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• W2045988498 abstract "Na+/Ca2+ exchange activity in Chinese hamster ovary cells expressing the bovine cardiac Na+/Ca2+ exchanger was inhibited by the short chain ceramide analogs N-acetylsphingosine andN-hexanoylsphingosine (5–15 μm). The sphingolipids reduced exchange-mediated Ba2+ influx by 50–70% and also inhibited the Ca2+ efflux mode of exchange activity. The biologically inactive ceramide analogN-acetylsphinganine had only modest effects on exchange activity. Cells expressing the Δ(241–680) and Δ(680–685) deletion mutants of the Na+/Ca2+ exchanger were not inhibited by ceramide; these mutants show defects in both Na+-dependent and Ca2+-dependent regulatory behavior. Another mutant, which was defective only in Na+-dependent regulation, was as sensitive to ceramide inhibition as the wild-type exchanger. Inhibition of exchange activity by ceramide was time-dependent and was accelerated by depletion of internal Ca2+ stores. Sphingosine (2.5 μm) also inhibited the Ca2+ influx and efflux modes of exchange activity in cells expressing the wild-type exchanger; sphingosine did not affect Ba2+ influx in the Δ(241–680) mutant. The effects of the exogenous sphingolipids were reproduced by blocking cellular ceramide utilization pathways, suggesting that exchange activity is inhibited by increased levels of endogenous ceramide and/or sphingosine. We propose that sphingolipids impair Ca2+-dependent activation of the exchanger and that in cardiac myocytes, this process serves as a feedback mechanism that links exchange activity to the diastolic concentration of cytosolic Ca2+. Na+/Ca2+ exchange activity in Chinese hamster ovary cells expressing the bovine cardiac Na+/Ca2+ exchanger was inhibited by the short chain ceramide analogs N-acetylsphingosine andN-hexanoylsphingosine (5–15 μm). The sphingolipids reduced exchange-mediated Ba2+ influx by 50–70% and also inhibited the Ca2+ efflux mode of exchange activity. The biologically inactive ceramide analogN-acetylsphinganine had only modest effects on exchange activity. Cells expressing the Δ(241–680) and Δ(680–685) deletion mutants of the Na+/Ca2+ exchanger were not inhibited by ceramide; these mutants show defects in both Na+-dependent and Ca2+-dependent regulatory behavior. Another mutant, which was defective only in Na+-dependent regulation, was as sensitive to ceramide inhibition as the wild-type exchanger. Inhibition of exchange activity by ceramide was time-dependent and was accelerated by depletion of internal Ca2+ stores. Sphingosine (2.5 μm) also inhibited the Ca2+ influx and efflux modes of exchange activity in cells expressing the wild-type exchanger; sphingosine did not affect Ba2+ influx in the Δ(241–680) mutant. The effects of the exogenous sphingolipids were reproduced by blocking cellular ceramide utilization pathways, suggesting that exchange activity is inhibited by increased levels of endogenous ceramide and/or sphingosine. We propose that sphingolipids impair Ca2+-dependent activation of the exchanger and that in cardiac myocytes, this process serves as a feedback mechanism that links exchange activity to the diastolic concentration of cytosolic Ca2+. Chinese hamster ovary N-acetylsphingosine N-hexanoylsphingosine N-acetylsphinganine dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol dl-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol sodium physiological salts solution potassium physiological salts solution 4-morpholinepropanesulfonic acid The Na+/Ca2+ exchanger is the principal Ca2+ efflux mechanism in cardiac myocytes and plays a critical role in regulating the force of cardiac muscle contraction (1Blaustein M.P. Lederer W.J. Physiol. Rev. 1999; 79: 763-854Crossref PubMed Scopus (1443) Google Scholar). Its stoichiometry is thought to be 3 Na+/Ca2+ (2Reeves J.P. Hale C.C. J. Biol. Chem. 1984; 259: 7733-7739Abstract Full Text PDF PubMed Google Scholar), although a recent report suggests that it may have a higher, or variable, stoichiometry (3Fujioka Y. Komeda M. Matsuoka S. J. Physiol. (Lond.). 2000; 523: 339-351Crossref Scopus (83) Google Scholar). Exchange activity is regulated by Ca2+-dependent and Na+-dependent processes; cytosolic Ca2+ activates exchange activity by binding to high affinity regulatory sites located within a large (546 residues) hydrophilic domain situated between the fifth and sixth transmembrane segments of the exchanger (4Nicoll D.A. Ottolia M. Lu L. Lu Y. Philipson K.D. J. Biol. Chem. 1999; 274: 910-917Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 5Iwamoto T. Nakamura T.Y. Pan Y. Uehara A. Imanaga I. Shigekawa M. FEBS Lett. 1999; 446: 264-268Crossref PubMed Scopus (90) Google Scholar). Cytosolic Na+ is thought to induce the time-dependent formation of an inactive state (Na+-dependent inactivation) after it binds to the Na+ translocation sites on the exchanger (6Hilgemann D.W. Matsuoka S. Nagel G.A. Collins A. J. Gen. Physiol. 1992; 100: 905-932Crossref PubMed Scopus (237) Google Scholar). Na+-dependent inactivation can be antagonized by ATP-dependent synthesis of phosphatidylinositol 4,5-bisphosphate, by elevated concentrations of cytosolic Ca2+ and by certain mutations within the “regulatory” hydrophilic domain of the exchanger (6Hilgemann D.W. Matsuoka S. Nagel G.A. Collins A. J. Gen. Physiol. 1992; 100: 905-932Crossref PubMed Scopus (237) Google Scholar, 7Hilgemann D.W. Collins A. Matsuoka S. J. Gen. Physiol. 1992; 100: 933-961Crossref PubMed Scopus (219) Google Scholar, 8Collins A. Somlyo A.V. Hilgemann D.W. J. Physiol. (Lond.). 1992; 454: 27-57Crossref Scopus (124) Google Scholar).The physiological significance of these regulatory mechanisms is uncertain (9Reeves J.P. J. Bioenerg. Biomembr. 1998; 30: 151-160Crossref PubMed Scopus (41) Google Scholar, 10Reeves, J. P., Condrescu, M., and Fang, Y. (1998) Internet Association for Biomedical Sciences 98: 5th Internet World Congress on Biomedical Sciences at McMaster Unversity.Google Scholar). In intact cells, the K d for Ca2+-dependent activation of the exchanger is ∼50 nm (11Fang Y. Condrescu M. Reeves J.P. Am. J. Physiol. 1998; 275: C50-C55Crossref PubMed Google Scholar, 12Noda M. Shepherd R.N. Gadsby D.C. Biophys. J. 1988; 53 (abstr.): 342Google Scholar, 13Miura Y. Kimura J. J. Gen. Physiol. 1989; 93: 1129-1145Crossref PubMed Scopus (173) Google Scholar), suggesting that the exchanger may be nearly fully activated under “resting” conditions. The low cytosolic Na+ concentration and the high levels of ATP and phosphatidyl 4,5-bisphosphate in healthy cells preclude a major role for Na+-dependent inactivation in regulating exchange activity under physiological conditions. Indeed, there is no direct evidence that Na+/Ca2+ exchange activity is in fact regulated in functioning cardiac myocytes. The present report addresses the possibility that sphingolipids such as ceramide and sphingosine serve as physiological regulators of Na+/Ca2+ exchange activity.Ceramide is a central component of the sphingomyelin cycle, a stress-activated signaling pathway that participates in the induction of apoptosis and growth arrest. The activating or inhibiting effects of ceramide on a host of intracellular signaling pathways have been described in several recent reviews (14Perry D.K. Hannun Y.A. Biochim. Biophys. Acta. 1998; 1436: 233-243Crossref PubMed Scopus (294) Google Scholar, 15Kolesnick R.N. Kronke M. Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (725) Google Scholar, 16Liu G. Kleine L. Hebert R.L. Crit. Rev. Clin. Lab Sci. 1999; 36: 511-573Crossref PubMed Scopus (66) Google Scholar). Ceramide can also be converted to sphingosine and sphingosine phosphate, two other signaling lipids with important regulatory effects of their own.Here we show that short chain ceramide analogs and sphingosine inhibit Na+/Ca2+ exchange activity in transfected Chinese hamster ovary (CHO)1cells expressing the bovine or canine cardiac Na+/Ca2+ exchangers. Similar effects were noted when the cells were treated for 60 min with an inhibitor of endogenous ceramide metabolism. The differential effects of ceramide on exchanger mutants defective in Na+- or Ca2+-dependent regulation suggest that ceramide blocks the conformational transitions associated with Ca2+-dependent activation of exchange activity. We propose that in functioning cardiac myocytes, sphingolipids and diastolic Ca2+ levels interact to control the distribution of exchangers between the Ca2+-activated and nonactivated forms.DISCUSSIONThe results presented here demonstrate that short chain ceramide analogs inhibited both the Ca2+ influx and Ca2+efflux modes of Na+/Ca2+ exchange activity (Figs. Figure 1, Figure 2, Figure 3). A biologically inactive ceramide analog, in which the essential double bond in the sphingosine moiety is hydrogenated, had only minor effects on exchange activity (Fig. 2). Moreover, certain mutant exchangers that are defective in their regulatory behavior were not inhibited by ceramide (Figs. 4 and 5). These data suggest that the effects of ceramide are not simply due to a generalized membrane perturbation but that ceramide acts in a biologically relevant manner and specifically targets the mechanisms that regulate exchange activity. Recent results indicate that 15 μm C2-ceramide also inhibits Na+/Ca2+ exchange currents in cardiac myocytes but does not inhibit either Na+ or Ca2+ channel activity. 2N. Shepherd, personal communication.Ceramide is a central component of the sphingomyelin signaling pathway and plays a critical role in the activation of the caspase cascade that leads to apoptosis (14Perry D.K. Hannun Y.A. Biochim. Biophys. Acta. 1998; 1436: 233-243Crossref PubMed Scopus (294) Google Scholar, 15Kolesnick R.N. Kronke M. Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (725) Google Scholar, 16Liu G. Kleine L. Hebert R.L. Crit. Rev. Clin. Lab Sci. 1999; 36: 511-573Crossref PubMed Scopus (66) Google Scholar). In cardiac myocytes, ceramide and sphingosine levels increase following ischemia/reperfusion (36Hernandez O.M. Discher D.J. Bishopric N.H. Webster K.A. Circ. Res. 2000; 86: 198-204Crossref PubMed Scopus (108) Google Scholar, 37Bielawska A.E. Shapiro J.P. Jiang L. Melkonyan H.S. Piot C. Wolfe C.L. Tomei L.D. Hannun Y.A. Umansky S.R. Am. J. Pathol. 1997; 151: 1257-1263PubMed Google Scholar) and after exposure of the cells to tumor necrosis factor α (38Meldrum D.R. Am. J. Physiol. 1998; 274: R577-R595Crossref PubMed Google Scholar). Ceramide induces a multitude of cellular responses, including induction of the stress-activated protein kinase pathway, activation or inhibition of various individual protein kinases, activation of protein phosphatase 2A, inhibition of mitochondrial respiration, activation of cytochromec release from mitochondria, and inhibition of phospholipase D (reviewed in Refs. 14Perry D.K. Hannun Y.A. Biochim. Biophys. Acta. 1998; 1436: 233-243Crossref PubMed Scopus (294) Google Scholar, 15Kolesnick R.N. Kronke M. Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (725) Google Scholar, 16Liu G. Kleine L. Hebert R.L. Crit. Rev. Clin. Lab Sci. 1999; 36: 511-573Crossref PubMed Scopus (66) Google Scholar). Moreover, ceramide can be rapidly converted to ceramide-1-phosphate, sphingosine, and sphingosine-1-phosphate, signaling lipids that have multiple effects of their own. The rapid time course of sphingolipid-induced inhibition of exchange activity in the present study suggests that these agents exert their effects directly on the exchanger rather than through one of the above signal transduction pathways. This conclusion is strongly supported by the recent finding that both sphingosine and C2-ceramide inhibit exchange activity when applied to excised patches from cardiac myocytes. 3D. Hilgemann, personal communication.Short chain ceramide analogs sometimes induce effects that are not mimicked by increases in endogenous ceramide (reviewed in Ref. 39Ghidoni R. Sala G. Giuliani A. Biochim. Biophys. Acta. 1999; 1439: 17-39Crossref PubMed Scopus (40) Google Scholar). Moreover, the short chain analogs may themselves bring about alterations in endogenous ceramide and/or sphingosine levels (35Lepple-Wienhues A. Belka C. Laun T. Jekle A. Walter B. Wieland U. Welz M. Heil L. Kun J. Busch G. Weller M. Bamberg M. Gulbins E. Lang F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13795-13800Crossref PubMed Scopus (152) Google Scholar, 40Cuvillier O. Edsall L. Spiegel S. J. Biol. Chem. 2000; 275: 15691-15700Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar,41Jaffrezou J.P. Maestre N. Mas-Mansat V. Bezombes C. Levade T. Laurent G. FASEB J. 1998; 12: 999-1006Crossref PubMed Scopus (67) Google Scholar). To address these issues, we preincubated the cells for 60 min with PPMP, an agent that blocks conversion of ceramide to glucosylceramide and has been used to elevate endogenous ceramide levels in several different cell types (30Negishi T. Chik C.L. Ho A.K. Endocrinology. 1999; 140: 5691-5697Crossref PubMed Scopus (10) Google Scholar, 31de Chaves E.I.P. Bussiere M. Vance D.E. Campenot R.B. Vance J.E. J. Biol. Chem. 1997; 272: 3028-3035Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 32Maceyka M. Machamer C.E. J. Cell Biol. 1997; 139: 1411-1418Crossref PubMed Scopus (31) Google Scholar, 33Ji L. Ito M. Zhang G. Hirabayashi Y. Inokuchi J. Yamagata T. Arch. Biochem. Biophys. 1998; 359: 107-114Crossref PubMed Scopus (4) Google Scholar). As shown in Fig. 8, treatment with PPMP mimicked the effects of the short chain ceramide analogs on Na+/Ca2+ exchange activity. We conclude that exchange activity is inhibited by increased endogenous ceramide and/or sphingosine as well as by the exogenous analogs.Ceramide and sphingosine did not inhibit exchange activity in certain regulatory-deficient mutants, and we therefore conclude that the sphingolipids target one of the mechanisms that regulate exchange activity. The best characterized regulatory mechanisms involve two time-dependent processes that promote inactive states of the exchanger (6Hilgemann D.W. Matsuoka S. Nagel G.A. Collins A. J. Gen. Physiol. 1992; 100: 905-932Crossref PubMed Scopus (237) Google Scholar, 7Hilgemann D.W. Collins A. Matsuoka S. J. Gen. Physiol. 1992; 100: 933-961Crossref PubMed Scopus (219) Google Scholar). The first process is called “Na+-dependent inactivation” and is observed in excised patches as an exponential decay of current to a steady-state value following application of cytosolic Na+. Na+-dependent inactivation is counteracted by the presence of phosphatidylinositol 4,5-bisphosphate (42Hilgemann D.W. Ball R. Science. 1996; 273: 956-959Crossref PubMed Scopus (557) Google Scholar), by high concentrations of cytosolic Ca2+ (6Hilgemann D.W. Matsuoka S. Nagel G.A. Collins A. J. Gen. Physiol. 1992; 100: 905-932Crossref PubMed Scopus (237) Google Scholar), and by mutations involving key basic residues or tyrosines in the XIP region (24Matsuoka S. Nicoll D.A. He Z. Philipson K.D. J. Gen. Physiol. 1997; 109: 273-286Crossref PubMed Scopus (144) Google Scholar, 43Pan Y. Iwamoto T. Uehara A. Nakamura T.Y. Imanaga I. Shigekawa M. Am. J. Physiol. 2000; 279: C393-C402Crossref PubMed Google Scholar). The second regulatory process involves the interaction of Ca2+ with high affinity regulatory sites within the central hydrophilic domain of the exchanger (7Hilgemann D.W. Collins A. Matsuoka S. J. Gen. Physiol. 1992; 100: 933-961Crossref PubMed Scopus (219) Google Scholar, 44Matsuoka S. Nicoll D.A. Hryshko L.V. Levitsky D.O. Weiss J.N. Philipson K.D. J. Gen. Physiol. 1995; 105: 403-420Crossref PubMed Scopus (204) Google Scholar); the binding of Ca2+ to these regulatory sites appears to be required for all modes of exchanger operation (1Blaustein M.P. Lederer W.J. Physiol. Rev. 1999; 79: 763-854Crossref PubMed Scopus (1443) Google Scholar, 9Reeves J.P. J. Bioenerg. Biomembr. 1998; 30: 151-160Crossref PubMed Scopus (41) Google Scholar).Na+-dependent inactivation does not appear to be involved in the effects of sphingolipids, because the XIPA mutant was as sensitive to inhibition by ceramide as the wild type (Fig.4 B); this mutant does not display Na+-dependent inactivation because of the alteration of critical basic residues in the XIP region. Moreover, ceramide inhibited wild-type exchange activity equally well at high (95 mm) and low (9 mm) Na+concentrations (data not shown). Because Na+-dependent inactivation requires high concentrations of cytosolic Na+ (6Hilgemann D.W. Matsuoka S. Nagel G.A. Collins A. J. Gen. Physiol. 1992; 100: 905-932Crossref PubMed Scopus (237) Google Scholar), these results provide another indication that ceramide does not promote this inactivation process.The findings with the various exchanger mutants and the effects of alterations in Ca2+ homeostasis suggest that sphingolipids interfere with the regulatory activation of the exchanger by Ca2+. C2-ceramide did not inhibit the activities of the deletion mutants Δ(680–685) and Δ(241–680) (Figs. 4 and 5), which are defective in both Ca2+-dependent activation and Na+-dependent inactivation. As mentioned above, C2-ceramide did inhibit the activity of the XIPA mutant (Fig.4 B), in which regulatory Ca2+ activation is intact, but Na+-dependent inactivation does not occur (24Matsuoka S. Nicoll D.A. He Z. Philipson K.D. J. Gen. Physiol. 1997; 109: 273-286Crossref PubMed Scopus (144) Google Scholar, 45Condrescu M. Reeves J.P. Biophys. J. 1999; 76 (abstr.): 253Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Thus, ceramide inhibited the activity of exchangers that display Ca2+-dependent activation but not in exchanger mutants in which this regulatory mechanism was defective.Experimental conditions that altered Ca2+ homeostatic processes also affected the response of exchange activity to ceramide. For example, the effects of ceramide developed slowly in cells with filled Ca2+ stores, as shown by the time-dependent decline in the rate of Ba2+influx in the presence of C2-ceramide (Fig. 1 A). When internal Ca2+ stores were depleted by prior treatment of the cells with thapsigargin or ionomycin, the development of the inhibition of ceramide was accelerated, and the traces for Ba2+ influx no longer displayed a downward curvature (Figs.2 and 6 A). The results suggest that there is a link between the filling state of intracellular Ca2+ stores and the susceptibility of the exchanger to ceramide inhibition. The basis for this observation is not known; perhaps filled Ca2+ stores generate local gradients of elevated [Ca2+]i in the vicinity of the exchanger, and this antagonizes ceramide inhibition. For PPMP-treated cells, inhibition of exchange activity in the absence of thapsigargin was immediate (Table I) and did not increase with time following Ba2+ addition (data not shown). This observation is entirely consistent with the observations described above, because internal Ca2+ stores in the PPMP-treated cells were already depleted, presumably because of an inhibition of Ca2+ influx (see discussion under “Results”).In several experiments, control cells showed a time-dependent increase in the rate of Ba2+influx, as shown by the upward curvature of the traces (Figs.4 B, 6 A, 7 A, and 8, A andC, trace b. Cont). This behavior was not seen with the Δ(241–680) mutant (Figs. 4 B, 7 B, and8 B), but it was observed with the XIPA mutant (Fig.4 B). We have previously suggested that the acceleration in Ba2+ influx is due to the auto-activation of the exchanger by cytosolic Ba2+ through its interaction with the Ca2+ regulatory sites (11Fang Y. Condrescu M. Reeves J.P. Am. J. Physiol. 1998; 275: C50-C55Crossref PubMed Google Scholar). In the presence of ceramide or sphingosine or in PPMP-treated cells, the corresponding traces for Ba2+ influx remained linear throughout the entire time course, indicating that the sphingolipids blocked this process.These considerations suggest that ceramide/sphingosine impairs the regulatory activation of exchange activity by Ca2+. Its precise mechanism of action is not known, however. It does not seem likely that these hydrophobic lipids would interact directly with high affinity Ca2+ regulatory sites, because these are located in the hydrophilic domain of the exchanger. A more plausible possibility is that they interact with the transmembrane segments of the exchanger and stabilize the inactive conformation that is attained upon dissociation of Ca2+ from its regulatory binding sites (Fig. 9). In this way, the sphingolipids might increase the K d for Ca2+activation, slow the conformational transitions involved, or block Ca2+ activation of the exchanger altogether in a subpopulation of exchangers. In any event, it is clear that ceramide does not completely block Ca2+ activation, because exchange activity was stimulated by increasing [Ca2+]i in the ionomycin experiments, both in the presence of C2-ceramide (Fig.6 C) and in the PPMP-treated cells (Fig. 8 D and Table I).In the experiments with ionomycin-treated cells (Figs. 6 and8 C), we sought to determine whether an increase in [Ca2+]i would antagonize the inhibitory effects of ceramide or PPMP treatment, as expected if a competitive effect were involved. In both cases, however, the degree of inhibition was similar at high and low [Ca2+]i. The highest value of [Ca2+]i attained in these experiments was only ∼110 nm, however, and it will be important to re-examine this issue in an experimental system (e.g. excised patches) that permits a broader range of [Ca2+]i values to be studied. These experiments also showed that for the ceramide- or PPMP-treated cells at the elevated [Ca2+]i, the rate of Ba2+ influx declined gradually following Ba2+ addition and eventually became equal to that seen at the lower [Ca2+]i (Figs. 6 B and8 C). This behavior probably reflects the gradual reduction in regulatory activation of exchange activity because the initially high level of [Ca2+]i declined following Ba2+ addition. In the corresponding experiments with control cells, the rate of Ba2+ influx did not decline in this manner, consistent with our hypothesis that the ability of Ca2+ and/or Ba2+ to activate exchange activity is impaired by the sphingolipids.What are the physiological implications of our results? Ceramide and sphingosine are elevated in cardiac myocytes during stress. The resulting inhibition of exchange activity could be viewed as a protective measure to preserve Ca2+ stores and maintain contractile strength under stressful conditions. Alternatively, when cytosolic [Na+] is elevated, as in ischemia, inhibition of exchange activity could be a means of protecting the cell against Ca2+ overload by reducing exchange-mediated Ca2+ influx. A more interesting possibility, however, is that under nonpathological conditions endogenous sphingolipids and the exchanger work coordinately as a Ca2+-dependent feedback mechanism to control the distribution of exchangers between active and inactive states, in the manner described below.The high affinity of the exchanger for regulatory Ca2+(K d ∼50 nm) (11Fang Y. Condrescu M. Reeves J.P. Am. J. Physiol. 1998; 275: C50-C55Crossref PubMed Google Scholar, 12Noda M. Shepherd R.N. Gadsby D.C. Biophys. J. 1988; 53 (abstr.): 342Google Scholar, 13Miura Y. Kimura J. J. Gen. Physiol. 1989; 93: 1129-1145Crossref PubMed Scopus (173) Google Scholar) would seem to provide no opportunity for meaningful regulation of exchange activity within a physiological range of [Ca2+]i values. In a functioning cardiac myocyte, the small fraction of exchangers that become inactive because of dissociation of regulatory Ca2+during diastole ([Ca2+]i ∼100 nm) would be rapidly reactivated by the ensuing rise in [Ca2+]i during the next systole. However, if endogenous ceramide/sphingosine were to interfere with Ca2+-dependent activation of the exchanger by any of the mechanisms suggested above (Fig. 9), a fraction of the inactive exchangers would be retained in the inactive state (I 2 in Fig. 9) despite the rise in [Ca2+]i. Over multiple contraction/relaxation cycles, the distribution of exchangers between active and inactive states would be determined by two principal factors: diastolic [Ca2+]i and endogenous levels of ceramide/sphingosine. A fall in diastolic [Ca2+]iwould increase the population of inactive exchangers, thereby reducing exchange-mediated Ca2+ efflux and eventually restoring diastolic [Ca2+]i to its normal level. An increase in endogenous ceramide or sphingosine, e.g. during stress, would have the same effect and establish a new steady-state relation between diastolic [Ca2+]i and exchange activity.This hypothesis, although speculative, provides a welcome framework for understanding the physiological role of Ca2+-dependent activation of exchange activity in light of the high affinity of the exchanger for regulatory Ca2+ (11Fang Y. Condrescu M. Reeves J.P. Am. J. Physiol. 1998; 275: C50-C55Crossref PubMed Google Scholar, 12Noda M. Shepherd R.N. Gadsby D.C. Biophys. J. 1988; 53 (abstr.): 342Google Scholar, 13Miura Y. Kimura J. J. Gen. Physiol. 1989; 93: 1129-1145Crossref PubMed Scopus (173) Google Scholar). In considering the physiological role of sphingolipid-exchanger interactions, it will be essential to define more precisely its effects on Ca2+-dependent activation and the influence of internal Ca2+ stores on this process. Work toward this end is currently in progress. The Na+/Ca2+ exchanger is the principal Ca2+ efflux mechanism in cardiac myocytes and plays a critical role in regulating the force of cardiac muscle contraction (1Blaustein M.P. Lederer W.J. Physiol. Rev. 1999; 79: 763-854Crossref PubMed Scopus (1443) Google Scholar). Its stoichiometry is thought to be 3 Na+/Ca2+ (2Reeves J.P. Hale C.C. J. Biol. Chem. 1984; 259: 7733-7739Abstract Full Text PDF PubMed Google Scholar), although a recent report suggests that it may have a higher, or variable, stoichiometry (3Fujioka Y. Komeda M. Matsuoka S. J. Physiol. (Lond.). 2000; 523: 339-351Crossref Scopus (83) Google Scholar). Exchange activity is regulated by Ca2+-dependent and Na+-dependent processes; cytosolic Ca2+ activates exchange activity by binding to high affinity regulatory sites located within a large (546 residues) hydrophilic domain situated between the fifth and sixth transmembrane segments of the exchanger (4Nicoll D.A. Ottolia M. Lu L. Lu Y. Philipson K.D. J. Biol. Chem. 1999; 274: 910-917Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 5Iwamoto T. Nakamura T.Y. Pan Y. Uehara A. Imanaga I. Shigekawa M. FEBS Lett. 1999; 446: 264-268Crossref PubMed Scopus (90) Google Scholar). Cytosolic Na+ is thought to induce the time-dependent formation of an inactive state (Na+-dependent inactivation) after it binds to the Na+ translocation sites on the exchanger (6Hilgemann D.W. Matsuoka S. Nagel G.A. Collins A. J. Gen. Physiol. 1992; 100: 905-932Crossref PubMed Scopus (237) Google Scholar). Na+-dependent inactivation can be antagonized by ATP-dependent synthesis of phosphatidylinositol 4,5-bisphosphate, by elevated concentrations of cytosolic Ca2+ and by certain mutations within the “regulatory” hydrophilic domain of the exchanger (6Hilgemann D.W. Matsuoka S. Nagel G.A. Collins A. J. Gen. Physiol. 1992; 100: 905-932Crossref PubMed Scopus (237) Google Scholar, 7Hilgemann D.W. Collins A. Matsuoka S. J. Gen. Physiol. 1992; 100: 933-961Crossref PubMed Scopus (219) Google Scholar, 8Collins A. Somlyo A.V. Hilgemann D.W. J. Physiol. (Lond.). 1992; 454: 27-57Crossref Scopus (124) Google Scholar). The physiological significance of these regulatory mechanisms is uncertain (9Reeves J.P. J. Bioenerg. Biomembr. 1998; 30: 151-160Crossref PubMed Scopus (41) Google Scholar, 10Reeves, J. P., Condrescu, M., and Fang, Y. (1998) Internet Association for Biomedical Sciences 98: 5th Internet World Congress on Biomedical Sciences at McMaster Unversity.Google Scholar). In intact cells, the K d for Ca2+-dependent activation of the exchanger is ∼50 nm (11Fang Y. Condrescu M. Reeves J.P. Am. J. Physiol. 1998; 275: C50-C55Crossref PubMed Google Scholar, 12Noda M. Shepherd R.N. Gadsby D.C. Biophys. J. 1988; 53 (abstr.): 342Google Scholar, 13Miura Y. Kimura J. J. Gen. Physiol. 1989; 93: 1129-1145Crossref PubMed Scopus (173) Google Scholar), suggesting that the exchanger may be nearly fully activated under “resting” conditions. The low cytosolic Na+ concentration and the high levels of ATP and phosphatidyl 4,5-bisphosphate in healthy cells preclude a major role for Na+-dependent inactivation in regulating exchange activity under physiological conditions. Indeed, there is no direct evidence that Na+/Ca2+ exchange activity is in fact regulated in functioning cardiac myocytes. The present report addresses the possibility that sphingolipids such as ceramide and sphingosine serve as physiological regulators of Na+/Ca2+ exchange activity. Ceramide is a central component of the sphingomyelin cycle, a stress-activated signaling pathway that participates in the induction of apoptosis and growth arrest. The activating or inhibiting effects of ceramide on a host of intracellular signaling pathways have been described in several recent reviews (14Perry D.K. Hannun Y.A. Biochim. Biophys. Acta. 1998; 1436: 233-243Crossref PubMed Scopus (294) Google Scholar, 15Kolesnick R.N. Kronke M. Annu. Rev. Physiol. 1998; 60: 643-665Crossref PubMed Scopus (725) Google Scholar, 16Liu G. Kleine L. Hebert R.L. Crit. Rev. Clin. Lab Sci. 1999; 36: 511-573Crossref PubMed Scopus (66) Google Scholar). Ceramide can also be converted to sphingosine and sphingosine phosphate, two other signaling lipids with important regulatory effects of their own. Here we show that short chain ceramide analogs and sphingosine inhibit Na+/Ca2+ exchange activity in transfected Chinese hamster ovary (CHO)1cells expressing the bovine or canine cardiac Na+/Ca2+ exchangers. Similar effects were noted when the cells were treated for 60 min with an inhibitor of endogenous cera" @default.
• W2045988498 created "2016-06-24" @default.
• W2045988498 creator A5032767292 @default.
• W2045988498 creator A5036375618 @default.
• W2045988498 date "2001-02-01" @default.
• W2045988498 modified "2023-09-27" @default.
• W2045988498 title "Inhibition of Sodium-Calcium Exchange by Ceramide and Sphingosine" @default.
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