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Thiol-Reactive Agents Biphasically Regulate Inositol 1,4,5-Trisphosphate Binding and Ca²⁺ Release Activities in Bovine Adrenal Cortex Microsomes¹

Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Sherbrooke Québec, Canada J1H 5N4

Address all correspondence and requests for reprints to: Dr. Gaétan Guillemette, Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, 3001, 12^e Avenue Nord, Sherbrooke, Québec, Canada J1H 5N4. E-mail: gguillem{at}courrier.usherb.ca

	Abstract

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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 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.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
CYTOSOLIC 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 mimicked by alkylation of the same thiol groups with thiol-reactive agents.

Experimental evidence suggests that the inositol 1,4,5-trisphosphate (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, 14, 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 consequences on InsP₃ receptor activity and thus on regulation of the intracellular level of Ca²⁺.

	Materials and Methods

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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).

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 x g for 15 min at 4 C, the supernatant was filtered on two layers of cheesecloth and centrifuged at 35,000 x g for 20 min at 4 C. The 35,000 x g pellet was resuspended in the same medium without EGTA (medium B) and centrifuged at 35,000 x 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 InsP₃-induced 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 ATP-dependent Ca²⁺ sequestering activity had reached a steady state. The free Ca²⁺ concentration of the medium was monitored with fura-2 (free acid; 1 µM) 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 (F_max and F_min) obtained by adding excess Ca²⁺ and EGTA (at pH 8.5), respectively, after treatment with 1 µM ionomycin. The equation used was [Ca²⁺] = 224 nM [(F - F_min)/(F_max - 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 µl with appropriate concentrations of [³H]InsP₃ (0.6–0.9 nM). Nonspecific binding was determined in the presence of 1 µM InsP₃. Incubations were performed for 30 min at 0 C and were terminated by centrifugation at 15,000 x g for 5 min at 4 C. The pellets were solubilized, and the receptor-bound radioactivity was analyzed by liquid scintillation spectrometry.

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 x 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 µg/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).

Data analysis
Results are presented as the mean ± SD. Binding curves, binding capacity (B_max), and K_d 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

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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 µM, respectively. Under these conditions, maximal effects were obtained with concentrations of NEM and thimerosal of 1 mM and 300 µM, 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 µM 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 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 µM NEM biphasically modulated [³H]InsP₃ binding activity (Fig. 2B). With both thiol-reactive 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 µM 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 proteins are incubated. To further emphasize the modulatory role of temperature on the NEM effect, microsomes were pretreated for 15 min with 500 µM 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.

View larger version (31K): [in this window] [in a new window]   Figure 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 µM NEM or 300 µM 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.

View larger version (14K): [in this window] [in a new window]   Figure 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 µM thimerosal (A) or 100 µM 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.

View larger version (17K): [in this window] [in a new window]   Figure 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 µM 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.

View larger version (22K): [in this window] [in a new window]   Figure 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 µM 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.

View larger version (16K): [in this window] [in a new window]   Figure 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 µM 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 µM 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 µM 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.

View larger version (12K): [in this window] [in a new window]   Figure 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 µM 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.

View larger version (11K): [in this window] [in a new window]   Figure 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 µM) 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 µM InsP3; C, 3 nmol CaCl2; Io, 1 µM ionomycin. This typical trace is representative of several such experiments performed with at least four different microsomal preparations.

View larger version (26K): [in this window] [in a new window]   Figure 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 µM NEM. I, 0.3 µM InsP3; C, 3 nmol CaCl2. B, The effect of a 30-sec treatment with 150 µM NEM on InsP3-induced Ca2+ release with different concentrations of InsP3. C, The dampening effect of a 15-min treatment with 150 µM NEM. I, 0.3 µM InsP3; C, 3 nmol CaCl2. D, The effect of a 15-min treatment with 150 µM 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.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

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 thiol-reactive 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 InsP₃induced Ca²⁺ release activity. In a previous study we showed that 100 µM 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 S-nitrosylation. 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. 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.

	Footnotes

 
¹ 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.

² Recipient of studentship from the Fonds de la Recherche en Santé du Québec.

Received January 12, 2001.

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