ToxSci Advance Access originally published online on May 12, 2004
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Toxicological Sciences 80, 60-68 (2004)
Toxicological Sciences vol. 80 no. 1 © Society of Toxicology 2004; all rights reserved.
Acute Exposure to Methylmercury Causes Ca2+ Dysregulation and Neuronal Death in Rat Cerebellar Granule Cells through an M3 Muscarinic Receptor-Linked Pathway
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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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Acute exposure to low concentrations of methylmercury (MeHg) causes a severe loss of intracellular calcium (
) homeostasis, which apparently contributes to neuronal death of cerebellar granule cells in culture. We examined the role of muscarinic receptors in MeHg-induced Ca2+ dysregulation and cell death in rat cerebellar granule cells in vitro using fura-2 single-cell microfluorimetry and viability assays, respectively. The nonspecific muscarinic receptor antagonist atropine significantly delayed the onset of MeHg-induced Ca2+ elevations and reduced the amount of Ca2+ released into the cytosol. Depletion of the smooth endoplasmic reticulum (SER) Ca2+ pool with thapsigargin or down-regulation of muscarinic receptors and inositol-1,3,4-triphosphate (IP3) receptors with bethanechol (BCh) caused similar reductions in the amplitude of the MeHg-induced Ca2+ increase, suggesting that MeHg interacts with muscarinic receptors to cause Ca2+ release from the SER through activation of the IP3 receptors. To determine whether this Ca2+ release plays a role in MeHg-induced cell death, cells were exposed to MeHg in the presence of specific muscarinic receptor inhibitors. Acute exposure to increasing concentrations of MeHg (0.2–1.0 µM) caused a corresponding increase in cell death at 24.5 h post-exposure. Prior down-regulation of muscarinic and IP3 receptors with BCh protected against cell death. Protection was ablated by atropine and the M3 receptor antagonist 4-diphenylacetoxyl-N-methylpiperidine methiodide (DAMP), but not by the neuronal nicotinic receptor antagonist dihydro-ß-erythroidine hydrobromide (DHE). Thus activation of M3 muscarinic receptors with subsequent generation of IP3 evidently contributes to elevated [Ca2+]i and subsequent cytotoxicity of cerebellar granule cells by MeHg. 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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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Methylmercury (MeHg) is an environmental contaminant that causes neurotoxicity in both the central and peripheral nervous system. A specific pattern of neurotoxicity occurs with MeHg poisoning; clinical signs include constriction of visual field, muscle weakness, impaired speech, and generalized gross ataxia (Chang, 1977
; Hunter and Russell, 1954). The ataxia is apparently caused by preferential loss of granule neurons from the cerebellar cortex, which are damaged without evidence of damage to other cerebellar neurons, despite their accumulating approximately equal or greater amounts of MeHg in vivo (Chang, 1977; Hunter and Russell, 1954). The basis for the selective vulnerability of cerebellar granule cells (CGCs) to MeHg is not yet understood.
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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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Materials and experimental solutions. Deoxyribonuclease I (DNase I) and type II trypsin were purchased from Worthington Biochemicals (Freehold, NJ). Cell culture supplies were purchased from Gibco BRL (Grand Island, NY). CellLocate coverslips were purchased from Eppendorf Scientific (Madison, WI). Fura-2 acetoxymethylester (fura-2 AM) and the Eukolight viability/cytotoxicity kit were purchased from Molecular Probes (Eugene, OR). Atropine, thapsigargin, carbamyl-ß-methylcholine chloride (bethanechol), 4-diphenylacetoxyl-N-methylpiperidine methiodide (4-DAMP), dihydro-ß-erythroidine hydrobromide (DHE), methoctramine hydrochloride (methoctramine), ryanodine, poly-D-lysine, HEPES, and cytosine-ß-arabinofuranoside were all purchased from Sigma (St. Louis, MO). Methyl mercuric chloride (MeHg) was purchased from ICN Biochemicals Inc. (Aurora, OH). Xestospongin C and U73122 were purchased from Calbiochem (La Jolla, CA).
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 23–25°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 2.0–2.2 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 6–8 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 3–4 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 (3–10) 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 0.2–1.0 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 24–30 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 (0–1.0 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 0–1.0 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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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The SER and Muscarinic Receptors Participate in MeHg-Induced Elevations of [Ca2+]i
To determine whether a muscarinic receptor-linked pathway contributes to the MeHg-induced elevations of [Ca2+]i, several inhibitors were utilized either to deplete the SER (10 µM thapsigargin), inhibit muscarinic receptors (10 µM atropine), down-regulate and desensitize both muscarinic receptors and IP3 receptors (1 mM BCh), or inhibit ryanodine receptors (10 µM ryanodine). The efficacy of each treatment was first verified by assaying for its ability to decrease or abolish the peak increase in [Ca2+]i produced in response to acute treatment with 1 mM BCh for 1.5 min in CGCs loaded with fura-2 (Fig. 1; Table 1). In the absence of MeHg, acute treatment with thapsigargin or atropine and desensitization of muscarinic receptors by 24 h treatment with BCh all abolished the BCh-mediated release of Ca2+ from the IP3 receptor. Previous studies have conclusively demonstrated that application of a muscarinic agonist for 24 h will effectively down-regulate and desensitize both muscarinic and IP3 receptors in CGCs. Thus the loss of response to an acute application of BCh is in agreement with those studies (Fohrman et al., 1993
; Fukamauchi et al., 1993; Simpson et al., 1996; Wojcikiewicz et al., 1994). Acute treatment with ryanodine significantly decreased the peak increase in [Ca2+]i; thus, application of these pharmacological agents was sufficient to deplete significantly specific pools of Ca2+.
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As described previously (Hare et al., 1993
; Marty and Atchison, 1997), MeHg causes a biphasic increase in the ratio of fura-2 fluorescence. The first phase elevation is due to an increase in cytosolic Ca2+ released from intracellular stores; the second phase is due to an influx of extracellular Ca2+ (see Limke and Atchison, 2002, for representative figures). The relative amplitude of the first-phase increase of [Ca2+]i can be determined by measuring the ratio of fura-2 fluorescence in a nominally 
solution ("EGTA-HBS") (see Limke and Atchison, 2002, for representative figures). Acute treatment with thapsigargin (Fig. 2A), or atropine (Fig. 2B) or 24 h pretreatment with BCh (Fig. 2C), resulted in significant reduction in the amplitude of this first-phase increase of [Ca2+]i caused by 0.2–1.0 µM MeHg. However ryanodine was not effective in this regard (results not shown). This implicates not only the SER but also muscarinic receptors and IP3 receptors as contributors to the first-phase increase in [Ca2+]i caused by MeHg.
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We then tested whether musarinic receptors are involved in the initial step(s) of MeHg-induced Ca2+ elevations using atropine to block all subtypes of muscarinic receptors. In the presence of
, application of 10 µM atropine for 10 min prior to, as well as during, exposure to MeHg significantly delayed the time-to-onset of the first-phase increase in fluorescence ratio caused by 0.2 and 0.5, but not 1.0 µM MeHg (Fig. 3A). Atropine caused a similar delay in the time-to-onset of the first-phase of increased fluorescence in cells exposed to 0.2–1.0 µM in the absence of extracellular Ca2+ (results not shown). However, atropine did not alter the time-to-onset of the second-phase of increased fluorescence, which results from influx of in response to 0.2, 0.5, or 1.0 µM MeHg (Fig. 3B). Thus muscarinic receptors appear to participate in the initial increase of [Ca2+]i caused by MeHg, but not the second-phase influx of .
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MeHg-Induced Cell Death Involves the IP3 Receptor and/or the M3 Muscarinic Receptor
Inhibition of muscarinic and/or IP3 receptors reduced the amplitude of MeHg-induced Ca2+ dysregulation. Next, we examined whether inhibition of these receptors would protect granule cells against, or delay the onset of, MeHg-induced cell death. We first sought to determine whether emptying the SER Ca2+ pool with thapsigargin was protective against cell death at 24.5 h post-exposure to 0–1 µM MeHg. However, a 5 min exposure to 10 µM thapsigargin was itself toxic at 24.5 h post-exposure (results not shown). It is important to note that this toxicity was not apparent for up to 60 min post-exposure, thus it did not affect the Ca2+ imaging experiments. However, it did preclude experiments examining the role of SER Ca2+ pools in MeHg-induced toxicity.
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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To determine whether the protection provided by BCh was due to action at muscarinic or nicotinic acetylcholine receptors, the nonspecific muscarinic antagonist atropine (10 µM) was applied for 10 min prior to BCh, as well as during the 24 h BCh treatment for 24 h prior to exposure to MeHg. As seen in Figure 5A, addition of atropine ablated the protection afforded by 1 mM BCh (see Figure 4), resulting in no significant difference in the number of cells surviving MeHg alone or MeHg plus BCh and atropine. The nicotinic receptor antagonist DHE (10 µM) did not alter the neuroprotective effects of BCh (Fig. 5B).
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CGCs express M2 and M3 receptors and do not express M1 or M4 receptors (Alonso et al., 1990
), thus the BCh-mediated protection could theoretically result from actions at either or both of these receptors. Preliminary tests revealed that the combination of the M2 receptor antagonist methoctramine (10 µM) with 1 mM BCh for 24 h caused significant cell death, thus preventing examination of whether the protective effect of BCh was due to action at the M2 receptors (results not shown). However, the M3 receptor antagonist 4-DAMP (10 µM) did prevent any protection by 1 mM BCh in a manner similar to atropine (Fig. 6), suggesting that BCh-mediated protection occurs through the M3 receptor.
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The 24 h BCh treatment regimen down-regulated both the M3 receptor and the IP3-receptor, thus the next experiment was designed to distinguish whether MeHg acts at the muscarinic and/or IP3 receptors. To this end, cells were exposed to atropine alone (10 µM) for 10 min prior to, as well as during, exposure to MeHg. Unlike the 24 h BCh pretreatment, atropine alone did not protect against cell death caused by 0.2–1.0 µM MeHg at 24.5 h post-exposure (results not shown). Similarly, acute application or 24 h pretreatment with the IP3 receptor inhibitor xestospongin C (1 µM) also did not prevent the MeHg-induced cell death. We hypothesized, but did not test, that this is due to MeHg binding the IP3 receptor with greater affinity than xestospongin (a reversible inhibitor). Inhibition of phospholipase C with U73122 was itself toxic, thereby preventing experiments using this compound (results not shown). Thus, we were unable to distinguish whether MeHg acts at muscarinic receptors and/or IP3 receptors to cause cell death. Finally, inhibition of Ca2+ release through the ryanodine receptors using 10 µM ryanodine proved ineffective against MeHg-induced neuronal death at all concentrations of MeHg examined (results not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MeHg-induced increases [Ca2+]i in rat CGCs in culture apparently contribute to MeHg-induced neuronal death (Castoldi et al., 2000
; Marty and Atchison, 1997, 1998). Because loss of Ca2+ regulation is a pivotal step in both apoptotic and necrotic death processes, experiments in the present study were designed to identify the mechanism by which MeHg causes release and cell death in CGCs in culture. MeHg interacts with muscarinic ACh receptors (Castoldi et al., 1996; Coccini et al., 2000), causes generation of IP3 in rat CGCs in culture (Sarafian, 1993), and causes Ca2+ release following activation of the IP3 receptor in NG108-15 cells (Hare and Atchison, 1995b) and rat T lymphocytes (Tan et al., 1993); thus, we examined the potential interaction between MeHg and these targets in the more sensitive CGCs.
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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The authors thank Aizhen Yao for her cell culture work; Jessica Hauptman, Ashley Bauer, and Mallory Koglin for expert word processing assistance; Ilyana Y. Martinez, Erica Fritz, and Scott Loiselle for their technical assistance; and Dr. Sue Marty for her input throughout this project. This work was supported by NIEHS grant ES03299. T.L.L. was supported by NIEHS grant T32-ES07255. Submitted in partial fulfillment of the requirement for the degree of Doctor of Philosophy (T.L.). J.J.B. was supported by a Merial Merck Summer Research Fellowship.
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NOTES |
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Portions of this study were presented in abstract form (The Toxicologist 2001, 60(1), 184; The Toxicologist 2002, 61(1), 122).
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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