Department of Pharmacology and Toxicology, Institute for Environmental Toxicology, and Neuroscience Program, Michigan State University, East Lansing, Michigan 48824
Received March 5, 2004; accepted March 8, 2004
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ABSTRACT |
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Key Words: methylmercury; neurotoxicity; inositol-1,4,5-triphosphate receptor; muscarinic receptor; Ca2+-mediated celldeath, cerebellum.
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INTRODUCTION |
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Among the early effects of MeHg in CGCs is a pronounced increase of intracellular [Ca2+] ([Ca2+]i) consisting of an initial release of stored intracellular Ca2+ followed by influx of extracellular Ca2+ (Marty and Atchison, 1997; Limke and Atchison, 2002
). This effect occurs at much lower MeHg concentrations in CGCs than in other, less sensitive cells (Hare and Atchison, 1995a
; Hare et al., 1993
). In CGCs in vitro, the incidence of MeHg-induced neuronal death is attenuated by chelating
(Atchison and Marty, 1998
), while in vivo, the neurological signs of MeHg toxicity in rats are attenuated by Ca2+ channel blockers (Sakamoto et al., 1996
), suggesting that loss of [Ca2+]i homeostasis is critical to MeHg-induced death of CGCs (Marty and Atchison, 1998
). Recent experiments indicate that the primary source of intracellular Ca2+ release during MeHg exposure of CGCs in culture is the mitochondria (Limke et al., 2003
). However, the mitochondrial Ca2+ originated in a nonmitochondrial, thapsigargin-sensitive intracellular source, implicating Ca2+ release from the smooth endoplasmic reticulum (SER) as the step preceding mitochondrial Ca2+ overload. Under physiological conditions, Ca2+ release from the SER into the cytosol occurs via the inositol-1,4,5-triphosphate (IP3) receptor and/or the ryanodine receptor. In NG108-15 cells, most of the Ca2+ responsible for the first-phase increase of the [Ca2+]i induced by MeHg originates from the IP3-sensitive pool in the SER, with some contribution from the ryanodine-sensitive pool (Hare and Atchison, 1995a
). However, the mechanism underlying this Ca2+ release is not yet known.
One potential route for MeHg-induced release of Ca2+ from the SER is via action at a cell-surface receptor linked to phospholipase C (PLC). In CGCs in culture, low micromolar concentrations of MeHg cause an increase in [IP3] (Sarafian, 1993), although this effect is not observed in all neuronal models (Hare and Atchison, 1995a
). The elevation of [IP3] could be due to binding of MeHg to muscarinic receptors, which has been observed in several neuronal systems (Abd-Elfattah and Shamoo, 1981
; Candura et al., 1997
; Castoldi et al., 1996
; Von Burg et al., 1980
). CGCs express a high density of M2 and M3 muscarinic receptors, and do not express M1 or M4 receptors; activation of the M3 receptor leads to generation of IP3 by activation of PLC (Alonso et al., 1990
; Fohrman et al., 1993
; Fukamauchi et al., 1993
; Simpson et al., 1994
). Muscarinic receptors of all subtypes contain two critical thiol groups within the receptor's active site, providing a potential site at which MeHg could bind and subsequently activate these receptors (Castoldi et al., 1996
; Coccini et al., 2000
). An interaction between MeHg and the muscarinic receptors could play a role in MeHg-induced cell death, because CGC death caused by a number of toxicants is mediated by the M3 receptors (Castoldi et al., 1998
; Gao et al., 1993
; Lin et al., 1997
; Yan et al., 1995
). Additionally, the underlying mechanism may involve both direct and indirect actions, as MeHg is reported to stimulate IP3 binding to its receptor in rat cerebellar membrane preparations (Chetty et al., 1996
).
The purpose of the present study was to determine whether inhibition of specific components of the muscarinic receptor-linked Ca2+ signaling pathways altered MeHg-induced elevation of [Ca2+]i and cell death in rat cerebellar granule cells in primary culture. Specifically, we examined the effects of inhibiting muscarinic receptors, PLC, IP3 receptors, and ryanodine receptors, as well as the effect of SER store depletion, on MeHg-induced cell death. Our results indicate that muscarinic receptors participate in the observed [Ca2+]i increase, with the M3 muscarinic receptor being involved in MeHg-induced cell death in rat granule cells in culture.
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MATERIALS AND METHODS |
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Unless otherwise noted, the standard physiological saline used for experimental solutions was HEPES Buffered Saline (HBS) which contains (mM): 150 NaCl, 5.4 KCl, 1.8 CaCl2, 0.8 MgSO4, 20 d-glucose, and 20 HEPES (free acid) (pH 7.3 at room temperature of 2325°C). The 40 mM K+ solution contains the same components as HBS except with 40 mM K+ and 115.4 mM NaCl. The low-[Ca2+], EGTA-containing buffer (EGTA-HBS) has the same components as HBS minus CaCl2 and plus 20 µM EGTA-final [Ca2+] approx. 60 nM (Marty and Atchison, 1997). For all experiments, pharmacological agents were dissolved in either HBS or EGTA-HBS, with appropriate controls for any additional solvents used (DMSO or ethanol, maximum final solvent of 0.01% [v/v], for all experiments). MeHg was prepared as a 5 mM stock solution in deionized water and diluted to working concentrations just prior to use.
Rat cerebellar granule cell isolation procedure. CGCs were isolated from seven-day-old Sprague-Dawley rats of either sex as described previously (Marty and Atchison, 1997). Cells were plated in medium that consisted of Dulbecco's Modified Eagle Medium with 10% (w/v) fetal bovine serum, 25 mM KCl, 50 µM GABA, 50 µM kainate, 5 µg/ml insulin, 100 U/ml penicillin, and 50 µg/ml streptomycin. Cells were plated at a density of 1.8 x 108 cells/35 mm2 dish, each containing a 13 mm CellLocate coverslip coated with 0.1 mg/ml poly-D-lysine, or at 2.02.2 x 106 cells/35 mm dish, each containing a 25 mm glass coverslip coated with 0.1 mg/ml poly-D-lysine. After 24 h, 10 µM cytosine-ß-arabinofuranoside was added to inhibit glial proliferation. Penicillin-streptomycin was not added at this or any subsequent medium changes, as streptomycin can alter ion channel function (Atchison et al., 1988
; Redman and Silinsky, 1994
). Cells were maintained at 37°C in 5% CO2 for 68 days in vitro to allow for cell maturation (Aronica et al., 1993
).
Fluorescence measurements of changes in [Ca2+]i. To measure changes in [Ca2+]i, cells were loaded with 34 µM fura-2 AM in HBS for 1 h at 37°C, followed by perfusion with HBS for 30 min. Digital fluorescent images were obtained using a Nikon Diaphot microscope (Nikon, Tokyo, Japan) or an Olympus IX70 microscope (Olympus Optical Co., Tokyo, Japan), coupled to an IonOptix system (Milton, MA) with an Open Perfusion Micro-Incubator (Harvard Apparatus, Holliston, MA), maintained at 37°C. For each experiment, changes in emitted fluorescence (505 nm) at excitation wavelengths of 340 and 380 nm were monitored simultaneously in multiple soma (310) within the same microscopic field. The fluorescence ratio (F340/380) indicated the approximate amount of [Ca2+]i, however the data were not converted to [Ca2+]i due to fura-2 interactions with other divalent cations, such as Zn2+, a cation known to contribute to the fluorescence response to MeHg (Denny and Atchison, 1994; Hare et al., 1993
), but which evidently plays a minor role in the response of CGCs to MeHg (Marty and Atchison, 1997
). Cells loaded with fura-2 were exposed to 0.21.0 µM MeHg dissolved in HBS or EGTA-HBS. All experiments began with a 1 min wash with HBS (to establish baseline fluorescence), followed by 1 min exposure to 40 mM K+ solution; only cells which exhibited a reversible increase in [Ca2+]i following the K+ exposure were considered to be viable for the experiment. The K+ solution was washed out for 3 min with HBS, then cells were exposed to thapsigargin (10 µM, 5 min immediately prior to MeHg) (Irving et al., 1992
; Simpson et al., 1996
); atropine (10 µM, 10 min prior to, as well as during, exposure to MeHg) (Yan et al., 1995
); ryanodine (10 µM, 10 min prior to, as well as during, exposure to MeHg) (Irving et al., 1992
); or bethanechol (BCh) (1 mM, added directly to the growth medium 2430 h prior to loading cells with fura-2) (Fohrman et al., 1993
; Fukamauchi et al., 1993
; Simpson et al., 1996
; Wojcikiewicz et al., 1994
). For BCh experiments, the fura-2 AM was added directly to the growth medium; thus cells were in BCh-free solution for the 30 min HBS rinse and the 5 min viability test with 40 mM K+. In these experiments, MeHg was perfused immediately following the 3 min K+ solution wash period.
Following exposure to MeHg, we measured the time-to-onset of the first and second phase increases in [Ca2+]i, where the first phase (from release of pools) was measured from the point at which the fluorescence ratio irreversibly left baseline, and the second phase (influx of extracellular Ca2+) began at the point at which the fluorescence evoked by stimulation at 380 nm dropped sharply. For experiments performed in the absence of extracellular Ca2+, we measured the amplitude of the increase in ratio of fluorescence of fura-2, normalized to the peak ratio of fura-2 fluorescence caused by the 1 min exposure to 40 mM K+ ("normalized ratio"). Representative figures for these measurements can be found elsewhere (Limke and Atchison, 2002
; Marty and Atchison, 1997
). For measurements of both time-to-onset and normalized fluorescence ratio, data from each cell observed in an experiment were averaged to provide mean time-to-onset for that dish of cells (n = 1). Comparisons of mean time-to-onset for "MeHg" vs. the corresponding "MeHg plus inhibitor" cells were made using Student's paired t-test, with values of p < 0.05 considered to be statistically significant. In order to minimize differences between cell isolates, experiments using MeHg alone and MeHg with the pharmacological agent were run on the same day, and experiments using the same agents were performed in at least two separate cell isolates. In a cell-free system, fura-2 did not interact with MeHg or any of the agents used at concentrations used in the present study (results not shown). The number of replicates (separate dishes of cells) for each experiment is given in the figure legend.
Measurement of granule cell viability. The protocol to assess cell viability in response to MeHg was designed to parallel experimental conditions in previous experiments, in which we studied the effects of MeHg on elevations of [Ca2+]i in CGCs in primary culture (Limke and Atchison, 2002; Marty and Atchison, 1997
). MeHg exposure times and concentrations were identical to those described previously (Limke and Atchison, 2002
; Marty and Atchison, 1998
). For all experiments, appropriate solvent and drug controls were performed at the same time; the duration of exposure for each of these controls was 76 min, as this was the maximum interval of exposure to MeHg. Following MeHg exposure, cells were returned to MeHg-free conditioned medium (to allow protein in the media to bind excess MeHg) for 24 h. At 24 h after treatment, cells were removed from conditioned medium, rinsed twice with HBS and overlaid with the Live/Dead Eukolight Viability/Cytotoxicity reagents (Molecular Probes, Eugene, OR) for 30 min at 37°C. As per the kit instructions, the reagent buffer contains 2 µM calcein acetoxymethylester (calcein-AM) and 4 µM ethidium homodimer both dissolved in HBS. Calcein-AM labels healthy cells green and ethidium homodimer labels dead cells red. Following the 30 min exposure, cells were examined using a Nikon Diaphot (Nikon Optics, Tokyo, Japan) epifluorescence microscope. The number of live and dead cells in eight randomly selected CellLocate grids was counted in order to determine percent viability for that coverslip. Because of the 30 min incubation period in Live/Dead reagent, cells were counted at 24.5 h after the cessation of MeHg treatment. A specific assay for apoptosis or necrosis was not used because MeHg causes both types of cell death in the concentration range used in these experiments.
To determine whether emptying of the SER Ca2+ pool protected against MeHg-induced neuronal death, cells were treated with the SER Ca2+ ATPase (SERCA) inhibitor thapsigargin (10 µM) for 5 min immediately prior to exposure to MeHg (01.0 µM; Simpson et al., 1996). Following MeHg treatment, cells were returned to conditioned medium for 24 h, followed by cell counting as above. To determine whether down-regulation of muscarinic and IP3 receptors (Fukamauchi et al., 1991
, 1993
) protected cells from MeHg-induced neuronal death, the muscarinic agonist BCh was added directly to the growth medium to give a final concentration of 1 mM. At 24 h after application of BCh, cells were treated with MeHg alone or MeHg plus 1 mM BCh, then returned to the conditioned medium for 24 h before counting. For each set of cells exposed to MeHg there were corresponding HBS, 1 mM BCh, and vehicle control cells which were treated and counted at the same time as the MeHg-treated cells. For cells treated with BCh, all HBS washes contained 1 mM BCh; additionally, the conditioned medium to which they were returned after MeHg treatment contained 1 mM BCh for the entire 24 h post-treatment period. In experiments in which BCh was added with a specific cholinergic receptor antagonist, the antagonist was added for 10 min prior to the initial application of 1 mM BCh, as well as during BCh treatment and HBS wash. The antagonists tested were atropine (nonspecific muscarinic receptor antagonist), methoctramine (M2 antagonist), 4-DAMP (M3 antagonist), and DHE (neuronal nicotinic ACh receptor antagonist), all at a final concentration of 10 µM (Lin et al., 1997
; Yan et al., 1995
). CGCs do not express M1 or M4 receptors; thus, specific antagonists of these receptors were not examined. Finally, acute treatment with pharmacological inhibitors of muscarinic receptors (atropine, 10 µM), phospholipase C (U73122, 1 µM), IP3 receptors (xestospongin C, 1 µM), and ryanodine receptors (ryanodine, 10 µM) was used (Irving et al., 1992
; Jin et al., 1994
; Netzeband et al., 1999
). All agents were applied for 10 min prior to, as well as during, exposure to 01.0 µM MeHg. For viability experiments, the percent viability (number of live cells/total number of cells x 100) was determined for each exposure group; values are presented as the mean ± SEM. Viability percentages were normalized using an angular transformation. Comparisons of cell viability were made using a repeated measures analysis of variance (ANOVA) followed by Tukey's procedure for post-hoc comparisons to compare MeHg to MeHg + inhibitor, with p < 0.05 considered to be statistically significant (Steel and Torrie, 1960
).
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RESULTS |
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Next, we examined whether down-regulation and desensitization of muscarinic and IP3 receptors, using a 24 h exposure to 1 mM BCh, protects against MeHg-induced cell death. BCh was also applied during MeHg exposure to maintain desensitization of these receptors. As seen in Figure 4, increasing concentrations of MeHg killed an increasing percentage of cells at 24.5 h post-exposure. The loss of cell viability was consistent with that observed in previous experiments (Limke and Atchison, 2002; Marty and Atchison, 1998
). Treatment with 1 mM BCh significantly protected cells from MeHg-induced cell death at all concentrations of MeHg examined (Fig. 4). In cells pretreated with BCh and exposed to 0.2 or 0.5 µM MeHg, the incidence of cell death was equal to that of untreated cells. However, at 1 µM MeHg, the protection provided by 1 mM BCh was not as great as at the lower MeHg concentrations.
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DISCUSSION |
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Ca2+ release from the SER by MeHg contributes approximately 30% to the observed increase in fura-2 fluorescence ratio in CGCs during the initial phase of exposure to MeHg. This result agrees with previous data indicating that the primary source of the increase in [Ca2+]i is mitochondrial in origin (Limke et al., 2003). However, this differs from results obtained in NG108-15 cells, in which emptying the SER with thapsigargin and bradykinin prior to MeHg treatment reduced the MeHg-induced increase in the first-phase ratio by 68% (Hare and Atchison, 1995b
). As was found in NG108-15 cells (Hare and Atchison, 1995b
), the ryanodine receptor Ca2+ pool in granule cells contributes minimal Ca2+ during MeHg exposure. Inhibition of muscarinic receptors (atropine), down-regulation and desensitization of muscarinic and IP3 receptors (BCh), and SER store depletion (thapsigargin) were all equally effective at decreasing the amplitude of the [Ca2+]i increase during the first-phase of response for all MeHg concentrations examined. This suggests that MeHg activates a common signaling pathway involving these components.
Atropine delayed the time-to-onset of the initial elevation of [Ca2+]i, suggesting that the initiating step in release is activation of muscarinic receptors by MeHg. Granule cell M3 receptors are coupled to Gq, which leads to generation of IP3 through PLC. M2 receptors are the other predominant muscarinic receptor subtype in cerebellar granule cells. These receptors are linked to Gi, which inhibits adenylate cyclase and thus is not directly involved in Ca2+ signaling. As such the effect of atropine is likely due to inhibition of M3 receptors (Contrera et al., 1993
; Doble et al., 1992
; Fohrman et al., 1993
; Whitham et al., 1991a
,b
). Viability experiments also implicate the M3 muscarinic receptor in MeHg-induced neuronal death. The protection provided by BCh is reversed by the M3-preferring antagonist 4-DAMP and is not due to an effect on nicotinic receptors, as indicated by the inability of DHE to protect against cell death-induced by MeHg. The specific contribution of M2 receptors to the cytotoxic action of MeHg could not be determined due to the cytotoxicity of the M2 antagonist, leaving open the possibility that actions at this receptor also contribute to cell death. However, in gastrointestinal smooth muscle, M2 receptors modulate the activity of M3 receptors (Ehlert et al., 1999
), so it seems unlikely that an action of MeHg on M2 receptors contributes significantly to the cytotoxic effect observed, unless MeHg exhibits agonistic actions at M3 and antagonistic actions at M2-type receptors. Thus the cytotoxicity observed in the present study with the M2 antagonist in the absence of MeHg might result from unchecked elevation of [Ca2+]i subsequent to stimulation of M3 receptors. Additionally, we did not consider other actions of BCh, such as activation of phosphatidylinositol-3-kinase, which could also mediate the observed protective effect. However, our results are consistent with previous observations that MeHg binds to multiple subtypes of muscarinic receptors in both the central and peripheral nervous systems (Abd-Elfattah and Shamoo, 1981
; Castoldi et al., 1996
; Coccini et al., 2000
) and causes increased [IP3] in granule neurons in vitro (Sarafian, 1993
). The binding of MeHg to muscarinic receptors could in part explain the selective neurotoxicity of specific cell types within the central nervous system to MeHg. In rats, CGCs express the highest density of muscarinic receptors in the cerebellar cortex, as assessed by [3H]quinuclidinyl benzilate binding (Neustadt et al., 1988
). In vivo studies indicate that MeHg causes an up-regulation of muscarinic receptors in both the cerebellum and hippocampus, regions that tend to accumulate MeHg and exhibit neuronal death following MeHg exposure. Thus receptor up-regulation may occur in these cells to compensate for loss of functional receptors as a result of irreversible binding by MeHg (Coccini et al., 2000
). Autoradiographic studies in mouse brain indicate that MeHg accumulates preferentially in these two brain regions (Berlin and Ullberg, 1963
), suggesting a possible correlation between the selective neurotoxicity of MeHg within specific areas of the central nervous system, and location of specific muscarinic receptor subtypes. However, given that MeHg can cause either apoptosis or necrosis, depending on the degree of exposure, the issue remains complex, and likely involves more than one pathway being affected simultaneously.
Experiments designed to differentiate between MeHg interactions with muscarinic receptors and with the IP3 receptor were inconclusive. Atropine alone did not protect against MeHg-induced cytotoxicity, which was surprising given the known interactions of MeHg with muscarinic receptors (Castoldi et al., 1996; Coccini et al., 2000
; Quandt et al., 1982
), and its ability to delay MeHg-induced elevations of [Ca2+]i. Perhaps, MeHg has a higher affinity for muscarinic receptors than does atropine, resulting in displacement of atropine by MeHg and subsequent promotion of neurotoxicity; however, we did not test this hypothesis. MeHg competes with M1- and M2-preferring antagonists in rat cortical membranes, and demonstrates higher affinity for the M1 receptor (IC50 = 3.4 µM) vs. the M2 receptor (IC50 = 149 µM) (Castoldi et al., 1996
). In guinea pig intestinal smooth muscle, MeHg inhibits contractions induced by stimulation of cholinergic nerves or by external application of ACh, suggesting that it has a high affinity for muscarinic receptors (Fukushi and Wakui, 1985
). MeHg cannot be removed from cholinergic receptors by mere wash with a MeHg-free solution. Thus it is conceivable that MeHg has higher affinity for muscarinic receptors than does atropine; however, the ability of MeHg to replace atropine on the muscarinic receptors in granule cells in culture was not examined directly.
Attempts to block the IP3 receptor with xestospongin C also did not prevent MeHg-induced cell death. Given the protective effect of BCh and its reversibility by atropine, this result was also surprising. The IP3 receptor has been implicated in MeHg-induced loss of [Ca2+]i homeostasis in NG108-15 neuroblastoma cells (Hare and Atchison, 1995a), T cells (Tan et al., 1993
), and CGCs (Sarafian, 1993
). Indeed, T cells that are deficient in IP3 receptors are resistant to apoptosis caused by a number of agents (Jayaraman and Marks, 1997
; Marks, 1997
). In cultures of granule cells, neurons which have a relative deficiency in IP3 receptors are resistant to apoptosis following exposure to low [K+] (Oberdorf et al., 1997
), suggesting that this receptor can participate in granule cell death. Given that MeHg increases [IP3] (Sarafian, 1993
), and that down-regulation of the M3 and IP3 receptors protects against MeHg-induced neuronal death, the lack of protection afforded by xestospognin C may reflect a higher affinity for the IP3 receptor by MeHg. Alternatively, the results suggest that MeHg-induced release of Ca2+ through the IP3 receptor does not contribute to MeHg-induced neurotoxicity, although this conclusion is not supported by the 24 h BCh data.
Overall, these results suggest that interactions with muscarinic receptors and, through this interaction, perturbation of SER Ca2+ regulation, may contribute to the selective vulnerability of CGCs. However, the importance of the SER as a target in MeHg neurotoxicity may lie in the effect of the released Ca2+ on nearby mitochondria. MeHg causes opening of the mitochondrial permeability transition pore (MTP) in cerebellar granule neurons in vitro, as indicated by the ability of cyclosporin A, which inhibits activation of the MTP, to delay MeHg-induced release of , loss of mitochondrial membrane potential, and MeHg-induced cell death (Limke and Atchison, 2002
). Under normal conditions, mitochondria do not store Ca2+, thus the mitochondria must first accumulate Ca2+ which is then released into the cytosol (Budd and Nicholls, 1996
). Recent experiments indicate that MeHg causes mitochondria in CGCs to accumulate Ca2+ rapidly in a thapsigargin- and cyclosporin A-sensitive manner, suggesting that the SER does contribute Ca2+ to the observed mitochondrial dysregulation and subsequent neuronal death via an MTP-dependent pathway (Limke et al., 2003
). Thus, the disruption of SER Ca2+ regulation caused by interaction of MeHg with M3 muscarinic receptors may play a significant triggering role in MeHg-induced neurotoxicity of granule cells, with the localization of specific muscarinic receptor subtypes contributing to the regional selectivity of MeHg neurotoxicity within the central nervous system.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Michigan State University, Dept. Pharmacology/Toxicology, B331 Life Sciences, East Lansing, MI 48824-1317. Fax: (517) 432-1341. E-mail: atchiso1{at}msu.edu.
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