, Debora A. Nicoll, Kenneth D. Philipson, and Larry V. Hryshko. "Functional Differences in Ionic Regulation between Alternatively Spliced Isoforms of the Na+-Ca2+ Exchanger from Drosophila melanogaster." Journal of General Physiology 111, no. 5 (May 1, 1998): 691–702. http://dx.doi.org/10.1085/jgp.111.5.691.
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Ion transport and regulation were studied in two, alternatively spliced isoforms of the Na+-Ca2+ exchanger from Drosophila melanogaster. These exchangers, designated CALX1.1 and CALX1.2, differ by five amino acids in a region where alternative splicing also occurs in the mammalian Na+-Ca2+ exchanger, NCX1. The CALX isoforms were expressed in Xenopus laevis oocytes and characterized electrophysiologically using the giant, excised patch clamp technique. Outward Na+-Ca2+ exchange currents, where pipette Ca2+o exchanges for bath Na+i, were examined in all cases. Although the isoforms exhibited similar transport properties with respect to their Na+i affinities and current–voltage relationships, significant differences were observed in their Na+i- and Ca2+i-dependent regulatory properties. Both isoforms underwent Na+i-dependent inactivation, apparent as a time-dependent decrease in outward exchange current upon Na+i application. We observed a two- to threefold difference in recovery rates from this inactive state and the extent of Na+i-dependent inactivation was approximately twofold greater for CALX1.2 as compared with CALX1.1. Both isoforms showed regulation of Na+-Ca2+ exchange activity by Ca2+i, but their responses to regulatory Ca2+i differed markedly. For both isoforms, the application of cytoplasmic Ca2+i led to a decrease in outward exchange currents. This negative regulation by Ca2+i is unique to Na+-Ca2+ exchangers from Drosophila, and contrasts to the positive regulation produced by cytoplasmic Ca2+ for all other characterized Na+-Ca2+ exchangers. For CALX1.1, Ca2+i inhibited peak and steady state currents almost equally, with the extent of inhibition being ≈80%. In comparison, the effects of regulatory Ca2+i occurred with much higher affinity for CALX1.2, but the extent of these effects was greatly reduced (≈20–40% inhibition). For both exchangers, the effects of regulatory Ca2+i occurred by a direct mechanism and indirectly through effects on Na+i-induced inactivation. Our results show that regulatory Ca2+i decreases Na+i-induced inactivation of CALX1.2, whereas it stabilizes the Na+i-induced inactive state of CALX1.1. These effects of Ca2+i produce striking differences in regulation between CALX isoforms. Our findings indicate that alternative splicing may play a significant role in tailoring the regulatory profile of CALX isoforms and, possibly, other Na+-Ca2+ exchange proteins.
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Lytton, Jonathan. "Na+/Ca2+ exchangers: three mammalian gene families control Ca2+ transport." Biochemical Journal 406, no. 3 (August 29, 2007): 365–82. http://dx.doi.org/10.1042/bj20070619.
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Mammalian Na+/Ca2+ exchangers are members of three branches of a much larger family of transport proteins [the CaCA (Ca2+/cation antiporter) superfamily] whose main role is to provide control of Ca2+ flux across the plasma membranes or intracellular compartments. Since cytosolic levels of Ca2+ are much lower than those found extracellularly or in sequestered stores, the major function of Na+/Ca2+ exchangers is to extrude Ca2+ from the cytoplasm. The exchangers are, however, fully reversible and thus, under special conditions of subcellular localization and compartmentalized ion gradients, Na+/Ca2+ exchangers may allow Ca2+ entry and may play more specialized roles in Ca2+ movement between compartments. The NCX (Na+/Ca2+ exchanger) [SLC (solute carrier) 8] branch of Na+/Ca2+ exchangers comprises three members: NCX1 has been most extensively studied, and is broadly expressed with particular abundance in heart, brain and kidney, NCX2 is expressed in brain, and NCX3 is expressed in brain and skeletal muscle. The NCX proteins subserve a variety of roles, depending upon the site of expression. These include cardiac excitation–contraction coupling, neuronal signalling and Ca2+ reabsorption in the kidney. The NCKX (Na2+/Ca2+–K+ exchanger) (SLC24) branch of Na+/Ca2+ exchangers transport K+ and Ca2+ in exchange for Na+, and comprises five members: NCKX1 is expressed in retinal rod photoreceptors, NCKX2 is expressed in cone photoreceptors and in neurons throughout the brain, NCKX3 and NCKX4 are abundant in brain, but have a broader tissue distribution, and NCKX5 is expressed in skin, retinal epithelium and brain. The NCKX proteins probably play a particularly prominent role in regulating Ca2+ flux in environments which experience wide and frequent fluctuations in Na+ concentration. Until recently, the range of functions that NCKX proteins play was generally underappreciated. This situation is now changing rapidly as evidence emerges for roles including photoreceptor adaptation, synaptic plasticity and skin pigmentation. The CCX (Ca2+/cation exchanger) branch has only one mammalian member, NCKX6 or NCLX (Na+/Ca2+–Li+ exchanger), whose physiological function remains unclear, despite a broad pattern of expression.
7
Schnetkamp, Paul P. M. "Functional expression of Na–Ca exchanger clones measured with the fluorescent Ca2+-indicating dye fluo-3." Biochemistry and Cell Biology 74, no. 4 (July 1, 1996): 535–39. http://dx.doi.org/10.1139/o96-457.
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The process of Ca2+ homeostasis is of prime importance to all cells because of the ubiquitous role of cytoplasmic Ca2+ as an intracellular messenger and the cytotoxicity of sustained elevated cytosolic Ca2+ concentrations. Two classes of plasma membrane proteins are responsible for maintaining cytosolic free Ca2+ in the submicromolar range against a very large electrochemical Ca2+ gradient across the plasma membrane, the ATP-driven Ca2+ pump and Na–Ca exchangers. Two types of Na–Ca exchangers are known, the 3Na:1Ca exchangers found in heart, brain, kidney, and most other tissues and the 4Na:1Ca+1K exchanger found in retinal rod and cone photoreceptors. Functional expression of Na–Ca(/K) exchangers is most often measured as 45Ca uptake in Na+-loaded cells or as Na–Ca exchange currents with the giant excised patch technique. In this study, two functional assays used to detect expression of the bovine heart Na–Ca exchanger in CHO cells are described. Both assays are based on measurements of cytosolic free Ca2+ with the fluorescent Ca2+-indicating dye fluo-3 and should be equally applicable in the study of functional expression of both Na–Ca and Na–Ca/K exchanger clones.Key words: Na–Ca exchange, Ca homeostasis, fluo-3, ion transport, functional expression.
8
Danaceau, Jonathan P., and Mary T. Lucero. "Electrogenic Na+/Ca2+ Exchange." Journal of General Physiology 115, no. 6 (June 1, 2000): 759–68. http://dx.doi.org/10.1085/jgp.115.6.759.
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Olfactory receptor neurons (ORNs) from the squid, Lolliguncula brevis, respond to the odors l-glutamate or dopamine with increases in internal Ca2+ concentrations ([Ca2+]i). To directly asses the effects of increasing [Ca2+]i in perforated-patched squid ORNs, we applied 10 mM caffeine to release Ca2+ 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 Ca2+-dependent nonselective cation current. The strict dependence on internal Ca2+ and external Na+ indicated that the inward current was due to an electrogenic Na+/Ca2+ exchanger. Block of the caffeine-induced current by an inhibitor of Na+/Ca2+ exchange (50–100 μM 2′,4′-dichlorobenzamil) and reversibility of the exchanger current, further confirmed its presence. We tested whether Na+/Ca2+ 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+/Ca2+ exchange was responsible for ∼26% of the total current associated with glutamate-induced odor responses. Although Na+/Ca2+ 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.
9
Rasgado-Flores, H., and M. P. Blaustein. "Na/Ca exchange in barnacle muscle cells has a stoichiometry of 3 Na+/1 Ca2+." American Journal of Physiology-Cell Physiology 252, no. 5 (May 1, 1987): C499—C504. http://dx.doi.org/10.1152/ajpcell.1987.252.5.c499.
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The portions of the 45Ca influx and 22Na efflux that were activated by physiological concentrations of intracellular free Ca2+, [Ca2+]i, were studied in internally perfused single giant barnacle muscle cells. Since both fluxes were activated by intracellular Ca2+ (Cai) and the Ca influx was dependent on internal Na+ (Nai), the fluxes appear to be coupled (Na/Ca exchange). Tracer Ca/Ca and Na/Na exchanges were eliminated by employing tris(hydroxymethyl)aminomethane (Tris) as the predominant external cation. Under these circumstances, the ratio of the external Ca2+ (Cao)-dependent, Cai-activated Na+ efflux to the Nai-dependent, Cai-activated Ca influx was 3.1-3.2 Na+/1 Ca2+, when the intracellular Na+ concentration, [Na+]i was either 30 or 46 mM. This is the first direct measurement of the Na/Ca exchange stoichiometry. In many types of cells, the Na/Ca exchange system appears to operate in parallel with a plasma membrane ATP-driven Ca pump that has a lower capacity (maximum velocity), but higher affinity for Ca2+ than the Na/Ca exchanger. The data on the stoichiometry and activation by internal Ca2+ imply that the turnover of the Na/Ca exchanger is modulated during periods of cell activity. When the cells are depolarized, the Na/Ca exchange system is activated by the rising [Ca2+]i, and Ca2+ entry via the exchanger is promoted. Then, at repolarization, Ca2+ exits rapidly, primarily via the exchanger. However, in resting cells, with a low [Ca2+]i, much (but not all) of the Ca2+ efflux is probably mediated by the ATP-driven Ca pump.
10
Blaustein, Mordecai P., and W. Jonathan Lederer. "Sodium/Calcium Exchange: Its Physiological Implications." Physiological Reviews 79, no. 3 (July 1, 1999): 763–854. http://dx.doi.org/10.1152/physrev.1999.79.3.763.
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The Na+/Ca2+exchanger, an ion transport protein, is expressed in the plasma membrane (PM) of virtually all animal cells. It extrudes Ca2+in parallel with the PM ATP-driven Ca2+pump. As a reversible transporter, it also mediates Ca2+entry in parallel with various ion channels. The energy for net Ca2+transport by the Na+/Ca2+exchanger and its direction depend on the Na+, Ca2+, and K+gradients across the PM, the membrane potential, and the transport stoichiometry. In most cells, three Na+are exchanged for one Ca2+. In vertebrate photoreceptors, some neurons, and certain other cells, K+is transported in the same direction as Ca2+, with a coupling ratio of four Na+to one Ca2+plus one K+. The exchanger kinetics are affected by nontransported Ca2+, Na+, protons, ATP, and diverse other modulators. Five genes that code for the exchangers have been identified in mammals: three in the Na+/Ca2+exchanger family ( NCX1, NCX2, and NCX3) and two in the Na+/Ca2+plus K+family ( NCKX1 and NCKX2). Genes homologous to NCX1 have been identified in frog, squid, lobster, and Drosophila. In mammals, alternatively spliced variants of NCX1 have been identified; dominant expression of these variants is cell type specific, which suggests that the variations are involved in targeting and/or functional differences. In cardiac myocytes, and probably other cell types, the exchanger serves a housekeeping role by maintaining a low intracellular Ca2+concentration; its possible role in cardiac excitation-contraction coupling is controversial. Cellular increases in Na+concentration lead to increases in Ca2+concentration mediated by the Na+/Ca2+exchanger; this is important in the therapeutic action of cardiotonic steroids like digitalis. Similarly, alterations of Na+and Ca2+apparently modulate basolateral K+conductance in some epithelia, signaling in some special sense organs (e.g., photoreceptors and olfactory receptors) and Ca2+-dependent secretion in neurons and in many secretory cells. The juxtaposition of PM and sarco(endo)plasmic reticulum membranes may permit the PM Na+/Ca2+exchanger to regulate sarco(endo)plasmic reticulum Ca2+stores and influence cellular Ca2+signaling.
11
Hilgemann, Donald W. "New insights into the molecular and cellular workings of the cardiac Na+/Ca2+ exchanger." American Journal of Physiology-Cell Physiology 287, no. 5 (November 2004): C1167—C1172. http://dx.doi.org/10.1152/ajpcell.00288.2004.
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The cardiac Na+/Ca2+ exchanger (NCX1) is almost certainly the major Ca2+ extrusion mechanism in cardiac myocytes, although the driving force for Ca2+ extrusion is quite small. To explain multiple recent results, it is useful to think of the exchanger as a slow Ca2+ buffer that can reverse its function multiple times during the excitation-contraction cycle (ECC). An article by the group of John Reeves brings new insights to this function by analyzing the role of regulatory domains of NCX1 that mediate its activation by a rise of cytoplasmic Ca2+. It was demonstrated that the gating reactions are operative just in the physiological range of Ca2+ changes, a few fold above resting Ca2+ level, and that they prevent the exchanger from damping out the influence of mechanisms that transiently increase Ca2+ levels. Furthermore, exchangers with deleted regulatory domains are shown to reduce resting Ca2+ to lower levels than achieved by wild-type exchangers. A study by the group of Kenneth Philipson demonstrated that the NCX1 regulatory domain can bind and respond to Ca2+ changes on the time scale of the ECC in rat myocytes. At the same time, studies of transgenic mice and NCX1 knockout mice generated by the Philipson group revealed that large changes of NCX1 activity have rather modest effects on ECC. Simple simulations predict these results very well: murine cardiac ECC is very sensitive to small changes of the Na+ gradient, very sensitive to changes of the sarcoplasmic reticulum Ca2+ pump activity, and very insensitive to changes of NCX1 activity. It is speculated that the NCX1 gating reactions not only regulate coupled 3Na+:1Ca2+ exchange but also control the exchanger’s Na+ leak function that generates background Na+ influx and depolarizing current in cardiac myocytes.
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White, K. E., F. A. Gesek, and P. A. Friedman. "Structural and functional analysis of Na+/Ca2+ exchange in distal convoluted tubule cells." American Journal of Physiology-Renal Physiology 271, no. 3 (September 1, 1996): F560—F570. http://dx.doi.org/10.1152/ajprenal.1996.271.3.f560.
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Renal distal convoluted tubules (DCT) are a major site of hormone-regulated, active calcium absorption. Calcium exit across basolateral plasma membranes is thought to be mediated by Na+/Ca2+ exchange and a Ca(2+)-ATPase. In this report the presence and function of Na+/Ca2+ exchangers in DCT cells were assessed. cDNAs encoding a conserved region and the variable regions of three alternatively spliced isoforms of the Na+/Ca2+ exchanger, NACA2, NACA3, and NACA6, were isolated in a ratio of 7:12:1 using homology-based reverse transcription-polymerase chain reaction (RT-PCR) with RNA from an immortalized mouse DCT cell line. Northern blots probed with a 32P-labeled PCR product from a conserved region of the exchanger were positive for a single transcript of 7 kb in primary cultures of distal tubule cells (cortical ascending limb + DCT cells), consistent with the reported size of the exchanger in other tissues. Na+/Ca2+ exchange was assessed by measuring sodium-dependent changes of intracellular calcium ([Ca2+]i), in single cells. In the presence of an outward Na+ gradient, [Ca2+]i increased by 240%. Collapsing the Na+ gradient with monensin inhibited the rise of [Ca2+]i. Removal of extracellular Ca2+ or the addition of an Na+ ionophore inhibited the rise of [Ca2+]i. The intracellular Na+ concentration decreased upon removal of extracellular Na+ in parallel with the rise of [Ca2+]i. Western analysis performed on membranes prepared from DCT cells or primary cultures of distal tubule cells with a polyclonal antibody revealed bands at approximately 125 and 85 kDa, consistent with reported sizes for exchanger protein. These findings show that Na+/Ca2+ exchanger transcripts, protein, and activity are present in DCT cells and that Na(+)-dependent Ca2+ efflux may be mediated by NACA2, NACA3, and NACA6.
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Zhang, Shen, Jason X. J. Yuan, Kim E. Barrett, and Hui Dong. "Role of Na+/Ca2+ exchange in regulating cytosolic Ca2+ in cultured human pulmonary artery smooth muscle cells." American Journal of Physiology-Cell Physiology 288, no. 2 (February 2005): C245—C252. http://dx.doi.org/10.1152/ajpcell.00411.2004.
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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.
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Linck, Bettina, Zhiyong Qiu, Zhaoping He, Qiusheng Tong, Donald W. Hilgemann, and Kenneth D. Philipson. "Functional comparison of the three isoforms of the Na+/Ca2+ exchanger (NCX1, NCX2, NCX3)." American Journal of Physiology-Cell Physiology 274, no. 2 (February 1, 1998): C415—C423. http://dx.doi.org/10.1152/ajpcell.1998.274.2.c415.
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Three distinct mammalian Na+/Ca2+exchangers have been cloned: NCX1, NCX2, and NCX3. We have undertaken a detailed functional comparison of these three exchangers. Each exchanger was stably expressed at high levels in the plasma membranes of 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, and exchange currents measured using giant patches of excised cell membrane. Apparent affinities for the transported ions Na+ and Ca2+ were markedly similar for the three exchangers at both membrane surfaces. Likewise, generally similar responses to changes in pH, chymotrypsin treatment, and application of various inhibitors were obtained. Depletion of cellular ATP inhibited NCX1 and NCX2 but did not affect the activity of NCX3. Exchange activities of NCX1 and NCX3 were modestly increased by agents that activate protein kinases A and C. All exchangers were regulated by intracellular Ca2+. NCX1-induced exchange currents were especially large in excised patches and, like the native myocardial exchanger, were stimulated by ATP. Results may be influenced by our choice of expression system and specific splice variants, but, overall, the three exchangers appear to have very similar properties.
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Reeves, John P., and Madalina Condrescu. "Allosteric Activation of Sodium–Calcium Exchange Activity by Calcium." Journal of General Physiology 122, no. 5 (October 27, 2003): 621–39. http://dx.doi.org/10.1085/jgp.200308915.
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The activity of the cardiac Na+/Ca2+ exchanger is stimulated allosterically by Ca2+, but estimates of the half-maximal activating concentration have varied over a wide range. In Chinese hamster ovary cells expressing the cardiac Na+/Ca2+ exchanger, the time course of exchange-mediated Ca2+ influx showed a pronounced lag period followed by an acceleration of Ca2+ uptake. Lag periods were absent in cells expressing an exchanger mutant that was not dependent on regulatory Ca2+ activation. We assumed that the rate of Ca2+ uptake during the acceleration phase reflected the degree of allosteric activation of the exchanger and determined the value of cytosolic Ca2+ ([Ca2+]i) at which the rate of Ca2+ influx was half-maximal (Kh). After correcting for the effects of mitochondrial Ca2+ uptake and fura-2 buffering, Kh values of ∼300 nM were obtained. After an increase in [Ca2+]i, the activated state of the exchanger persisted following a subsequent reduction in [Ca2+]i to values <100 nM. Thus, within 30 s after termination of a transient increase in [Ca2+]i, exchange-mediated Ca2+ entry began without a lag period and displayed a linear rate of Ca2+ uptake in most cells; a sigmoidal time course of Ca2+ uptake returned 60–90 s after the transient increase in [Ca2+]i was terminated. Relaxation of the activated state was accelerated by the activity of the endoplasmic reticulum Ca2+ pump, suggesting that local Ca2+ gradients contribute to maintaining exchanger activation after the return of global [Ca2+]i to low values.
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Matsuoka, Satoshi, Debora A. Nicoll, Zhaoping He, and Kenneth D. Philipson. "Regulation of the Cardiac Na+-Ca2+ Exchanger by the Endogenous XIP Region." Journal of General Physiology 109, no. 2 (February 1, 1997): 273–86. http://dx.doi.org/10.1085/jgp.109.2.273.
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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+.
17
Kahn, A. M., J. C. Allen, and H. Shelat. "Na+-Ca2+ exchange in sarcolemmal vesicles from bovine superior mesenteric artery." American Journal of Physiology-Cell Physiology 254, no. 3 (March 1, 1988): C441—C449. http://dx.doi.org/10.1152/ajpcell.1988.254.3.c441.
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These studies were designed to determine whether a Na+-Ca2+ exchanger is present in sarcolemmal vesicles from bovine superior mesenteric artery and, if so, to determine whether this transport system is qualitatively similar to that found in other excitable tissues. Vesicles, preferentially enriched in sarcolemma, were prepared by a Mg2+ aggregation and differential centrifugation technique. An inwardly directed Ca2+ gradient stimulated 22Na+ efflux and an outwardly directed Ca2+ gradient stimulated 22Na+ uptake. Similarly, an inwardly directed Na+ gradient stimulated 45Ca2+ efflux, and an outwardly directed Na+ gradient stimulated 45Ca2+ uptake. Ca2+ gradient-stimulated Na+ transport and Na+ gradient-stimulated Ca2+ transport were not due to voltage coupling between the two ions. Hence, a Na+-Ca2+ exchanger is present in these vesicles. The Na+ gradient-dependent component of Ca2+ uptake (Na+-Ca2+ exchange) was stimulated by rendering the vesicles electropositive inside, and Na+-Ca2+ exchange activity was inhibited by amiloride and quinidine in a dose-dependent fashion. These data demonstrate similarities between this mesenteric arterial smooth muscle Na+-Ca2+ exchanger and that found in other excitable tissues. In the absence of added Ca2+, amiloride-sensitive 22Na+ uptake in the vesicles was stimulated by an outwardly directed proton gradient, and an inwardly directed Na+ gradient stimulated proton efflux. Thus these vesicles also contain a Na+-H+ exchanger, which has been found in the sarcolemma of other vascular smooth muscle cells. When Na+ uptake was stimulated via Na+-H+ exchange, the subsequent uptake of Ca2+ via Na+-Ca2+ exchange was tripled. In conclusion, these studies unequivocally demonstrate that sarcolemmal-enriched vesicles from bovine superior mesenteric artery contain a Na+-Ca2+ exchanger.(ABSTRACT TRUNCATED AT 250 WORDS)
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Ledeen, Robert W., and Gusheng Wu. "GM1 ganglioside: another nuclear lipid that modulates nuclear calcium. GM1 potentiates the nuclear sodium–calcium exchangerThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell." Canadian Journal of Physiology and Pharmacology 84, no. 3-4 (March 2006): 393–402. http://dx.doi.org/10.1139/y05-133.
Full textAbstract:
The nuclear envelope (NE) enclosing the cell nucleus, although morphologically and chemically distinct from the plasma membrane, has certain features in common with the latter including the presence of GM1 as an important modulatory molecule. This ganglioside influences Ca2+ flux across both membranes, but by quite different mechanisms. GM1 in the NE contributes to regulation of nuclear Ca2+ through potentiation of a Na+/Ca2+ exchanger in the inner nuclear membrane, whereas in the cell membrane, it regulates cytosolic Ca2+ through modulation of a nonvoltage-gated Ca2+ channel. Studies with neuroblastoma cells suggest GM1 concentration becomes elevated in the NE with onset of axonogenesis. However, the nuclear GM1/exchanger complex is not limited to neuronal cells but also occurs in NE of astrocytes, C6 cells, and certain non-neural cells. Immunoprecipitation and immunoblot experiments have shown high affinity association of the nuclear Na+/Ca2+ exchanger with GM1, in contrast to Na+/Ca2+ exchangers of the plasma membrane, which bind GM1 less avidly or not at all. This is believed to be due to different isoforms of the exchanger and a difference in topology of GM1 relative to the large inner loop of the exchanger in the 2 membranes. Cultured neurons from mice genetically engineered to lack GM1 suffered Ca2+ dysregulation as seen in their high vulnerability to Ca2+-induced apoptosis. They were rescued by GM1 and more effectively by LIGA20, a membrane-permeant derivative of GM1. The mutant animals were highly susceptible to kainate-induced seizures, which are also a reflection of Ca2+ dysregulation. The seizures were effectively attenuated by LIGA20 in parallel with the ability of this agent to enter brain cells, insert into the NE, and potentiate Na+/Ca2+ exchange activity in the nucleus. The Na+/Ca2+ exchanger of the NE, in association with nuclear GM1, is thus seen contributing to independent regulation of Ca2+ by the nucleus in a manner that provides cytoprotection against Ca2+-induced apoptosis.
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Dyck, Chris, Alexander Omelchenko, Chadwick L. Elias, Beate D. Quednau, Kenneth D. Philipson, Mark Hnatowich, and Larry V. Hryshko. "Ionic Regulatory Properties of Brain and Kidney Splice Variants of the Ncx1 Na+–Ca2+ Exchanger." Journal of General Physiology 114, no. 5 (October 25, 1999): 701–11. http://dx.doi.org/10.1085/jgp.114.5.701.
Full textAbstract:
Ion transport and regulation of Na+–Ca2+ exchange were examined for two alternatively spliced isoforms of the canine cardiac Na+–Ca2+ exchanger, NCX1.1, to assess the role(s) of the mutually exclusive A and B exons. The exchangers examined, NCX1.3 and NCX1.4, are commonly referred to as the kidney and brain splice variants and differ only in the expression of the BD or AD exons, respectively. Outward Na+–Ca2+ exchange activity was assessed in giant, excised membrane patches from Xenopus laevis oocytes expressing the cloned exchangers, and the characteristics of Na+i- (i.e., I1) and Ca2+i- (i.e., I2) dependent regulation of exchange currents were examined using a variety of experimental protocols. No remarkable differences were observed in the current–voltage relationships of NCX1.3 and NCX1.4, whereas these isoforms differed appreciably in terms of their I1 and I2 regulatory properties. Sodium-dependent inactivation of NCX1.3 was considerably more pronounced than that of NCX1.4 and resulted in nearly complete inhibition of steady state currents. This novel feature could be abolished by proteolysis with α-chymotrypsin. It appears that expression of the B exon in NCX1.3 imparts a substantially more stable I1 inactive state of the exchanger than does the A exon of NCX1.4. With respect to I2 regulation, significant differences were also found between NCX1.3 and NCX1.4. While both exchangers were stimulated by low concentrations of regulatory Ca2+i, NCX1.3 showed a prominent decrease at higher concentrations (>1 μM). This does not appear to be due solely to competition between Ca2+i and Na+i at the transport site, as the Ca2+i affinities of inward currents were nearly identical between the two exchangers. Furthermore, regulatory Ca2+i had only modest effects on Na+i-dependent inactivation of NCX1.3, whereas I1 inactivation of NCX1.4 could be completely eliminated by Ca2+i. Our results establish an important role for the mutually exclusive A and B exons of NCX1 in modulating the characteristics of ionic regulation and provide insight into how alternative splicing tailors the regulatory properties of Na+–Ca2+ exchange to fulfill tissue-specific requirements of Ca2+ homeostasis.
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Pijuan, V., Y. Zhuang, L. Smith, C. Kroupis, M. Condrescu, J. F. Aceto, J. P. Reeves, and J. B. Smith. "Stable expression of the cardiac sodium-calcium exchanger in CHO cells." American Journal of Physiology-Cell Physiology 264, no. 4 (April 1, 1993): C1066—C1074. http://dx.doi.org/10.1152/ajpcell.1993.264.4.c1066.
Full textAbstract:
A line of Chinese hamster ovary (CHO) cells called CK1.4 was produced by transfection with the gene for the bovine cardiac Na(+)-Ca2+ exchanger. CK1.4 cells stably expressed substantial exchange activity and exchanger protein as shown by immunoprecipitation. Exchange activity was quantified as 45Ca2+ influx that depended on both increasing intracellular Na+ and lowering the concentration of external Na+. Replacing external Na+ with K+ slightly increased 45Ca2+ uptake by CK1.4 cells with basal Na+ and greatly increased 45Ca2+ uptake by Na(+)-loaded cells. Neither exchange activity nor exchanger protein was detected in the nontransfected parental line. By contrast to CK1.4 cells, replacing external Na+ with K+ decreased 45Ca2+ uptake in the nontransfected cells whether or not they were Na+ loaded. Changes in cytosolic free Ca2+ determined with fura-2 were consistent with the 45Ca2+ uptake data. Analysis of poly(A)(+)-RNA by Northern blot confirmed that CK1.4 cells, but not the parental line, expressed the exchangerx. Expression of the exchanger was also observed in aortic myocytes and a renal epithelial cell line (LLC-MK2) but not in other lines of renal epithelial cells (MDCK, LLC-PK1) or human dermal fibroblasts. The cardiac exchanger produced substantial 45Ca2+ efflux from CK1.4 cells in response to hormone-evoked release of stored Ca2+. CK1.4 cells are an attractive model for studies of the regulation of the cardiac exchanger because they stably express sufficient exchanger for biochemical and immunological analysis.
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Chernysh, Olga, Madalina Condrescu, and John P. Reeves. "Calcium-dependent regulation of calcium efflux by the cardiac sodium/calcium exchanger." American Journal of Physiology-Cell Physiology 287, no. 3 (September 2004): C797—C806. http://dx.doi.org/10.1152/ajpcell.00176.2004.
Full textAbstract:
Allosteric regulation by cytosolic Ca2+ of Na+/Ca2+ exchange activity in the Ca2+ efflux mode has received little attention because it has been technically difficult to distinguish between the roles of Ca2+ as allosteric activator and transport substrate. In this study, we used transfected Chinese hamster ovary cells to compare the Ca2+ efflux activities in nontransfected cells and in cells expressing either the wild-type exchanger or a mutant, Δ(241–680), that operates constitutively; i.e., its activity does not require allosteric Ca2+ activation. Expression of the wild-type exchanger did not significantly lower the cytosolic Ca2+ concentration ([Ca2+]i) compared with nontransfected cells. During Ca2+ entry through store-operated Ca2+ channels, Ca2+ efflux by the wild-type exchanger became evident only after [Ca2+]i approached 100–200 nM. A subsequent decline in [Ca2+]i was observed, suggesting that the activation process was time dependent. In contrast, Ca2+ efflux activity was evident under all experimental conditions in cells expressing the constitutive exchanger mutant. After transient exposure to elevated [Ca2+]i, the wild-type exchanger behaved similarly to the constitutive mutant for tens of seconds after [Ca2+]i had returned to resting levels. We conclude that Ca2+ efflux activity by the wild-type exchanger is allosterically activated by Ca2+, perhaps in a time-dependent manner, and that the activated state is briefly retained after the return of [Ca2+]i to resting levels.
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Lyu, R. M., L. Smith, and J. B. Smith. "Ca2+ influx via Na(+)-Ca2+ exchange in immortalized aortic myocytes. II. Feedback inhibition by [Ca2+]i." American Journal of Physiology-Cell Physiology 263, no. 3 (September 1, 1992): C635—C641. http://dx.doi.org/10.1152/ajpcell.1992.263.3.c635.
Full textAbstract:
Depolarization with 50 mM K+ evoked a spike in cytosolic free Ca2+ ([Ca2+]i) and increased 45Ca2+ uptake in immortalized aortic myocytes. The following evidence indicates that the electrogenic Na(+)-Ca2+ exchanger caused the Ca2+ influx that was evoked by K+ depolarization. First, K+ depolarization had no effect on [Ca2+]i and 45Ca2+ uptake in cells with basal Na+ but strikingly increased both in Na(+)-loaded cells. Second, the [Ca2+]i increases produced by K+ depolarization depended hyperbolically on external Ca2+ (50% maximum concentration = 1.5 mM). Third, the increases in [Ca2+]i and 45Ca2+ uptake were greater when external Na+ was replaced with K+ rather than with N-methyl-D-glucamine or choline. A series of K+ depolarizations elicited a sequence of [Ca2+]i spikes, provided there was a short incubation at 5 mM K+ between the depolarizations. A prior K+ depolarization almost abolished the 45Ca2+ uptake response to K+ depolarization. The inhibition of exchange activity by a prior K+ depolarization required external Ca2+ and was completely reversible. A prior incubation with angiotensin II, platelet-derived growth factor, or ionomycin also inhibited exchange activity. Moderate [Ca2+]i increases probably feedback inhibit Ca2+ influx via the exchanger by a kinetic mechanism. Inactivation of the exchanger, together with Ca2+ extrusion or sequestration, causes the rapid decrease in [Ca2+]i from the peak evoked by depolarization.
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Trac, Michael, Christopher Dyck, Mark Hnatowich, Alexander Omelchenko, and Larry V. Hryshko. "Transport and Regulation of the Cardiac Na+-Ca2+ Exchanger, NCX1." Journal of General Physiology 109, no. 3 (March 1, 1997): 361–69. http://dx.doi.org/10.1085/jgp.109.3.361.
Full textAbstract:
Cardiac muscle fails to relax upon replacement of extracellular Ca2+ with Ba2+. Among the manifold consequences of this intervention, one major possibility is that Na+-Ba2+ exchange is inadequate to support normal relaxation. This could occur due to reduced transport rates of Na+-Ba2+ exchange and/or by failure of Ba2+ to activate the exchanger molecule at the high affinity regulatory Ca2+ binding site. In this study, we examined transport and regulatory properties for Na+-Ca2+ and Na+-Ba2+ exchange. Inward and outward Na+-Ca2+ or Na+-Ba2+ exchange currents were examined at 30°C in giant membrane patches excised from Xenopus oocytes expressing the cloned cardiac Na+-Ca2+ exchanger, NCX1. When excised patches were exposed to either cytoplasmic Ca2+ or Ba2+, robust inward Na+-Ca2+ exchange currents were observed, whereas Na+-Ba2+ currents were absent or barely detectable. Similarly, outward currents were greatly reduced when pipette solutions contained Ba2+ rather than Ca2+. However, when solution temperature was elevated from 30°C to 37°C, a substantial increase in outward Na+-Ba2+ exchange currents was observed, but not so for inward currents. We also compared the relative abilities of Ca2+ and Ba2+ to activate outward Na+-Ca2+ exchange currents at the high affinity regulatory Ca2+ binding site. While Ba2+ was capable of activating the exchanger, it did so with a much lower affinity (KD ∼ 10 μM) compared with Ca2+ (KD ∼ 0.3 μM). Moreover, the efficiency of Ba2+ regulation of Na+-Ca2+ exchange is also diminished relative to Ca2+, supporting ∼60% of maximal currents obtainable with Ca2+. Ba2+ is also much less effective at alleviating Na+i-induced inactivation of NCX1. These results indicate that the reduced ability of NCX1 to adequately exchange Na+ and Ba2+ contributes to failure of the relaxation process in cardiac muscle.
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Nicoll, D. A., B. R. Barrios, and K. D. Philipson. "Na(+)-Ca2+ exchangers from rod outer segments and cardiac sarcolemma: comparison of properties." American Journal of Physiology-Cell Physiology 260, no. 6 (June 1, 1991): C1212—C1216. http://dx.doi.org/10.1152/ajpcell.1991.260.6.c1212.
Full textAbstract:
The properties of the Na(+)-Ca2+ exchangers from cardiac sarcolemma (SL) and rod outer segments (ROS) were studied in parallel by measuring the Na+ gradient-dependent Ca2+ uptake into SL or ROS vesicles. The ROS exchanger, but not the SL exchanger, has a striking specific dependence on K+. The ROS exchanger is stimulated by K+ with an apparent concentration at half-maximum of 1 mM. The addition of valinomycin, to produce an inside-positive membrane potential, stimulates the SL exchanger 1.8-fold and the ROS exchanger 1.2-fold. The Michaelis constant for half-maximal transport rate for Ca2+ and the maximal transport rate for the exchangers, in the absence of valinomycin, are estimated to be 20 microM and 8 nmol.mg-1.2.2 s-1 in SL and 5 microM and 1 nmol.mg-1.2.2 s-1 in ROS. Both exchangers are modulated by the same regulatory influences. For example, both are stimulated by proteases, phospholipase D, and intravesicular Ca2+.
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Iwamoto, Takahiro. "Vascular Na+/Ca2+ exchanger: implications for the pathogenesis and therapy of salt-dependent hypertension." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 290, no. 3 (March 2006): R536—R545. http://dx.doi.org/10.1152/ajpregu.00592.2005.
Full textAbstract:
The Na+/Ca2+ exchanger is an ion transporter that exchanges Na+ and Ca2+ in either Ca2+ efflux or Ca2+ influx mode, depending on membrane potential and transmembrane ion gradients. In arterial smooth muscle cells, the Na+/Ca2+ exchanger is thought to participate in the maintenance of vascular tone by regulating cytosolic Ca2+ concentration. Recent pharmacological and genetic engineering studies have revealed that the Ca2+ influx mode of vascular Na+/Ca2+ exchanger type-1 (NCX1) is involved in the pathogenesis of salt-dependent hypertension. SEA0400, a specific Na+/Ca2+ exchange inhibitor that preferentially blocks the Ca2+ influx mode, lowers arterial blood pressure in salt-dependent hypertensive models, but not in normotensive rats or other types of hypertensive rats. Furthermore, heterozygous mice with reduced expression of NCX1 are resistant to development of salt-dependent hypertension, whereas transgenic mice with vascular smooth muscle-specific overexpression of NCX1 readily develop hypertension after high-salt loading. SEA0400 reverses the cytosolic Ca2+ elevation and vasoconstriction induced by nanomolar ouabain, as well as humoral factors in salt-loaded animals. One possibility is that circulating endogenous cardiotonic steroids may be necessary for NCX1-mediated hypertension. These findings help to explain how arterial smooth muscle cells in blood vessels contribute to salt-elicited blood pressure elevation and suggest that NCX1 inhibitors might be therapeutically useful for salt-dependent hypertension.
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Kimura, Masayuki, Elisabeth M. Jeanclos, Robert J. Donnelly, Jonathan Lytton, John P. Reeves, and Abraham Aviv. "Physiological and molecular characterization of the Na+/Ca2+exchanger in human platelets." American Journal of Physiology-Heart and Circulatory Physiology 277, no. 3 (September 1, 1999): H911—H917. http://dx.doi.org/10.1152/ajpheart.1999.277.3.h911.
Full textAbstract:
In this report, we have demonstrated that Na+/Ca2+exchanger activity in a human megakaryocytic cell line (CHRF-288 cells) is K+ dependent, similar to the properties previously described for Na+/Ca2+exchange activity in human platelets. With the use of RT-PCR techniques and mRNA, the exchanger expressed in CHRF-288 cells was found to be identical to that expressed in human retinal rods. Northern blot analysis of the mRNA for the human retinal rod exchanger in CHRF-288 cells revealed a major transcript at 5.8 kb with two minor bands at 4.9 and 6.8 kb. mRNA for the retinal rod exchanger was also identified in human platelets. Using Ba2+ influx as a measure of Na+/Ca2+exchange activity in human platelets, we have demonstrated that exchange activity is driven by the transmembrane gradient for K+ as well as that for Na+. We propose that the K+ dependence of the platelet Na+/Ca2+exchanger could make platelets especially sensitive to daily fluctuations in salt intake.
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Almilaji, Ahmad, Jing Yan, Zohreh Hosseinzadeh, Evi Schmid, Meinrad Gawaz, and Florian Lang. "Up-Regulation of Na+/Ca2+ Exchange in Megakaryocytes Following TGFβ1 Treatment." Cellular Physiology and Biochemistry 39, no. 2 (2016): 693–99. http://dx.doi.org/10.1159/000445660.
Full textAbstract:
Background: Blood platelets are activated by increase of cytosolic Ca2+ activity ([Ca2+]i). Ca2+ entry is accomplished in part by store operated Ca2+ entry (SOCE) involving Ca2+ release activated Ca2+-channel (CRAC) moiety Orai1 and its regulator STIM1, which are stimulated by depletion of intracellular Ca2+ stores. An increase of [Ca2+]i is terminated by Na+/Ca2+-exchange. The expression of both, Orai1 and STIM1 in megakaryocytes is up-regulated by tumor growth factor TGFβ1, a powerful regulator of megakaryocyte differentiation. The present study explored whether TGFβ1 similarly modifies megakaryocyte Na+/Ca2+-exchanger activity. Methods: [Ca2+]i was determined utlizing Fura-2 fluorescence, SOCE from increase of [Ca2+]i, following readdition of extracellular Ca2+ after store depletion, and Na+/Ca2+-exchanger activity from increase of [Ca2+]i and whole cell currents following removal of extracellular Na+. Results: TGFβ1 treatment not only augments the increase of [Ca2+]i following store depletion and SOCE, but significantly up-regulates Na+/Ca2+-exchanger activity as apparent from [Ca2+]i measurements and whole cell currents. Conclusions: TGFβ1 is a powerful stimulator of both, SOCE and Na+/Ca2+-exchanger activity in megakaryocytes.
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Wu, C., and C. H. Fry. "Na+/Ca2+ exchange and its role in intracellular Ca2+ regulation in guinea pig detrusor smooth muscle." American Journal of Physiology-Cell Physiology 280, no. 5 (May 1, 2001): C1090—C1096. http://dx.doi.org/10.1152/ajpcell.2001.280.5.c1090.
Full textAbstract:
The role of Na+/Ca2+ exchange in regulating intracellular Ca2+ concentration ([Ca2+]i) in isolated smooth muscle cells from the guinea pig urinary bladder was investigated. Incremental reduction of extracellular Na+ concentration resulted in a graded rise of [Ca2+]i; 50–100 μM strophanthidin also increased [Ca2+]i. A small outward current accompanied the rise of [Ca2+]i in low-Na+ solutions (17.1 ± 1.8 pA in 29.4 mM Na+). The quantity of Ca2+ influx through the exchanger was estimated from the charge carried by the outward current and was ∼30 times that which is necessary to account for the rise of [Ca2+]i, after correction was made for intracellular Ca2+ buffering. Ca2+ influx through the exchanger was able to load intracellular Ca2+ stores. It is concluded that the level of resting [Ca2+]i is not determined by the exchanger, and under resting conditions (membrane potential −50 to −60 mV), there is little net flux through the exchanger. However, a small rise of intracellular Na+ concentration would be sufficient to generate significant net Ca2+ influx.
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Nazer, Mark A., and Cornelis Van Breemen. "A role for the sarcoplasmic reticulum in Ca2+ extrusion from rabbit inferior vena cava smooth muscle." American Journal of Physiology-Heart and Circulatory Physiology 274, no. 1 (January 1, 1998): H123—H131. http://dx.doi.org/10.1152/ajpheart.1998.274.1.h123.
Full textAbstract:
Ca2+extrusion from rabbit inferior vena cava smooth muscle was studied using ratiometric fura 2 fluorimetry. Concomitant blockade of the plasma membrane Ca2+-adenosinetriphosphatase (ATPase; PCMA), Na+-Ca2+exchanger, and sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) completely prevented the decline in intracellular Ca2+ concentration ([Ca2+]i) normally observed when Ca2+ is removed from the extracellular space (ECS) after stimulated Ca2+ influx. Blockade of the Na+-Ca2+exchanger by removal of external Na+ reduced the rate of [Ca2+]idecline by 47%. Blockade of SERCA with cyclopiazonic acid reduced it by 23%, and this was not additive to the effects of Na+ removal. Exposure to nominally Ca2+-free solution prevented the sarcoplasmic reticulum (SR) from reloading only if the Na+-Ca2+exchanger was operational. Our results can be explained by an SR contribution to Ca2+ extrusion in which SERCA is arranged in series with Na+-Ca2+exchange.
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Unlap, M. Tino, Janos Peti-Peterdi, and P. Darwin Bell. "Cloning of mesangial cell Na+/Ca2+exchangers from Dahl/Rapp salt-sensitive/resistant rats." American Journal of Physiology-Renal Physiology 279, no. 1 (July 1, 2000): F177—F184. http://dx.doi.org/10.1152/ajprenal.2000.279.1.f177.
Full textAbstract:
The Dahl/Rapp rat model of hypertension is characterized by a marked increase in blood pressure and a progressive fall in glomerular filtration rate when salt-sensitive (S) rats are placed on an 8% NaCl diet. On the same diet, the salt-resistant (R) rat does not exhibit these changes. In previous studies we found that protein kinase C (PKC) upregulates Na+/Ca2+ exchanger activity in afferent arterioles and mesangial cells from R but not S rats. One possible reason for the difference in PKC sensitivity may be due to differences in the S and R Na+/Ca2+ exchanger protein. We now report the cloning of Na+/Ca2+ exchangers from R (RNCX1) and S (SNCX1) mesangial cells. At the amino acid level, SNCX1 differs from RNCX1 at position 218 in the NH2-terminal domain where it is isoleucine in RNCX1 but phenylalanine in SNCX1. These two exchangers also differ by 23 amino acids at the alternative splice site within the cytosolic domain. RNCX1 and SNCX1 were expressed in OK-PTH cells and45Ca2+-uptake studies were performed. Acute phorbol 12-myristate 13-acetate (PMA) treatment (300 nM, 20 min) upregulated exchanger activity in cells expressing RNCX1 but failed to stimulate exchanger activity in SNCX1 expressing cells. Upregulation of RNCX1 could be prevented by prior 24-h pretreatment with PMA, which downregulates PKC. These results demonstrate a difference in PKC-Na+/Ca2+ exchange activity between the isoform of the exchanger cloned from the R vs. the S rat. Lack of PKC activation of SNCX1 may contribute to a dysregulation of intracellular Ca2+ concentration and enhanced renal vasoreactivity in this model of hypertension.
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Chen, F., G. Mottino, T. S. Klitzner, K. D. Philipson, and J. S. Frank. "Distribution of the Na+/Ca2+ exchange protein in developing rabbit myocytes." American Journal of Physiology-Cell Physiology 268, no. 5 (May 1, 1995): C1126—C1132. http://dx.doi.org/10.1152/ajpcell.1995.268.5.c1126.
Full textAbstract:
The Na+/Ca2+ exchanger is a major pathway for transmembrane flux of Ca2+ in cardiac cells. Immunolabeling in adult rabbit myocytes showed localization of the Na+/Ca2+ exchanger to the peripheral sarcolemma and especially in the T tubules. Previous studies have also demonstrated higher Na+/Ca2+ exchanger activity in fetal and newborn rabbit hearts in which the T tubular system is not completely developed. Indirect immunofluorescent studies were performed to localize the Na+/Ca2+ exchanger in isolated myocytes from immature (5, 11, 17, and 30 days) and adult rabbits. Cells were incubated with a monoclonal antibody to the exchanger followed by fluorescein-labeled goat anti-mouse antibody. It is found that at 5 days of age the immunofluorescent labeling was very intense and confined to the peripheral sarcolemma. After 11 days of age, localization of labeling followed the development of the T tubules. The exchanger appeared in the T tubules as soon as they were formed. The Na+/Ca2+ exchange protein is abundantly localized to the peripheral sarcolemma before and during the development of T tubule system.
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Loo, T. W., and D. M. Clarke. "Functional expression of human renal Na+/Ca2+ exchanger in insect cells." American Journal of Physiology-Renal Physiology 267, no. 1 (July 1, 1994): F70—F74. http://dx.doi.org/10.1152/ajprenal.1994.267.1.f70.
Full textAbstract:
The cDNA coding for the human renal isoform of the Na+/Ca2+ exchanger was cloned from a human embryonic kidney cell line (HEK 293) using a polymerase chain reaction strategy. It was found by sequence analysis that the cDNA encoded for a human kidney isoform of the Na+/Ca2+ exchanger. The kidney isoform is nearly identical to the human cardiac Na+/Ca2+ exchanger except for a segment of amino acids within the predicted cytoplasmic loop region of the molecule. Within this region, the kidney isoform contains a deletion of 36 amino acids, as well as a segment of 33 amino acids that is only 33% identical to the equivalent region of the cardiac exchanger. The cDNA was then tested for function by expression in insect cells. We constructed a recombinant baculovirus containing renal Na+/Ca2+ exchanger cDNA under control of the polyhedrin promoter. High levels of expression and Na+/Ca2+ exchange activity were found in Sf9 insect cells infected with the recombinant virus. Our results indicate that human kidney expresses a functional and alternatively spliced variant of the human cardiac Na+/Ca2+ exchanger.
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Matsuoka, Satoshi. "Forefront of Na+/Ca2+ Exchanger Studies: Regulation Kinetics of Na+/Ca2+ Exchangers." Journal of Pharmacological Sciences 96, no. 1 (2004): 12–14. http://dx.doi.org/10.1254/jphs.fmj04002x2.
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Artman, M. "Sarcolemmal Na(+)-Ca2+ exchange activity and exchanger immunoreactivity in developing rabbit hearts." American Journal of Physiology-Heart and Circulatory Physiology 263, no. 5 (November 1, 1992): H1506—H1513. http://dx.doi.org/10.1152/ajpheart.1992.263.5.h1506.
Full textAbstract:
It has been postulated that as a consequence of an underdeveloped sarcoplasmic reticulum, sarcolemmal Na(+)-Ca2+ exchange assumes relatively greater importance in modulating Ca2+ fluxes in the developing heart. To explore this concept, cardiac sarcolemmal vesicles were prepared from late fetal (28-day gestation), newborn (24-48 h), immature (14-16 days), and adult New Zealand White rabbits. Na(+)-dependent Ca2+ uptake was measured by diluting Na(+)-loaded (140 mM) vesicles into Na(+)-free buffer and measuring 45Ca2+ uptake (40 microM Ca2+) by timed quenching and rapid filtration. Vesicles from all four age groups demonstrated Ca2+ uptake curves characteristic of Na(+)-Ca2+ exchange with stimulation by valinomycin and inhibition by amiloride. Initial uptake velocity (measured at 2 s and corrected for the fraction of competent vesicles) was significantly higher in fetal (23.2 +/- 5.5 nmol/mg) and newborn (26.2 +/- 5.9 nmol/mg) than in adult sarcolemmal preparations (7.3 +/- 1.2 nmol/mg). Uptake was intermediate in the 2-wk-old group (13.7 +/- 1.7 nmol/mg). The relative amounts of exchanger protein were compared by quantitating immunoreactivity using a polyclonal antibody to the Na(+)-Ca2+ exchanger. Densitometric scanning of protein slot blots demonstrated approximately 2.5 times more exchanger protein in fetal and newborn sarcolemma than in adult preparations. The relative amount of exchanger protein detected immunologically corresponded with the age-related differences observed in exchanger activity. Thus the cardiac sarcolemmal Na(+)-Ca2+ exchanger is abundant and functionally well-developed in the late fetal/early newborn rabbit heart and appears to decline postnatally.(ABSTRACT TRUNCATED AT 250 WORDS)
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Al-Maghout, Tamer, Lisann Pelzl, Itishri Sahu, Basma Sukkar, Zohreh Hosseinzadeh, Ravi Gutti, Stefan Laufer, et al. "P38 Kinase, SGK1 and NF-κB Dependent Up-Regulation of Na+/Ca2+ Exchanger Expression and Activity Following TGFß1 Treatment of Megakaryocytes." Cellular Physiology and Biochemistry 42, no. 6 (2017): 2169–81. http://dx.doi.org/10.1159/000479992.
Full textAbstract:
Background: TGFβ1, a decisive regulator of megakaryocyte maturation and platelet formation, has previously been shown to up-regulate both, store operated Ca2+ entry (SOCE) and Ca2+ extrusion by Na+/Ca2+ exchange. The growth factor thus augments the increase of cytosolic Ca2+ activity ([Ca2+]i) following release of Ca2+ from intracellular stores and accelerates the subsequent decline of [Ca2+]i. The effect on SOCE is dependent on a signaling cascade including p38 kinase, serum & glucocorticoid inducible kinase SGK1, and nuclear factor NFκB. The specific Na+/Ca2+ exchanger isoforms involved and the signalling regulating the Na+/Ca2+ exchangers remained, however elusive. The present study explored, whether TGFβ1 influences the expression and function of K+ insensitive (NCX) and K+ sensitive (NCKX) Na+/Ca2+ exchangers, and aimed to shed light on the signalling involved. Methods: In human megakaryocytic cells (MEG01) RT-PCR was performed to quantify NCX/NCKX isoform transcript levels, [Ca2+]i was determined by Fura-2 fluorescence, and Na+/Ca2+ exchanger activity was estimated from the increase of [Ca2+]i following switch from an extracellular solution with 130 or 90 mM Na+ and 0 mM Ca2+ to an extracellular solution with 0 Na+ and 2 mM Ca2+. K+ concentration was 0 mM for analysis of NCX and 40 mM for analysis of NCKX. Results: TGFβ1 (60 ng/ml, 24 h) significantly increased the transcript levels of NCX1, NCKX1, NCKX2 and NCKX5. Moreover, TGFβ1 (60 ng/ml, 24 h) significantly increased the activity of both, NCX and NCKX. The effect of TGFβ1 on NCX and NCKX transcript levels and activity was significantly blunted by p38 kinase inhibitor Skepinone-L (1 µM), the effect on NCX and NCKX activity further by SGK1 inhibitor GSK-650394 (10 µM) and NFκB inhibitor Wogonin (100 µM). Conclusions: TGFβ1 markedly up-regulates transcription of NCX1, NCKX1, NCKX2, and NCKX5 and thus Na+/Ca2+ exchanger activity, an effect requiring p38 kinase, SGK1 and NFκB.
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Beaugé, Luis, and Reinaldo DiPolo. "Dual effect of Nai+ on Ca2+ influx through the Na+/Ca2+ exchanger in dialyzed squid axons. Experimental data confirming the validity of the squid axon kinetic model." American Journal of Physiology-Cell Physiology 294, no. 1 (January 2008): C118—C125. http://dx.doi.org/10.1152/ajpcell.00341.2007.
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We propose a steady-state kinetic model for the squid Na+/Ca2+ exchanger that differs from other current models of regulation in that it takes into account, within a single kinetic scheme, all ionic [intracellular Ca2+ (Cai2+)-intracellular Na+ (Nai+)-intracellular Hi+] and metabolic (ATP) regulations of the exchanger in which the Cai2+-regulatory pathway plays the central role in regulation. Although the integrated ionic-metabolic model predicts all squid steady-state experimental data on exchange regulation, a critical test for the validity of it is the predicted dual effect of Nai+ on steady-state Ca2+ influx through the exchanger. To test this prediction, an improved technique for the estimation of isotope fluxes in squid axons was developed, which allows sequential measurements of ion influx and effluxes. With this method, we report here two novel observations of the squid axon Na+/Ca2+ exchanger. First, at intracellular pH (7.0) and in the absence of MgATP, Nai+ has a dual effect on Ca2+ influx: inhibition at low concentrations followed by stimulation at high Nai+ concentrations, reaching levels higher than those seen without Nai+. Second, in the presence of MgATP, the biphasic response to Nai+ disappears and is replaced by a sigmoid activation. Furthermore, the model predicts that Ca2+ efflux is monotonically inhibited by Nai+, more pronouncedly without than with MgATP. These results are predicted by the proposed kinetic model. Although not fully applicable to all exchangers, this scheme might provide some insights on expected net Ca2+ movements in other tissues under a variety of intracellular ionic and metabolic conditions.
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Micci, M. A., and Burgess N. Christensen. "Na+/Ca2+exchange in catfish retina horizontal cells: regulation of intracellular Ca2+ store function." American Journal of Physiology-Cell Physiology 274, no. 6 (June 1, 1998): C1625—C1633. http://dx.doi.org/10.1152/ajpcell.1998.274.6.c1625.
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The role of the Na+/Ca2+exchanger in intracellular Ca2+regulation was investigated in freshly dissociated catfish retinal horizontal cells (HC). Ca2+-permeable glutamate receptors and L-type Ca2+ channels as well as inositol 1,4,5-trisphosphate-sensitive and caffeine-sensitive intracellular Ca2+ stores regulate intracellular Ca2+ in these cells. We used the Ca2+-sensitive dye fluo 3 to measure changes in intracellular Ca2+ concentration ([Ca2+]i) under conditions in which Na+/Ca2+exchange was altered. In addition, the role of the Na+/Ca2+exchanger in the refilling of the caffeine-sensitive Ca2+ store following caffeine-stimulated Ca2+ release was assessed. Brief applications of caffeine (1–10 s) produced rapid and transient changes in [Ca2+]i. Repeated applications of caffeine produced smaller Ca2+ transients until no further Ca2+ was released. Store refilling occurred within 1–2 min and required extracellular Ca2+. Ouabain-induced increases in intracellular Na+ concentration ([Na+]i) increased both basal free [Ca2+]iand caffeine-stimulated Ca2+release. Reduction of external Na+concentration ([Na+]o) further and reversibly increased [Ca2+]iin ouabain-treated HC. This effect was not abolished by the Ca2+ channel blocker nifedipine, suggesting that increases in [Na+]ipromote net extracellular Ca2+influx through a Na+/Ca2+exchanger. Moreover, when [Na+]owas replaced by Li+, caffeine did not stimulate release of Ca2+ from the caffeine-sensitive store after Ca2+ depletion. The Na+/Ca2+exchanger inhibitor 2′,4′-dimethylbenzamil significantly reduced the caffeine-evoked Ca2+response 1 and 2 min after store depletion.
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Tibbits, G. F., H. Kashihara, and K. O'Reilly. "Na+-Ca2+ exchange in cardiac sarcolemma: modulation of Ca2+ affinity by exercise." American Journal of Physiology-Cell Physiology 256, no. 3 (March 1, 1989): C638—C643. http://dx.doi.org/10.1152/ajpcell.1989.256.3.c638.
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The high activity of the cardiac Na+-Ca2+ exchanger has led to the suggestion that it plays an important role in the regulation of myocardial contractility. We have proposed that exercise training increases stroke volume as a consequence of an enhanced contractility caused by an adaptation in Ca2+ transport across the cardiac plasma membrane (sarcolemma). The present study examined the possibility that the Na+-Ca2+ exchanger in heart muscle is modified in response to training. Sprague-Dawley rats (female, n = 72) were randomly divided into exercise-trained (T) and sedentary control (C) groups. As a result of the 11-wk treadmill-training paradigm, group T had a 7.6% higher (P less than 0.005) heart-to-body weight ratio and a 36% increase (P less than 0.01) in gastrocnemius mitochondrial enzyme activity. Na+-Ca2+ exchange was studied in highly purified sarcolemmal vesicles using rapid-quenching techniques. The absolute initial rate of uptake was significantly higher in T vs. C at calcium concentrations [( Ca2+]) ranging from 10 to 80 microM. This increased uptake appears to be due solely to the fact that the apparent Km of the myocardial Na+-Ca2+ exchanger for Ca2+ was significantly lower in T vs. C (15.7 +/- 1.1 vs. 36.1 +/- 2.6 microM), since the maximum velocity was unchanged. The observed increase in the affinity of the exchanger for Ca2+ is not attributable to group differences in vesicular purity, cross-contamination, or passive Ca2+ efflux. This observation is consistent with observed alterations in sarcolemmal composition in response to exercise training. We propose that the modification of the Na+-Ca2+ exchanger may play an important role in the adaptation of the heart to exercise.
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Plasman, P. O., P. Lebrun, and A. Herchuelz. "Characterization of the process of sodium-calcium exchange in pancreatic islet cells." American Journal of Physiology-Endocrinology and Metabolism 259, no. 6 (December 1, 1990): E844—E850. http://dx.doi.org/10.1152/ajpendo.1990.259.6.e844.
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Na(+)-Ca2+ exchange may play a role in Ca2+ extrusion from the pancreatic B-cell. The characteristics of the process working in its reverse mode were examined in normal rat pancreatic islet cells. Isosmotical replacement of extracellular Na+ by sucrose induced a concentration-dependent increase in 45Ca uptake, displaying a pharmacological sensitivity compatible with an uptake mediated by Na(+)-Ca2+ exchange. Glucose, up to 2.8 mM, stimulated reverse Na(+)-Ca2+ exchange. Likewise, membrane depolarization activated the process but only under raised intracellular Na+ activity. In conclusion, the B-cell Na(+)-Ca2+ exchange displays properties similar to those observed in other cells: reversibility and sensitivity to membrane potential. When working in its reverse mode the exchanger displays a quite large capacity. The role played by the exchanger in the process of insulin release warrants further investigation.
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Schoenmakers, T. J. M., P. M. Verbost, G. Flik, and S. E. Wendelaar Bonga. "TRANSCELLULAR INTESTINAL CALCIUM TRANSPORT IN FRESHWATER AND SEAWATER FISH AND ITS DEPENDENCE ON SODIUM/CALCIUM EXCHANGE." Journal of Experimental Biology 176, no. 1 (March 1, 1993): 195–206. http://dx.doi.org/10.1242/jeb.176.1.195.
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Transepithelial calcium uptake and transcellular calcium uptake mechanisms were compared in the proximal intestine of freshwater- and seawater-adapted tilapia, Oreochromis mossambicus. Stripped intestinal epithelium of seawater fish showed a higher paracellular permeability to calcium in vitro. Net transepithelial calcium uptake was 71 % lower, reflecting a physiological response to the increased inward calcium gradient. Na+/K+-ATPase activity was significantly enhanced in enterocytes of seawater-adapted fish, in line with the water transport function of the intestine in seawater fish. The Vmax and the Km values for Ca2+ of the ATP-dependent Ca2+ pump in seawater fish enterocytes were 28 % and 27 %, respectively, lower than in freshwater fish. The Km for Ca2+ of the Na+/Ca2+ exchanger was 22 % lower, and a 57 % decrease in the Vmax for Ca2+ of the exchanger was observed. Apparently, the density of exchanger molecules in the basolateral plasma membrane is reduced in seawater fish. From the correlation between the differences in net intestinal calcium uptake and Na+/Ca2+ exchange activity we conclude that Na+/Ca2+ exchange is the main basolateral effector of transcellular calcium uptake.
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Elias, Chadwick L., Anton Lukas, Sabin Shurraw, Jason Scott, Alexander Omelchenko, Gil J. Gross, Mark Hnatowich, and Larry V. Hryshko. "Inhibition of Na+/Ca2+ exchange by KB-R7943: transport mode selectivity and antiarrhythmic consequences." American Journal of Physiology-Heart and Circulatory Physiology 281, no. 3 (September 1, 2001): H1334—H1345. http://dx.doi.org/10.1152/ajpheart.2001.281.3.h1334.
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The Na+/Ca2+ exchanger plays a prominent role in regulating intracellular Ca2+ levels in cardiac myocytes and can serve as both a Ca2+ influx and efflux pathway. A novel inhibitor, KB-R7943, has been reported to selectively inhibit the reverse mode (i.e. , Ca2+ entry) of Na+/Ca2+ exchange transport, although many aspects of its inhibitory properties remain controversial. We evaluated the inhibitory effects of KB-R7943 on Na+/Ca2+exchange currents using the giant excised patch-clamp technique. Membrane patches were obtained from Xenopus laevis oocytes expressing the cloned cardiac Na+/Ca2+exchanger NCX1.1, and outward, inward, and combined inward-outward currents were studied. KB-R7943 preferentially inhibited outward (i.e., reverse) Na+/Ca2+ exchange currents. The inhibitory mechanism consists of direct effects on the transport machinery of the exchanger, with additional influences on ionic regulatory properties. Competitive interactions between KB-R7943 and the transported ions were not observed. The antiarrhythmic effects of KB-R7943 were then evaluated in an ischemia-reperfusion model of cardiac injury in Langendorff-perfused whole rabbit hearts using electrocardiography and measurements of left ventricular pressure. When 3 μM KB-R7943 was applied for 10 min before a 30-min global ischemic period, ventricular arrhythmias (tachycardia and fibrillation) associated with both ischemia and reperfusion were almost completely suppressed. The observed electrophysiological profile of KB-R7943 and its protective effects on ischemia-reperfusion-induced ventricular arrhythmias support the notion of a prominent role of Ca2+ entry via reverse Na+/Ca2+ exchange in this process.
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Tibbits, G. F., K. D. Philipson, and H. Kashihara. "Characterization of myocardial Na(+)-Ca2+ exchange in rainbow trout." American Journal of Physiology-Cell Physiology 262, no. 2 (February 1, 1992): C411—C417. http://dx.doi.org/10.1152/ajpcell.1992.262.2.c411.
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This study compared Na(+)-Ca2+ exchange from the hearts of rainbow trout with that from canines. In several respects, trout cardiac Na(+)-Ca2+ exchange is functionally similar to that from dogs and other mammals. Trout cardiac Na(+)-Ca2+ exchange is stimulated approximately 200% after 30-min incubation with 10 micrograms/ml chymotrypsin at 21 degrees C, similar to mammals. On the other hand, both the temperature and pH dependencies are strikingly different between the trout and canine myocardial Na(+)-Ca2+ exchange. While canine heart Na(+)-Ca2+ exchange exhibits a Q10 of greater than 2 (similar to values observed in other mammals), that from trout is relatively insensitive to temperature with a Q10 of approximately 1.2. The absolute rates of Na(+)-Ca2+ exchange in trout heart are four- to sixfold higher than that in mammals when measured at 7 degrees C. Furthermore, the temperature insensitivity of trout myocardial Na(+)-Ca2+ exchange is retained when the exchanger is reconstituted into an asolectin bilayer, suggesting that this property is intrinsic to the protein and not dependent on species differences in lipid bilayer composition. Trout Na(+)-Ca2+ exchange is not markedly stimulated by alkaline pH, in contrast to mammals, and this characteristic is also maintained after reconstitution. Western blots of trout cardiac sarcolemma run on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis react with antibodies raised against the canine Na(+)-Ca2+ exchanger with a similar pattern of bands (70, 120, and 160 kDa). Furthermore, a cDNA probe from canine Na(+)-Ca2+ exchanger hybridizes on Northern blots of trout heart mRNA to a 7-kb band, similar to that in mammals. Thus, while important functional differences in Na(+)-Ca2+ exchange exist between trout and mammalian hearts, the molecular basis is not yet known.
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Watanabe, Yasuhide, Yuuki Koide, and Junko Kimura. "Topics on the Na+/Ca2+ Exchanger: Pharmacological Characterization of Na+/Ca2+ Exchanger Inhibitors." Journal of Pharmacological Sciences 102, no. 1 (2006): 7–16. http://dx.doi.org/10.1254/jphs.fmj06002x2.
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Pelzl, Lisann, Zohreh Hosseinzadeh, Tamer al-Maghout, Yogesh Singh, Itishri Sahu, Rosi Bissinger, Sebastian Schmidt, et al. "Role of Na+/Ca2+ Exchangers in Therapy Resistance of Medulloblastoma Cells." Cellular Physiology and Biochemistry 42, no. 3 (2017): 1240–51. http://dx.doi.org/10.1159/000478953.
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Background/Aims: Alterations of cytosolic Ca2+-activity ([Ca2+]i) are decisive in the regulation of tumor cell proliferation, migration and survival. Transport processes participating in the regulation of [Ca2+]i include Ca2+ extrusion through K+-independent (NCX) and/or K+-dependent (NCKX) Na+/Ca2+-exchangers. The present study thus explored whether medulloblastoma cells express Na+/Ca2+-exchangers, whether expression differs between therapy sensitive D283 and therapy resistant UW228-3 medulloblastoma cells, and whether Na+/Ca2+-exchangers participate in the regulation of cell survival. Methods: In therapy sensitive D283 and therapy resistant UW228-3 medulloblastoma cells transcript levels were estimated by RT-PCR, protein abundance by Western blotting, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, Na+/ Ca2+-exchanger activity from the increase of [Ca2+]i (Δ[Ca2+]i) and from whole cell current (Ica) following abrupt replacement of Na+ containing (130 mM) and Ca2+ free by Na+ free and Ca2+ containing (2 mM) extracellular perfusate as well as cell death from PI -staining and annexin-V binding in flow cytometry. Results: The transcript levels of NCX3, NCKX2, and NCKX5, protein abundance of NCX3, slope and peak of Δ[Ca2+]i as well as Ica were significantly lower in therapy sensitive D283 than in therapy resistant UW228-3 medulloblastoma cells. The Na+/Ca2+-exchanger inhibitor KB-R7943 (10 µM) significantly blunted Δ[Ca2+]i, and augmented the ionizing radiation-induced apoptosis but did not significantly modify clonogenicity of medulloblastoma cells. Apoptosis was further enhanced by NCX3 silencing. Conclusions: Na+/Ca2+-exchanger activity significantly counteracts apoptosis but does not significantly affect clonogenicity after radiation of medulloblastoma cells.
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Condrescu, Madalina, Galina Chernaya, Vijay Kalaria, and John P. Reeves. "Barium Influx Mediated by the Cardiac Sodium-Calcium Exchanger in Transfected Chinese Hamster Ovary Cells." Journal of General Physiology 109, no. 1 (January 1, 1997): 41–51. http://dx.doi.org/10.1085/jgp.109.1.41.
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We examined Ba2+ influx using isotopic and fura-2 techniques in transfected Chinese hamster ovary cells expressing the bovine cardiac Na+/Ca2+ exchanger (CK1.4 cells). Ba2+ competitively inhibited exchange-me diated 45Ca2+ uptake with a Ki ∼ 3 mM. Ba2+ uptake was stimulated by pretreating the cells with ouabain and by removing extracellular Na+, as expected for Na+/Ba2+ exchange activity. The maximal velocity of Ba2+ accumulation was estimated to be 50% of that for Ca2+. When the monovalent cation ionophore gramicidin was used to equilibrate internal and external concentrations of Na+, Ba2+ influx was negligible in the absence of Na+ and increased to a maximum at 20–40 mM Na+. At higher Na+ concentrations, Ba2+ influx declined, presumably due to the competition between Na+ and Ba2+ for transport sites on the exchanger. Unlike Ca2+, Ba2+ did not appear to be taken up by intracellular organelles: Thus, 133Ba2+ uptake in ouabain-treated cells was not reduced by mitochondrial inhibitors such as Cl-CCP or oligomycin-rotenone. Moreover, intracellular Ca2+ stores that had been depleted of Ca2+ by pretreatment of the cells with ionomycin (a Ca2+ ionophore) remained empty during a subsequent period of Ba2+ influx. Ca2+ uptake or release by intracellular organelles secondarily regulated exchange activity through alterations in [Ca2+]i. Exchange-mediated Ba2+ influx was inhibited when cytosolic [Ca2+] was reduced to 20 nM or less and was accelerated at cytosolic Ca2+ concentrations of 25–50 nM. We conclude that (a) Ba2+ substitutes for Ca2+ as a transport substrate for the exchanger, (b) cytosolic Ba2+ does not appear to be sequestered by intracellular organelles, and (c) exchange-mediated Ba2+ influx is accelerated by low concentrations of cytosolic Ca2+.
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Omelchenko, Alexander, Ron Bouchard, Sabin Shurraw, Michael Trac, Mark Hnatowich, and Larry V. Hryshko. "Frequency-dependent regulation of cardiac Na+/Ca2+ exchanger." American Journal of Physiology-Heart and Circulatory Physiology 289, no. 4 (October 2005): H1594—H1603. http://dx.doi.org/10.1152/ajpheart.01094.2004.
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The activity of the cardiac Na+/Ca2+ exchanger (NCX1.1) undergoes continuous modulation during the contraction-relaxation cycle because of the accompanying changes in the electrochemical gradients for Na+ and Ca2+. In addition, NCX1.1 activity is also modulated via secondary, ionic regulatory mechanisms mediated by Na+ and Ca2+. 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[Formula: see text] and Ca[Formula: see text]). Na+/Ca2+ exchange activity was measured by the giant excised patch-clamp technique with conditions chosen to maximize the extent of Na+- and Ca2+-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+/Ca2+ 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 Ca2+, or exposure of patches to limited proteolysis by α-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[Formula: see text]-dependent inactivation was not apparent. The current approach provides a novel means for assessment of Na+/Ca2+ exchange ionic regulation that may ultimately prove useful in understanding its role under physiological and pathophysiological conditions.
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Vornanen, M. "Na+/Ca2+ exchange current in ventricular myocytes of fish heart: contribution to sarcolemmal Ca2+ influx." Journal of Experimental Biology 202, no. 13 (July 1, 1999): 1763–75. http://dx.doi.org/10.1242/jeb.202.13.1763.
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Influx of extracellular Ca2+ plays a major role in the activation of contraction in fish cardiac cells. The relative contributions of Na+/Ca2+ exchange and L-type Ca2+ channels to Ca2+ influx are, however, unknown. Using a physiological action potential as the command pulse in voltage-clamped heart cells, we examined sarcolemmal Ca2+ influx through Na+/Ca2+ exchange and L-type Ca2+ channels in crucian carp (Carassius carassius L.) ventricular myocytes. When other cation conductances were blocked, a Ni2+-sensitive current with the characteristic voltage- and time-dependent properties of the Na+/Ca2+ exchange current could be distinguished. At the maximum overshoot voltage of the ventricular action potential (+40 mV; [Na+]i=10 mmol l-1), the density of the Na+/Ca2+ exchange current was 2.99+/−0.27 pA pF-1 for warm-acclimated fish (23 degrees C) and 2.38+/−0.42 pA pF-1 for cold-acclimated fish (4 degrees C) (means +/− s.e.m., N=5-6; not significantly different, P=0.26). The relative contributions of the Na+/Ca2+ exchanger and L-type Ca2+ channels to Ca2+ influx were estimated using two partly different methods. Integration of the Ni2+-sensitive Na+/Ca2+ exchange current and the verapamil- and Cd2+-sensitive L-type Ca2+ current suggests that, during the action potential, approximately one-third of the activating Ca2+ comes through Na+/Ca2+ exchange and approximately two-thirds through L-type Ca2+ channels. An alternative method of analysis, using the inward tail current as a measure of the total sarcolemmal Ca2+ flux from which the Ni2+-sensitive Na+/Ca2+ exchange current was subtracted to obtain the Ca2+ influx through the channels, suggests that L-type Ca2+ channels and Na+/Ca2+ exchange are almost equally important in the activation of contraction. Furthermore, the time course of cell shortening is not adequately explained by sarcolemmal Ca2+ influx through the channels alone, but is well approximated by the sum of Ca2+ influx through the channels and the exchanger. The present results indicate that reverse Na+/Ca2+ exchange in crucian carp ventricular myocytes has sufficient capacity to trigger contraction and suggest that the exchange current makes a significant contribution to contractile Ca2+ during the physiological action potential. The relative significance of channels and exchanger molecules in sarcolemmal Ca2+ entry into crucian carp ventricular myocytes was unaffected by thermal acclimation when determined at 22 degrees C.
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Pelzl, Lisann, Zohreh Hosseinzadeh, Kousi Alzoubi, Tamer Al-Maghout, Sebastian Schmidt, Christos Stournaras, and Florian Lang. "Impact of Na+/Ca2+ Exchangers on Therapy Resistance of Ovary Carcinoma Cells." Cellular Physiology and Biochemistry 37, no. 5 (2015): 1857–68. http://dx.doi.org/10.1159/000438547.
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Background/Aims: According to previous observations, enhanced store-operated Ca2+-entry (SOCE) accomplished by the pore forming ion channel unit Orai1 and its regulator STIM1 contribute to therapy resistance of ovary carcinoma cells. Ca2+ signaling is further shaped by Ca2+ extrusion through K+-independent (NCX) and/or K+-dependent (NCKX) Na+/Ca2+-exchangers. The present study thus explored whether therapy resistance is further paralleled by altered expression and/or function of Na+/Ca2+-exchangers. Methods: In therapy resistant (A2780cis) and therapy sensitive (A2780sens) ovary carcinoma cells transcript levels were estimated from RT-PCR, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, Na+/Ca2+-exchanger activity from the increase of [Ca2+]i (Δ[Ca2+]i) and from whole cell current (Ica) following abrupt replacement of Na+ containing (130 mM) and Ca2+ free extracellular perfusate by Na+ free and Ca2+ containing (2 mM) extracellular perfusate, as well as cell death from PI -staining in flow cytometry. Results: The transcript levels of NCX3, NCKX4, NCKX5, and NCKX6, slope and peak of Δ[Ca2+]i as well as Ica were significantly higher in therapy resistant than in therapy sensitive ovary carcinoma cells. The Na+/Ca2+-exchanger inhibitor KB-R7943 (10 µM) significantly blunted Δ[Ca2+]i and significantly augmented the cisplatin-induced cell death of therapy resistant ovary carcinoma cells without significantly modifying cisplatin-induced cell death of therapy sensitive ovary carcinoma cells. Conclusion: Enhanced Na+/Ca2+-exchanger activity may contribute to the therapy sensitivity of ovary carcinoma cells.
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Dipolo, Reinaldo, and Luis Beaugé. "Sodium/Calcium Exchanger: Influence of Metabolic Regulation on Ion Carrier Interactions." Physiological Reviews 86, no. 1 (January 2006): 155–203. http://dx.doi.org/10.1152/physrev.00018.2005.
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The Na+/Ca2+ exchanger's family of membrane transporters is widely distributed in cells and tissues of the animal kingdom and constitutes one of the most important mechanisms for extruding Ca2+ from the cell. Two basic properties characterize them. 1) Their activity is not predicted by thermodynamic parameters of classical electrogenic countertransporters (dependence on ionic gradients and membrane potential), but is markedly regulated by transported (Na+ and Ca2+) and nontransported ionic species (protons and other monovalent cations). These modulations take place at specific sites in the exchanger protein located at extra-, intra-, and transmembrane protein domains. 2) Exchange activity is also regulated by the metabolic state of the cell. The mammalian and invertebrate preparations share MgATP in that role; the squid has an additional compound, phosphoarginine. This review emphasizes the interrelationships between ionic and metabolic modulations of Na+/Ca2+ exchange, focusing mainly in two preparations where most of the studies have been carried out: the mammalian heart and the squid giant axon. A surprising fact that emerges when comparing the MgATP-related pathways in these two systems is that although they are different (phosphatidylinositol bisphosphate in the cardiac and a soluble cytosolic regulatory protein in the squid), their final target effects are essentially similar: Na+-Ca2+-H+ interactions with the exchanger. A model integrating both ionic and metabolic interactions in the regulation of the exchanger is discussed in detail as well as its relevance in cellular Cai2+ homeostasis.
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Iwamoto, Takahiro. "Forefront of Na+/Ca2+ Exchanger Studies: Molecular Pharmacology of Na+/Ca2+ Exchange Inhibitors." Journal of Pharmacological Sciences 96, no. 1 (2004): 27–32. http://dx.doi.org/10.1254/jphs.fmj04002x6.
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