Produção Nacional
Revisado por pares
Cyclosporin A Inhibits Inositol 1,4,5-Trisphosphate-dependent Ca2+ Signals by Enhancing Ca2+ Uptake into the Endoplasmic Reticulum and Mitochondria
2001; Elsevier BV; Volume: 276; Issue: 26 Linguagem: Inglês
10.1074/jbc.m100989200
ISSN1083-351X
AutoresSoraya S. Smaili, Kerri Anne Stellato, P. J. Burnett, Andrew P. Thomas, Lawrence D. Gaspers,
Tópico(s)Ion channel regulation and function
ResumoCytosolic Ca2+([Ca2+]i) oscillations may be generated by the inositol 1,4,5-trisphosphate receptor (IP3R) driven through cycles of activation/inactivation by local Ca2+feedback. Consequently, modulation of the local Ca2+gradients influences IP3R excitability as well as the duration and amplitude of the [Ca2+]ioscillations. In the present work, we demonstrate that the immunosuppressant cyclosporin A (CSA) reduces the frequency of IP3-dependent [Ca2+]ioscillations in intact hepatocytes, apparently by altering the local Ca2+ gradients. Permeabilized cell experiments demonstrated that CSA lowers the apparent IP3 sensitivity for Ca2+ release from intracellular stores. These effects on IP3-dependent [Ca2+]isignals could not be attributed to changes in calcineurin activity, altered ryanodine receptor function, or impaired Ca2+fluxes across the plasma membrane. However, CSA enhanced the removal of cytosolic Ca2+ by sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), lowering basal and inter-spike [Ca2+]i. In addition, CSA stimulated a stable rise in the mitochondrial membrane potential (ΔΨm), presumably by inhibiting the mitochondrial permeability transition pore, and this was associated with increased Ca2+ uptake and retention by the mitochondria during a rise in [Ca2+]i. We suggest that CSA suppresses local Ca2+ feedback by enhancing mitochondrial and endoplasmic reticulum Ca2+ uptake, these actions of CSA underlie the lower IP3 sensitivity found in permeabilized cells and the impaired IP3-dependent [Ca2+]i signals in intact cells. Thus, CSA binding proteins (cyclophilins) appear to fine tune agonist-induced [Ca2+]i signals, which, in turn, may adjust the output of downstream Ca2+-sensitive pathways. Cytosolic Ca2+([Ca2+]i) oscillations may be generated by the inositol 1,4,5-trisphosphate receptor (IP3R) driven through cycles of activation/inactivation by local Ca2+feedback. Consequently, modulation of the local Ca2+gradients influences IP3R excitability as well as the duration and amplitude of the [Ca2+]ioscillations. In the present work, we demonstrate that the immunosuppressant cyclosporin A (CSA) reduces the frequency of IP3-dependent [Ca2+]ioscillations in intact hepatocytes, apparently by altering the local Ca2+ gradients. Permeabilized cell experiments demonstrated that CSA lowers the apparent IP3 sensitivity for Ca2+ release from intracellular stores. These effects on IP3-dependent [Ca2+]isignals could not be attributed to changes in calcineurin activity, altered ryanodine receptor function, or impaired Ca2+fluxes across the plasma membrane. However, CSA enhanced the removal of cytosolic Ca2+ by sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), lowering basal and inter-spike [Ca2+]i. In addition, CSA stimulated a stable rise in the mitochondrial membrane potential (ΔΨm), presumably by inhibiting the mitochondrial permeability transition pore, and this was associated with increased Ca2+ uptake and retention by the mitochondria during a rise in [Ca2+]i. We suggest that CSA suppresses local Ca2+ feedback by enhancing mitochondrial and endoplasmic reticulum Ca2+ uptake, these actions of CSA underlie the lower IP3 sensitivity found in permeabilized cells and the impaired IP3-dependent [Ca2+]i signals in intact cells. Thus, CSA binding proteins (cyclophilins) appear to fine tune agonist-induced [Ca2+]i signals, which, in turn, may adjust the output of downstream Ca2+-sensitive pathways. cyclosporin A inositol 1,4,5-trisphosphate inositol 1,4,5-trisphosphate receptor ryanodine receptors endoplasmic reticulum phenylephrine vasopressin N-methylvaline-cyclosporin cyclophilin D mitochondrial matrix-free Ca2+ concentration cytosolic-free Ca2+ concentration medium-free Ca2+concentration proton motive force mitochondrial membrane potential FK-506 binding protein permeability transition pore 4-morpholineethanesulfonic acid intracellular-like media tetramethylrhodamine ethyl ester protein kinase C thapsigargin sarco-endoplasmic reticulum Ca2+-ATPase 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tacrolimus carbonyl cyanidep-(tri-fluoromethoxy)phenyl-hydrazone glycine,N-[3-(2-benzothiazolyl)- 6-[2-[2-[bis(carboxymethyl)amino]-5-methylphenoxy]ethoxy]-2-oxo-2H-1-benzopyran-7-yl]-N-(carboxymethyl)-tetrapotassium salt the mitochondrial inhibitors FCCP, rotenone, and oligomycin Immunosuppressants exert their activity by binding to immunophilins, an evolutionary conserved, but structurally heterogeneous family of proteins that shares a common enzymatic activity and pharmacological profile (1Galat A. Eur. J. Biochem. 1993; 216: 689-707Crossref PubMed Scopus (316) Google Scholar, 2Fruman D.A. Burakoff S.J. Bierer B.E. FASEB J. 1994; 8: 391-400Crossref PubMed Scopus (235) Google Scholar, 3Marks A.R. Physiol. Rev. 1996; 76: 631-649Crossref PubMed Scopus (336) Google Scholar). All immunophilins described to date possesscis-trans-peptidylprolyl isomerase or rotamase activity, which has been implicated in the folding, assembly, and trafficking of target proteins in vivo (1Galat A. Eur. J. Biochem. 1993; 216: 689-707Crossref PubMed Scopus (316) Google Scholar, 2Fruman D.A. Burakoff S.J. Bierer B.E. FASEB J. 1994; 8: 391-400Crossref PubMed Scopus (235) Google Scholar). Thecis-trans-peptidylprolyl isomerase activity is inhibited by low concentrations of immunosuppressants. Based upon binding criteria, immunophilins are divided into two classes: (a) the cyclophilin family that selectively binds cyclosporin A (CSA)1 and (b) the FK-506 binding proteins (FKBP), which bind to FK-506, its analogues, and rapamycin. Although the precise functions of immunophilins and their downstream targets remain to be determined, they have frequently been implicated in regulating a variety of Ca2+-dependent pathways. The best characterized therapeutic action of CSA or FK-506 is the suppression of interleukin-2 gene transcription during antigen-induced T-lymphocyte activation. Specifically, CSA·cyclophilin or FK-506·FKBP complexes are targeted to and inhibit the catalytic activity of the Ca2+/calmodulin-dependent protein phosphatase, calcineurin (4Liu J. Farmer Jr., J.D. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3567) Google Scholar), thereby blocking the translocation of nuclear factor of activated T cells (3Marks A.R. Physiol. Rev. 1996; 76: 631-649Crossref PubMed Scopus (336) Google Scholar, 5Snyder S.H. Sabatini D.M. Lai M.M. Steiner J.P. Hamilton G.S. Suzdak P.D. Trends Pharmacol. Sci. 1998; 19: 21-26Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar).In isolated mitochondria, CSA binds to cyclophilin D (CyP-D), which is believed to be a component of the mitochondrial permeability transition pore (PTP). Displacing CyP-D from its binding site favors the closed state of the pore (6Zoratti M. Szabo I. Biochim. Biophys. Acta. 1995; 1241: 139-176Crossref PubMed Scopus (2182) Google Scholar, 7Crompton M. Virji S. Ward J.M. Eur. J. Biochem. 1998; 258: 729-735Crossref PubMed Scopus (403) Google Scholar, 8Woodfield K. Ruck A. Brdiczka D. Halestrap A.P. Biochem. J. 1998; 336: 287-290Crossref PubMed Scopus (321) Google Scholar). Recent evidence suggests that the PTP may be involved in Ca2+ signaling (9Ichas F. Jouaville L.S. Mazat J.P. Cell. 1997; 89: 1145-1153Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar, 10Ichas F. Mazat J.P. Biochim. Biophys Acta. 1998; 1366: 33-50Crossref PubMed Scopus (493) Google Scholar), as well as necrotic and apoptotic cell death (11Kroemer G. Dallaporta B. Resche-Rigon M. Annu. Rev. Physiol. 1998; 60: 619-642Crossref PubMed Scopus (1750) Google Scholar, 12Lemasters J.J. Nieminen A.L. Qian T. Trost L.C. Elmore S.P. Nishimura Y. Crowe R.A. Cascio W.E. Bradham C.A. Brenner D.A. Herman B. Biochim. Biophys. Acta. 1998; 1366: 177-196Crossref PubMed Scopus (1218) Google Scholar, 13Crompton M. Biochem. J. 1999; 341: 233-249Crossref PubMed Scopus (2100) Google Scholar, 14Duchen M.R. J. Physiol. (Lond.). 1999; 516: 1-17Crossref Scopus (528) Google Scholar, 15Smaili S.S. Hsu Y.-H. Youle R.J. Russell J.T. J. Bioenerg. Biomembr. 2000; 32: 35-46Crossref PubMed Scopus (140) Google Scholar). Operating in a low conductance mode, the PTP is also proposed to furnish mitochondria with a fast Ca2+-efflux mechanism (i.e. mitochondrial calcium-induced calcium-release) that, in turn, amplifies inositol 1,4,5-trisphosphate (IP3)-dependent cytosolic calcium ([Ca2+]i) signals (9Ichas F. Jouaville L.S. Mazat J.P. Cell. 1997; 89: 1145-1153Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar, 10Ichas F. Mazat J.P. Biochim. Biophys Acta. 1998; 1366: 33-50Crossref PubMed Scopus (493) Google Scholar).Immunophilins may also modulate the Ca2+ release channels of the internal stores directly. FKBP12 forms tight complexes with both ryanodine receptors (RyRs) and IP3 receptors (IP3Rs) that are perturbed by FK-506 or rapamycin (16Brillantes A.B. Ondrias K. Scott A. Kobrinsky E. Ondriasova E. Moschella M.C. Jayaraman T. Landers M. Ehrlich B.E. Marks A.R. Cell. 1994; 77: 513-523Abstract Full Text PDF PubMed Scopus (698) Google Scholar, 17Cameron A.M. Steiner J.P. Roskams A.J. Ali S.M. Ronnett G.V. Snyder S.H. Cell. 1995; 83: 463-472Abstract Full Text PDF PubMed Scopus (446) Google Scholar, 18Cameron A.M. Steiner J.P. Sabatini D.M. Kaplin A.I. Walensky L.D. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1784-1788Crossref PubMed Scopus (269) Google Scholar, 19Cameron A.M. Nucifora Jr., F.C. Fung E.T. Livingston D.J. Aldape R.A. Ross C.A. Snyder S.H. J. Biol. Chem. 1997; 272: 27582-27588Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Dissociation of the FKBP from the channels results in increased Ca2+ fluxes in response to caffeine or IP3 (16Brillantes A.B. Ondrias K. Scott A. Kobrinsky E. Ondriasova E. Moschella M.C. Jayaraman T. Landers M. Ehrlich B.E. Marks A.R. Cell. 1994; 77: 513-523Abstract Full Text PDF PubMed Scopus (698) Google Scholar,18Cameron A.M. Steiner J.P. Sabatini D.M. Kaplin A.I. Walensky L.D. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1784-1788Crossref PubMed Scopus (269) Google Scholar). This enhanced Ca2+ release can either be explained by destabilization of the channel following FKBP12 dissociation (3Marks A.R. Physiol. Rev. 1996; 76: 631-649Crossref PubMed Scopus (336) Google Scholar, 16Brillantes A.B. Ondrias K. Scott A. Kobrinsky E. Ondriasova E. Moschella M.C. Jayaraman T. Landers M. Ehrlich B.E. Marks A.R. Cell. 1994; 77: 513-523Abstract Full Text PDF PubMed Scopus (698) Google Scholar) or a change in the phosphorylation state, because FKBP12 also anchors calcineurin to the channel (17Cameron A.M. Steiner J.P. Roskams A.J. Ali S.M. Ronnett G.V. Snyder S.H. Cell. 1995; 83: 463-472Abstract Full Text PDF PubMed Scopus (446) Google Scholar). Moreover, two ubiquitously expressed members of the cyclophilin family, s-cyclophilin and cyclophilin A, colocalize with or bind to the Ca2+storage protein of the ER, calreticulin (20Arber S. Krause K.H. Caroni P. J. Cell Biol. 1992; 116: 113-125Crossref PubMed Scopus (98) Google Scholar, 21Reddy P.A. Atreya C.D. Int. J. Biol. Macromol. 1999; 25: 345-351Crossref PubMed Scopus (14) Google Scholar). Therefore, this raises the possibility that cyclophilins may modulate Ca2+storage properties of the ER and, indirectly, Ca2+ flux through the RyR and/or IP3R. Taken together, there is a wealth of evidence that immunophilins can potentially activate or inhibit Ca2+-dependent signal transduction by modifying the activity of diverse cellular targets.Periodic oscillations or spikes in [Ca2+]i are utilized by a wide range of extracellular stimuli (22Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6148) Google Scholar, 23Berridge M.J. Bootman M.D. Lipp P. Nature. 1998; 395: 645-648Crossref PubMed Scopus (1756) Google Scholar, 24Thomas A.P. Bird G.S. Hajnóczky G. Robb-Gaspers L.D. Putney Jr., J.W. FASEB J. 1996; 10: 1505-1517Crossref PubMed Scopus (418) Google Scholar) and are decoded by a host of downstream sensors (25Li W. Llopis J. Whitney M. Zlokarnik G. Tsien R.Y. Nature. 1998; 392: 936-941Crossref PubMed Scopus (770) Google Scholar, 26Gu X. Spitzer N.C. Nature. 1995; 375: 784-787Crossref PubMed Scopus (470) Google Scholar, 27Dolmetsch R.E. Xu K. Lewis R.S. Nature. 1998; 392: 933-936Crossref PubMed Scopus (1668) Google Scholar, 28Hajnóczky G. Robb-Gaspers L.D. Seitz M.B. Thomas A.P. Cell. 1995; 82: 415-424Abstract Full Text PDF PubMed Scopus (946) Google Scholar). In non-excitable tissues, extracellular agonists mediate increases in [Ca2+]i through the formation of the second messenger IP3 and activation of intracellular Ca2+-release channels (22Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6148) Google Scholar, 23Berridge M.J. Bootman M.D. Lipp P. Nature. 1998; 395: 645-648Crossref PubMed Scopus (1756) Google Scholar, 24Thomas A.P. Bird G.S. Hajnóczky G. Robb-Gaspers L.D. Putney Jr., J.W. FASEB J. 1996; 10: 1505-1517Crossref PubMed Scopus (418) Google Scholar, 29Patel S. Joseph S.K. Thomas A.P. Cell Calcium. 1999; 25: 247-264Crossref PubMed Scopus (369) Google Scholar), which may in turn stimulate the influx of external Ca2+ to sustain the agonist signal and refill the internal stores (30Putney Jr., J.W. Bird G.S. Endocr. Rev. 1993; 14: 610-631Crossref PubMed Scopus (483) Google Scholar). In hepatocytes, the agonist dose, which presumably determines the intracellular [IP3], sets the frequency of [Ca2+]i spikes (i.e. frequency modulation). However, the positive feedback effect of [Ca2+]i on IP3R generates the rapid-rising phase of the [Ca2+]i spike, which is essentially independent of the agonist dose. This Ca2+-positive feedback is also thought to underlie the propagation of regenerative intracellular Ca2+ waves (24Thomas A.P. Bird G.S. Hajnóczky G. Robb-Gaspers L.D. Putney Jr., J.W. FASEB J. 1996; 10: 1505-1517Crossref PubMed Scopus (418) Google Scholar). We have previously suggested that the minimal prerequisites for generating oscillatory [Ca2+]i signals are the concordant actions of Ca2+ and IP3 on IP3R function (31Hajnoczky G. Thomas A.P. EMBO J. 1997; 16: 3533-3543Crossref PubMed Scopus (154) Google Scholar). Because Ca2+ is involved in both the activation and inactivation of the IP3R (22Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6148) Google Scholar, 23Berridge M.J. Bootman M.D. Lipp P. Nature. 1998; 395: 645-648Crossref PubMed Scopus (1756) Google Scholar, 24Thomas A.P. Bird G.S. Hajnóczky G. Robb-Gaspers L.D. Putney Jr., J.W. FASEB J. 1996; 10: 1505-1517Crossref PubMed Scopus (418) Google Scholar), other Ca2+ transport mechanisms could exert profound effects on the dynamics and frequency of [Ca2+]isignals by altering local Ca2+ gradients. Indeed, mitochondrial Ca2+ uptake can modulate IP3sensitivity by suppressing the local, positive feedback effects of Ca2+ on the IP3R in hepatocytes (32Hajnoczky G. Hager R. Thomas A.P. J. Biol. Chem. 1999; 274: 14157-14162Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar).In the present work, we have investigated the effects of CSA on IP3-dependent [Ca2+]ioscillations in hepatocytes. We demonstrate that CSA or its non-immunosuppressive analogue, N-methylvaline-cyclosporin (MeVal-CS), inhibits the frequency of IP3-dependent [Ca2+]ioscillations by simultaneously activating ER and mitochondrial Ca2+ uptake. We provide evidence that this activation suppresses local, positive Ca2+ feedback on the IP3R and lowers IP3R sensitivity. Thus, cyclophilins may play an essential role in determining the shape and frequency of IP3-dependent [Ca2+]i signals and, ultimately, the activity of downstream Ca2+-sensitive targets by regulating cellular Ca2+ transport mechanisms. Immunosuppressants exert their activity by binding to immunophilins, an evolutionary conserved, but structurally heterogeneous family of proteins that shares a common enzymatic activity and pharmacological profile (1Galat A. Eur. J. Biochem. 1993; 216: 689-707Crossref PubMed Scopus (316) Google Scholar, 2Fruman D.A. Burakoff S.J. Bierer B.E. FASEB J. 1994; 8: 391-400Crossref PubMed Scopus (235) Google Scholar, 3Marks A.R. Physiol. Rev. 1996; 76: 631-649Crossref PubMed Scopus (336) Google Scholar). All immunophilins described to date possesscis-trans-peptidylprolyl isomerase or rotamase activity, which has been implicated in the folding, assembly, and trafficking of target proteins in vivo (1Galat A. Eur. J. Biochem. 1993; 216: 689-707Crossref PubMed Scopus (316) Google Scholar, 2Fruman D.A. Burakoff S.J. Bierer B.E. FASEB J. 1994; 8: 391-400Crossref PubMed Scopus (235) Google Scholar). Thecis-trans-peptidylprolyl isomerase activity is inhibited by low concentrations of immunosuppressants. Based upon binding criteria, immunophilins are divided into two classes: (a) the cyclophilin family that selectively binds cyclosporin A (CSA)1 and (b) the FK-506 binding proteins (FKBP), which bind to FK-506, its analogues, and rapamycin. Although the precise functions of immunophilins and their downstream targets remain to be determined, they have frequently been implicated in regulating a variety of Ca2+-dependent pathways. The best characterized therapeutic action of CSA or FK-506 is the suppression of interleukin-2 gene transcription during antigen-induced T-lymphocyte activation. Specifically, CSA·cyclophilin or FK-506·FKBP complexes are targeted to and inhibit the catalytic activity of the Ca2+/calmodulin-dependent protein phosphatase, calcineurin (4Liu J. Farmer Jr., J.D. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3567) Google Scholar), thereby blocking the translocation of nuclear factor of activated T cells (3Marks A.R. Physiol. Rev. 1996; 76: 631-649Crossref PubMed Scopus (336) Google Scholar, 5Snyder S.H. Sabatini D.M. Lai M.M. Steiner J.P. Hamilton G.S. Suzdak P.D. Trends Pharmacol. Sci. 1998; 19: 21-26Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). In isolated mitochondria, CSA binds to cyclophilin D (CyP-D), which is believed to be a component of the mitochondrial permeability transition pore (PTP). Displacing CyP-D from its binding site favors the closed state of the pore (6Zoratti M. Szabo I. Biochim. Biophys. Acta. 1995; 1241: 139-176Crossref PubMed Scopus (2182) Google Scholar, 7Crompton M. Virji S. Ward J.M. Eur. J. Biochem. 1998; 258: 729-735Crossref PubMed Scopus (403) Google Scholar, 8Woodfield K. Ruck A. Brdiczka D. Halestrap A.P. Biochem. J. 1998; 336: 287-290Crossref PubMed Scopus (321) Google Scholar). Recent evidence suggests that the PTP may be involved in Ca2+ signaling (9Ichas F. Jouaville L.S. Mazat J.P. Cell. 1997; 89: 1145-1153Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar, 10Ichas F. Mazat J.P. Biochim. Biophys Acta. 1998; 1366: 33-50Crossref PubMed Scopus (493) Google Scholar), as well as necrotic and apoptotic cell death (11Kroemer G. Dallaporta B. Resche-Rigon M. Annu. Rev. Physiol. 1998; 60: 619-642Crossref PubMed Scopus (1750) Google Scholar, 12Lemasters J.J. Nieminen A.L. Qian T. Trost L.C. Elmore S.P. Nishimura Y. Crowe R.A. Cascio W.E. Bradham C.A. Brenner D.A. Herman B. Biochim. Biophys. Acta. 1998; 1366: 177-196Crossref PubMed Scopus (1218) Google Scholar, 13Crompton M. Biochem. J. 1999; 341: 233-249Crossref PubMed Scopus (2100) Google Scholar, 14Duchen M.R. J. Physiol. (Lond.). 1999; 516: 1-17Crossref Scopus (528) Google Scholar, 15Smaili S.S. Hsu Y.-H. Youle R.J. Russell J.T. J. Bioenerg. Biomembr. 2000; 32: 35-46Crossref PubMed Scopus (140) Google Scholar). Operating in a low conductance mode, the PTP is also proposed to furnish mitochondria with a fast Ca2+-efflux mechanism (i.e. mitochondrial calcium-induced calcium-release) that, in turn, amplifies inositol 1,4,5-trisphosphate (IP3)-dependent cytosolic calcium ([Ca2+]i) signals (9Ichas F. Jouaville L.S. Mazat J.P. Cell. 1997; 89: 1145-1153Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar, 10Ichas F. Mazat J.P. Biochim. Biophys Acta. 1998; 1366: 33-50Crossref PubMed Scopus (493) Google Scholar). Immunophilins may also modulate the Ca2+ release channels of the internal stores directly. FKBP12 forms tight complexes with both ryanodine receptors (RyRs) and IP3 receptors (IP3Rs) that are perturbed by FK-506 or rapamycin (16Brillantes A.B. Ondrias K. Scott A. Kobrinsky E. Ondriasova E. Moschella M.C. Jayaraman T. Landers M. Ehrlich B.E. Marks A.R. Cell. 1994; 77: 513-523Abstract Full Text PDF PubMed Scopus (698) Google Scholar, 17Cameron A.M. Steiner J.P. Roskams A.J. Ali S.M. Ronnett G.V. Snyder S.H. Cell. 1995; 83: 463-472Abstract Full Text PDF PubMed Scopus (446) Google Scholar, 18Cameron A.M. Steiner J.P. Sabatini D.M. Kaplin A.I. Walensky L.D. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1784-1788Crossref PubMed Scopus (269) Google Scholar, 19Cameron A.M. Nucifora Jr., F.C. Fung E.T. Livingston D.J. Aldape R.A. Ross C.A. Snyder S.H. J. Biol. Chem. 1997; 272: 27582-27588Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Dissociation of the FKBP from the channels results in increased Ca2+ fluxes in response to caffeine or IP3 (16Brillantes A.B. Ondrias K. Scott A. Kobrinsky E. Ondriasova E. Moschella M.C. Jayaraman T. Landers M. Ehrlich B.E. Marks A.R. Cell. 1994; 77: 513-523Abstract Full Text PDF PubMed Scopus (698) Google Scholar,18Cameron A.M. Steiner J.P. Sabatini D.M. Kaplin A.I. Walensky L.D. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1784-1788Crossref PubMed Scopus (269) Google Scholar). This enhanced Ca2+ release can either be explained by destabilization of the channel following FKBP12 dissociation (3Marks A.R. Physiol. Rev. 1996; 76: 631-649Crossref PubMed Scopus (336) Google Scholar, 16Brillantes A.B. Ondrias K. Scott A. Kobrinsky E. Ondriasova E. Moschella M.C. Jayaraman T. Landers M. Ehrlich B.E. Marks A.R. Cell. 1994; 77: 513-523Abstract Full Text PDF PubMed Scopus (698) Google Scholar) or a change in the phosphorylation state, because FKBP12 also anchors calcineurin to the channel (17Cameron A.M. Steiner J.P. Roskams A.J. Ali S.M. Ronnett G.V. Snyder S.H. Cell. 1995; 83: 463-472Abstract Full Text PDF PubMed Scopus (446) Google Scholar). Moreover, two ubiquitously expressed members of the cyclophilin family, s-cyclophilin and cyclophilin A, colocalize with or bind to the Ca2+storage protein of the ER, calreticulin (20Arber S. Krause K.H. Caroni P. J. Cell Biol. 1992; 116: 113-125Crossref PubMed Scopus (98) Google Scholar, 21Reddy P.A. Atreya C.D. Int. J. Biol. Macromol. 1999; 25: 345-351Crossref PubMed Scopus (14) Google Scholar). Therefore, this raises the possibility that cyclophilins may modulate Ca2+storage properties of the ER and, indirectly, Ca2+ flux through the RyR and/or IP3R. Taken together, there is a wealth of evidence that immunophilins can potentially activate or inhibit Ca2+-dependent signal transduction by modifying the activity of diverse cellular targets. Periodic oscillations or spikes in [Ca2+]i are utilized by a wide range of extracellular stimuli (22Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6148) Google Scholar, 23Berridge M.J. Bootman M.D. Lipp P. Nature. 1998; 395: 645-648Crossref PubMed Scopus (1756) Google Scholar, 24Thomas A.P. Bird G.S. Hajnóczky G. Robb-Gaspers L.D. Putney Jr., J.W. FASEB J. 1996; 10: 1505-1517Crossref PubMed Scopus (418) Google Scholar) and are decoded by a host of downstream sensors (25Li W. Llopis J. Whitney M. Zlokarnik G. Tsien R.Y. Nature. 1998; 392: 936-941Crossref PubMed Scopus (770) Google Scholar, 26Gu X. Spitzer N.C. Nature. 1995; 375: 784-787Crossref PubMed Scopus (470) Google Scholar, 27Dolmetsch R.E. Xu K. Lewis R.S. Nature. 1998; 392: 933-936Crossref PubMed Scopus (1668) Google Scholar, 28Hajnóczky G. Robb-Gaspers L.D. Seitz M.B. Thomas A.P. Cell. 1995; 82: 415-424Abstract Full Text PDF PubMed Scopus (946) Google Scholar). In non-excitable tissues, extracellular agonists mediate increases in [Ca2+]i through the formation of the second messenger IP3 and activation of intracellular Ca2+-release channels (22Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6148) Google Scholar, 23Berridge M.J. Bootman M.D. Lipp P. Nature. 1998; 395: 645-648Crossref PubMed Scopus (1756) Google Scholar, 24Thomas A.P. Bird G.S. Hajnóczky G. Robb-Gaspers L.D. Putney Jr., J.W. FASEB J. 1996; 10: 1505-1517Crossref PubMed Scopus (418) Google Scholar, 29Patel S. Joseph S.K. Thomas A.P. Cell Calcium. 1999; 25: 247-264Crossref PubMed Scopus (369) Google Scholar), which may in turn stimulate the influx of external Ca2+ to sustain the agonist signal and refill the internal stores (30Putney Jr., J.W. Bird G.S. Endocr. Rev. 1993; 14: 610-631Crossref PubMed Scopus (483) Google Scholar). In hepatocytes, the agonist dose, which presumably determines the intracellular [IP3], sets the frequency of [Ca2+]i spikes (i.e. frequency modulation). However, the positive feedback effect of [Ca2+]i on IP3R generates the rapid-rising phase of the [Ca2+]i spike, which is essentially independent of the agonist dose. This Ca2+-positive feedback is also thought to underlie the propagation of regenerative intracellular Ca2+ waves (24Thomas A.P. Bird G.S. Hajnóczky G. Robb-Gaspers L.D. Putney Jr., J.W. FASEB J. 1996; 10: 1505-1517Crossref PubMed Scopus (418) Google Scholar). We have previously suggested that the minimal prerequisites for generating oscillatory [Ca2+]i signals are the concordant actions of Ca2+ and IP3 on IP3R function (31Hajnoczky G. Thomas A.P. EMBO J. 1997; 16: 3533-3543Crossref PubMed Scopus (154) Google Scholar). Because Ca2+ is involved in both the activation and inactivation of the IP3R (22Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6148) Google Scholar, 23Berridge M.J. Bootman M.D. Lipp P. Nature. 1998; 395: 645-648Crossref PubMed Scopus (1756) Google Scholar, 24Thomas A.P. Bird G.S. Hajnóczky G. Robb-Gaspers L.D. Putney Jr., J.W. FASEB J. 1996; 10: 1505-1517Crossref PubMed Scopus (418) Google Scholar), other Ca2+ transport mechanisms could exert profound effects on the dynamics and frequency of [Ca2+]isignals by altering local Ca2+ gradients. Indeed, mitochondrial Ca2+ uptake can modulate IP3sensitivity by suppressing the local, positive feedback effects of Ca2+ on the IP3R in hepatocytes (32Hajnoczky G. Hager R. Thomas A.P. J. Biol. Chem. 1999; 274: 14157-14162Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). In the present work, we have investigated the effects of CSA on IP3-dependent [Ca2+]ioscillations in hepatocytes. We demonstrate that CSA or its non-immunosuppressive analogue, N-methylvaline-cyclosporin (MeVal-CS), inhibits the frequency of IP3-dependent [Ca2+]ioscillations by simultaneously activating ER and mitochondrial Ca2+ uptake. We provide evidence that this activation suppresses local, positive Ca2+ feedback on the IP3R and lowers IP3R sensitivity. Thus, cyclophilins may play an essential role in determining the shape and frequency of IP3-dependent [Ca2+]i signals and, ultimately, the activity of downstream Ca2+-sensitive targets by regulating cellular Ca2+ transport mechanisms. We thank Dr. Anthony J. Morgan for the decisive interaction and discussion with the permeabilized hepatocytes experiments and Drs. Basal Hantash, Sandip Patel, and John Reeves for helpful discussion during the execution of this work.
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