Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2014 Jun 4.
Published in final edited form as: J Neurochem. 2009 Feb 7;109(2):525–538. doi: 10.1111/j.1471-4159.2009.05969.x

Muscarinic receptors prevent oxidative stress-mediated apoptosis induced by domoic acid in mouse cerebellar granule cells

G Giordano 1, L Li 1, CC White 1, FM Farin 1, HW Wilkerson 1, TJ Kavanagh 1, LG Costa 1,2
PMCID: PMC4045406  NIHMSID: NIHMS586688  PMID: 19200344

Abstract

In mouse cerebellar granule neurons (CGNs) low concentrations of domoic acid (DomA) induce apoptotic cell death, which is mediated by oxidative stress; apoptosis is more pronounced in CGNs from Gclm (−/−) mice, which lack the modifier subunit of glutamate cysteine ligase (GCL) and have very low glutathione (GSH) levels. By activating M3 muscarinic receptors, the cholinergic agonist carbachol inhibits DomA-induced apoptosis, and the anti-apoptotic action of carbachol is more pronounced in CGNs from Gclm (+/+) mice. Carbachol does not prevent DomA-induced increase in reactive oxygen species (ROS), suggesting that its anti-apoptotic effect is downstream of ROS production. Carbachol inhibits DomA-induced activation of JNK and p38 kinases, increased translocation to mitochondria of the pro-apoptotic protein Bax, and activation of caspase-3. Carbachol activates Erk 1/2 mitogen-activated protein kinase (MAPK) and phospahtidylinositol-3 kinase (PI3K) in CGNs from both genotypes. However, while the protective effect of carbachol is mediated by Erk1/2 MAPK in CGNs from both mouse genotypes, inhibitors of PI3K are only effective at antagonizing the action of carbachol in CGNs from Gclm (+/+) mice. In CGNs from both Gclm (+/+) and (−/−) mice, carbachol induces a MAPK-dependent increase in the level of the anti-apoptotic protein Bcl-2. In contrast, carbachol causes a PI3K-dependent increase in GCL activity and of GSH levels only in CGNs from Gclm (+/+) mice. Such increase in GCL is not due to a transcriptionally-mediated increase in GCLC (the catalytic subunit) or GCLM, but rather to an increase in the formation of the GCL holoenzyme. The results indicate that multiple pathways may contribute to the protective action of carbachol toward DomA-induced apoptosis. Compromised GCLM expression, which is also found in a common genetic polymorphism in humans, leads to lower GSH levels, which can exacerbate the neurotoxicity of DomA, and decreases the anti-apoptotic effectiveness of muscarinic agonists.

Keywords: domoic acid, muscarinic receptors, carbachol, apoptosis, phosphoinositide-3-kinase, glutamate cysteine ligase

Introduction

Cholinergic muscarinic receptors are a family of five subtypes (M1-M5) of G-protein-coupled receptors (Caulfield and Birdsall 1998). Activation of Gq/11-coupled muscarinic receptors (M1, M3, M5) has been shown to protect cells against apoptosis induced by a variety of stimuli. In rat cerebellar granule neurons (CGNs), muscarinic agonists protect against apoptosis induced by non-depolarizing conditions (low K+) (Yan et al. 1995; Copani et al. 1995; Castoldi et al. 1998). The cholinergic agonist carbachol protects PC12 cells expressing M1 receptors (PC12M1) from apoptosis induced by serum deprivation (Lindenboim et al. 1995; Leloup et al. 2000), while oxotremorine-M, another muscarinic agonist, antagonizes apoptosis induced by camptothecin and H2O2 in SH-SY5Y neuroblastoma cells (De Sarno et al. 2003; 2005). Additionally, carbachol was shown to protect cortical neurons from apoptosis induced by the β-amyloid fragment 31–35 (Yan et al. 2000). Protection by carbachol of CHO (Chinese hamster ovary) cells transfected with M1, M3 or M5 receptors toward apoptosis induced by etoposide (Budd et al. 2003; 2004), and of oligodendrocyte progenitors undergoing apoptosis due to growth factor withdrawal (Cui et al. 2006), have also been observed. Moreover, choline protects myocardial cells from apoptosis induced by ischemia in vivo, and by H2O2 in vitro, by activating M3 receptors (Liu et al. 2004; Yang et al. 2005). Finally, activation of muscarinic M1 receptors was reported to inhibit apoptosis induced by UV irradiation in COS-7 cells (Murga et al. 1998), and by glutamate in rat retinal neurons (Zhou et al. 2008). Thus, the anti-apoptotic response of Gq/11-coupled muscarinic receptors appears to be generalized to a number of cell types and apoptotic stimuli (Tobin and Budd 2003).

Despite these observations, the intracellular signaling mechanisms which may mediate the protective effects of muscarinic agonists are still unclear, as apparently contrasting findings have been reported. For example, a role for phosphatidylinositol-3 kinase (PI-3K) has been shown in some studies (Murga et al. 1998; Cui et al. 2006), but not in others (Leloup et al. 2000; Budd et al. 2003; De Sarno et al. 2005). Similar contrasting results were reported with regard to Erk 1/2 mitogen activated kinases (MAPK) (Yang et al. 2005; Leloup et al. 2000; Budd et al. 2003; De Sarno et al. 2005), and tyrosine kinases (Cui et al. 2006; De Sarno et al. 2005). Rho kinase was shown in one study to mediate the anti-apoptotic effect of muscarinic receptors (De Sarno et al. 2005). On the other hand, there is agreement that down–stream effects of muscarinic receptor activation are an increase in the anti-apoptotic protein Bcl-2 (Itano et al. 1996; Yan et al. 2000; De Sarno et al. 2003; Li et al. 2003; Budd et al. 2004; Yang et al. 2005; Zhou et al. 2008), and the inhibition of pro-apoptotic proteins such as Bax, Bim and Bad (Yan et al. 2000; Li et al. 2003; De Sarno et al. 2003; Styles et al. 2005).

Independent of the underlying intracellular mechanisms, cell survival mediated by muscarinic receptors may be of relevance during brain development and/or aging, in neurodegenerative diseases, or in the event of exposure to neurotoxic chemicals. Following a 1987 outbreak of toxicity in Eastern Canada, due to consumption of mussels contaminated with the potent neuroexcitatory toxin domoic acid (DomA) (Perl et al. 1990; Teitelbaum et al. 1990), increasing attention has been devoted to the features and mechanism of DomA neurotoxicity (Jeffery et al. 2004). DomA is a structural analog of kainic acid (KA), an excitatory amino acid that exerts it toxicity by activating the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid)/KA subtype of glutamate receptors (Hampson and Manalo 1998). Exposure of mouse CGNs to DomA induces cell death, either by apoptosis or necrosis, depending on its concentration (Giordano et al. 2006; 2007). At the concentration of 100 nM, cell death induced by DomA is primarily apoptotic (Giordano et al. 2007). DomA-induced apoptosis is due to activation of AMPA/KA receptors, involves activation of caspase-3, is mediated by oxidative stress, and is more pronounced in CGNs from Gclm (−/−) mice than in CGNs from wild-type mice (Giordano et al. 2007). Gclm (−/−) mice lack the modifier subunit of glutamate cysteine ligase (GCL), the first and rate-limiting enzyme in the synthesis of glutathione (GSH) (Giordano et al. 2007). In the absence of GCLM, the ability of the catalytic subunit (GCLC) to synthesize GSH is drastically reduced, resulting in significantly lower GSH levels (Yang et al. 2002; Giordano et al. 2006; McConnachie et al. 2007). DomA also causes an increase in the phosphorylation of p38 and JNK kinases, which are preferentially activated by cell stress-inducing signals (Raman et al., 2007), and a decrease of phosphorylated Erk 1/2 (Giordano et al. 2008), thus shifting the balance of these MAPKs toward an apoptotic milieu (Xia et al. 1995).

The aim of this study was to examine the ability of muscarinic receptors to provide protection against DomA-induced apoptosis in mouse CGNs. We show that this is indeed the case, and that muscarinic receptors provide protection in part by a novel mechanism that involves an increase in the antioxidant capacity of the cells.

Materials and Methods

Materials

Neurobasal-A medium, B27 Minus AO, Hanks’ balanced salt solution, Glutamax, Superscript reverse transcriptase III, 2,7’-dichlorofluorescin diacetate (DCF-DA), Hoechst 33342 trihydrochloride and ethidium bromide were from Invitrogen (Carlsbad, CA). Domoic acid (DomA), carbamoylcholine chloride (carbachol), mecamylamine, atropine, N-2-chloroethyl-4-piperidinyl diphenylacetate (4-DAMP), pirenzepine, methoctramine, poly-D-lysine, glucose, potassium chloride, horderadish peroxidase-conjugated anti-mouse IgG, mouse anti-β-actin, paraformaldehyde (PFA), dimethyl sulfoxide (DMSO), 4-ethylmorpholine, phenylmethylsulfonylfluoride (PMSF), and sodium orthovanadate were from Sigma-Aldrich (St. Louis, MO). Anti-JNK, anti-p-JNK (46 KDa isoform), anti-p38, anti-p-p38, anti-Akt, anti-p-Akt, anti-Erk1/2, anti-p-Erk antibodies, horseradish peroxidase -conjugated anti-rabbit IgG, and 1,4-Diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene (UO126) were from Cell Signaling (Danvers, MA). The caspase 3 inhibitor, DEVD-FMK, fluorescein-isothiocyanate (FITC)- conjugated, monobromobimane (MBB) and (5-isoquinolinesulfonyl) homopiperazine (HA-1077) were from Calbiochem (La Jolla, CA). Agarose was from Cambrex Bio Science (Rockland, ME). Protease and phosphatase inhibitors were from Roche Diagnostics Corp. (Hague Road, IN).

Animals

Experiments were carried out in CGNs from Gclm (+/+) and Gclm (−/−) mice. Generation of Gclm null mice and genotyping has been previously described in detail (Giordano et al. 2006).

Cultures of cerebellar granule neurons and cell treatments

Cultures of cerebellar granule neurons (CGN) were prepared from 7 day-old mice sacrificed by decapitation after CO2 narcosis, as described by Giordano et al. (2006). Neurons were grown for 10–12 days before treatments. All compounds were dissolved in Locke’s solution. 24 hours before the experiment the culture medium was replaced with fresh insulin-free media consisting of neurobasal medium, 1% glutamax-I and the components of the B18 supplement (Brewer and Cotman, 1989) with some modifications, omitting antioxidants (retinol, retinyl acetate, superoxide dismutase, catalase, reduced glutathione, tocopherol) and hormones ( insulin, triiodothyronine and corticosterone). Unless indicated, in all experiments, CGNs were incubated with DomA for 1 h in Locke’s buffer, followed by washout and an additional 23 h incubation. Carbachol and/or antagonists were added 30 min before DomA. Insulin was added to cell cultures at final concentrations of 100 nM.

Measurements of apoptosis

To visualize nuclear morphology following DomA treatment, cells were fixed with paraformaldehyde and stained in 10 µg/mL Hoechst DNA binding dye for 15 min. Five fields placed across the diameter of each well were assessed using a 20× objective on a fluorescence microscope. DNA fragmentation was detected with a commercial kit. Briefly, cells were washed twice with Locke’s buffer, and incubated for 10 minutes with an equal volume of lysis buffer containing 10 mM Tris-HCl (pH 7.4), 6 M guanidine-HCl, 10 mM urea plus EDTA, and 0.2% Triton X-100. The samples were passed through glass fiber fleece by centrifugation and the nucleic acid bound to the glass fibers was eluted. The DNA was applied to a 1.5% agarose gel, and the bands were visualized by ethidium bromide staining.

Assay of Reactive Oxygen Species (ROS) formation

ROS formation was determined by fluorescence using 2,7’-dichlorofluorescin diacetate (DCF-DA) (Giordano et al. 2006). Upon entering cells DCF-DA is de-esterified to DCFH, which is then oxidized by ROS to form the fluorescent 2,7’-dichlorofluorescein (DCF). In a typical experiment, cells were first washed with Locke’s solution, and then preincubated for 30 min (37°C) with DCF-DA (50 nmol/mg cell protein) in Locke’s solution. DCF-DA was added from a stock solution in DMSO; the quantity of DMSO never exceeded 0.1% and was also added to the blank. Cells were then washed with Locke’ solution to remove extracellular DCF-DA and fluorescence was immediately read using a fluorescence microplate reader (excitation 488 nm, emission 538 nm).

Fluorescence imaging of cytoplasmic free Ca2+ in single cells

CGNs were loaded with the Ca2+ -sensitive fluorescent dye fluo-3/AM (3 µM) at 37°C for 60 min in culture medium. Cells were then washed and incubated for an additional 30 min in a fluo-3/AM-free Locke’s buffer to remove extracellular traces of the dye and to complete intracellular de-esterification. The plates were placed on the stage of a fluorescence microscope. The dye in the cytoplasmic portion of the cells was excited, and fluorescence images were captured at 20-s intervals by a MicroMax cooled CCD camera (Princeton Instruments, Trenton, NJ ) using Metamorph software.

Immunoblotting analysis

Neurons were scraped in lysis buffer [Tris 50 mM pH 7.5, 2 mM EDTA, 0.5mM dithiothreitol, 0.5 mM PMSF, 10 µg/ml leupeptin, and 2 µg/ml aprotinin, 1mM sodium orthovanadate, 1 mM NaF, 1% Triton and 0.1% SDS], and whole homogenates were subjected to SDS-PAGE and immunoblotting using rabbit antibodies against p-JNK (1:1000), p-p38 (1:1000) p-Akt (1:500), p-Erk1/2 (1:1000), GCLC (1:2000), GCLM (1:1000) Bcl-2 (1:250 ), Bax (1:500), or mouse anti-β-actin (1:2000). After electrophoresis, proteins were transferred to polyvinylidene difluoride (PDVF) membranes and incubated with the above antibodies. Membranes were rinsed in Tris-buffered saline Tween (TBS-T) and incubated with horseradish peroxidase-conjugated anti-rabbit IgG or with horseradish peroxidase-conjugated anti-mouse IgG (for actin) at the appropriate dilutions (1:2000, 1:5000 respectively).

Assay of caspase-3 activation

The activation of caspase 3 was examined using a caspase 3 inhibitor (DEVD-FMK) conjugated to fluorescein-isothiocyanate (FITC) as the fluorescent in situ marker in living cells, as described in detail by Giordano et al. (2007).

Isolation of mitochondria and cytosol

Mitochondria were isolated from mouse CGNs by differential centrifugation. The cells, washed with Locke's buffer, were removed from the petri dishes in the mitochondria isotonic buffer containing 20mM HEPES-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 200 mM mannitol, and 75 mM sucrose and gently homogenized with a glass homogenizer. The homogenate was centrifuged at 750 × g for 10 min at 4°C to remove nuclei and unbroken cells, and the supernatant was centrifuged at 15,000 × g for 10 min. The supernatant was separated from the mitochondrial pellet and centrifuged again to obtain the cytosolic fraction. The mitochondrial pellet was lysed in 50 µl of 20 mM Tris (pH 7.4), 100 mM NaCl, 1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Protein was determined in both mitochondrial and cytosolic fractions by the bicinchoninic acid assay.

Measurement of GSH

CGNs were homogenized in Locke's buffer and an aliquot was taken to measure the protein concentration, whereas a second aliquot was diluted (1:1) in 10% 5-sulfosalicylic acid (SSA) for the measurement of GSH (Giordano et al. 2006). The SSA fraction was centrifuged at 12,000 rpm for 5 min at 4°C, and the supernatant was used for GSH determinations. Aliquots from the SSA fraction were added to a black flat-bottomed 96-well plate, and pH was adjusted to 7 with 0.2 M N-ethylmorpholine/0.02 M KOH. Oxidized glutathione was reduced by adding 10 µl of 10 mM TCEP for 15 min at room temperature. The pH was then adjusted to 12.5 by using 0.5 N NaOH before derivatizing the samples with 10 mM naphthalene dicarboxaldehyde for 30 min. Finally, the samples were analyzed on a spectrofluorometric plate reader (λEX = 472 and λEM = 528 nm). After incubation, the total amount of GSH in the sample was expressed as nanomoles per milligram of protein.

Measurement of GCL activity

GCL activity was assayed by a HPLC-based method, as previously described (White et al. 1999). CGNs were scraped and sonicated on ice in TES buffer (20 mM Tris, 1 mM EDTA, 250 mM sucrose, pH 7.4) containing 20 mM serine, 1 mM boric acid and peptidase inhibitors. Protein levels were measured in the supernatant by the bis-cinchoninic acid method (Pierce, Rockford, IL). Samples were dissolved in Tris/EDTA buffer containing 21.3 mM L-glutamate, 10.67 mM ATP, 10.7 mM MgCl2 and 10 mM of cysteine at 37°C. The reaction was stopped by ice-cold SSA solution and baseline GSH levels were determined. Precipitated proteins were removed and the supernatant was analyzed after monobromobimane derivatization by HPLC. Gamma-glutamylcysteine (γ-GC) and GSH peaks were identified in the chromatographs; GCL activity level was normalized to protein concentrations, and expressed as nmol γ-GC formed per mg protein per min.

Measurement of Gclc and Gclm mRNA levels

mRNA levels of Gclc and Gclm were determined by quantitative RT-PCR, using a fluorogenic 5' nuclease-based assay developed by the Functional Genomics Laboratory at the University of Washington. Briefly, reverse transcription was performed according to the manufacturer's established protocol using total RNA and the SuperScript® III First-Strand Synthesis System (Invitrogen, Carlsbad, CA.). For gene expression measurements, 4 µL of cDNA were included in a PCR reaction (25 µL final volume) that also consisted of the appropriate forward (FP) and reverse (RP) primers at 360 nM each, 80 nM TaqMan probe and TaqMan Gene Expression Master Mix (Applied Biosystems Inc., Foster City, CA). The PCR primers and the dual-labeled probes [6-carboxy-fluorescein (FAM) and 6-carboxy-tetramethyl-rhodamine (TAMRA)] for all genes were designed using ABI Primer Express v.1.5 software (Applied Biosystems Inc., Foster City, CA). Amplification and detection of PCR amplicons were performed with the ABI PRISM 7900 system (Applied Biosystems Inc., Foster City, CA) with the following PCR reaction profile: 1 cycle of 95°C for 10 min., 40 cycles of 95°C for 30sec, and 62°C for 1 min. β-actin amplification plots derived from serial dilutions of an established reference sample were used to create a linear regression formula in order to calculate expression levels, and β-actin gene expression levels were utilized as an internal control to normalize the data.

Measurements of GCLC and GCLM levels and of GCLholo

GCLC and GCLM, as well as GCL holoenzyme (GCLholo), were measured by Western-blotting as described by Lee et al. (2006). Standard curves were generated by loading incremental amounts of purified mouse GCLC and GCLM on to each gel. Aliquots of supernatant containing 40 µg of total soluble proteins were separated by one-dimensional SDS/PAGE using 10% (w/v) gels. SDS/PAGE was run under non-reducing conditions without adding DTT or heat before loading. The presence of complex was detected adding increasing amounts of GCLM to GCLC and followed the formation of holoenzyme by nondenaturing PAGE. Upon the addition of GCLM to GCLC, a more slowly migrating complex was formed (data not shown), representing GCLholo; antibodies to either GCLM or GCLC recognized this complex and confirmed its identity.

Statistical Analysis

Data are expressed as the mean ± SD of at least three independent experiments. Statistical analysis was performed by one way ANOVA followed by Bonferroni's Multiple Comparison Test.

Results

In a previous study (Giordano et al. 2007) we had shown that a low concentration of DomA (100 nM) induced apoptotic cell death of mouse CGNs. We have confirmed and extended this finding by showing that at concentrations as low as 50 nM DomA induced apoptosis in CGNs from Gclm (+/+) mice (Fig. 1). Neurons from Gclm (−/−) mice were more sensitive to DomA toxicity, and at 10 nM DomA induced a significant increase in apoptotic cells. As 100 nM DomA caused maximal apoptosis in CGNs from both genotypes, this concentration was used in all other experiments.

Fig. 1.

Fig. 1

Open in a new tab

DomA causes apoptotic cell death of mouse CGNs. CGNs from Gclm (+/+) or Gclm (−/−) mice were exposed to different concentrations of DomA for 1 h. After washout and additional 23 h incubation, neurons were stained with Hoechst 33342, and nuclei of apoptotic cells were counted and expressed as a percentage of the total number of nuclei. Results represent the mean (± SD) of at least three experiments. *Significantly different from untreated control (p < 0.05). **Significantly different from untreated control (p < 0.01). aSignificantly different from Gclm (+/+) (p < 0.001).

The cholinergic agonist carbachol caused a concentration-dependent inhibition of DomA-induced apoptosis, as measured by Hoechst staining (Figg. 2A and 2D). This was confirmed by analysis of DNA laddering (Fig. 2B). This effect of carbachol was maximal at a concentration of 1 mM, which was thus used in all further experiments. A comparison of CGNs from Gclm (+/+) and Gclm (−/−) mice suggests a differential ability of carbachol to inhibit DomA-induced apoptosis (Fig. 2A). For example, 1 mM carbachol afforded complete protection in Gclm (+/+) CGNs, but only partial protection in Gclm (−/−) neurons. As CGNs express cholinergic nicotinic receptors as well as several subtypes of muscarinic receptors (Fukamauchi et al, 1993), we sought to determine the receptor mediating the anti-apoptotic effect of carbachol. As shown in Fig. 2C, atropine and the M3 antagonist 4-DAMP were the only compounds capable of antagonizing the action of carbachol, indicating that the anti-apoptotic effect in CGNs is mediated by activation of M3 muscarinic receptors. The differential effect of carbachol in CGNs from Gclm (+/+) and (−/−) mice was not due to a differential expression of muscarinic M3 receptors, as suggested by the finding that carbachol (1 mM) caused the same increase of intracellular calcium in cells from both mouse genotypes (not shown).

Fig. 2.

Fig. 2

Fig. 2

Open in a new tab

A. Carbachol inhibits DomA-induced apoptosis. CGNs from Gclm (+/+) and Gclm (−/−) mice were pretreated with 0, 10, 100, or 1000 µM carbachol for 30min and then treated with 100 nM DomA for 1 h. After an additional 23 hr of incubation, apoptosis was scored based on nuclear morphology (Hoechst staining). Results represent the mean (± SD) of at least three separate experiments. *Significantly different from DomA treated CGNs (p < 0.05). **Significantly different from DomA treated CGNs (p < 0.01) aSignificantly different from Gclm (+/+) (p < 0.05). bSignificantly different from Gclm (+/+) (p < 0.01). cSignificantly different from Gclm (+/+) (p < 0.001). B. Carbachol (1 mM) prevents internucleosomal DNA fragmentation induced by DomA in CGNs from Gclm (+/+) mice. Ethidium bromide staining was used to visualize DNA extracted after 100 nM DomA treatment. C. The protective effect of carbachol is mediated by muscarinic M3 receptors. All antagonists were added 30 min before DomA (100 nM) at the concentration of 10 µM. Only atropine (Atr), and the M3 antagonist 4-DAMP were effective, while the M2 antagonist methroctamine (Meth), the M1 antagonist pirenzepine (Pir), and the nicotinic antagonist mecamylamine (Mec) were ineffective. Results shown are for CGNs from Gclm (+/+) mice. Apoptosis was scored using the Hoechst staining assay. Results represent the mean (± SD) from three separate experiments *Significantly different from DomA+carb treated CGNs, P<0.001. D. Carbachol (1 mM) prevents apoptosis induced by 100 nM DomA in CGNs from Gclm (+/+) and Gclm (−/−) mice. Microphotographs were taken after Hoechst staining using a 20× objective on a fluorescence microscope.

DomA-induced apoptosis is mediated by oxidative stress and involves an initial increase in reactive oxygen species (ROS) (Giordano et al. 2007). As shown in Fig. 3A, carbachol did not affect the early increase in ROS caused by DomA (nor did it have any effect on its own), suggesting that its anti-apoptotic action occurs downstream of ROS production. DomA-induced apoptosis has been shown to involve activation of caspase-3, following cytochrome c release from mitochondria (Giordano et al. 2007). Figg. 3B and 3C show that in CGNs from Gclm (+/+) mice, activation of caspase-3 by DomA is completely prevented by carbachol. We had also shown that DomA-induced apoptosis was mediated by activation of p38 and JNK kinases (Giordano et al. 2008). Figg. 3D and 3E show that carbachol prevents activation of these two kinases, an effect which is mediated by muscarinic receptors. DomA also promoted the translocation of Bax, a pro-apoptotic member of the Bcl-2 family of proteins (Leber et al. 2007), from the cytosol to the mitochondria (Fig. 3F). Bax can promote apoptosis by forming oligomers onto the mitochondrial outer membrane and creating a channel for the release of cytochrome c and other pro-apoptotic substances (Leber et al. 2007; Lalier et al. 2007). Carbachol completely inhibited Bax translocation, while having no effect on its own (Fig. 3F).

Fig. 3.

Fig. 3

Fig. 3

Open in a new tab

A. Carbachol does not prevent the early DomA-induced increase in ROS levels. CGNs from Gclm (+/+) mice were exposed to DomA (100 nM) with or without a 30 min carbachol (1 mM) pretreatment, and ROS were measured 30 min after DomA. Values are the mean (± SD) of three separate determinations.*Significantly different from control (p < 0.05) B. Carbachol prevents activation of caspase-3 by DomA. CGNs from Gclm (+/+) mice were pretreated with 1 mM carbachol for 30min, followed by 100 nM DomA for 1 h. After 12hr, caspase 3 activity was assessed as described in Materials and Methods. Values are the mean (± SD) of three separate determinations. * Significantly different from control (p < 0.05). C. CGNs were treated as in B, and microphotographs were taken using a caspase-3 inhibitor (DEVD-FMK) conjugated to fluorescein-isothiocyanate (FITC) as the fluorescent in situ marker in living cells using a 20× objective on a fluorescence microscope. D. Carbachol inhibits the activation of JNK and p38 induced by DomA. CGNs were pretreated with carbachol alone or together with 10 µM atropine (Atr) or 10 µM mecamylamine (Mec) for 30 min, and then exposed to DomA (100 nM) for 1hr. After 3 hrs, cell lysates were harvested and subjected to Western blot analysis using antibodies detecting phosphorylated forms of JNK and p38. Total JNK, total p38 and β-actin were used as loading controls. Experiments were repeated three times with similar results. Representative blot of pJNK are showing the 46 Kda isoform. E. Quantitation of results shown by representative blot in D. Bars show quantitation of pJNK (46 Kda isoform) and p-p38 normalized to total JNK and p38 by densitometric analysis. Data represent the mean (± SD) of three separate determinations. *Significantly different from control, p<0.05; **p<0.01. F. Carbachol inhibits Bax translocation induced by DomA. CGNs from Gclm (+/+) mice were pretreated with 1 mM carbachol for 30min, followed by 100 nM DomA for 1 h. After 9 hours cells were collected and subjected to fractionation by differential centrifugation as described in Methods. Bax levels in the mitochondrial and cytosolic fractions was assessed by Western blot as described in Methods. Blot is from one experiment which was repeated three times with similar results.

As Erk 1/2 MAPK and PI3K are two known effectors of muscarinic receptors (Lanzafame et al. 2003), and have been shown by some, but not all previous studies to possibly mediate the anti-apoptotic effect of muscarinic receptor agonists, we investigated the role of these two signal transduction pathways in the protective effect of carbachol on DomA-induced apoptosis. In CGNs from both Gclm (+/+) and Gclm (−/−) mice, carbachol caused phosphorylation of Erk 1/2 MAPK and of Akt (Figg. 4A, 4B and 4C). The MEK inhibitor UO126 inhibited the anti-apoptotic effect of carbachol in CGNs from both genotypes (Fig. 4D). In contrast, the PI3K inhibitor LY294002 inhibited the protective effect of carbachol in CGNs from Gclm (+/+) mice, but not in neurons from Gclm (−/−) mice (Fig. 4E). Akt inhibitor III, a phosphatidylinositol analog that acts by competing with phosphatidylinositol 3,4-bisphosphate (Cui et al. 2006), also inhibited carbachol’s effects in a genotype-dependent manner (Fig. 4E). These findings raised the possibility that carbachol may exert its anti-apoptotic effect by a dual mechanism, one present in CGNs of both Gclm (+/+) and Gclm (−/−) mice and involving Erk 1/2 MAPK, and another, present only in Gclm (+/+) CGNs, involving the PI3K/Akt pathway.

Fig. 4.

Fig. 4

Open in a new tab

Effect of carbachol on the phosphorylation of Erk 1/2 (A) and Akt (B). CGNs were treated with 1 mM carbachol and cells were harvested at indicated times and subjected to Western blot analysis using antibodies detecting phosphorylated forms of Akt (Ser473), and Erk 1/2. β–actin was used as a loading control. Results are the mean (± SD) of three separate experiments. *Significantly different from the respective controls, p<0.05. C. Representative blots from A an B showing the effect of carbachol on phosphorylation of Erk 1/2 and Akt. D. The MEK inhibitor UO126 (10 µM) inhibited the anti-apoptotic effect of carbachol in CGNs from both genotypes. E. The PI3K inhibitor LY294002 (10 µM) and Akt inhibitor III (20 µM) inhibited the protective effect of carbachol in CGNs from Gclm (+/+) mice, but not in neurons from Gclm (−/−) mice. In both D and E cells were pretreated with carbachol or with carbachol ± inhibitors, and then exposed to 100 nM DomA for 1h. After an additional 23h, Hoechst staining was used to assess apoptotic cells. Results represent the mean (± SD) of three separate experiments. *Significantly different from DomA-treated GCNs, P<0.05. **Significantly different from-DomA treated GCNs, P<0.01. a Significantly different from Gclm (+/+) (p < 0.05).

The anti-apoptotic protein Bcl-2 has been previously shown to be induced by carbachol (Yan et al. 2000; De Sarno et al. 2003; Li et al. 2003; Budd et al. 2004; Yang et al. 2005). We confirmed that carbachol increased Bcl-2 levels, and this occurs to the same degree in CGNs of both genotypes (Figg. 5A and 5B). DomA caused a decrease of Bcl-2 levels, and this decrease was antagonized by carbachol, again similarly in all CGNs (Figg. 5A and 5B). UO126 antagonized the effect of carbachol on Bcl-2, while LY294002 had no effect (Figg. 5A and 5B), suggesting that stimulation of Bcl-2 occurs through activation of Erk 1/2 MAPK, as previously reported (Li et al. 2003). It had been previously reported that inhibition of Rho kinase antagonizes the anti-apoptotic effect of carbachol (De Sarno et al. 2005). We confirmed this finding, showing that the RhoA inhibitor HA-1077 antagonizes the protective effect of carbachol toward DomA-induced apoptosis. Fig. 5C shows the results obtained in CGNs from Gclm (+/+) mice, and similar results were obtained in CGNs from Gclm (−/−) mice (not shown).

Fig. 5.

Fig. 5

Open in a new tab

A. Effect of carbachol on Bcl-2 levels. Carbachol (1 mM) caused a significant increase in Bcl-2 levels, which was similar in CGNs from both mouse genotypes, and was antagonized by the MEK inhibitor UO126 (UO, 10 µM), but not by the PI3K inhibitor LY294002 (LY, 10 µM). DomA (100 nM) caused a significant decrease of Bcl-2, which was antagonized by carbachol. Bcl-2 was measured by Western blot at the 6 hour time-point. Results are expressed as optical density (OD) after normalization with β-actin, and represent the mean (± SD) of three separate experiments. *Significantly different from control, p<0.05. B. Representative blot of A, showing the effect of carbachol on Bcl-2. C. A RhoA kinase inhibitor antagonizes the inhibitory effect of carbachol on DomA-induced apoptosis. CGNs from Gclm (+/+) mice were pretreated with carbachol (1 mM) alone or together with the RhoA kinase inhibitor HA-1077 (10, 25 or 50 µM) for 30 min, and then exposed to 100 nM DomA for 1 hr. After an additional 23 hr, Hoechst staining was used to assess apoptotic cells. Results represent the mean (± SD) of three separate experiments. *Significantly different from DomA+carb, p<0.01.

As indicated earlier, inhibitors of the PI3K/Akt pathway antagonize the protective effect of carbachol in CGNs from Gclm (+/+), but not Gclm (−/−) mice, implicating an additional anti-apoptotic pathway in neurons from wild-type animals. It has been previously shown that insulin protects brain endothelial cells from hyperglycemia-induced apoptosis through a PI3K/Akt-mediated increase in GCLC expression (Okouchi et al. 2006). PI3K/Akt was also shown to mediate insulin-mediated increases in GCL activity and GCLC expression in rat hepatocytes (Kim et al. 2004). To determine whether a similar mechanism was involved in the anti-apoptotic effect of carbachol, we measured its ability to induce GCL activity. As shown in Fig. 6A, carbachol caused a time-dependent increase of GCL activity in CGNs from Gclm (+/+) mice, which was paralleled by an increase in intracellular GSH levels (Fig. 6B), and a decrease in ROS levels (Fig. 6C). No such changes in GCL activity or GSH levels were found in CGNs from Gclm (−/−) mice (Fig. 6A, B). Carbachol-induced increases in GCL activity and GSH levels were inhibited by LY294002 but not by UO126 (Fig. 6D,E), indicating that they are mediated by activation of the PI3K/Akt pathway.

Fig. 6.

Fig. 6

Open in a new tab

Carbachol increases GCL activity (A) and GSH level (B) in a time-dependent manner in CGNs from Gclm (+/+) mice. Cells were incubated with carbachol (1 mM) for the indicated times. GCL activity and GSH intracellular levels were measured as described in Materials and Methods. *Significantly different from control, p<0.05. **Significantly different from control, p<0.01. C. Carbachol prevents the late DomA- induced increase in ROS levels. CGNs from Gclm (+/+) mice were exposed to 100 nM DomA with or without a 30 min carbachol (1 mM) pretreatment, and ROS levels were measured at the indicated times. Values are the mean (± SD) of three separate determinations.*Significantly different from untreated control (p<0.05) **Significantly different from untreated control (p < 0.01). aSignificantly different from Gclm (+/+) (p < 0.05). bSignificantly different from Gclm (+/+) (p < 0.01). D, E. Inhibition of the PI3K/Akt pathway, but not of the Erk1/2 MAPK pathway, prevents carbachol-induced increase of GCL activity and GSH levels. CGNs from Gclm (+/+) mice were treated with carbachol alone, or with carbachol together with the PI3K inhibitor LY294002 (LY, 10 µM), or the MEK inhibitor UO126 (UO, 10 µM) for 30 min, and then returned to the medium for 16 hr. Values are the mean (± SD) of three separate determinations. *Significantly different from carbachol treated CGNs, p<0.05. **Significantly different from carbachol treated CGNs, p<0.01.

Pre-treatment of CGNs with actinomycin (2 ug/ml for 30 min) followed by carbachol exposure, did not alter the increase of GCL induced by the cholinergic agonist (not shown), suggesting that the increase in GCL activity was not mediated by increased transcription. This was confirmed by the fact that carbachol did not increase Gclc or Gclm mRNA levels (Fig. 7A,B). Accordingly, there were no changes in the levels of expression of GCLC or GCLM proteins (Fig. 7C), and the ratio of the two GCL subunits (about 0.15) was constant across time after exposure to carbachol (Fig. 7D). In contrast, insulin caused an increase in GCLC mRNA and protein (not shown), as previously reported in other cell types.

Fig. 7.

Fig. 7

Open in a new tab

Effect of carbachol on GCLC and GCLM mRNA and protein levels. A. The levels of Gclc mRNA are not affected by carbachol (1 mM) in CGNs from both genotypes. Similarly (B), carbachol did not alter levels of Gclm mRNA in CGNs from Gclm (+/+) mice. Levels of mRNA were measured by quantitative RT-PCR as described in Methods. Values are the mean (± SD) of three separate determinations. C. Carbachol (1 mM) does not alter levels of GCLC and GCLM proteins in CGNs from Gclm (+/+) mice. Blot is representative of two other experiments with similar results. D. Ratio of GCLM/GCLC proteins at different time intervals following treatment of Gclm (+/+) CGNs with 1 mM carbachol. Values are the mean (± SD) of three separate determinations. E. Carbachol increases GCLholo levels. CGNs from Gclm (+/+) mice were treated with carbachol (1 mM) alone or together with the PI3K inhibitor LY294002 (LY, 10 µM) or the MEK inhibitor UO126 (UO, 10 µM), and levels of GCLholo were measured as described in Methods. The blot is representative of two other experiments providing similar results.

As the catalytic efficiency of GCL is known to be increased by holoenzyme formation, i.e. by the formation of a heterodimer between GCLC and GCLM (Lee et al. 2006), we investigated the effect of carbachol on the levels of GCLholo. As shown in Fig. 7E, carbachol increased GCLholo, and this effect was mediated by PI3K/Akt, but not by the Erk1/2 MAPK pathway.

Discussion

The main findings of this study are that activation of muscarinic M3 receptors inhibits DomA-induced apoptosis in mouse CGNs, and that this protective effect is mediated by at least two intracellular pathways. A particularly significant finding is that the cholinergic agonist carbachol can stimulate activity of GCL by a novel, non-transcriptional mechanism.

Muscarinic agonists have been shown to exert an anti-apoptotic effect in several cell types expressing endogenous or transfected muscarinic receptors, independent of the apoptotic stimulus (Yan et al. 1995; Lindenboim et al. 1995; Copani et al. 1995; Castoldi et al. 1998; Murga et al. 1998; Yan et al. 2000; Leloup et al. 2000; De Sarno et al. 2003; Budd et al. 2004; Yang et al. 2005; Cui et al. 2006; Zhou et al. 2008). In this study we present evidence that carbachol can exert an inhibitory effect on apoptosis induced by the marine neurotoxin DomA in mouse CGNs. The mechanisms of DomA neurotoxicity have been recently characterized (Giordano et al. 2006; 2007). At relatively high concentrations (> 1 uM), DomA induces neuronal cell death which is primarily necrotic in nature. By activating AMPA/KA receptors, DomA causes an increase in [Ca2+]i which then results in a release of L-glutamate. This released glutamate in turn activates NMDA receptors and promotes further glutamate release. This combined action causes a rapid accumulation of [Ca2+]i, promotes GSH efflux, and causes a concomitant decrease in intracellular GSH. High [Ca2+]i and low GSH lead to production of ROS, which, because of the insufficient GSH for scavenging, increase lipid peroxidation and lead to neuronal cell death (Giordano et al. 2006).

In contrast, at a lower concentration (100 nM), and even lower, as shown in the present study (Fig. 1), DomA causes primarily apoptotic cell death of mouse CGNs (Giordano et al. 2007). Apoptosis is only due to activation of AMPA/KA receptors, as these lower concentrations of DomA do not stimulate L-glutamate release (Giordano et al. 2007). A small and transient increase in [Ca2+]i is followed by sequestration of calcium in the mitochondria, which leads to a loss of mitochondrial membrane potential, an increase in mitochondrial oxidative stress and the opening of the permeability transition pore. This is then followed by the release of cytochrome c, activation of caspase-3 and degradation of poly (ADP-ribose) polymerase (Giordano et al. 2007). Both DomA-induced necrosis and apoptosis thus involve oxidative stress, are inhibited by antioxidants, and are more pronounced in GCNs from Gclm (−/−) mice which have very low GSH levels (Giordano et al. 2006; 2007).

Results shown in Fig. 1 confirm the ability of DomA to induce apoptotic neuronal death and the differential sensitivity of CGNs from Gclm (+/+) and Gclm (−/−) mice. By activating muscarinic M3 receptors, carbachol caused a concentration-dependent inhibition of DomA induced apoptosis. Additionally, carbachol also inhibited DomA-induced caspase-3 activation (Giordano et al. 2007), phosphorylation of p38 and JNK kinases (Giordano et al. 2008), and translocation of Bax. Interestingly, 1 mM carbachol afforded complete protection toward DomA-induced apoptosis in CGNs from Gclm (+/+) mice, while only partial protection was observed in neurons from Gclm (−/−) mice (Fig. 2A). At the concentration of 100 nM, DomA increases ROS levels by about two-fold, thirty min after exposure (Giordano et al. 2007; Fig. 2A). Carbachol did not affect this initial ROS increase, suggesting that down-stream targets are involved in its anti-apoptotic effect.

Several of the signalling pathways stimulated by Gq/11-coupled muscarinic receptors have been investigated for their involvement in their anti-apoptotic action. For example, the phospholipase C/calcium/protein kinase C pathway has been consistently shown not to be involved in this effect (Lindenboim et al. 1995; Budd et al. 2004: De Sarno et al. 2005). In contrast, studies on the involvement of Erk1/2 MAPK and of PI3K/Akt have been contradictory, with both positive (Murga et al. 1998; Cui et al. 2006; Yang et al. 2005) and negative (Leloup et al. 2000; Budd et al. 2003; De Sarno et al. 2005) findings reported. We found that inhibitors of both the Erk 1/2 MAPK and the PI3K/Akt pathways inhibited the anti-apoptotic effect of carbachol in CGNs from Gclm (+/+) mice; however, inhibitors of PI3K/Akt had no effect in CGNs from Gclm (−/−) mice. Muscarinic agonists have been shown to cause an increase in the anti-apoptotic protein Bcl-2 and to antagonize the decrease in Bcl-2 caused by apoptotic stimuli (Itano et al. 1996; Yan et al. 2000; De Sarno et al. 2003; Li et al. 2003; Budd et al. 2004; Yang et al. 2005; Zhou et al. 2008); we confirmed both findings with regard to DomA. We also showed that the increase of Bcl-2 was mediated by Erk 1/2 MAPK, confirming a previous observation (Li et al. 2003), but not by PI3K/Akt. We also confirmed the finding by De Sarno et al. (2005) of an involvement of Rho kinase in the anti-apoptotic effect of carbachol. Rho kinase is the effector of the small GTPase Rho, and RhoA has been shown to prevent apoptosis through activation of the Erk1/2 MAPK pathway (Kobayashi et al. 2004; Zhu et al. 2008). These finding support the notion that the RhoA→Rho kinase→MAPK→Bcl-2 pathway represents a process by which carbachol exerts its anti-apoptotic effect toward DomA. As shown, this pathway is present, and is equally activated by carbachol in CGNs from both Gclm (+/+) and Gclm (−/−) mice.

The inability of PI3K/Akt inhibitors to affect the anti-apoptotic action of carbachol in CGNs from Gclm (−/−) mice, suggests that these cells may lack a pathway that instead contributes to the protective effect of carbachol in CGNs from Gclm (+/+) mice. Gclm (−/−) mice lack GCLM, the modifier subunit of GCL, the first and rate-limiting enzyme in the synthesis of GSH (Yang et al. 2002). In the absence of GCLM, the ability of the catalytic subunit GCLC to synthesize GSH is drastically reduced (Dalton et al. 2004). Indeed, in CGNs from Gclm (−/−) mice, GCLM is absent, and GSH levels are only 20% of those present in CGNs from wild-type animals, despite an up-regulation of GCLC (Giordano et al. 2006). Such low GSH levels render cells more susceptible to the toxicity of agents such as DomA that elicit oxidative stress (Giordano et al. 2006; 2007). It was previously reported that insulin protects brain endothelial cells from hyperglycemia-induced apoptosis through a PI3K/Akt-mediated increase in GCL activity and GCLC expression (Okouchi et al. 2006). Insulin was also shown to increase GSH levels, GCL activity and GCLC expression in rat hepatocytes through a PI3K-dependent pathway (Kim et al. 2004). Furthermore, adrenomedullin was shown to protect lung epithelial cells from hypoxia-induced apoptosis, by a PI3K/Akt-dependent mechanism that involved increases of GSH levels, GCL activity and GCLC expression (Kim et al. 2006). We therefore investigated whether a similar mechanism was involved in carbachol’s anti-apoptotic effect toward DomA. Carbachol caused a time-dependent increase in GCL activity and GSH levels in CGNs from Gclm (+/+) mice, and such effects were blocked by LY294002, but not by UO126, implicating the PI3K/Akt pathway. However, somewhat surprisingly, these effects were not inhibited by an inhibitor of transcription, and no changes were found in the levels of GCLC or GCLM proteins or mRNAs. The ratio of GCLM/GCLC was 0.15, similar to that reported by others in brain tissue (Chen et al. 2005; Lee et al. 2006), and was not altered by carbachol. Additionally, carbachol did not alter GCL activity, GSH levels or GCLC protein and mRNA in CGNs from Gclm (−/−) mice.

The regulation of GCL has been the subject of several investigations (Soltaninassab et al. 2000; Huang et al. 2000; Krzywanski et al. 2004; Iles and Liu 2005; Toroser et al. 2006; Dasgupta et al. 2007). In all cases, increases in GCL activity were due to a transcriptional mechanism, involving an increased level of GCLC, and in some cases of GCLM. Transcription of GCL subunits occurs through the sequence-specific binding of NF-E2-related factor (Nrf2) to antioxidant response elements (AREs) present in the promoters of these two genes (Huang et al. 2000; Zipper and Mulkahy 2000). The lack of effects of carbachol on GCL subunits mRNAs and proteins suggested, however, that another mechanism should be involved in the observed increase in GCL activity. GCL activity has been shown to be regulated both by changes in the amount of GCLC and by changes in the kcat for the enzyme due to formation of the holoenzyme (GCLholo) between GCLC and GCLM (Chen et al. 2005; Lee et al. 2006). The catalytic efficiency of GCLholo is more than 4-fold higher than that of GCLC (Chen et al. 2005), providing an explanation for the low activity of GCL in Gclm (−/−) mice, despite an up-regulation of GCLC (Yang et al. 2002; Giordano et al. 2006). We found that carbachol increased the amount of GCLholo in CGNs from Gclm (+/+) mice, an effect that was mediated by the PI3K/Akt pathway. This represents a novel, non transcriptionally-mediated mechanism of up regulation of GCL activity, which is non-operational in CGNs from Gclm (−/−) mice, given the absence of GCLM. In contrast, insulin increased GCL activity in CGNs of both genotypes by increasing GCLC mRNA and protein levels. The mechanism(s) by which carbachol would increase formation of GCLholo by a PI3K/Akt-mediated pathway is still elusive. Additional kinases, downstream of PI3K, such as p70S kinase may also play a role. GCLC can be phosphorylated by protein kinases A and C and by Ca2+/calmodulin kinase (Sun et al. 1996), however, this phosphorylation results in a decrease of enzyme activity, while GCLM does not seem to undergo phosphorylation (Sun et al. 1996). Further studies will be needed to investigate molecular mechanisms responsible for increased formation of GCLholo.

In summary, the present study showed that activation of muscarinic M3 receptors provides protection against DomA-induced apoptosis in mouse CGNs. Such protection appears to involve two pathways: one mediated by Rho kinase and Erk 1/2 MAPK, leading to an increase in the anti-apoptotic protein Bcl-2; the other, mediated by PI3K/Akt, leading to an increase in GCL activity and GSH levels. The latter is due to a novel mechanism, the enhancement of GCLholo formation by carbachol. The Gclm (−/−) mouse used in this study represents a model for a relatively common polymorphism (C588T) in the 5’-flanking region of the GCLM gene, which has been associated with low plasma levels of GSH (Nakamura et al. 2002). If individuals with the T allele display lower levels of GSH also in the central nervous system, they may be more susceptible to the neurotoxicity of DomA, as well as to other excitatory amino acids, including glutamate itself, that activate AMPA/KA and NMDA receptors. The present findings would also suggest that a possible mechanism for counteracting such neurotoxicity (inhibition of apoptosis by increased signalling of endogenous acetylcholine-activated M3 muscarinic receptors) may be only partially effective in these individuals, given the reduced levels of GCLM.

Acknowledgments

This study was supported in part by grants from the National Institute of Environmental Health Sciences and the National Science Foundation (ES012762/NSF-OCE-0434087, R01ES10849 and P30ES07033).

References

  1. Budd DC, McDonald J, Emsley N, Cain K, Tobin AB. The C-terminal of the M3-muscarinic receptor possesses anti-apoptotic properties. J. Biol. Chem. 2003;278:19565–19573. doi: 10.1074/jbc.M211670200. [DOI] [PubMed] [Google Scholar]
  2. Budd DC, Spragg EJ, Ridd K, Tobin AB. Signalling of the M3-muscarinic receptor to the anti-apoptotic pathway. Biochem. J. 2004;381:43–49. doi: 10.1042/BJ20031705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brewer GJ, Cotman CW. Survival and growth of hippocampal neurons in defined medium at low density: advantages of a sandwich culture technique or low oxygen. Brain Res. 1989;494:65–74. doi: 10.1016/0006-8993(89)90144-3. [DOI] [PubMed] [Google Scholar]
  4. Castoldi AF, Barni S, Randine G, Costa LG, Manzo L. Ethanol selectively interferes with the trophic action of NMDA and carbachol on cultured cerebellar granule neurons undergoing apoptosis. Dev. Brain Res. 1998;111:279–289. doi: 10.1016/s0165-3806(98)00135-7. [DOI] [PubMed] [Google Scholar]
  5. Caulfield MP, Birdsall NJ. International Union of Paharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacol. Rev. 1998;50:279–290. [PubMed] [Google Scholar]
  6. Chen Y, Shertzer HG, Schneider SN, Nebert DW, Dalton TP. Glutamate cysteine ligase catalysis. Dependence on ATP and modifier subunit for regulation of tissue glutathione levels. J. Biol. Chem. 2005;280:33766–33774. doi: 10.1074/jbc.M504604200. [DOI] [PubMed] [Google Scholar]
  7. Copani A, Bruno VMG, Barresi V, Battaglia G, Condorelli DF, Nicoletti F. Activation of metabotropic glutamate receptors prevents neuronal apoptosis in culture. J. Neurochem. 1995;64:101–108. doi: 10.1046/j.1471-4159.1995.64010101.x. [DOI] [PubMed] [Google Scholar]
  8. Cui QL, Fogle E, Almazan G. Muscarinic acetylcholine receptors mediate oligodendrocyte progenitor survival through Src-like tyrosine kinases and PI3K/Akt pathways. Neurochem. Int. 2006;48:383–393. doi: 10.1016/j.neuint.2005.11.014. [DOI] [PubMed] [Google Scholar]
  9. Dalton TP, Chen Y, Schneider SN, Nebert DW, Schertzer HG. Genetically altered mice to evaluate glutathione homeostasis in health and disease. Free Radic. Biol. Med. 2004;37:1511–1526. doi: 10.1016/j.freeradbiomed.2004.06.040. [DOI] [PubMed] [Google Scholar]
  10. Dasgupta A, Das S, Sarkar PK. Thyroid hormone promotes glutathione synthesis in astrocytes by up regulation of glutamate cysteine ligase through differential stimulation of its catalytic and modulator subunit mRNAs. Free Radic. Biol. Med. 2007;42:617–626. doi: 10.1016/j.freeradbiomed.2006.11.030. [DOI] [PubMed] [Google Scholar]
  11. De Sarno P, Shestopal SA, King TD, Zmijewska A, Song L, Jope RS. Muscarinic receptor activation protects cells from apoptotic effects of DNA damage, oxidative stress, and mitochondrial inhibition. J. Biol. Chem. 2003;278:11086–11093. doi: 10.1074/jbc.M212157200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. De Sarno P, Shestopal SA, Zmijewska AA, Jope RS. Anti-apoptotic effects of muscarinic receptor activation are mediated by Rho kinase. Brain Res. 2005;1041:112–115. doi: 10.1016/j.brainres.2005.01.081. [DOI] [PubMed] [Google Scholar]
  13. Fukamauchi F, Saunders PA, Hough C, Chuang DM. Agonist-induced down-regulation and antagonist-induced up-regulation of m2- and m3-muscarinic acetylcholine receptor mRNA and protein in cultured cerebellar granule cells. Mol. Pharmacol. 1993;44:940–949. [PubMed] [Google Scholar]
  14. Giordano G, White CC, McConnachie LA, Fernandez C, Kavanagh TJ, Costa LG. Neurotoxicity of domoic acid in cerebellar granule neurons in a genetic model of glutathione deficiency. Mol. Pharmacol. 2006;70:2116–2126. doi: 10.1124/mol.106.027748. [DOI] [PubMed] [Google Scholar]
  15. Giordano G, White CC, Mohar I, Kavanagh TJ, Costa LG. Glutathione levels modulate domoic acid-induced apoptosis in mouse cerebellar granule neurons. Toxicol. Sci. 2007;100:433–444. doi: 10.1093/toxsci/kfm236. [DOI] [PubMed] [Google Scholar]
  16. Giordano G, Klintworth HM, Kavanagh TJ, Costa LG. Apoptosis induced by domoic acid in mouse cerebellar granule neurons involves activation of p38 and JNK MAP kinases. Neurochem. Int. 2008;52:1100–1105. doi: 10.1016/j.neuint.2007.11.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hampson DR, Manalo JL. The activation of glutamate receptors by kainic acid and domoic acid. Nat. Toxins. 1998;6:153–158. doi: 10.1002/(sici)1522-7189(199805/08)6:3/4<153::aid-nt16>3.0.co;2-1. [DOI] [PubMed] [Google Scholar]
  18. Huang ZZ, Yang H, Chen C, Zeng Z, Lu SC. Inducers of γ-glutamylcysteine synthetase and their effects on glutathione synthetase expression. Biochim. Biophys. Acta. 2000;1493:48–55. doi: 10.1016/s0167-4781(00)00156-1. [DOI] [PubMed] [Google Scholar]
  19. Iles KE, Liu RM. Mechanisms of glutamate cysteine ligase (GCL) induction by 4-hydroxynonenal. Free Radic. Biol. Med. 2005;38:547–556. doi: 10.1016/j.freeradbiomed.2004.11.012. [DOI] [PubMed] [Google Scholar]
  20. Itano Y, Ito A, Uehara T, Nomura Y. Regulation of Bcl-2 protein expression in human neuroblastoma SH-SY5Y cells: positive and negative effects of protein kinases C and A, respectively. J. Neurochem. 1996;67:131–137. doi: 10.1046/j.1471-4159.1996.67010131.x. [DOI] [PubMed] [Google Scholar]
  21. Jeffery B, Barlow T, Moizer K, Paul S, Boyle C. Amnesic shellfish poison. Food Chem Toxicol. 2004;42:545–557. doi: 10.1016/j.fct.2003.11.010. [DOI] [PubMed] [Google Scholar]
  22. Kim JY, Yim JH, Cho JH, Kim JH, Ko JH, Kim SM, Park S, Park JH. Adrenomedullin regulates cellular glutathione content via modulation of γ-glutamate-cysteine ligase catalytic subunit expression. Endocrinology. 2006;147:1357–1364. doi: 10.1210/en.2005-0895. [DOI] [PubMed] [Google Scholar]
  23. Kim SK, Woodcroft KJ, Khodadadeh SS, Novak RF. Insulin signaling regulates γ-glutamylcysteine ligase catalytic subunit expression in primary cultures rat hepatocytes. J. Exp. Pharmacol. Ther. 2004;311:99–108. doi: 10.1124/jpet.104.070375. [DOI] [PubMed] [Google Scholar]
  24. Kobayashi K, Takahashi M, Matsushita N, Miyakazi J, Koike M, Yaginuma H, Osumi N, Kaibuchi K, Kobayashi K. Survival of developing motor neurons mediated by Rho GTPase signaling pathway through Rho-kinase. J. Neurosci. 2004;24:3480–3488. doi: 10.1523/JNEUROSCI.0295-04.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Krzywanski DM, Dickinson DA, Iles KE, Wigley AF, Franklin CC, Liu RM, Kavanagh TJ, Forman HJ. Variable regulation of glutamate cysteine ligase subunit proteins affects glutathione biosynthesis in response to oxidative stress. Arch. Biochem. Biophys. 2004;423:116–125. doi: 10.1016/j.abb.2003.11.004. [DOI] [PubMed] [Google Scholar]
  26. Lalier L, Cartron PF, Juin P, Nedelkina S, Manon S, Bechinger B, Vallette FM. Bax activation and mitochondrial insertion during apoptosis. Apoptosis. 2007;12:887–896. doi: 10.1007/s10495-007-0749-1. [DOI] [PubMed] [Google Scholar]
  27. Lanzafame AA, Christopoulos A, Mitchelson F. Cellular signaling mechanisms for muscarinic acetylcholine receptors. Receptors Channels. 2003;9:241–260. [PubMed] [Google Scholar]
  28. Leber B, Lin J, Andrews DW. Embedded together: the life and death consequences of interaction of th eBcl-2 family with membranes. Apoptosis. 2007;12:897–211. doi: 10.1007/s10495-007-0746-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lee JI, Kang J, Stipanuk MH. Differential regulation of glutamate-cysteine ligase subunit expression and increased holoenzyme formation in response to cysteine deprivation. Biochem J. 2006;393:181–190. doi: 10.1042/BJ20051111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Leloup C, Michaelson DM, Fisher A, Hartman T, Beyreuther K, Stein R. M1 muscarinic receptors block caspase activation by phosphoinositide 3-kinase and MAPK/ERK-independent pathways. Cell Death Different. 2000;7:825–833. doi: 10.1038/sj.cdd.4400713. [DOI] [PubMed] [Google Scholar]
  31. Li JL, Zhu JH, Jing ZZ, Chen ZC, Xiao ZQ. G-protein-coupled muscarinic acetylcholine receptor activation up-regulates Bcl-2 and phospho-bad via Ras-ERK-1/2 signaling pathway. ShangWu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shangai) 2003;35:41–48. [PubMed] [Google Scholar]
  32. Lindenboim L, Pinkas-Kramarski R, Sokolovsky M, Stein R. Activation of muscarinic receptors inhibits apoptosis in PC12M1 cells. J. Neurochem. 1995;64:2491–2499. doi: 10.1046/j.1471-4159.1995.64062491.x. [DOI] [PubMed] [Google Scholar]
  33. Liu Y, Sun HL, Wu H, Gao YH, Li HL, Yang BF. Protective effect of M3 receptor on H2O2-induced apoptosis of rat myocardial cells in vitro. Yao Xue Xuee Bao. 2004;39:887–891. [PubMed] [Google Scholar]
  34. McConnachie LA, Mohar I, Hudson FN, Ware CB, Ladiges WC, Fernandz C, Chatterton-Kirchmeier S, White CC, Pierce RH, Kavanagh TJ. Glutamate cysteine ligase modifier subunit deficiency and gender determinants of acetaminophen-induced hepatotoxicity in mice. Toxicol. Sci. 2007;99:628–636. doi: 10.1093/toxsci/kfm165. [DOI] [PubMed] [Google Scholar]
  35. Murga C, Laguinge L, Wetzker R, Cuadrado A, Gutkind JS. Activation of Akt/protein kinase B by G protein-coupled receptors. J. Biol. Chem. 1998;273:19080–19088. doi: 10.1074/jbc.273.30.19080. [DOI] [PubMed] [Google Scholar]
  36. Nakamura S, Kugiyama S, Sugiyama S, Miyamoto S, Koides S, Fukushima H, Honda O, Yoshimura M, Ogawa H. Polymorphism in the 5’-flanking region of human glutamate-cysteine ligase modifier subunit gene is associated with myocardial infarction. Circulation. 2002;105:2968–2973. doi: 10.1161/01.cir.0000019739.66514.1e. [DOI] [PubMed] [Google Scholar]
  37. Okouchi M, Okayama N, Alexander JS, Aw TY. NRF2-dependent glutamate-L-cysteine ligase catalytic subunit expression mediates insulin protection against hyperglycemia-induced brain endothelial cell apoptosis. Curr. Neurovasc. Res. 2006;3:249–261. doi: 10.2174/156720206778792876. [DOI] [PubMed] [Google Scholar]
  38. Perl TM, Bedard L, Kosatsky T, Hockin JC, Todd EC, Remis RS. An outbreak of toxic encephalopathy caused by eating mussels contaminated with domoic acid. New Eng. J. Med. 1990;322:1775–1780. doi: 10.1056/NEJM199006213222504. [DOI] [PubMed] [Google Scholar]
  39. Raman M, Chen W, Cobb MH. Differential regulation and properties of MAPKs. Oncogene. 2007;26:3100–3112. doi: 10.1038/sj.onc.1210392. [DOI] [PubMed] [Google Scholar]
  40. Soltaninassab SR, Sekhar KR, Meredith MJ, Freeman ML. Multi-faceted regulation of γ-glutamylcysteine synthetase. J. Cell. Physiol. 2000;182:163–170. doi: 10.1002/(SICI)1097-4652(200002)182:2<163::AID-JCP4>3.0.CO;2-1. [DOI] [PubMed] [Google Scholar]
  41. Styles NA, Zhu W, Li X. Phosphorylation and down-regulation of Bim by muscarinic cholinergic receptor activation via protein kinase C. Neurochem Int. 2005;47:519–527. doi: 10.1016/j.neuint.2005.08.001. [DOI] [PubMed] [Google Scholar]
  42. Sun WM, Huang ZZ, Lu SC. Regulation of gamma-glutamylcysteine-synthetase by protein phosphorylation. Biochem. J. 1996;320:321–328. doi: 10.1042/bj3200321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Teitelbaum JS, Zatorre RJ, Carpenter S, Gendron D, Evans AC, Gjedde A, Cashman NR. Neurologic sequelae of domoic acid intoxication due to ingestion of contaminated mussels. New Engl. J. Med. 1990;322:1781–1787. doi: 10.1056/NEJM199006213222505. [DOI] [PubMed] [Google Scholar]
  44. Tobin A, Budd DC. The anti-apoptotic response of the Gq/11-coupled muscarinic receptor family. Biochem. Soc. Transact. 2003;31:1182–1185. doi: 10.1042/bst0311182. [DOI] [PubMed] [Google Scholar]
  45. Toroser D, Yarian CS, Orr WC, Sohal RS. Mechanisms of γ-glutamylcysteine ligase regulation. Biochim. Biophys. Acta. 2006;1760:233–244. doi: 10.1016/j.bbagen.2005.10.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. White CC, Krejsa CM, Eaton DL, Kavanagh TJ. HPLC-based assay for enzyme of glutathione biosynthesis. Current Protocols in Toxicology. 1999:6.5.1–6.5.14. doi: 10.1002/0471140856.tx0605s00. [DOI] [PubMed] [Google Scholar]
  47. Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science. 1995;270:1326–1331. doi: 10.1126/science.270.5240.1326. [DOI] [PubMed] [Google Scholar]
  48. Yan GM, Lin SZ, Irwin RP, Paul SM. Activation of muscarinic cholinergic receptors blocks apoptosis of cultured cerebellar granule neurons. Mol. Pharmacol. 1995;47:248–257. [PubMed] [Google Scholar]
  49. Yan XZ, Xiao R, Dou Y, Wang SD, Qiao ZD, Qiao JT. Carbachol blocks β-amyloid fragment 31–35-induced apoptosis in cultured cortical neurons. Brain Res. Bull. 2000;51:465–470. doi: 10.1016/s0361-9230(99)00255-5. [DOI] [PubMed] [Google Scholar]
  50. Yang B, Lin H, Xu C, Liu Y, Wang H, Han H, Wang Z. Choline produces cytoprotective effects against ischemic myocardial injuries: evidence for the role of cardiac M3 subtype of muscarinic acetylcholine receptors. Cell. Physiol. Biochem. 2005;16:163–174. doi: 10.1159/000089842. [DOI] [PubMed] [Google Scholar]
  51. Yang Y, Dieter MZ, Chen Y, Shertzer HG, Nebert DW, Dalton TP. Initial characterization of the glutamate-cysteine ligase modifier subunit Gclm (−/−) mouse. Novel model system for a severely compromised oxidative stress response. J. Biol. Chem. 2002;277:49446–49452. doi: 10.1074/jbc.M209372200. [DOI] [PubMed] [Google Scholar]
  52. Zhou W, Zhu X, Zhu L, Cui YY, Wang H, Qi H, Ren QS, Chen HZ. Neuroprotection of muscarinic receptor agonist pilocarpine against glutamate-induced apoptosis in retinal neurons. Cell. Mol. Neurobiol. 2008;28:263–275. doi: 10.1007/s10571-007-9251-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zhu S, Korzh V, Gong Z, Low BC. RhoA prevents apoptosis during zebrafish embryogenesis through activation of Mek/Erk pathway. Oncogene. 2008;27:1580–1589. doi: 10.1038/sj.onc.1210790. [DOI] [PubMed] [Google Scholar]
  54. Zipper L, Mulcahy RT. Inhibition of ERK and p38 MAP kinases inhibits binding of Nrf2 and induction of GCS genes. Biochem. Biophys. Res. Commun. 2000;278:884–492. doi: 10.1006/bbrc.2000.3830. [DOI] [PubMed] [Google Scholar]

RESOURCES