0013-7227/01/$03.00/0 Endocrinology Copyright © 2001 by The Endocrine Society
Vol. 142, No. 6 Printed in U.S.A.
Thiol-Reactive Agents Biphasically Regulate Inositol 1,4,5-Trisphosphate Binding and Ca2ⴙ Release Activities in Bovine Adrenal Cortex Microsomes* ´ PHANE N. POIRIER†, MARC POITRAS, KARINA LAFLAMME, STE ´ TAN GUILLEMETTE GAE
AND
Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Sherbrooke Que´bec, Canada J1H 5N4 ABSTRACT Within all endocrine cells, the inositol 1,4,5-trisphosphate (InsP3) receptor plays an important role in regulation of the intracellular Ca2⫹ concentration. In the present study we showed that a single short-term treatment with either N-ethylmaleimide (known to decrease InsP3 receptor activity) or thimerosal (known to increase InsP3 receptor activity) caused time-dependent biphasic effects on the InsP3 binding activity of bovine adrenal cortex microsomes. The early potentiating effect of thiol-reactive agents translated into a 2-fold increase in binding affinity and Ca2⫹ release efficiency. The late dampening effect of thiol-reactive agents translated into a continuous reduction of the maximal binding
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YTOSOLIC Ca2⫹ signals control a vast array of cellular functions, including contraction, secretion, cell growth, and cell proliferation (1). These Ca2⫹ signals generated during cell activation frequently display a complex pattern consisting of repetitive spikes also known as Ca2⫹ oscillations. Recently, several ion channels have been shown to be regulated by thiol nitrosylation, a process emerging as a prototype redox-related signal that modifies the properties of regulatory proteins (2). S-Nitrosylation of target proteins is a direct effect, independent of activation of guanylyl cyclase, which is a major target for nitric oxide (NO) and a known mediator of the actions of NO (3, 4). Studies have revealed that nitrosothiol formation underlies the direct modifying action of NO on a number of important plasma membrane and intracellular Ca2⫹ channels, including the N-methyl-d-aspartate receptor (5), the l-type Ca2⫹ channel (6), the ryanodine receptor Ca2⫹ release channel (7, 8), and the store-operated Ca2⫹ channel (9). S-Nitrosylation also regulates the activity of other ions channels, such as the cyclic nucleotide-gated cation channel (10, 11) and the Ca2⫹-activated K⫹ channel (12). For several of these channels, NO donor-induced S-nitrosylation results in channel activation, and this activation is Received January 12, 2001. Address all correspondence and requests for reprints to: Dr. Gae´tan Guillemette, Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, Que´bec, Canada J1H 5N4. E-mail:
[email protected]. * This work was supported by a grant from the Canadian Institutes of Health Research. This work is part of the Ph.D. thesis of S.N.P. † Recipient of studentship from the Fonds de la Recherche en Sante´ du Que´bec.
capacity of the microsomes with a concomitant decrease in Ca2⫹ release efficiency. Under these conditions, Western blot analyses demonstrated that the level of InsP3 receptor protein was not modified. Sequential treatments with thimerosal and the reducing agent dithiothreitol showed that the InsP3 receptor can readily oscillate between high and low affinity states that are related to its alkylation state. Our results suggest a common mode of action of thiol-reactive agents on the InsP3 receptor. These results also support the contention that cellular mechanisms of thiol group modification could play important roles in regulation of the intracellular Ca2⫹ concentration. (Endocrinology 142: 2614 –2621, 2001)
mimicked by alkylation of the same thiol groups with thiol-reactive agents. Experimental evidence suggests that the inositol 1,4,5trisphosphate (InsP3) receptor plays a central role in the initiation and propagation of intracellular Ca2⫹ spikes. The thiol-reactive agents t-butyl-hydroperoxide and thimerosal have been shown to promote repetitive Ca2⫹ oscillations in several cell types (13–15). It was suggested that the effects of these agents were due to the modification of some thiol groups on the InsP3 receptor, thus increasing InsP3-induced Ca2⫹ release. This interpretation was supported by direct results showing that thimerosal (16, 17) and oxidized glutathione (18, 19) increase InsP3 receptor activity. These results suggested that thiol-reactive agents modify the conformational state of the InsP3 receptor, which adopts a higher affinity functional state. Intriguingly, other studies have demonstrated that thiol-reactive agents, such as N-ethylmaleimide (NEM) (20), p-chloromercuribenzoic acid (21), Ag⫹ (22), and mersalyl (17), decrease InsP3 receptor activity. It thus appears that different thiol groups on the InsP3 receptor can be preferentially modified by different thiol-reactive agents, leading to opposite regulatory effects on InsP3 receptor activity. The purpose of the present study was to investigate the divergent effects of thiol-reactive agents on InsP3 receptor activity. We used the thiol-reactive agents NEM (known to decrease InsP3 receptor activity) and thimerosal (known to increase InsP3 receptor activity) to demonstrate that the modification of thiol groups on the InsP3 receptor of bovine adrenal cortex causes significant biphasic changes in its binding and Ca2⫹ release activities. Our results suggest that the intracellular redox state may have profound
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consequences on InsP3 receptor activity and thus on regulation of the intracellular level of Ca2⫹. Materials and Methods
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nm). Nonspecific binding was determined in the presence of 1 m InsP3. Incubations were performed for 30 min at 0 C and were terminated by centrifugation at 15,000 ⫻ g for 5 min at 4 C. The pellets were solubilized, and the receptor-bound radioactivity was analyzed by liquid scintillation spectrometry.
Material The chemicals used in the present study were obtained from the following sources: InsP3 (trilithium salt) from LC Services Corp. (Woburn, MA); fura-2 (free acid) and ionomycin from Calbiochem (San Diego, CA); [3H]InsP3 (33 or 48 Ci/mmol) and ECL Plus Western blotting detection system from Amersham Pharmacia Biotech (Arlington Heights, IL); NEM and thimerosal from Sigma (St. Louis, MO); polyvinylidene difluoride membranes Immobilon-P (PVDF) from Millipore Corp. (Bedford, MA); Tween-20 from Bio-Rad Laboratories, Inc. (Hercules, CA); horseradish peroxidase-conjugated donkey antirabbit IgG antibody and protease inhibitor cocktail Complete from Roche Molecular Biochemicals (Laval, Canada). All other reagents were purchased from Sigma or Fisher Scientific, Inc. (Fairlawn, NJ).
Antibodies Rabbit polyclonal antibodies were raised against the carboxyl-terminal of type 1 InsP3 receptor. The antibody was affinity purified, and its selectivity was established as described previously (23).
Electrophoresis and immunoblotting Proteins were solubilized in Laemmli’s buffer [60 mm Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, 125 mm DTT, and 0.3% bromophenol blue], boiled for 5 min, and then centrifuged at 15,000 ⫻ g for 5 min. Aliquots of supernatant were subjected to SDS-PAGE on 4 – 6% (wt/vol) gels for 85 min at a constant voltage of 200 V. Proteins were electrotransferred to a PVDF membrane at a constant current of 0.5 A for 4 h in a cold room. The blots were incubated in PBST (3.5 mm NaH2PO4, 8.7 mm Na2HPO4, 2.7 mm KCl, 137 mm NaCl, and 0.1% Tween-20) containing 5% (wt/vol) nonfat dried milk for 1 h at room temperature. The blots were then incubated for 3 h at room temperature with the anti-InsP3 receptor antibody (1 g/ml). After three washes with PBST containing 5% (wt/ vol) nonfat dried milk, the membranes were incubated for 1 h at room temperature with donkey antirabbit IgG antibody (1:2000). Blots were then washed once by incubating them for 10 min in PBST containing 5% (wt/vol) nonfat dried milk and washed twice more by incubating them for 10 min in PBST. The immunoreactivity was detected with ECL Plus (Amersham Pharmacia Biotech) on a Bio-Max ML film (Eastman Kodak Co., Rochester, NY).
Preparation of microsomes Bovine adrenal glands were obtained at a nearby slaughterhouse. Bovine adrenal cortexes (dissected free of medullary tissue) were homogenized with eight strokes of a Dounce homogenizer (Kontes Co., Vineland, NJ; loose pestle) in a medium (medium A) containing 25 mm Tris-HCl buffered at pH 7.2, 110 mm KCl, 10 mm NaCl, 5 mm KH2PO4, 1 mm dithiothreitol (DTT), 2 mm EGTA, and the protease inhibitor cocktail Complete. After centrifugation at 500 ⫻ g for 15 min at 4 C, the supernatant was filtered on two layers of cheesecloth and centrifuged at 35,000 ⫻ g for 20 min at 4 C. The 35,000 ⫻ g pellet was resuspended in the same medium without EGTA (medium B) and centrifuged at 35,000 ⫻ g for 20 min at 4 C. The pellet was resuspended in medium B supplemented with glycerol (14%, vol/vol) and sorbitol (1.4%, wt/vol) at a concentration of 30 – 40 mg protein/ml. The protein concentration was evaluated by the method of Lowry using BSA as standard. This preparation was aliquoted and stored at ⫺80 C until used for InsP3induced Ca2⫹ release and InsP3 binding studies.
Ca2⫹ uptake and Ca2⫹ release studies Bovine adrenal cortex microsomes (8 –10 mg protein) were incubated in a medium containing 20 mm Tris-HCl buffered at pH 7.2, 110 mm KCl, 10 mm NaCl, 5 mm KH2PO4, 2 mm MgCl2, 40 mm phosphocreatine, and 20 U/ml creatine kinase in a final volume of 1.5 ml. Under our experimental conditions, the Ca2⫹ in the medium was exclusively contaminating Ca2⫹. Ca2⫹ uptake was initiated by the addition of ATP (2 mm) to the bathing medium containing the microsomes. The Ca2⫹-releasing effects of InsP3 and other reagents were measured shortly after ATPdependent Ca2⫹ sequestering activity had reached a steady state. The free Ca2⫹ concentration of the medium was monitored with fura-2 (free acid; 1 m) on a Hitachi F-2000 spectrofluorometer (Hialeah, FL). The excitation wavelength was 340 nm (slit 10), and the emission was recorded at 510 nm (slit 10). Incubations were performed at 37 C. Each record was calibrated by the addition of a known amount of Ca2⫹ (CaCl2) to the mixture. The actual free Ca2⫹ concentration of the medium was calculated from the maximum and minimum fluorescence values (Fmax and Fmin) obtained by adding excess Ca2⫹ and EGTA (at pH 8.5), respectively, after treatment with 1 m ionomycin. The equation used was [Ca2⫹] ⫽ 224 nm [(F ⫺ Fmin)/(Fmax ⫺ F)].
[3H]InsP3 binding assays Bovine adrenal cortex microsomes (1 mg protein) were incubated in a medium containing 25 mm Tris-HCl buffered at pH 8.5, 110 mm KCl, 10 mm NaCl, 5 mm KH2PO4, and 1 mm EDTA (medium C) in a final volume of 500 l with appropriate concentrations of [3H]InsP3 (0.6 – 0.9
Data analysis Results are presented as the mean ⫾ sd. Binding curves, binding capacity (Bmax), and Kd values were analyzed with the Kell program (Biosoft, Ferguson, MO), which uses a weighted nonlinear curve-fitting routine. Statistical significance (P ⬍ 0.05) was determined by unpaired Student’s t test.
Results Effects of thimerosal and NEM on InsP3 binding activity
Most of the previous studies investigating the regulation of InsP3 receptor activity by thiol-reactive agents were performed at 0 C. The temperature greatly influences the apparent effect of thiol-reactive agents on InsP3-binding activity. Figure 1A shows that treatment with NEM or thimerosal for 15 min at 0 C enhanced [3H]InsP3 binding activity by about 2-fold compared with that in untreated microsomes. We had shown in a previous study that pretreatment at 37 C in the presence of NEM completely abolished [3H]InsP3 binding (20). Concurrently, Fig. 1B shows that treatment with thimerosal or NEM for 15 min at 37 C completely abolished [3H]InsP3 binding activity. Figure 1C demonstrates that the potentiation of [3H]InsP3 binding activity after treatment with NEM or thimerosal at 0 C is dose dependent. Threshold effects were observed with concentrations of NEM and thimerosal of 100 and 10 m, respectively. Under these conditions, maximal effects were obtained with concentrations of NEM and thimerosal of 1 mm and 300 m, respectively. Interestingly, Fig. 1D shows that when pretreatments were performed for 15 min at 37 C with increasing concentrations of thiol-reactive agents, [3H]InsP3 binding activity was potentiated at low concentrations and dampened at higher concentrations of thiol-reactive agents. The biphasic effects of thimerosal (Fig. 2A) and NEM (Fig. 2B) were also observed in kinetic studies performed at 37 C. Figure 2A shows that pretreatment of microsomes with 300 m thimerosal at 37 C potentiated [3H]InsP3 binding activity to a maximal level (⬃2-fold above control level), reached within 2 min. This rapid potentiating effect was followed by an opposite dampening
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FIG. 1. Effects of NEM and thimerosal on InsP3 binding activity. Bovine adrenal cortex microsomes (1 mg protein) were treated for 15 min at 0 or at 37 C in the presence of 300 M NEM or 300 M thimerosal (TM; A and B). C and D, Microsomes were treated for 15 min at 0 or 37 C in the presence of increasing concentrations of NEM or thimerosal. The microsomes were then washed and assayed for [3H]InsP3 binding as described in Materials and Methods. These experiments were performed in triplicate (mean ⫾ SD). Similar results were obtained with three different microsomal preparations.
effect that rapidly brought (within 5 min) the binding activity below its initial level. The binding activity then slowly declined to an almost undetectable level within 30 min. Similarly, pretreatment of microsomes with 100 m NEM biphasically modulated [3H]InsP3 binding activity (Fig. 2B). With both thiolreactive agents, the maximal potentiating effects increased the InsP3 binding activity by about 2-fold. Together, these results demonstrate that thimerosal and NEM have similar biphasic temperature-sensitive effects on InsP3 binding activity. To assess whether the opposite (potentiating and dampening) effects of NEM were due to alkylation of different thiol groups, microsomes were pretreated for 15 min with 500 m NEM at 0 C (Fig. 3A). After a wash by centrifugation to remove NEM, microsomes were incubated for 15 min at different temperatures, and their InsP3 binding activities were finally assessed. As expected, pretreatment at 0 C with NEM, followed by a 15-min incubation period at 0 C, potentiated InsP3 binding activity by about 2-fold. Similar results were obtained when pretreated microsomes were incubated at 22 C before assessing their InsP3 binding activity. Interestingly, after a 15-min pretreatment with NEM at 0 C, incubation of the washed microsomes for 15 min at 30 C produced only a slight potentiating effect on InsP3 binding activity, whereas incubation of the washed microsomes for 15 min at 37 C almost completely abolished the InsP3 binding activity. These results demonstrate that the alkylation of a unique set of thiol groups (or of a single thiol group) can cause the whole spectrum of potentiating and dampening effects, depending on the conditions under which the alkylated pro-
teins are incubated. To further emphasize the modulatory role of temperature on the NEM effect, microsomes were pretreated for 15 min with 500 m NEM at 0 C (Fig. 3B). After a wash by centrifugation to remove NEM, microsomes were incubated for different periods at 22, 30, or 37 C, and their InsP3 binding activity was then assessed. These time-course experiments demonstrated that the potentiating effect of NEM pretreatment at 0 C slowly disappears upon further incubation at 22 C, rapidly disappears upon further incubation at 30 C, and very rapidly disappears upon further incubation at 37 C. Again, these results indicate that the alkylation of a unique set of thiol groups is responsible for the whole spectrum of potentiating and dampening effects of NEM on InsP3 binding activity. Clearly, the temperature influences the rate at which the sequential effects occur. These results suggest that the alkylation of a unique set of thiol groups on the InsP3 receptor initiates a series of conformational changes that confer to the receptor an early gain of binding activity, followed by a slow decline, leading to a complete loss of binding activity. These sequential conformational changes are slowed at low temperature and accelerated at high temperature. Reversibility of the effect of thimerosal
If the effect of thiol-reactive agents has any physiological significance, it should be reversible. In other words, dealkylation of the InsP3 receptor should bring its binding activity back to the basal level. Figure 4 shows that a 10-min pre-
BIPHASIC EFFECT OF THIOL REAGENTS ON InsP3 RECEPTOR
FIG. 2. Time course of NEM and thimerosal effects on InsP3 binding activity. Microsomes (1 mg protein) were incubated for the indicated time at 37 C in the presence of 300 M thimerosal (A) or 100 M NEM (B). Microsomes were then washed and assayed for [3H]InsP3 binding as described in Materials and Methods. These experiments were performed in triplicate (mean ⫾ SD). Similar results were obtained with three different microsomal preparations.
treatment of the microsomes with 2 mm DTT did not significantly modify their InsP3 binding activity. A 15-min pretreatment of the microsomes with 100 m thimerosal at 37 C increased by about 2-fold the InsP3 binding activity, whereas a 15-min pretreatment of the microsomes with 500 m thimerosal at 37 C completely abolished the InsP3 binding activity. Figure 4 also shows that DTT could reverse the potentiating effect of 100 m thimerosal on InsP3 binding activity, but was unable to restore the InsP3 binding activity that had been abolished by previous treatment with 500 m thimerosal. Finally, Fig. 4 further demonstrates that a subsequent treatment with 100 m thimerosal could realkylate and thus repotentiate the InsP3 binding activity of microsomes that had been dealkylated with DTT. These results suggest that the InsP3 receptor can readily oscillate between two binding states that are dependent on its alkylated or dealkylated forms. Moreover, they suggest that the conformational state adopted by the InsP3 receptor after a drastic treatment with a thiol-reactive agent is irreversible and corresponds to a nonbinding conformation. The effect of NEM (potentiating and dampening) on the InsP3 binding activity of the microsomes was not reversible by DTT treatment (data not shown). Thermodynamically, the strength of the chem-
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FIG. 3. Effect of temperature on the potentiation of InsP3 binding activity by NEM. Microsomes (1 mg protein) were incubated at 0 C for 15 min in the presence of 500 M NEM. The microsomes were then washed and incubated at different temperatures for 15 min (A) or for longer periods of time (B). At the end of the pretreatment period, the microsomes were washed and assayed for [3H]InsP3 binding as described in Materials and Methods. The experiments were performed in triplicate (mean ⫾ SD) and are representative of at least three similar experiments performed with different microsomal preparations.
ical sulfur-carbon bond between NEM and the thiol group of cysteine is such that it cannot be reduced by DTT under our experimental conditions, whereas the weaker sulfur-sulfur bond between thimerosal and the thiol group of cysteine is reducible by DTT. NEM increases the affinity of InsP3 receptor
To better characterize the pharmacological effect of NEM on InsP3 binding activity, [3H]InsP3 dose-displacement experiments were performed on microsomes that had been pretreated for 10 min with 300 m NEM at different temperatures. The Scatchard analyses of these results are presented at Fig. 5. At 0 C, pretreatment with NEM did not modify the maximal amount of binding sites (Bmax of 570 ⫾ 67 fmol/mg in the absence of NEM; Bmax of 533 ⫾ 84 fmol/mg in the presence of NEM; mean ⫾ sd of five experiments; intercept on the abscissa), but significantly enhanced by about 2-fold the InsP3 binding affinity
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FIG. 4. Effect of sequential treatments with thimerosal and DTT on InsP3 binding activity. Microsomes (1 mg protein) were incubated for 15 min at 37 C in the absence or presence of thimerosal. Microsomes were then washed and incubated for 10 min at 37 C in the absence or presence of 2 mM DTT as indicated. Microsomes were washed again and incubated for 15 min at 37 C in the absence or presence of 100 M thimerosal. After these incubations, the microsomes were washed again and assayed for [3H]InsP3 binding as described in Materials and Methods. This experiment was performed in triplicate (mean ⫾ SD) and is representative of three similar experiments.
(Kd of 12.9 ⫾ 3.5 nm in the absence of NEM; Kd of 5.0 ⫾ 2.2 nm in the presence of NEM; mean ⫾ sd of five experiments; reciprocal of the slope), as illustrated in Fig. 5A. When pretreatment with 300 m NEM was performed at higher temperatures, significant changes were observed for both the Kd and Bmax values (Fig. 5B). After pretreatment at 30 C, a high affinity state was obtained with a Kd value of 2.8 ⫾ 1.2 nm, and the total amount of binding sites was reduced to 300 ⫾ 43 fmol/mg protein (mean ⫾ sd of three experiments). After pretreatment at 37 C, a condition previously shown to considerably dampen the InsP3 binding activity (see Fig. 1), a high affinity state was still maintained (Kd of 2.4 ⫾ 1.2 nm; mean ⫾ sd of three experiments), but the total amount of binding sites was considerably reduced (Bmax of 91 ⫾ 23 fmol/mg protein; mean ⫾ sd of three experiments). These results suggest that the early potentiating effect of NEM is due to a conformational change conferring a high affinity state to the InsP3 receptor, whereas the secondary dampening effect of NEM is due to a decrease in the total number of InsP3 receptors. Classically, a Bmax reduction is interpreted as a loss of receptors. To verify the integrity of the InsP3 receptor protein after treatment with NEM, Western blot analyses were performed with a specific anti-InsP3 receptor antibody. Figure 6 shows that InsP3 receptor type 1 (the most abundant type in bovine adrenal cortex) remained intact under all of the conditions used to treat the microsomes with NEM. Some of these conditions were previously shown to abolish InsP3 binding activity (see Fig. 1D). Similar results were obtained with anti-type 2 and anti-type 3 InsP3 receptor antibodies (data not shown). These results suggest that the loss of binding
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FIG. 5. Effect of NEM on InsP3 binding affinity. Microsomes (1 mg protein) were treated at different temperatures for 10 min in the absence or presence of 300 M NEM. Microsomes were then washed and assayed for [3H]InsP3 binding in the presence of increasing concentrations of nonradioactive InsP3. The dose-displacement experiments were then resolved by Scatchard analyses. A, Scatchard analyses obtained with microsomes that had been pretreated for 10 min at 0 C in the absence (square) or presence of 300 M NEM (circle). B, Scatchard analyses obtained with microsomes that had been pretreated at 0 C (circle), 30 C (square), or 37 C (triangle) with 300 M NEM. The dotted line in B represents the Scatchard plot obtained with untreated microsomes (same as in A, square). These experiments were performed in triplicate (mean ⫾ SD) and are representative of at least three independent experiments.
FIG. 6. Time-course effect of NEM on the integrity of the InsP3 receptor. Microsomes were treated at 37 or 4 C for the indicated period of time in the absence or presence of 300 M NEM. Microsomes were then solubilized in Laemmli’s buffer, loaded on a 4 – 6% (wt/vol) SDS-PAGE column, subjected to electrophoresis, and transferred to a PVDF membrane. The blot was developed with an anti-InsP3 receptor type 1 antibody as described in Materials and Methods. The arrow indicates the InsP3 receptor migration position. This typical experiment is representative of three experiments with different microsomal preparations.
activity (decreased Bmax) is not due to degradation of the InsP3 receptor but, rather, to spontaneous acquisition of a nonbinding conformation. Functional relevance of NEM treatment
We showed that NEM produces a biphasic effect on InsP3 binding activity. As the InsP3 receptor constitutes a Ca2⫹ channel, we investigated the effect of NEM on the InsP3-
BIPHASIC EFFECT OF THIOL REAGENTS ON InsP3 RECEPTOR
induced Ca2⫹ release activity of our microsomal preparation. Figure 7 shows a typical experiment; upon incubation at 37 C in the presence of 2 mm ATP, bovine adrenal cortex microsomes (8 –10 mg protein) exhibited a high Ca2⫹-sequestering activity, thus decreasing the ambient Ca2⫹ concentration to a low nanomolar level. Successive doses of 0.3, 1, and 10 m InsP3 released, respectively, 2.5, 3.8, and 4.5 nmol Ca2⫹. These InsP3-induced Ca2⫹ responses were quantified by the addition of a known amount of exogenous Ca2⫹ (3 nmol). As InsP3 is rapidly degraded under our experimental conditions, Ca2⫹ was sequestered back into its reservoir, thus decreasing the ambient Ca2⫹ concentration to its initial basal level. Addition of 1 m ionomycin immediately released all of the accumulated Ca2⫹, indicating the vesicular nature of the Ca2⫹-sequestering process. With this approach, we investigated the effect of NEM on InsP3-induced Ca2⫹ release activity. Figure 8A shows that after a 30-sec treatment with NEM (150 m), a low dose of InsP3 (0.3 m) released 2.7 nmol Ca2⫹, an amount about 1.5-fold higher than that released by the same dose of InsP3 (1.9 nmol Ca2⫹) before treatment with NEM. Figure 8B shows dose-dependent curves for InsP3induced Ca2⫹ release measured before or after a 30-sec treatment with NEM (150 m). Under these conditions, InsP3induced Ca2⫹ release activity was significantly potentiated at low doses of InsP3. This modest potentiating effect (1.5- to 2.5-fold) was consistently observed in at least three independent experiments. Moreover, the potentiating effect of this NEM treatment correlated with the potentiating effect previously observed on InsP3 binding affinity (2-fold increase in the affinity). That the effects of the highest concentrations of InsP3 were not significantly affected by a 30-sec treatment with NEM supports the idea that NEM increases the affinity of InsP3 receptor without affecting its maximal capacity. We also investigated the effect of a 15-min treatment with NEM (150 m), a condition known to dampen InsP3 binding activity. Figure 8C shows that a 15-min treatment with 150 m NEM caused a significant reduction in the InsP3-induced Ca2⫹ release activity observed at low InsP3
FIG. 7. ATP-dependent Ca2⫹-sequestering activity and InsP3induced Ca2⫹ release activity of adrenal cortex microsomes. Microsomes (8 –10 mg protein) were incubated at 37 C, and their Ca2⫹ uptake and release activities were monitored using fura-2 (free acid; 1 M) under the conditions described in Materials and Methods. The Ca2⫹ sequestered by an ATP-dependent process was partially released by InsP3. The amount of Ca2⫹ released was calibrated by the addition of a known amount of Ca2⫹ (3 nmol). ATP, 2 mM ATP; I, 0.3, 1, and 10 M InsP3; C, 3 nmol CaCl2; Io, 1 M ionomycin. This typical trace is representative of several such experiments performed with at least four different microsomal preparations.
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concentrations. The amount of Ca2⫹ released by 0.3 m InsP3 was significantly lower after treatment with NEM (1.3 nmol) than before treatment with NEM (2.1 nmol). Figure 8D shows that this 15-min treatment with 150 m NEM significantly reduced the Ca2⫹-releasing effect of high doses of InsP3. Under the same conditions, the maximal amount of Ca2⫹ released with 10 m InsP3 was reduced from 4.3 ⫾ 0.7 to 2.1 ⫾ 0.2 nmol Ca2⫹. Again these results correlate very well with those obtained in InsP3 binding studies under similar conditions. With our InsP3-induced Ca2⫹ release approach, we could reproduce the biphasic effect of NEM observed in InsP3 binding studies. Discussion
In the present study we demonstrated that two thiolreactive agents produced biphasic effects on the InsP3 binding activity of bovine adrenal cortex microsomes. NEM and thimerosal both alkylated a unique set of thiol groups (or a single thiol group) that is essential for the binding properties of the InsP3 receptor. The alkylation of this unique set of thiol groups conferred to the receptor an early gain of affinity, followed by a progressive and complete loss of binding activity. The rate at which the biphasic effect occurred after alkylation was dependent on the temperature (higher rate at high temperature). These results can reconcile the apparently discrepant results obtained in numerous studies on the effect of thiol-reactive agents on InsP3 binding activity. We previously reported that a 15-min treatment of adrenal cortex microsomes at 37 C with 10 mm NEM completely abolished their InsP3 binding activity (20). Identical results were obtained after treatment of rat liver membranes with 500 m NEM under similar conditions (24). We clearly showed in the present study that these conditions are sufficiently drastic to abolish the InsP3 binding activity. In more recent studies, 100 m thimerosal (15-min treatment at 0 C) was shown to increase InsP3 binding activity in adrenal cortex microsomes (16) and in permeabilized hepatocytes (17). As demonstrated in the present study, under these milder conditions thiolreactive agents increased InsP3 binding affinity. Interestingly, Hilly et al. (25) showed that a short treatment (5 min) at 37 C with 100 m thimerosal increased InsP3 binding activity in permeabilized hepatocytes and cerebellar membranes. Again, the results of the present study indicate that these conditions correspond to a mild treatment causing a potentiation of InsP3 binding activity. Taken together these results suggest that thiol-reactive agents initiate a time- and dose-dependent (accelerated at high temperature) series of conformational changes conferring to the InsP3 receptor an early gain of affinity followed by a progressive and complete loss of binding activity. The InsP3 receptor is the intracellular Ca2⫹ release channel in nonexcitable cells. It was thus important to verify the functional significance of InsP3 receptor treatment with thiolreactive agents. In the present study we showed that the InsP3-induced Ca2⫹ release activity of adrenal cortex microsomes was biphasically regulated by NEM. We demonstrated that a mild treatment with NEM significantly increased (enhanced apparent affinity), whereas a severe treatment with NEM considerably diminished the
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FIG. 8. Effect of NEM on InsP3-induced Ca2⫹ release activity. Microsomes (8 –10 mg protein) were incubated as described in Fig. 7. A, The potentiating effect of a 30-sec treatment with 150 M NEM. I, 0.3 M InsP3; C, 3 nmol CaCl2. B, The effect of a 30-sec treatment with 150 M NEM on InsP3-induced Ca2⫹ release with different concentrations of InsP3. C, The dampening effect of a 15-min treatment with 150 M NEM. I, 0.3 M InsP3; C, 3 nmol CaCl2. D, The effect of a 15-min treatment with 150 M NEM on InsP3-induced Ca2⫹ release with different concentrations of InsP3. Each value is the mean ⫾ SD of three to six separate experiments. Results were reproduced with at least four different microsomal preparations. *, P ⬍ 0.05 vs. control.
InsP3induced Ca2⫹ release activity. In a previous study we showed that 100 m thimerosal potentiated (enhanced apparent affinity) the release of Ca2⫹ induced by InsP3 in adrenal cortex microsomes (16). In permeabilized hepatocytes, thimerosal was also shown to potentiate InsP3-induced Ca2⫹ release, and under similar conditions, mersalyl (a more effective alkylating reagent) blocked InsP3-induced Ca2⫹ release (17). A biphasic effect of thimerosal on InsP3-induced Ca2⫹ release was also observed in permeabilized A7r5 smooth muscle cells (26). These results suggest that the alkylation of InsP3 receptor biphasically modulates its InsP3 binding activity and its functional Ca2⫹ release activity. Could the modification of thiol groups be considered a genuine mechanism of regulation of InsP3 receptor activity? If this is the case, as regulatory mechanisms imply a controlled balance between positive and negative influences, the effect of thiol-reactive agents should be reversible. In the present study we showed that the reducing agent DTT could reverse the potentiating effect of thimerosal, and we further showed that thimerosal could repotentiate the InsP3 binding activity of a DTT-treated adrenal cortex microsomal preparation. These results suggest that at early time points after alkylation, the InsP3 receptor can oscillate between two affinity states that are related to its alkylation state. Interestingly, the endogenous antioxidant glutathione was shown to
increase the latency between thimerosal treatment and the sensitization of InsP3-induced Ca2⫹ release in single HeLa cell video imaging experiments (14). S-Nitrosylation is a newly recognized cellular mechanism of thiol group modification that has been shown to regulate the activity of several Ca2⫹ channels, including N-methyl-d-aspartate receptor (5), ryanodine receptor (7, 8), and store-operated Ca2⫹ channel (9). A recent study demonstrated that S-nitrosylation is a dynamic regulatory modification of ryanodine receptor 1 and proposed that S-nitrosylation may be involved as a mechanism for control of the protein function in the truest sense analogous to phosphorylation (8). It is tempting to suggest that InsP3 receptor could also be a target for Snitrosylation. Further studies are needed to substantiate this hypothesis and to identify the cellular pathway responsible for the denitrosylation of InsP3 receptor. In the present study we have shown that the potentiating effect of thimerosal could be reversed by dealkylation with DTT. However, we also showed that at late time points after alkylation, once the InsP3 receptor had acquired a nonbinding conformation, a dealkylating treatment with DTT did not rescue any InsP3 binding activity. These results suggest that unless the alkylated state of the InsP3 receptor is reversed before it acquires a nonbinding conformation, the receptor will irreversibly lose its binding and Ca2⫹ release activities.
BIPHASIC EFFECT OF THIOL REAGENTS ON InsP3 RECEPTOR
Under extreme conditions, such as during an oxidative stress, this irreversible loss of InsP3 receptor function could represent a self-protective mechanism for the cells. This idea is consistent with the conclusion of a recent study that showed that ischemia insults (a condition known to generate reactive oxygen species) in certain regions of rat brain caused conformational changes in InsP3 receptor that led to reduced binding activity without a reduction in receptor protein level (27). What happens to InsP3 receptors that have acquired a nonbinding conformation? It is tempting to propose that these receptors will eventually be degraded. Further work is needed to verify this possibility in intact bovine adrenal cells. In conclusion, we have shown that thiol-reactive agents biphasically regulate InsP3 receptor activity. We also demonstrated that InsP3 receptor can alternate between different affinity states that are related to its alkylation state. Our results reconcile the conclusions of several others studies performed under different experimental conditions with different thiol-reactive agents. Further studies are needed to extend these observations in the context of intact adrenal cells and under conditions where endogenous thiol-reactive agents are produced. References 1. Berridge MJ, Bootman MD, Lipp P 1998 Calcium: a life and death signal. Nature 395:645– 648 2. Broillet MC 1999 S-Nitrosylation of proteins. Cell Mol Life Sci 55:1036 –1042 3. Bredt DS, Snyder SH 1998 Nitric oxide: a physiologic messenger molecule. Annu Rev Biochem 63:175–195 4. McDonald LJ, Murad F 1996 Nitric oxide and cyclic GMP signaling. Proc Soc Exp Biol Med 211:1– 6 5. Lipton SA, Choi YB, Pan ZH, Lei SZ, Chen HS, Sucher NJ, Loscalzo J, Singel DJ, Stamler JS 1993 A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds. Nature 364:626 – 632 6. Campbell DL, Stamler JS, Strauss HC 1996 Redox modulation of L-type calcium channels in ferret ventricular myocytes. Dual mechanism regulation by nitric oxide and S-nitrosothiols. J Gen Physiol 108:277–293 7. Xu L, Eu JP, Meissner G, Stamler JS 1998 Activation of the cardiac calcium release channel (ryanodine receptor) by poly-S-nitrosylation. Science 279:234 –237 8. Eu JP, Sun J, Xu L, Stamler JS, Meissner G 2000 The skeletal muscle calcium release channel: coupled O2 sensor and NO signaling functions. Cell 102:499 –509 9. Ma HT, Favre CJ, Patterson RL, Stone MR, Gill DL 1999 Ca2⫹ entry activated by S-nitrosylation. Relationship to store-operated Ca2⫹ entry. J Biol Chem 274:35318 –35324
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