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• W2079355407 abstract "The coupling between Ca2+ pools and store-operated Ca2+ entry channels (SOCs) remains an unresolved question. Recently, we revealed that Ca2+ entry could be activated in response to S-nitrosylation and that this process was stimulated by Ca2+ pool emptying (Favre, C. J., Ufret-Vincenty, C. A., Stone, M. R., Ma, H-T., and Gill, D. L. (1998) J. Biol. Chem. 273, 30855–30858). In DDT1MF-2 smooth muscle cells and DC-3F fibroblasts, Ca2+ entry activated by the lipophilic NO donor, GEA3162 (5-amino-3-(3,4-dichlorophenyl)1,2,3,4-oxatriazolium), or the alkylator, N-ethylmaleimide, was observed to be strongly activated by transient external Ca2+ removal, closely resembling activation of SOC activity in the same cells. The nonadditivity of SOC and NO donor-activated Ca2+ entry suggested a single entry mechanism. Calyculin A-induced reorganization of the actin cytoskeleton prevented SOC but had no effect on GEA3162-induced Ca2+ entry. However, a single entry mechanism could account for both SOC and NO donor-activated entry if the latter reflected direct modification of the entry channel byS-nitrosylation, bypassing the normal coupling process between channels and pools. Small differences between SOC and GEA3162-activated Ba2+ entry and sensitivity to blockade by La3+ were observed, and in HEK293 cells SOC activity was observed without a response to thiol modification. It is concluded that in some cells, S-nitrosylation modifies an entry mechanism closely related to SOC and/or part of the regulatory machinery for SOC-mediated Ca2+ entry. The coupling between Ca2+ pools and store-operated Ca2+ entry channels (SOCs) remains an unresolved question. Recently, we revealed that Ca2+ entry could be activated in response to S-nitrosylation and that this process was stimulated by Ca2+ pool emptying (Favre, C. J., Ufret-Vincenty, C. A., Stone, M. R., Ma, H-T., and Gill, D. L. (1998) J. Biol. Chem. 273, 30855–30858). In DDT1MF-2 smooth muscle cells and DC-3F fibroblasts, Ca2+ entry activated by the lipophilic NO donor, GEA3162 (5-amino-3-(3,4-dichlorophenyl)1,2,3,4-oxatriazolium), or the alkylator, N-ethylmaleimide, was observed to be strongly activated by transient external Ca2+ removal, closely resembling activation of SOC activity in the same cells. The nonadditivity of SOC and NO donor-activated Ca2+ entry suggested a single entry mechanism. Calyculin A-induced reorganization of the actin cytoskeleton prevented SOC but had no effect on GEA3162-induced Ca2+ entry. However, a single entry mechanism could account for both SOC and NO donor-activated entry if the latter reflected direct modification of the entry channel byS-nitrosylation, bypassing the normal coupling process between channels and pools. Small differences between SOC and GEA3162-activated Ba2+ entry and sensitivity to blockade by La3+ were observed, and in HEK293 cells SOC activity was observed without a response to thiol modification. It is concluded that in some cells, S-nitrosylation modifies an entry mechanism closely related to SOC and/or part of the regulatory machinery for SOC-mediated Ca2+ entry. endoplasmic reticulum nitric oxide fura-2 acetoxymethylester 5-amino-3-(3,4-dichlorophenyl)1,2,3,4-oxatriazolium N-ethylmaleimide Cytosolic Ca2+ signals control a vast array of cellular functions ranging from short term responses such as contraction and secretion to longer term regulation of cell growth and proliferation (1Berridge M.J. Bootman M.D. Lipp P. Nature. 1998; 395: 645-648Crossref PubMed Scopus (1754) Google Scholar). The generation of receptor-induced cytosolic Ca2+ signals is complex, involving two closely coupled components: rapid, transient release of Ca2+ stored in the endoplasmic reticulum (ER),1followed by slowly developing extracellular Ca2+ entry (1Berridge M.J. Bootman M.D. Lipp P. Nature. 1998; 395: 645-648Crossref PubMed Scopus (1754) Google Scholar, 2Putney J.W. Bird G.S. Cell. 1993; 75: 199-201Abstract Full Text PDF PubMed Scopus (392) Google Scholar, 3Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2254) Google Scholar, 4Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1285) Google Scholar, 5Putney J.W. McKay R.R. Bioessays. 1999; 21: 38-46Crossref PubMed Scopus (357) Google Scholar). G protein-coupled receptors and tyrosine kinase receptors, through activation of phospholipase C, generate the second messenger, inositol 1,4,5-trisphosphate. This chemical message diffuses rapidly within the cytosol to interact with inositol 1,4,5-trisphosphate receptors located on the ER, which serve as Ca2+ channels to release luminal stored Ca2+ and generate the initial Ca2+ signal phase (1Berridge M.J. Bootman M.D. Lipp P. Nature. 1998; 395: 645-648Crossref PubMed Scopus (1754) Google Scholar, 3Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2254) Google Scholar). The resulting depletion of Ca2+ stored within the ER lumen serves as the primary trigger for a message that is returned to the plasma membrane, resulting in the slow activation of “store-operated” Ca2+ entry channels (2Putney J.W. Bird G.S. Cell. 1993; 75: 199-201Abstract Full Text PDF PubMed Scopus (392) Google Scholar, 4Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1285) Google Scholar, 5Putney J.W. McKay R.R. Bioessays. 1999; 21: 38-46Crossref PubMed Scopus (357) Google Scholar, 6Gill 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 (67) Google Scholar). This second Ca2+entry phase of Ca2+ signals serves to mediate longer term cytosolic Ca2+ elevations and provides a means to replenish intracellular stores (2Putney J.W. Bird G.S. Cell. 1993; 75: 199-201Abstract Full Text PDF PubMed Scopus (392) Google Scholar, 4Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1285) Google Scholar). Whereas receptor-induced generation of inositol 1,4,5-trisphosphate and the function of Ca2+release channels to mediate the initial Ca2+-signaling phase is well understood, the mechanism for coupling ER Ca2+ store depletion with Ca2+ entry remains a crucial but unresolved question (4Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1285) Google Scholar, 5Putney J.W. McKay R.R. Bioessays. 1999; 21: 38-46Crossref PubMed Scopus (357) Google Scholar, 6Gill 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 (67) Google Scholar).Recently, several major channels have been shown to be regulated by thiol nitrosylation, a process becoming recognized as an important nitric oxide (NO)-mediated posttranslational modification affecting control over a diverse array of signaling and regulatory proteins (7Stamler J.S. Singel D. Loscalzo J. Science. 1992; 258: 1898-1902Crossref PubMed Scopus (2435) Google Scholar, 8Lipton S.A. Choi Y.-B. Pan Z.-H. Lei S.Z. Chen H.-S.V. Sucher N.J. Loscalzo J. Singel D. Stamler J.S. Nature. 1993; 364: 626-632Crossref PubMed Scopus (2286) Google Scholar, 9Stamler J.S. Toone E.J. Lipton S.A. Sucher N.J. Neuron. 1997; 18: 691-696Abstract Full Text Full Text PDF PubMed Scopus (612) Google Scholar, 10Stamler J.S. Cell. 1994; 78: 931-936Abstract Full Text PDF PubMed Scopus (1629) Google Scholar, 11Stamler J.S. Hausladen A. Nat. Struct. Biol. 1998; 5: 247-249Crossref PubMed Scopus (244) Google Scholar, 12McVey M. Hill J. Howlett A. Klein C. J. Biol. Chem. 1999; 274: 18887-18892Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Such S-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 (13Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1998; 63: 175-185Crossref Scopus (2125) Google Scholar, 14McDonald 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 the N-methyl-d-aspartate receptor (8Lipton S.A. Choi Y.-B. Pan Z.-H. Lei S.Z. Chen H.-S.V. Sucher N.J. Loscalzo J. Singel D. Stamler J.S. Nature. 1993; 364: 626-632Crossref PubMed Scopus (2286) Google Scholar), cyclic nucleotide-gated cation channel (15Broillet M.-C. Firestein S. Neuron. 1996; 16: 377-385Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 16Broillet M.-C. Firestein S. Neuron. 1997; 18: 951-958Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), Ca2+-activated K+ channel (17Bolotina V.M. Najibi S. Palacino J.J. Pagano P.J. Cohen R.A. Nature. 1994; 368: 850-853Crossref PubMed Scopus (1504) Google Scholar), L-type Ca2+ channel (18Campbell D.L. Stamler J.S. Strauss H.C. J. Gen. Physiol. 1996; 108: 277-293Crossref PubMed Scopus (399) Google Scholar), and the ryanodine receptor Ca2+ release channel (19Xu 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 (15Broillet M.-C. Firestein S. Neuron. 1996; 16: 377-385Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 16Broillet M.-C. Firestein S. Neuron. 1997; 18: 951-958Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 17Bolotina V.M. Najibi S. Palacino J.J. Pagano P.J. Cohen R.A. Nature. 1994; 368: 850-853Crossref PubMed Scopus (1504) Google Scholar, 18Campbell D.L. Stamler J.S. Strauss H.C. J. Gen. Physiol. 1996; 108: 277-293Crossref PubMed Scopus (399) Google Scholar, 19Xu 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 and, rather than diffusion-dependent, NO (or an equivalent of the nitrosonium ion, NO+) may be donated and exchanged between neighboring protein thiols by local transnitrosation events (7Stamler J.S. Singel D. Loscalzo J. Science. 1992; 258: 1898-1902Crossref PubMed Scopus (2435) Google Scholar, 9Stamler J.S. Toone E.J. Lipton S.A. Sucher N.J. Neuron. 1997; 18: 691-696Abstract Full Text Full Text PDF PubMed Scopus (612) Google Scholar, 10Stamler J.S. Cell. 1994; 78: 931-936Abstract Full Text PDF PubMed Scopus (1629) Google Scholar, 11Stamler J.S. Hausladen A. Nat. Struct. Biol. 1998; 5: 247-249Crossref PubMed Scopus (244) Google Scholar, 15Broillet M.-C. Firestein S. Neuron. 1996; 16: 377-385Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 16Broillet M.-C. Firestein S. Neuron. 1997; 18: 951-958Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar).We recently 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 (20Favre C.J. Ufret-Vincenty C.A. Stone M.R. Ma H.-T. Gill D.L. J. Biol. Chem. 1998; 273: 30855-30858Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). A novel class of lipophilic NO donors, including the oxatriazole-5-imine derivative, GEA3162, activated Ca2+ entry independent of the well defined NO target, guanylyl cyclase. Strikingly similar Ca2+ entry induced by cell permeant alkylators indicated that this Ca2+ entry process was activated through thiol modification. Significantly, Ca2+ entry activated by NO donors or alkylators was stimulated by Ca2+ pool depletion, which increased the rate and size of the Ca2+ response and the sensitivity to thiol modifiers. These results led us to postulate that S-nitrosylation may underlie activation of an important store-operated Ca2+ entry mechanism. Here we have examined the relationship between store-operated Ca2+ entry occurring independently of S-nitrosylation and Ca2+ entry activated in response toS-nitrosylation.RESULTS AND DISCUSSIONOur previous results revealed that NO donors including nitroprusside, nitrite, and the lipophilic donor, GEA3162, were effective in directly inducing Ca2+ entry (20Favre C.J. Ufret-Vincenty C.A. Stone M.R. Ma H.-T. Gill D.L. J. Biol. Chem. 1998; 273: 30855-30858Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). A highly similar entry of Ca2+ was induced with alkylators including NEM and 4-vinylpyridine, indicating that the activation of Ca2+ entry resulted from thiol modification, either nitrosylation or alkylation (20Favre C.J. Ufret-Vincenty C.A. Stone M.R. Ma H.-T. Gill D.L. J. Biol. Chem. 1998; 273: 30855-30858Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The Ca2+ entry observed with NO donors or with alkylators, in both cases, was substantially enhanced by emptying Ca2+ pools before administration of the activator. Pool emptying increased three parameters of thiol modifier-induced Ca2+ entry: the time-dependence of entry, the size of the Ca2+ entry response, and the sensitivity to thiol modifier (20Favre C.J. Ufret-Vincenty C.A. Stone M.R. Ma H.-T. Gill D.L. J. Biol. Chem. 1998; 273: 30855-30858Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The most prominent of these effects was the time dependence. Thus, in normal cells with filled pools, there was a pronounced lag in the Ca2+ entry response to thiol modifiers of at least 1 min; after pool emptying, the Ca2+entry response was extremely rapid, suggesting that pool emptying had allowed the Ca2+ entry channel to alter its configuration to expose a thiol group that was important in modifying channel activity (20Favre C.J. Ufret-Vincenty C.A. Stone M.R. Ma H.-T. Gill D.L. J. Biol. Chem. 1998; 273: 30855-30858Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The question of whether this putative entry channel was indeed the store-operated Ca2+ channel was important to address.Store-operated Ca2+ entry channels display a further important characteristic. In many cells, the entry of Ca2+, activated after pool depletion, becomes deactivated with time, and transient removal and readdition of extracellular Ca2+ is a well described means for reactivating the entry mechanism (24Waldron 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, 28Missiaen L. DeSmedt H. Parys J.B. Oike M. Casteels R. J. Biol. Chem. 1994; 269: 5817-5823Abstract Full Text PDF PubMed Google Scholar, 29Zweifach A. Lewis R.S. J. Biol. Chem. 1995; 270: 14445-14451Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 30Ufret-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, 31Louazo C.M. Ribeiro C.M.P. Bird G. St J. Putney J.W. J. Biol. Chem. 1996; 271: 14807-14813Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The effect is frequently referred to as the Ca2+ “overshoot” response, since it results in a transiently high reactivation of Ca2+ entry, which then deactivates once again with time (24Waldron 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, 28Missiaen L. DeSmedt H. Parys J.B. Oike M. Casteels R. J. Biol. Chem. 1994; 269: 5817-5823Abstract Full Text PDF PubMed Google Scholar). The results in Fig. 1 reveal that transient removal of external Ca2+ has a dramatic enhancing effect on the operation of Ca2+ entry activated by GEA3162. Untreated DDT1MF-2 cells exposed briefly to nominally Ca2+-free medium then returned to medium containing normal Ca2+ showed no change in cytosolic Ca2+ (Fig. 1 A). The addition of GEA3162 at 25 μm, a submaximal concentration under normal conditions (20Favre C.J. Ufret-Vincenty C.A. Stone M.R. Ma H.-T. Gill D.L. J. Biol. Chem. 1998; 273: 30855-30858Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), in the continued presence of external Ca2+ induced a modest rise of Ca2+ after a lag of approximately 2 min. If external Ca2+ was removed before the addition of 25 μmGEA3162 (Fig. 1 B), no significant change in Ca2+occurred for several minutes, confirming the lack of any effect of the NO donor on release of internal Ca2+. However, upon readdition of external Ca2+, a rapid entry of Ca2+ occurred, resulting in a considerably larger peak of Ca2+ (Fig. 1 B) than that observed in the continuous presence of external Ca2+ (Fig. 1 A). Thus the removal and readdition of Ca2+ considerably enhanced the effectiveness of GEA3162. Control experiments revealed that prolonged (10 min) removal of external Ca2+ did not cause any release of Ca2+ from pools and that following such prolonged external Ca2+ removal, no entry of Ca2+ was observed upon the readdition of Ca2+in the absence of GEA3162. The stimulatory effect of transient Ca2+ removal on the action of GEA3162 was further characterized as shown in Fig. 2. In this experiment external Ca2+ was transiently removed after the addition of different GEA3162 concentrations. After adding GEA3162 at 1 μm, a 3-min period of external Ca2+ removal resulted in only a very slight entry of Ca2+ (Fig. 2 A). However, after the addition of 10 μmGEA3162, the transient removal of external Ca2+ triggered a much more significant and rapid increase in Ca2+ following Ca2+ readdition (Fig. 2 B). Under normal conditions of external Ca2+, GEA3162 at 10 μmwas below its effective threshold and induced almost no Ca2+ entry (20Favre C.J. Ufret-Vincenty C.A. Stone M.R. Ma H.-T. Gill D.L. J. Biol. Chem. 1998; 273: 30855-30858Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Therefore, the entry of Ca2+observed after the brief removal of external Ca2+represents a real potentiation of the effect of GEA3162. The resultant increase in Ca2+ was rapid but transient and began to decline after 1 min. Removal of Ca2+ prevented any further entry of Ca2+, and the Ca2+ level fell rapidly. After a further 3 min, readdition of Ca2+ resulted again in a rapid and transient entry of Ca2+. The entry of Ca2+ could be repeatedly reactivated by transient removal of Ca2+ (Fig. 2 B). Although the peak size of the response to 10 μm GEA3162 after the initial transient Ca2+ depletion was smaller, the peaks following subsequent brief periods of Ca2+ removal were larger and approached the maximal size attainable. Thus, external Ca2+ removal followed by the readdition in the presence of 25 μmGEA3162 (Fig. 2 C) resulted in a rapid and maximal activation of Ca2+ entry. Again, the activation rapidly deactivated with time, and cycling of reactivation of Ca2+ entry in response to transient Ca2+ removal could be repeated several times in succession.FIG. 2Repeated transient Ca2+ depletion induces a large potentiation of Ca2+ entry activated by varying concentrations of the NO donor, GEA3162 , in DDT1MF-2 cells. Bars indicate times of replacement of medium with nominally Ca2+-free medium (no Ca2+). GEA3162 (GEA) was added at either 1 μm (A), 10 μm (B), or 25 μm (C) at the times indicated (arrows) and maintained at these levels throughout the remainder of traces.View Large Image Figure ViewerDownload (PPT)This pattern of deactivation and reactivation by transient removal of Ca2+ is highly similar to the operation of store-operated Ca2+ entry channels. As shown in Fig. 3 A, after thapsigargin-induced pool emptying in the absence of external Ca2+, readdition of Ca2+ caused a large increase in cytosolic Ca2+, reflecting a high level of store-operated Ca2+ entry. The entry of Ca2+ rapidly deactivated with time, and subsequent removal of external Ca2+ prevented any further Ca2+ entry. Upon the readdition of external Ca2+, maximal store-operated Ca2+ entry was restored. This overshoot response pattern classically reflects the operation of store-operated Ca2+entry and is believed to represent the function of Ca2+-binding sites, which negatively control store-operated Ca2+ entry (24Waldron 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, 28Missiaen L. DeSmedt H. Parys J.B. Oike M. Casteels R. J. Biol. Chem. 1994; 269: 5817-5823Abstract Full Text PDF PubMed Google Scholar, 29Zweifach A. Lewis R.S. J. Biol. Chem. 1995; 270: 14445-14451Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 31Louazo C.M. Ribeiro C.M.P. Bird G. St J. Putney J.W. J. Biol. Chem. 1996; 271: 14807-14813Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). According to such a model, as Ca2+ increases in the cytosol, binding of Ca2+to such regulatory sites inhibits entry; transient external Ca2+ removal prevents Ca2+ entry, allowing cytosolic Ca2+ to fall rapidly as Ca2+ is pumped out of the cell. As a result, Ca2+ dissociates from the regulatory site, permitting the channel to become fully reactivated; upon readdition of Ca2+, a high level of Ca2+ entry is again observed. As shown in Fig. 3 A, this process could be repeated many times. However, it is important to reiterate that the entry observed was completely dependent on pool depletion. Thus, transient removal of Ca2+ at the beginning of the trace before pools were emptied induced no entry of Ca2+. Indeed, in experiments with normal, pool-filled cells, repeated transient removal and readdition of Ca2+ over a period of 30 min induced no change in cytosolic Ca2+ (not shown). The means of activation, the appearance, and the size of the overshoot responses after pool emptying were all remarkably similar to those described above, activated in response to the NO donor. Yet in the case of the NO donor, pools were not emptied. This point is reinforced from the data shown in Fig. 3 B. Thus addition of 15 μmGEA3162 before thapsigargin had no effect on the size of the Ca2+ pool released by subsequent addition of thapsigargin. Moreover, in this experiment the size of overshoots induced after application of both GEA3162 and thapsigargin was not measurably different from that induced by each agent alone. Also, addition of 15 μm GEA3162 to cells after pool depletion with thapsigargin resulted in little significant change in the size of overshoots induced by repeated transient Ca2+ removal (Fig. 3 A). Thus, thapsigargin and NO donor induced similar shaped and sized overshoots that did not appear to be additive. This suggested they were activating either the same or a closely coupled entry mechanism.FIG. 3Thapsigargin-induced pool emptying in DDT1MF-2 cells induces overshoots of store-operated Ca2+ entry, which are similar to and nonadditive with the effects of GEA3162 . Bars indicate times of replacement of medium with nominally Ca2+-free medium (no Ca 2+ ). A, 2 μmthapsigargin (TG) and 15 μm GEA3162 (GEA) were added at the times shown. B, as forA except 15 μm GEA3162 was added before 1 μm thapsigargin. In each case, thapsigargin and GEA3162, once added, were maintained throughout the experiment.View Large Image Figure ViewerDownload (PPT)As described above and earlier (20Favre C.J. Ufret-Vincenty C.A. Stone M.R. Ma H.-T. Gill D.L. J. Biol. Chem. 1998; 273: 30855-30858Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), thiol modification by either nitrosylation or alkylation activated a very similar entry of Ca2+. We therefore examined whether the stimulatory action of transient Ca2+ removal also activated Ca2+entry induced by alkylators. Experiments utilized the DC-3F fibroblast cell line in which responses to NO donors and alkylators were similar to DDT1MF-2 cells. As shown in Fig. 4 A, the addition of the alkylator, NEM, at 10 μm induced only a slight increase in cytosolic Ca2+ (Fig. 4 A). However, if extracellular Ca2+ was transiently removed for just a short (2 min) period, a substantial entry of Ca2+ immediately followed the readdition of Ca2+ (Fig. 4 B). As with DDT1MF-2 cells, transient Ca2+ removal without alkylator or NO donor present had no effect on cytosolic Ca2+ in DC-3F cells (24Waldron 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). The shape and time dependence of the transient Ca2+ removal-induced entry response seen after NEM treatment (Fig. 4 B) and after pool emptying (24Waldron 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) were impressively similar in the DC-3F cells. NEM-induced entry of Ca2+ into DDT1MF-2 cells was similarly potentiated by transient removal of Ca2+ (data not shown).FIG. 4Transient Ca2+ depletion induces a large potentiation of N-ethylmaleimide-activated Ca2+ entry in DC-3F cells. A, 10 μm NEM was added under standard external conditions.B, 10 μm NEM addition was followed by replacement of medium with nominally Ca2+-free medium for 2 min as shown by the bar (no Ca 2+ ) followed by return of standard external Ca2+ medium. NEM was maintained after the addition.View Large Image Figure ViewerDownload (PPT)Taken together, the above results and those published previously (20Favre C.J. Ufret-Vincenty C.A. Stone M.R. Ma H.-T. Gill D.L. J. Biol. Chem. 1998; 273: 30855-30858Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) revealed that Ca2+ entry activated byS-nitrosylation was stimulated by both Ca2+ pool depletion and transient external Ca2+ removal, the two primary conditions for activating store-operated Ca2+ entry channels. From this it could be concluded thatS-nitrosylation was affecting a process closely linked with store-operated Ca2+ entry. However, other approaches to determining the relationship between the Ca2+ entry mechanisms have revealed some interesting differences. In recent work, we determined that the operation of store-operated Ca2+entry appears to involve trafficking of the ER toward the plasma membrane (32Patterson R.L. van Rossum D.B. Gill D.L. Cell. 1999; 98: 487-499Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). One of the approaches to this work was to utilize the phosphatase inhibitor, calyculin A, which in two distinct cell types induced a profound redistribution of actin resulting in the formation of a tight ring of cortical actin filaments subjacent to the plasma membrane. This cortical actin appeared to act as a physical barrier to prevent close interaction between the ER and plasma membrane (32Patterson R.L. van Rossum D.B. Gill D.L. Cell. 1999; 98: 487-499Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). Under this condition, the activation of store-operated Ca2+entry by pool emptying with thapsigargin was blocked in both cell types. We therefore compared the action of calyculin A on Ca2+ entry activated by thapsigargin-induced Ca2+ pool depletion with its effects on Ca2+entry activated by NO donor.As shown in Fig. 5, we were able to directly observe the two means of Ca2+ entry activation in a single trace. In normal DDT1MF-2 cells, typical activation of Ca2+ entry via pool depletion and application of GEA3162 is shown in Fig. 5 A. After the addition of 2 μm thapsigargin, rapid release of Ca2+ from pools was observed. The slower secondary peak was due to entry of Ca2+ dependent on pool depletion; this became deactivated with time, and cytosolic Ca2+ decreased to a reduced level. The basal Ca2+ level reached was slightly higher than normal resting Ca2+, reflecting a small level of residual Ca2+ entry. After removal of external Ca2+, this low level of entry was abolished, and upon subsequent readdition of Ca2+, the typical large overshoot of Ca2+entry was observed, consistent with that described above. After the store-operated entry had once again deactivated, the addition of a 100 μm GEA3162 clearly activated a large increase in Ca2+ entry, and this entry again deactivated with time. In similar experiments, lower GEA3162 concentrations also induced entry of Ca2+, although the entry was less prolonged (not shown). Note that in these experiments, deactivation of store-operated Ca2+ entry occurred for a longer period of time compared with that in Fig. 3, and there was no further removal of external Ca2+. Thus, if GEA3162 was activating the same Ca2+ entry pathway as pool emptying, then this result would suggest that GEA3162 was able to reverse the deactivation process occurring as a result of Ca2+ inhibition.FIG. 5Calyculin A treatment blocks store-operated Ca2+ entry in response to thapsigargin-induced pool emptying in DDT1MF-2 cells but does not block Ca2+ entry activated by the NO donor, GEA3162 . Bars indicate times of replacement of medium with nominally Ca2+-free medium (no Ca 2+ ). A, 2 μm thapsigargin (TG) and 100 μm GEA3162 (GEA) were added at the times shown. B, cells were initially treated with the phosphatase inhibitor calyculin A (CalyA) at 100 nm for 10 min before the addition of 2 μmthapsigargin and 100 μm GEA3162 at the times indicated by the respective arrows. Each of the agents was maintained in medium after addition throughout successive changes of medium with or without Ca2+.View Large Image Figure ViewerDownload (PPT)Clearly, the picture was very different in the presence of 100 nm calyculin A. The experiment shown in Fig. 5 Bincluded a 10-min pretreatment with the phosphatase inhibitor that was sufficient to rearrange cortical actin into a tight band closely associated with the plasma membrane (32Patterson R.L. van Rossum D.B. Gi" @default.
• W2079355407 created "2016-06-24" @default.
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• W2079355407 title "Ca2+ Entry Activated byS-Nitrosylation" @default.
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