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• W2022187477 abstract "The entry of Ca2+ following Ca2+ pool release is a major component of Ca2+ signals; yet despite intense study, how “store-operated” entry channels are activated is unresolved. Because S-nitrosylation has become recognized as an important regulatory modification of several key channel proteins, its role in Ca2+ entry was investigated. A novel class of lipophilic NO donors activated Ca2+ entry independent of the well defined NO target, guanylate cyclase. Strikingly similar entry of Ca2+ induced by cell permeant alkylators indicated that this Ca2+ entry process was activated through thiol modification. Significantly, Ca2+ entry activated by either NO donors or alkylators was highly stimulated by Ca2+ pool depletion, which increased both the rate of Ca2+ release and the sensitivity to thiol modifiers. The results indicate thatS-nitrosylation underlies activation of an important store-operated Ca2+ entry mechanism. The entry of Ca2+ following Ca2+ pool release is a major component of Ca2+ signals; yet despite intense study, how “store-operated” entry channels are activated is unresolved. Because S-nitrosylation has become recognized as an important regulatory modification of several key channel proteins, its role in Ca2+ entry was investigated. A novel class of lipophilic NO donors activated Ca2+ entry independent of the well defined NO target, guanylate cyclase. Strikingly similar entry of Ca2+ induced by cell permeant alkylators indicated that this Ca2+ entry process was activated through thiol modification. Significantly, Ca2+ entry activated by either NO donors or alkylators was highly stimulated by Ca2+ pool depletion, which increased both the rate of Ca2+ release and the sensitivity to thiol modifiers. The results indicate thatS-nitrosylation underlies activation of an important store-operated Ca2+ entry mechanism. (5-amino-3-(3, 4-dichlorophenyl)1,2,3,4-oxatriazolium) 2,3,4-oxatriazolium) vinylpyridine sodium nitroprusside N-ethylmaleimide 2,5-di-tert-butylhydroquinone 8-bromo-guanosine 3′,5′-cyclic monophosphate. Ca2+ signals in cells are complex events involving both intracellular Ca2+ pool release and extracellular Ca2+ entry. Emptying of intracellular Ca2+ pools is the major trigger for activation of Ca2+ entry during the generation of receptor-mediated Ca2+ signals (1Putney Jr., J.W. Bird G.S. Cell. 1993; 75: 199-201Abstract Full Text PDF PubMed Scopus (393) Google Scholar, 2Berridge M.J. Biochem. J. 1995; 312: 1-11Crossref PubMed Scopus (1047) Google Scholar, 3Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1289) Google Scholar). However, the mechanism by which Ca2+ pool depletion is coupled to activation of “store-operated” Ca2+ entry channels remains an important but unsolved question (1Putney Jr., J.W. Bird G.S. Cell. 1993; 75: 199-201Abstract Full Text PDF PubMed Scopus (393) Google Scholar, 2Berridge M.J. Biochem. J. 1995; 312: 1-11Crossref PubMed Scopus (1047) Google Scholar, 3Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1289) Google Scholar, 4Favre C.J. Nüße O. Lew D.P. Krause K. J. Lab. Clin. Med. 1996; 128: 19-26Abstract Full Text PDF PubMed Scopus (34) Google Scholar, 5Gill D.L. Waldron R.T. Rys-Sikora K.E. Ufret-Vincenty C.A. Graber M.N. Favre C.J. Alfonso A. Biosci. Rep. 1996; 16: 139-157Crossref PubMed Scopus (68) Google Scholar). Recently, several major channels have been shown to be regulated by thiol nitrosylation, a process becoming recognized as an important NO-mediated post-translational modification effecting control over a diverse array of signaling and regulatory proteins (6Stamler J.S. Singel D. Loscalzo J. Science. 1992; 258: 1898-1902Crossref PubMed Scopus (2449) Google Scholar, 7Stamler J.S. Toone E.J. Lipton S.A. Sucher N.J. Neuron. 1997; 18: 691-696Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar, 8Stamler J.S. Cell. 1994; 78: 931-936Abstract Full Text PDF PubMed Scopus (1633) Google Scholar, 9Stamler J.S. Hausladen A. Nat. Struct. Biol. 1998; 5: 247-249Crossref PubMed Scopus (247) Google Scholar). SuchS-nitrosylation-mediated effects are direct and independent of activation of guanylyl cyclase, which is a major target for NO and a frequent mediator of the actions of NO (10Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1998; 63: 175-185Crossref Scopus (2131) Google Scholar, 11McDonald L.J. Murad F. Proc. Soc. Exp. Biol. Med. 1996; 211: 1-6Crossref PubMed Google Scholar). Studies have revealed that nitrosothiol formation underlies the direct modifying action of NO on a number of important plasma membrane and intracellular channels for Ca2+ and other ions including theN-methyl-d-aspartate receptor (12Lipton S.A. Choi Y. Pan Z. Lei S.Z. Chen H.V. Sucher N.J. Loscalzo J. Singel D. Stamler J.S. Nature. 1993; 364: 626-632Crossref PubMed Scopus (2300) Google Scholar), cyclic nucleotide-gated cation channel (13Broillet M. Firestein S. Neuron. 1996; 16: 377-385Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 14Broillet M. Firestein S. Neuron. 1997; 18: 951-958Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), Ca2+-activated K+ channel (15Bolotina V.M. Najibi S. Palacino J.J. Pagano P.J. Cohen R.A. Nature. 1994; 368: 850-853Crossref PubMed Scopus (1510) Google Scholar), l-type Ca2+channel (16Campbell D.L. Stamler J.S. Strauss H.C. J. Gen. Physiol. 1996; 108: 277-293Crossref PubMed Scopus (399) Google Scholar), and most recently, the ryanodine receptor Ca2+ release channel (17Xu L. Eu J.P. Meissner G. Stamler J.S. Science. 1998; 279: 234-237Crossref PubMed Scopus (857) Google Scholar). For several of these channels, NO donor-induced S-nitrosylation results in channel activation, and this activation is mimicked by alkylation of the same thiol groups (13Broillet M. Firestein S. Neuron. 1996; 16: 377-385Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 14Broillet M. Firestein S. Neuron. 1997; 18: 951-958Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 15Bolotina V.M. Najibi S. Palacino J.J. Pagano P.J. Cohen R.A. Nature. 1994; 368: 850-853Crossref PubMed Scopus (1510) Google Scholar, 16Campbell D.L. Stamler J.S. Strauss H.C. J. Gen. Physiol. 1996; 108: 277-293Crossref PubMed Scopus (399) Google Scholar, 17Xu L. Eu J.P. Meissner G. Stamler J.S. Science. 1998; 279: 234-237Crossref PubMed Scopus (857) Google Scholar). Because of the reactivity of thiols toward NO, the sphere of influence of NO can be highly restricted; hence, rather than being diffusion-dependent, NO (or an equivalent of the nitrosonium ion, NO+) may be donated and exchanged between neighboring protein thiols by local transnitrosation events (6Stamler J.S. Singel D. Loscalzo J. Science. 1992; 258: 1898-1902Crossref PubMed Scopus (2449) Google Scholar, 7Stamler J.S. Toone E.J. Lipton S.A. Sucher N.J. Neuron. 1997; 18: 691-696Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar, 8Stamler J.S. Cell. 1994; 78: 931-936Abstract Full Text PDF PubMed Scopus (1633) Google Scholar, 9Stamler J.S. Hausladen A. Nat. Struct. Biol. 1998; 5: 247-249Crossref PubMed Scopus (247) Google Scholar, 13Broillet M. Firestein S. Neuron. 1996; 16: 377-385Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 14Broillet M. Firestein S. Neuron. 1997; 18: 951-958Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Here, we have utilized a combination of membrane-permeant NO donors and alkylators to probe the role of S-nitrosylation in the process of Ca2+ entry and its relationship to Ca2+ pool depletion. The DDT1MF-2 hamster smooth muscle and DC-3F Chinese hamster lung fibroblast lines were cultured as described previously (20Waldron R.T. Short A.D. Gill D.L. J. Biol. Chem. 1995; 270: 11955-11961Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 21Waldron R.T. Short A.D. Gill D.L. J. Biol. Chem. 1997; 272: 6440-6447Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Cells grown on coverslips for 1 day were loaded with fura-2/acetoxymethylester as described previously (22Short A.D. Klein M.G. Schneider M.F. Gill D.L. J. Biol. Chem. 1993; 268: 25887-25893Abstract Full Text PDF PubMed Google Scholar, 23Short A.D. Bian J. Ghosh T.K. Waldron R.T. Rybak S.L. Gill D.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4986-4990Crossref PubMed Scopus (248) Google Scholar). Fluorescence measurements (505 nm emission) are shown as 340/380 nm (excitation) ratios obtained from groups of 10–12 cells. Details of Ca2+ measurements were recently described for DDT1MF-2 (24Graber M.N. Alfonso A. Gill D.L. J. Biol. Chem. 1997; 272: 29546-29553Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) and DC-3F cells (21Waldron R.T. Short A.D. Gill D.L. J. Biol. Chem. 1997; 272: 6440-6447Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Resting Ca2+ levels were approximately 60–90 nm in DDT1MF-2 cells and 25–50 nm in DC-3F cells; maximal activation by GEA3162 resulted in up to 600 nmCa2+. Measurements shown are representative of at least three and, in most cases a larger number, of independent experiments. GEA3162,1GEA5024, and LY83583 were from Alexis Corp. (San Diego, CA). 2,5-Di-tert-butylhydroquinone (DBHQ), and 4-vinylpyridine (4-VP), were from Aldrich. Thapsigargin was from LC Services (Woburn, MA). Fura-2/acetoxymethylester was from Molecular Probes (Eugene, OR). 8-Br-cGMP was from Calbiochem (San Diego, CA).N-Ethylmaleimide (NEM) and all other compounds were from Sigma. Measurements of cGMP were made using the standard protocol of the NEN Life Science Products RIA kit. The action of different NO-donating molecules on Ca2+ entry was examined using intact fura-2-loaded cells (24Graber M.N. Alfonso A. Gill D.L. J. Biol. Chem. 1997; 272: 29546-29553Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) in which the coupling process between intracellular Ca2+ pools and Ca2+ entry channels itself remains functionally intact. Cells selected for study included the DDT1MF-2 smooth muscle and DC-3F lung fibroblast cell lines, which have been extensively used to study function and distribution of Ca2+ pools (18Ghosh T.K. Mullaney J.M. Tarazi F.I. Gill D.L. Nature. 1989; 340: 236-239Crossref PubMed Scopus (135) Google Scholar, 19Ghosh T.K. Bian J. Gill D.L. Science. 1990; 248: 1653-1656Crossref PubMed Scopus (328) Google Scholar, 22Short A.D. Klein M.G. Schneider M.F. Gill D.L. J. Biol. Chem. 1993; 268: 25887-25893Abstract Full Text PDF PubMed Google Scholar, 23Short A.D. Bian J. Ghosh T.K. Waldron R.T. Rybak S.L. Gill D.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4986-4990Crossref PubMed Scopus (248) Google Scholar, 24Graber M.N. Alfonso A. Gill D.L. J. Biol. Chem. 1997; 272: 29546-29553Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 25Graber M.N. Alfonso A. Gill D.L. J. Biol. Chem. 1996; 271: 883-888Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) and their relationship to Ca2+ entry (5Gill D.L. Waldron R.T. Rys-Sikora K.E. Ufret-Vincenty C.A. Graber M.N. Favre C.J. Alfonso A. Biosci. Rep. 1996; 16: 139-157Crossref PubMed Scopus (68) Google Scholar, 20Waldron R.T. Short A.D. Gill D.L. J. Biol. Chem. 1995; 270: 11955-11961Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 21Waldron R.T. Short A.D. Gill D.L. J. Biol. Chem. 1997; 272: 6440-6447Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 26Ufret-Vincenty C.A. Short A.D. Alfonso A. Gill D.L. J. Biol. Chem. 1995; 270: 26790-26793Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). A profound, dose-dependent increase in cytosolic Ca2+ was induced by application of the NO-donating oxatriazole derivative, GEA3162, as shown in Fig. 1 A. An unusually lipophilic NO-releasing agent, GEA3162, was recently characterized as a highly effective NO donor in vitro and in mediating the actions of NO on intact cells (27Kankaanranta H. Rydell E. Petersson A.-S. Holm P. Moilanen E. Corell T. Karup G. Vuorinen P. Pedersen S.B. Wennmalm A. Metsa-Ketela T. Br. J. Pharmacol. 1996; 117: 401-406Crossref PubMed Scopus (55) Google Scholar, 28Kosonen O. Kankaanranta H. Vuorinen P. Moilanen E. Eur. J. Pharmacol. 1997; 337: 55-61Crossref PubMed Scopus (28) Google Scholar). Although lipophilic, this mesoionic 3-aryl-substituted oxatriazole-5-imine derivative is sufficiently amphipathic that it may preferentially localize to donate NO in close proximity to the membrane surface. The increase in Ca2+ after application of GEA3162 was preceded by a lag, which itself was dose-dependent and of at least 1 min in duration. The GEA3162-induced rise in Ca2+ was clearly due to entry; in the absence of extracellular Ca2+, 100 μm GEA3162 induced no change in cytosolic Ca2+ (Fig. 1 B), indicating that no release from pools occurred. An immediate and large increase in cytosolic Ca2+ was observed upon Ca2+ readdition, indicating that entry had become fully activated. The action of GEA3162 was not attributable to any change in Ca2+ efflux because experiments (not shown) revealed no effect on the ability of the plasma membrane Ca2+ pump to pump down Ca2+ in the cells. As seen in Fig. 1 (A and B), the GEA3162-induced Ca2+ entry mechanism became deactivated with time; after reaching a maximum within a few minutes, the entry of Ca2+ always decreased. In other experiments, reapplication of 100 μm GEA3162 after deactivation caused no further increase in Ca2+; removal of GEA3162 for 5 min and subsequent readdition also did not cause reactivation of the entry process. Other structurally diverse NO donors activated similar Ca2+ entry; application of sodium nitroprusside (SNP) or sodium nitrite (NO2−) each induced increases in cytosolic Ca2+ (Fig. 2, A and C). In both cases, relatively high levels of the donors were required (likely due to lower efficiency at physiological pH), and the rise in Ca2+ was smaller and more variable than with GEA3162 but again occurred after a significant lag period. As with GEA3162, no significant changes in Ca2+ were observed with either SNP or NO2− in the absence of external Ca2+; however, Ca2+ entry again commenced immediately upon readdition of external Ca2+ (Fig. 2,B and D).Figure 2Ca2+ entry activated by other NO donors. A, cells were treated with 1.5 mm SNP added at the arrow. B, medium was replaced with Ca2+-free medium as shown by thebars, and then following a brief intervening exposure to standard Ca2+-containing medium, 1.5 mm SNP was added in the presence of Ca2+-free medium before reapplication of Ca2+-containing medium. C, cells were treated with 15 mm sodium nitrite (NO2−) added at the arrow. D, medium was replaced with Ca2+-free medium as shown by the bars, followed by addition of 15 mm NO2− at the arrow and return of cells to normal Ca2+-containing medium.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Crucial to investigate was the relationship between this NO donor-induced entry process and the operation of intracellular Ca2+ pools. The results shown in Fig. 3 reveal that pool emptying has a major stimulatory action on the Ca2+ entry pathway. Pools were emptied with either of two distinct intracellular Ca2+ pump blockers, thapsigargin (29Thastrup O. Cullen P.J. Drobak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (2998) Google Scholar) and DBHQ (30Moore G.A. McConkey D.J. Kass G.E. O'Brien P.J. Orrenius S. FEBS Lett. 1987; 224: 331-336Crossref PubMed Scopus (164) Google Scholar). As shown in Fig. 3 A, 10 μm DBHQ caused a rapid release of pool Ca2+ followed by a later rise of Ca2+representing store-operated Ca2+ entry (26Ufret-Vincenty C.A. Short A.D. Alfonso A. Gill D.L. J. Biol. Chem. 1995; 270: 26790-26793Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Upon application of GEA3162 there was a large and almost instantaneous rise in cytosolic Ca2+. Thus, pool emptying had completely eliminated the lag seen with normal pool-filled cells (Fig. 1 A). The effect of pool emptying was even more profound at lower GEA3162 levels (Fig. 3, B and C). 10 μm GEA3162 had no effect on normal cells (Fig. 1 A) but was able to induce a substantial and rapid effect after pool emptying (Fig. 3 C). At 25 μm (Fig. 3 B) the long (>4 min) delay in onset of Ca2+entry was almost completely eliminated after pool emptying. Emptying of pools with either thapsigargin or the ionophore, ionomycin, gave identical stimulation of the GEA3162-induced influx. The enhancement of NO donor-induced Ca2+ entry after pool emptying was not due to increased cytosolic Ca2+; at longer times following pool emptying with DBHQ or thapsigargin (up to 3 h) at which time cytosolic Ca2+ had returned to a level indistinguishable from basal levels (yet pools remained completely empty), the sensitivity to and rapidity of action of GEA3162 were exactly as observed upon addition immediately following pool emptying. The potentiation of the effect of GEA by pool emptying was not a reflection of the inability of the intracellular Ca2+ pumps to buffer Ca2+ in the cytosol. Thus, as shown in Fig. 3 A(inset), GEA3162-induced entry of Mn2+, monitored by quenching of fura-2 excited at its isosbestic wavelength, 360 nm, revealed identical kinetics and stimulation by pool emptying as seen for changes in cytosolic Ca2+ measured by ratio fluorimetry. Mn2+ is not a substrate for Ca2+pumps and hence reliably reports influx without being pumped into organelles or out of the cell. When 100 μm GEA3162 was added in the presence of 1 mm Mn2+, a significant entry of Mn2+ occurred. However, the onset of Mn2+ entry was slow to develop, and it took almost 90 s before maximal entry was occurring. After the pools had been emptied with 1 μm thapsigargin, 100 μm GEA induced an immediate entry of Mn2+, which remained at this rate for approximately 45 s before declining. Under this condition the contribution of endogenous store-operated entry without GEA was almost negligible. Thus, the kinetics of GEA-dependent Mn2+ influx were almost identical to the kinetics of Ca2+ entry induced by 100 μm GEA as shown in Figs. 1 A and 3 A. Experiments revealed almost identical NO donor-induced Ca2+ entry in the unrelated DC-3F fibroblast cell line, which again was highly stimulated by the emptying of Ca2+ pools. These results indicate operation of an important and potentially widespread NO donor-induced Ca2+ entry mechanism that undergoes striking stimulation by pool emptying. Entry is activated at μm NO donor concentrations that may correspond to NO levels in the physiological nm range (27Kankaanranta H. Rydell E. Petersson A.-S. Holm P. Moilanen E. Corell T. Karup G. Vuorinen P. Pedersen S.B. Wennmalm A. Metsa-Ketela T. Br. J. Pharmacol. 1996; 117: 401-406Crossref PubMed Scopus (55) Google Scholar, 28Kosonen O. Kankaanranta H. Vuorinen P. Moilanen E. Eur. J. Pharmacol. 1997; 337: 55-61Crossref PubMed Scopus (28) Google Scholar). A major target for NO is the heme group of the guanylyl cyclase enzyme, and many effects of NO are mediated through the ensuing increased cGMP levels (10Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1998; 63: 175-185Crossref Scopus (2131) Google Scholar, 11McDonald L.J. Murad F. Proc. Soc. Exp. Biol. Med. 1996; 211: 1-6Crossref PubMed Google Scholar). However, no changes in Ca2+entry could be observed with application of 8-Br-cGMP over a broad range (10 μm to 1 mm). 8-Br-cGMP also did not modify NO donor-induced Ca2+ influx. Additionally, the guanylyl cyclase inhibitor, LY83583 (31Schmidt M.J. Sawyer B.D. Truex L.L. Marshall W.S. Fleisch J.H. J. Pharmacol. Exp. Ther. 1998; 232: 764-769Google Scholar), had no effect on NO donor-induced Ca2+ entry. Measurements of cGMP did not reveal any significant changes in cGMP levels associated with Ca2+ entry activated by GEA3162. This latter result is significant in indicating that global NO elevation within the cells was not occurring and that the NO-donating activity of GEA3162 may be spatially restricted as a result of the lipophilic character of the molecule. Earlier studies suggested that NO-induced cGMP changes might mediate store-operated Ca2+ entry and that pool emptying could activate synthesis of NO (32Bahnson T.D. Pandol S.J. Dionne V.E. J. Biol. Chem. 1993; 268: 10808-10812Abstract Full Text PDF PubMed Google Scholar, 33Xu X. Start R.A. Tortorici G. Muallem S. J. Biol. Chem. 1994; 269: 12645-12653Abstract Full Text PDF PubMed Google Scholar). Subsequent work has suggested that such an effect may occur in only certain cell types and that increased cGMP may be dependent on, rather than the cause of, increased Ca2+ levels (34Gilon P. Obie J.F. Bian X. Bird G.S. Putney Jr., J.W. Biochem. J. 1995; 331: 649-656Crossref Scopus (38) Google Scholar, 35Bischof G. Serwold T.F. Machen T.E. Cell Calcium. 1997; 21: 135-142Crossref PubMed Scopus (22) Google Scholar, 36Clementi E. Meldolesi J. Trends Pharmacol. Sci. 1997; 18: 266-269Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In contrast, the action of NO donors on Ca2+ entry described here appears to be entirely independent of cGMP, and instead may reflect an important direct action of NO. Recently, much attention has focused onS-nitrosylation events as major directprotein-modifying regulatory responses induced by NO that are independent of changes in cGMP (6Stamler J.S. Singel D. Loscalzo J. Science. 1992; 258: 1898-1902Crossref PubMed Scopus (2449) Google Scholar, 7Stamler J.S. Toone E.J. Lipton S.A. Sucher N.J. Neuron. 1997; 18: 691-696Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar, 8Stamler J.S. Cell. 1994; 78: 931-936Abstract Full Text PDF PubMed Scopus (1633) Google Scholar, 9Stamler J.S. Hausladen A. Nat. Struct. Biol. 1998; 5: 247-249Crossref PubMed Scopus (247) Google Scholar). Indeed, as described above, several major channels for Ca2+ and other ions are revealed to be activated by S-nitrosylation (13Broillet M. Firestein S. Neuron. 1996; 16: 377-385Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 14Broillet M. Firestein S. Neuron. 1997; 18: 951-958Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 15Bolotina V.M. Najibi S. Palacino J.J. Pagano P.J. Cohen R.A. Nature. 1994; 368: 850-853Crossref PubMed Scopus (1510) Google Scholar, 16Campbell D.L. Stamler J.S. Strauss H.C. J. Gen. Physiol. 1996; 108: 277-293Crossref PubMed Scopus (399) Google Scholar, 17Xu L. Eu J.P. Meissner G. Stamler J.S. Science. 1998; 279: 234-237Crossref PubMed Scopus (857) Google Scholar). In the present studies, the lack of involvement of cGMP in mediating the action of NO was consistent with a direct S-nitrosylation event mediating Ca2+ entry but certainly not proof. The role of thiol modification could only be ascertained by comparing the actions of known sulfhydryl-modifying reagents. The results shown in Fig. 4 reveal that the actions of two quite different membrane-permeant alkylating agents, 4-VP and N-ethylmaleimide (NEM), were impressively similar to the effects of NO donors. Added to normal cells, 1 mm 4-VP induced a modest increase in cytosolic Ca2+ but only after a delay of approximately 2 min (Fig. 4 A). After pool emptying with the pump blocker, DBHQ, the action of 4-VP was greatly stimulated, inducing a rapid, large, and transient increase in Ca2+ almost identical to the NO donor-induced response. Again, in the absence of extracellular Ca2+, even at 10 mm, 4-VP induced no release of Ca2+, but immediately upon Ca2+ readdition, the increased level of cytosolic Ca2+ reflected a large, transient entry of extracellular Ca2+ (Fig. 4 C). Significantly, following the complete response to 4-VP, the effect of 100 μm GEA3162 was entirely blocked (Fig. 4 C), indicating that the alkylating agent and NO donor were activating the same Ca2+ entry mechanism. Reversed addition of the agents (GEA3162 followed by 4-VP) resulted in blockade of the action of 4-VP. If submaximally effective 4-VP concentrations were used, subsequently added GEA3162 induced an effect that corresponded inversely in size with that induced by 4-VP. From these results it is concluded that there is a stoichiometric activation of a finite number of entry channels by either NO donors or alkylators and that pool emptying profoundly stimulates the same mechanism of Ca2+ entry induced by either type of agent. The more powerful alkylator, NEM, at 10 μm induced effects that were very similar to 4-VP, activating a slight increase in Ca2+ alone that was greatly stimulated by pool emptying, in this case with thapsigargin (Fig. 4 B). Concentrations of NEM above 10 μm could not be used because they induced nonselective modification of the Ca2+ handling machinery of cells (especially Ca2+ pool release) not seen with 4-VP. As with 4-VP the action of NEM was clearly on Ca2+ entry and was again able to completely prevent the action of subsequently added GEA (Fig. 4 D). The competition between the actions of either of the two alkylators and GEA3162 is interesting. 4-VP and NEM are both membrane permeant. Of many NO donors tested, GEA3162 and the close structural analogue, GEA5024 (27Kankaanranta H. Rydell E. Petersson A.-S. Holm P. Moilanen E. Corell T. Karup G. Vuorinen P. Pedersen S.B. Wennmalm A. Metsa-Ketela T. Br. J. Pharmacol. 1996; 117: 401-406Crossref PubMed Scopus (55) Google Scholar), were most effective in activating Ca2+ entry. As mentioned above, these compounds differ from other NO donors in being lipophilic enough to penetrate the membrane; yet by virtue of weak charge on the oxatriazole ring, they may be sufficiently amphipathic to selectively donate NO at the surface of the membrane in the vicinity of reactive thiols of the entry channel or an associated protein. The results presented here reveal a novel and significant regulatory mechanism involved in the coupling of pool emptying to Ca2+ entry. Nitrosylation of thiols is becoming recognized as a widespread post-translational protein modification controlling the activity of a spectrum of major regulatory proteins (6Stamler J.S. Singel D. Loscalzo J. Science. 1992; 258: 1898-1902Crossref PubMed Scopus (2449) Google Scholar, 7Stamler J.S. Toone E.J. Lipton S.A. Sucher N.J. Neuron. 1997; 18: 691-696Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar, 8Stamler J.S. Cell. 1994; 78: 931-936Abstract Full Text PDF PubMed Scopus (1633) Google Scholar, 9Stamler J.S. Hausladen A. Nat. Struct. Biol. 1998; 5: 247-249Crossref PubMed Scopus (247) Google Scholar). The data indicate that Ca2+ entry is activated as a consequence of direct modification of one or more thiols either on the channel itself or a protein involved in its coupling to pool emptying. Importantly, activation via thiol nitrosylation provides a strong analogy with at least three other major Ca2+ channels, the ryanodine-sensitive Ca2+ release channel (17Xu L. Eu J.P. Meissner G. Stamler J.S. Science. 1998; 279: 234-237Crossref PubMed Scopus (857) Google Scholar), thel-type Ca2+ channel (16Campbell D.L. Stamler J.S. Strauss H.C. J. Gen. Physiol. 1996; 108: 277-293Crossref PubMed Scopus (399) Google Scholar), and the cyclic nucleotide-gated channel (13Broillet M. Firestein S. Neuron. 1996; 16: 377-385Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 14Broillet M. Firestein S. Neuron. 1997; 18: 951-958Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). In all cases, increased channel activity induced by NO-donors results from S-nitrosylation, and this stimulatory action is mimicked by modification of the presumed same thiol group (or groups) by alkylating agents. Whereas nitrosylation of the three other Ca2+ channels is of uncertain physiological role, in the present study it appears that the major physiological activating condition, namely emptying of pools, facilitates an increase in the susceptibility of the channel to activation by thiol modification. Such modification does not necessarily require a generalized increase in NO levels within the cytosol and indeed may reflect a localized transnitrosation event from a nearby donor nitrosothiol (7Stamler J.S. Toone E.J. Lipton S.A. Sucher N.J. Neuron. 1997; 18: 691-696Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar, 8Stamler J.S. Cell. 1994; 78: 931-936Abstract Full Text PDF PubMed Scopus (1633) Google Scholar); this event may be stimulated by pool emptying and intimately involved in the process of coupling pool emptying to Ca2+ entry. Pool emptying appears therefore to induce a significant conformational change in a Ca2+ entry channel or associated protein, increasing the availability of a key thiol, modification of which greatly enhances channel activity. Lastly, a conformational alteration in the availability of thiols on the entry channel specifically induced by pool emptying provides a direct means to selectively label and identify the channel protein itself. We greatly thank Dr. Kim Collins for invaluable assistance in the completion of this work. We also thank Dr. Alison Short for help in the early part of these studies." @default.
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