J Biol Chem, Vol. 273, Issue 47, 30855-30858, November 20, 1998

COMMUNICATION
Ca²⁺ Pool Emptying Stimulates Ca²⁺ Entry Activated by S-Nitrosylation^*

Cécile J. Favre, Carmen A. Ufret-Vincenty, Michele R. Stone, Hong-Tao Ma, and Donald L. Gill^§

From the Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201

	ABSTRACT

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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 that S-nitrosylation underlies activation of an important store-operated Ca²⁺ entry mechanism.

	INTRODUCTION

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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 (1-3). However, the mechanism by which Ca²⁺ pool depletion is coupled to activation of "store-operated" Ca²⁺ entry channels remains an important but unsolved question (1-5). 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 (6-9). 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 (10, 11). 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 (12), cyclic nucleotide-gated cation channel (13, 14), Ca²⁺-activated K⁺ channel (15), L-type Ca²⁺ channel (16), and most recently, the ryanodine receptor Ca²⁺ release channel (17). 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 (13-17). 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 (6-9, 13, 14). 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.

	EXPERIMENTAL PROCEDURES

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Intracellular Calcium Measurements-- The DDT₁MF-2 hamster smooth muscle and DC-3F Chinese hamster lung fibroblast lines were cultured as described previously (20, 21). Cells grown on coverslips for 1 day were loaded with fura-2/acetoxymethylester as described previously (22, 23). 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 (24) and DC-3F cells (21). 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 nM Ca²⁺. Measurements shown are representative of at least three and, in most cases a larger number, of independent experiments.

Materials and Miscellaneous Procedures-- GEA3162,¹ GEA5024, 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.

	RESULTS AND DISCUSSION

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The action of different NO-donating molecules on Ca²⁺ entry was examined using intact fura-2-loaded cells (24) 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 (18, 19, 22-25) and their relationship to Ca²⁺ entry (5, 20, 21, 26). A profound, dose-dependent increase in cytosolic Ca²⁺ was induced by application of the NO-donating oxatriazole derivative, GEA3162, as shown in Fig. 1A. 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 (27, 28). 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. 1B), 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).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Entry of Ca2+ into DDT1MF-2 cells activated by the NO donor GEA3162. Cytosolic Ca2+ was measured in fura-2-loaded DDT1MF-2 cells attached to glass coverslips as described (11). A, the NO donor GEA3162 was added at 10, 25, 50, or 100 µM at the time indicated by the arrow. B, 100 µM GEA3162 was added as indicated by the arrow shortly after replacing the bathing medium with nominally Ca2+-free medium; Ca2+-free conditions were maintained as shown by the bar, after which medium was replaced with standard (1 mM) Ca2+-containing medium.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Ca2+ 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 the bars, 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.

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 (29) and DBHQ (30). As shown in Fig. 3A, 10 µM DBHQ caused a rapid release of pool Ca²⁺ followed by a later rise of Ca²⁺ representing store-operated Ca²⁺ entry (26). 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. 1A). 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. 1A) but was able to induce a substantial and rapid effect after pool emptying (Fig. 3C). At 25 µM (Fig. 3B) 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. 3A (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. 1A and 3A. 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 (27, 28).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Ca2+ pool depletion stimulates NO donor-activated Ca2+ entry into DDT1MF-2 cells. A, Ca2+ pools were depleted by adding the Ca2+ pump blocker, DBHQ (10 µM), at the first arrow; after a further 8 min, 100 µM GEA3162 was added (second arrow). B and C, upper traces, Ca2+ pools were depleted with 10 µM DBHQ (first arrow) followed 8 min later by either 25 µM (B) or 10 µM GEA3162 (C) added at the second arrow. Lower traces, cells were treated at the arrow with either 25 µM (B) or 10 µM GEA3162 (C) with no addition of DBHQ. Inset, GEA3162-induced Mn2+ entry measured by quenching of fura-2 fluorescence (23). Normal cells (pool-filled) or cells treated with 1 µM thapsigargin (pool-emptied) were incubated under standard conditions for 8 min followed by addition of 100 µM GEA3162 in the presence of 1 mM MnCl2. Fluoresence emission at 510 nm was monitored by quenching of fura-2 excited at its Ca2+-independent isosbestic wavelength, 360 nm.

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 (10, 11). 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 (31), 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 (32, 33). 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 (34-36). 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 on S-nitrosylation events as major direct protein-modifying regulatory responses induced by NO that are independent of changes in cGMP (6-9). Indeed, as described above, several major channels for Ca²⁺ and other ions are revealed to be activated by S-nitrosylation (13-17). 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. 4A). 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. 4C). Significantly, following the complete response to 4-VP, the effect of 100 µM GEA3162 was entirely blocked (Fig. 4C), 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. 4B). 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. 4D). 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 (27), 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.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Alkylating agents activate Ca2+ entry, which is highly stimulated by Ca2+ pool depletion. A, upper trace, pools were emptied with 10 µM DBHQ (first arrow) followed by addition of 1 mM 4-VP at the second arrow. Lower trace, cells were treated at the arrow with 1 mM 4-VP with no addition of DBHQ. B, upper trace, pools were emptied with 2 µM thapsigargin (TG, first arrow) followed by addition of 10 µM NEM at the second arrow. Lower trace, cells were treated at the arrow with 10 µM NEM with no addition of DBHQ. C, Ca2+-free medium was applied to cells for the duration of the bar; 10 mM 4-VP was applied (first arrow) followed later by 100 µM GEA3162 (second arrow). D, Ca2+-free medium was applied to cells for the duration of the bar; 10 µM NEM was applied (first arrow) followed later by 100 µM GEA3162 (second arrow).

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 (6-9). 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 (17), the L-type Ca²⁺ channel (16), and the cyclic nucleotide-gated channel (13, 14). 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 (7, 8); 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.

	ACKNOWLEDGEMENTS

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.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant HL55426, National Science Foundation Grant MCB 9307746, a Grant-In-Aid from the American Heart Association (Maryland Affiliate), and a fellowship from the Swiss Federal Research Foundation (to C. J. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Anatomy, University of California at San Francisco, 513 Parnassus Ave., San Francisco, CA 94143.

^§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene St., Baltimore, MD 21201. Tel.: 410-706-2593; Fax: 410-706-6676; E-mail: dgill{at}umaryland.edu.

The abbreviations used are: GEA3162, (5-amino-3-(3, 4-dichlorophenyl)1,2,3,4-oxatriazolium); GEA5024 (5-amino-3-(3-chloro-2-methylphenyl)1, 2,3,4-oxatriazolium); 4-VP, vinylpyridine; SNP, sodium nitroprusside; NEM, N-ethylmaleimide; DBHQ, 2,5-di-tert- butylhydroquinone; 8-Br-cGMP, 8-bromo-guanosine 3',5'-cyclic monophosphate.

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