Activation of GABA_A receptors suppresses ethanol-induced upregulation of type 1 IP₃ receptors

Abstract

Although Type 1 inositol 1,4,5-trisphosphate receptors (IP₃Rs-1) are one of the major calcium channels to regulate intracellular Ca²⁺ concentration, there have been few available data how their expression is modified by long-term exposure to ethanol. The present study attempted to clarify mechanisms of modification of IP₃R-1 expression during long-term ethanol exposure by γ-aminobutyric acid (GABA)A receptors using mouse cerebral cortical neurons. Long-term exposure to ethanol induced IP₃R-1 protein upregulation following increased expression of its mRNA. Pretreatment with muscimol, a selective GABA_A receptor agonist, significantly suppressed the ethanol-induced upregulation of IP₃R-1 protein and its mRNA, which was significantly abolished by bicuculline, a selective GABA_A receptor antagonist. These results indicate that GABA_A receptors negatively regulate the ethanol-induced upregulation of IP₃R-1 protein expression via the suppression of gene transcription. Synapse, 2013. © 2012 Wiley Periodicals, Inc.

INTRODUCTION

Several actions of ethanol are mediated via modulation of the function of Type A γ-aminobutyric acid (GABA_A) receptors. Previous studies have shown that effects of short- or long-term exposure to ethanol on the mammalian central nervous system (CNS) are exhibited by enhancement or impairment of inhibitory synaptic transmission at the level of the GABA_A receptors (Grobin et al., 1998). The development of ethanol dependence is associated with alterations in functional properties of GABA_A receptors throughout the brain, which is attributed to altered expression or composition of GABA_A receptors on the neuronal surface. Chronic ethanol administration differentially alters the expression of distinct GABA_A receptor subunit mRNA and peptide levels as well as the receptor function in cerebral cortex or hippocampus (Cagetti et al., 2003; Liang et al., 2007; Marutha Ravindran et al., 2007; Werner et al., 2009). Previous in vivo studies also showed that GABA_A receptor agonists modulated ethanol self-administration (Kemppainen et al., in press; Samson and Chappell, 2001).

Type 1 inositol 1,4,5-trisphosphate receptors (IP₃Rs-1), together with ryanodine receptors, are major calcium channels to regulate intracellular Ca²⁺ concentration by releasing Ca²⁺ release into cytoplasm from its intracellular store and localized predominantly on endoplasmic reticulum. They are one of neuronal members of the IP₃R family in the CNS, and predominantly enriched in cerebellar Purkinje cells and also abundant in neurons in cerebral cortex (Faure et al., 2001; Mikoshiba, 2007). Because calcium signals regulate cellular functions such as neurotransmitter release, synaptic plasticity, neurite outgrowth, and neuronal degeneration (Berridge, 1998; Ciccolini et al., 2003), functional abnormalities in endoplasmic calcium channels could result in disturbance in cellular calcium homeostasis and, in turn, to produce pathophysiological conditions. Previous studies have shown that IP₃R-knockdown mice show antidepressant behavior, and that IP₃R-1 deficient mice exhibit ataxia and epileptic seizures (Galeotti et al., 2008; Matsumoto et al., 1996). In addition, our recent in vivo study demonstrated that IP₃Rs-1 are upregulated in the mouse brain with cocaine-induced place preference and that selective IP₃Rs antagonists significantly suppressed these behavioral changes (Kurokawa et al., 2012).

Although the most important regulators of IP₃R channel functions are considered to be changes in intracellular Ca²⁺ concentration and phosphorylation of IP₃Rs by numerous kinases and calcium modulators (Foskett et al., 2007), changes of IP₃R-1 expression and its regulatory mechanisms during chronic ethanol exposure have not yet been clarified. The present study therefore attempted to investigate the effect of chronic ethanol exposure on IP₃R-1 expression and the regulatory role of GABA_A receptors on IP₃R-1 expression during chronic ethanol exposure using primary culture of mouse cerebral cortical neurons having functional GABA_A receptors (Kuriyama et al., 1987).

Primary culture of mouse cerebral cortical neurons were carried out according to the method described previously (Ohkuma et al., 1986) with a minor modification. In brief, the cortex excluding the meninges was removed from 15-day-old fetus of ddY strain mouse (Japan SLC, Hamamatsu, Japan), deeply anesthetized with sodium pentobarbital, minced, dispersed with trypsin, and centrifuged at 1000g at 4°C. The dissociated cells were suspended with Dulbecco's modified Eagle's medium (DMEM) containing 15% fetal bovine serum, placed on a poly-L-lysine-coated culture dish, and cultured at 37°C in humidified 95% air/5% CO₂ for 3 days. Thereafter, the cells were treated with 10 μM cytosine arabinoside in DMEM containing 10% horse serum (HS) for 24 h to suppress non-neuronal cell proliferation followed by the culture in fresh DMEM with 10% HS. The culture medium was exchanged to fresh DMEM with 10% HS every 3 days and the neurons were used for the following experiments on the 13th day in culture (DIV). Immunocytochemical studies revealed that more than 95% of cells used here were neurons (Mohri et al., 2003; Ohkuma et al., 1986) and that a part of these neurons had tyrosine hydroxylase and L-amino-aromatic acid decarboxylase, neurochemical marker for dopaminergic neurons (Kurokawa et al., 2011). All animal experiments carried out in this study were approved by the Animal Research Committee of Kawasaki Medical School and conducted according to the “Guide for Care and Use of Laboratory Animals” of Kawasaki Medical School that is based on the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised 1996.

In order to investigate the effect of continuous exposure to ethanol on IP₃R-1 protein expression, the culture medium was exchanged to fresh one with ethanol (50 mM) on the 10th DIV and then the exposure continued for up to 72 h. In the case of the experiment to examine the effect of ethanol on IP₃R-1 mRNA expression, the neurons were cultured on the 13th DIV with ethanol for 1, 3, 6, and 24 h.

Considering that effects of ethanol are mediated by modulation of GABA_A receptor function, we investigated whether GABA_A receptors played a role to regulate ethanol-induced IP₃R-1 upregulation. On the 10th DIV, the culture medium was exchanged to the fresh one, and bicuculline (a selective GABA_A receptor antagonist; 10 μM) dissolved in DMSO was added into the culture medium 1 h before the exposure to both muscimol and ethanol. Muscimol (a selective GABA_A receptor agonist; 10 μM) dissolved in distilled water was added into the culture medium 1 h before the exposure to ethanol. Thereafter, the neurons were incubated with ethanol, bicuculline, and muscimol for 72 h to measure IP₃R-1 protein expression and for 6 h to examine IP₃R-1 mRNA level.

After the treatment of the neurons with agents described above, the neurons were washed twice with ice-cold phosphate buffer saline (PBS: pH 7.4), scraped off with ice-cold lysis buffer containing 10 mM Tris-HCl (pH 7.4), 0.15 M NaCl, 0.5 mM EDTA, 10 mM NaF, 0.5% Triton X-100 with a protease inhibitor cocktail, and homogenized using a Potter-Elvehjem tissue grinder with a Teflon pestle. The homogenate was centrifuged at 1000g for 10 min at 4°C. The supernatant was further centrifuged at 100,000g for 60 min at 4°C and the pellets thus obtained were retained as the membrane fractions for the subsequent analysis. The protein content in the samples prepared from the neurons was determined using a Micro BCA Protein Assay Kit (Thermo; Rockford, IL).

Proteins (10 μg protein applied onto each lane of 3–8% Tris-acetate gels (Invitrogen, Carlsbad, CA) were separated by electrophoresis (150 V, 60 min). After the separated proteins were transferred to a nitrocellulose membrane with a wet type transblotter (90 V, 60 min), the nitrocellulose membrane was then blocked with 5% nonfat milk, incubated with each of 1st antibodies for IP₃R-1 (ab5804, Abcam, Cambridge, UK) (1:1000 dilution) and β actin (sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA) (1:5000) in PBS supplemented with 0.05% Tween 20 (T-PBS) at room temperature for 2 h, rinsed twice with T-PBS followed by the incubation with anti-rabbit IgG or anti-mouse IgG conjugated horseradish peroxidase (1:5000 dilution) in T-PBS at room temperature for 1 h, and washed twice with T-PBS. The membrane was finally treated with SuperSignal West Femto Maximum Sensitivity Substrate, and separated proteins were detected with chemiluminescence. In order to determine the quantity of IP₃R-1 expression, the expression of β actin was used as internal standard.

Total RNA was prepared using TRIzol reagent (Invitrogen life technologies; CA) according to the standard protocol for RNA extraction. The amount of total RNA was quantified by measuring OD260 using a Nanodrop spectrophotometer (Wilimington, NC). Total RNA (1 μg) was reacted with PrimeScript II 1st strand cDNA Synthesis Kit (Takara, Kyoto, Japan). Quantitative PCR was performed with QuantiFast SYBR Green PCR Kit (Qiagen, Tokyo, Japan) using 7500 real-time PCR system (Applied Biosystems, Foster city, CA) according to the protocol supplied by the manufacturer, and the data were analyzed by 7500 system SDS Software 1.3.1 (Applied Biosystems) using the standard curve method. The sequences of the primers for IP₃R-1 were as follows; forward primer: 5′-TCCGCTCCCATCCTAACGGAACG-3′ and reverse primer: 5′-CACGCCGGTTTGGGGGATCA-3′ (NM_010585.4), and of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as follows; forward primer: 5′-GAACCACGAGAAATATGACAAC-3′ and reverse primer: 5′-ATGAGCCCTTCCACAATG-3′ (NM_ 008084.2).

The data were analyzed using Image J (ver. 1.43u). The data were represented as the mean ± SEM. The statistical significance was assessed by the method described in each figure legend after the application of one-way ANOVA.

For determining the optimal conditions of continuous ethanol exposure of the neurons to change IP₃R-1 expression, the neurons were continuously treated with 50 mM ethanol for various durations of the incubation (for IP₃R-1 protein: 6, 12, 24, 48, and 72 h; for its mRNA: 1, 3, 6, and 24 h) and were continuously treated with various concentrations (10, 30, and 50 mM) of ethanol for 72 h. As shown in Figure 1A, ethanol time-dependently increased IP₃R-1 protein expression and the increase of the expression attained its plateau 48 and 72 h after the initiation of ethanol exposure. Ethanol dose-dependently increased IP₃R-1 protein expression and showed its maximal effect at 30 mM (Fig. 1B). On the other hand, ethanol (50 mM) significantly increased the expression of IP₃R-1 mRNA at 6 h after the initiation of ethanol exposure (Fig. 1C). Therefore, we treated the neurons with 50 mM ethanol for 72 h as the experimental conditions to upregulate IP₃Rs-1 protein and for 6 h to upregulate IP₃R-1 mRNA in the following experiments.

Figure 1. Effect of ethanol on expression of IP₃R-1 protein and its mRNA in the cerebral cortical neurons. A: Time course of ethanol-induced IP₃R-1 protein expression. The neurons were continuously treated with ethanol (50 mM) for 6, 12, 24, 48, and 72 h and then the samples to measure IP₃R-1 expression by Western blot analysis were prepared. Each column represents the mean ± S.E.M. of four separate experiments. F(5, 23) = 17.01, *P < 0.05, **P < 0.01, ***P < 0.001 vs. control (Dunnett's multiple comparison test). B: Effects of various concentrations of ethanol on IP₃R-1 protein expression. The neurons were continuously treated with ethanol (10, 30, or 50 mM) for 72 h. F(3, 15) = 13.04, ***P < 0.001 vs. control (Dunnett's multiple comparison test). C: Time course of ethanol-induced IP₃R-1 mRNA expression. The neurons were continuously treated with ethnol (50 mM) for 1, 3, 6, and 24 h and then samples to measure IP₃R-1 mRNA expression by real time PCR analysis were prepared. Each column represents the mean ± S.E.M. of five separate experiments. F(4, 24) = 6.944, *P < 0.05 vs. control (Dunnett's multiple comparison test).

We examined the effect of muscimol, a selective GABA_A receptor agonist, on the IP₃R-1 upregulation by ethanol. Figure 2A shows that muscimol suppresses the upregulation of IP₃R-1 protein induced by ethanol, though muscimol alone has no significant potential to modify IP₃R-1 expression. In addition, bicuculline, a selective GABA_A receptor antagonist, rescued the suppression of the ethanol-induced IP₃R-1 protein upregulation by muscimol. Furthermore, muscimol completely suppressed the expression of IP₃R-1 mRNA increased by ethanol, and bicuculline abolished the change of mRNA expression by muscimol as same in the case of the protein expression (Fig. 2B).

Figure 2. Effect of GABA_A receptor ligands on ethanol-induced IP₃R-1 expression in the cerebral cortical neurons. A: Effect of muscimol and bicuculline on ethanol-induced IP₃R-1 protein expression. The neurons were exposed to muscimol (10 μM) for 1 h after the treatment with bicuculline (10 μM) for 1 h, and then exposed to bicuculline, muscimol, and ethanol (50 mM) for 72 h. Thereafter, samples to measure IP₃R-1 expression by Western blot analysis were prepared. Each column represents the mean ± S.E.M. of five separate experiments. F(7, 39) = 18.49, *P < 0.05, *P < 0.01, ***P < 0.001 (Bonferroni's multiple comparison test). B: Effect of muscimol and bicuculline on ethanol-induced IP₃R-1 mRNA expression. The neurons were exposed to muscimol (10 μM) for 1 h after the treatment with bicuculline (10 μM) for 1 h, and then exposed to bicuculline, muscimol, and ethanol (50 mM) for 6 h. Thereafter, samples to measure IP₃R-1 mRNA expression by real time PCR analysis were prepared. Each column represents the mean ± S.E.M. of five separate experiments. F(7, 39) = 14.10, *P < 0.05, ***P < 0.001 (Bonferroni's multiple comparison test).

This study was carried out for the purpose to define mechanisms of increase in IP₃R-1 protein using the primary cultures of mouse cerebral cortical neurons chronically exposed to ethanol. As demonstrated here, the ethanol-induced increase of IP₃R-1 protein was accompanied with the increase of their mRNA, indicating that the upregulation of these receptor proteins is mediated via increased transcription of their genes.

Muscimol, a selective agonist of GABA_A receptors, significantly suppressed the ethanol-induced upregulation of IP₃R-1 proteins. It is noteworthy that muscimol produces a similar complete inhibition to the increased expression of IP₃R-1 mRNA. Furthermore, the suppression of increased expression of both IP₃R-1 protein and mRNA by muscimol was reversed by bicuculline. The previous investigations have reported the suppressive action of ethanol in vivo on GABA_A receptor function (Cagetti et al., 2003; Liang et al., 2007; Werner et al., 2009) and in vitro on GABA_A receptor expression on the cell surface in cultured hippocampal neurons (Shen et al., 2011). In addition, several reports have also shown that chronic ethanol administration differently alters the expression of distinct GABA_A receptor subunits and such changes cause to decrease GABA_A function (Kralic et al., 2002a,b; Whittemore et al., 1996). Taken together with these data, it is considered that chronic ethanol exposure may suppress GABA_A receptor function and delete, in turn, negative regulatory potential of GABA_A receptors on IP₃R-1 expression. In addition, bicuculline, an antagonist selective to GABA_A receptors, did not show any effects on the ethanol-induced IP₃R-1 upregulation, suggesting that the inhibitory action of ethanol on GABA_A receptors may attain its maximal level and that GABA_A receptors may not any longer show the regulatory potential to modify IP₃R-1 expression. Considering the effect of bicuculline on IP₃R-1 expression, it is also supposed that there is no tonic GABA_A receptor function that is affecting IP₃R-1 expression under the conditions without ethanol. However, as ethanol has multiple effects on various functional proteins in various types of cells including neurons, functional modification of GABA_A receptor to regulate IP₃R-1 expression may occur under the conditions in the presence of ethanol, which is supported by the observation on the modification of the ethanol-induced IP₃R-1 expression by GABA_A receptors.

On the other hand, we reported the stimulatory effect of dopamine D1 receptor activation on IP₃R-1 upregulation (Mizuno et al., in press). Furthermore, previous investigations reported that ethanol had stimulatory potential to release dopamine (Boileau et al., 2003; Tupala and Tiihonen, 2004) and to increase dopamine D1 receptor expression in cortical neurons (Lee et al., 2008). Therefore, the inhibition of the ethanol-induced IP₃R-1 upregulation by GABA_A receptor activation with muscimol may be due to direct inhibitory interaction of GABA_A receptors with dopamine D1 receptors or suppressive action of GABA_A receptors on dopamine release by ethanol, though the exact mechanisms remain to be elucidated.

Acknowledgements

The authors would like to thank to Junko Katayama and Noriko Ohtsuki for their excellent technical assistance.