Elsevier

Neuropharmacology

Volume 44, Issue 4, March 2003, Pages 493-502
Neuropharmacology

Protein kinase Cϵ is involved in ethanol potentiation of glycine-gated Cl current in rat neurons of ventral tegmental area

https://doi.org/10.1016/S0028-3908(02)00409-4Get rights and content

Abstract

Previously, we demonstrated that ethanol potentiates glycine current (IGly) in 35% of neurons freshly isolated from the ventral tegmental area (VTA) of rats (J. Pharmacol. Exp. Ther. 296 (2001) 77). In the present study, we examined the role of protein kinase C (PKC) in this action of ethanol on VTA neurons from young rats. Extracellular ethanol and intracellular ATP-γ-S when applied separately potentiated IGly. However, ethanol potentiation of IGly was significantly reduced in neurons dialyzed with 2 mM ATP-γ-S. Phorbol-12-myristate-13-acetate (PMA, 10 nM), a PKC activator also increased IGly and reduced ethanol potentiation of IGly. In addition, GF109203X (0.2 μM), a PKC inhibitor antagonized the potentiation effects produced either by PMA or by ethanol. Thus, ethanol potentiation of IGly may be associated with PKC activation. While intracellular application of 1,2-bis(aminophenoxy)-ethane-N,N,N,N′-tetraacetic acid, a Ca2+ chelator or Gö6976, an inhibitor of Ca2+-dependent PKC had no appreciable effect on ethanol potentiation of IGly, translocation inhibitor peptide (PKCϵ-TIP) (500 nM) significantly reduced ethanol potentiation, an action the translocation inhibitor peptide negative control (PKCϵ-TIP-NC) (500 nM) did not have. These results suggest that the activation of PKCϵ isoenzyme contributes to ethanol-induced potentiation of GlyR function.

Introduction

Glycine is one of the most important inhibitory neurotransmitters in the adult mammalian CNS (Krnjevic’, 1974, Nicoll et al., 1990). The glycine receptor (GlyR) consists of two main subunits, α (48 KDa) and β (58 KDa), forming a pentameric channel structure (Betz, 1991). Phosphorylation of GlyRs is an important mechanism of modulation of inhibitory synapses. Recent physiological data suggest that GlyRs may be phosphorylated by several protein kinases. For example, a role for PKA is noted in CNS neurons (Agopyan et al., 1993, Gu and Huang, 1998, Ren et al., 1998, Song and Huang, 1990), while α1 subunits of GlyRs can be phosphorylated by protein kinase C (PKC) in vitro and in intact cells (Gu and Huang, 1998, Vaello et al., 1994, Ruiz-Gomez et al., 1991). Furthermore, the β subunit of the GlyRs contains a consensus site for phosphorylation by PKC and protein tyrosine kinases on its major intracellular loop (Huganir and Greengard, 1990, Caraiscos et al., 2002). Functional studies of the phosphorylation of the GlyRs show that the effect of PKC on glycine responses varies for different preparations. For example, activation of PKC inhibits GlyR expressed in Xenopus oocytes (Uchiyama et al., 1994, Vaello et al., 1994, Nishizaki and Ikeuchi, 1995), but potentiates glycine current (IGly) in various neurons (Gu and Huang, 1998, Jeong et al., 2001, Albarran et al., 2001, Ruiz-Gomez et al., 1991, Schonrock and Bormann, 1995, Tapia et al., 1997).

The ventral tegmental area (VTA) contains the cells of origin of the mesolimbic system, which plays a pivotal role in the mediation of the rewarding effects of drugs of abuse, including ethanol (Gatto et al., 1994). Our recent experiments have revealed that glycine elicits responses in the majority of VTA neurons and these currents are sensitive to pharmacologically relevant concentrations of ethanol (Ye et al., 2001a, Ye et al., 2001b). Modulation of GlyRs of VTA neurons may therefore be responsible for some behavioral effects of ethanol.

Studies of ethanol effects on GlyR function are increasing. Mounting evidence indicates that GlyRs contribute to some behavioral actions of ethanol. For example, a most recent in vivo study in transgenic mice showed that mice with a mutant GlyR subunit are insensitive to the potentiating effects of ethanol (Findlay et al., 2002). More data regarding ethanol effects on GlyR function are from in vitro studies. Several electrophysiological experiments have shown that ethanol enhances the function of GlyRs (Aguayo et al., 1996, Aguayo and Pancetti, 1994, Celentano et al., 1988, Eggers et al., 2000, Engblom and Akerman, 1991, Mascia et al., 1996a, Mascia et al., 1996b, Valenzuela et al., 1998). We have also reported that ethanol (1–40 mM) enhanced glycine-elicited responses in 35% of VTA neurons (Ye et al., 2001a), but the mechanisms causing this effect are unclear. In the current report we demonstrated that the PKC pathway of signal transduction, more specifically the protein kinase Cϵ isoform, is involved in ethanol-induced potentiation of IGly in neurons freshly dissociated from the VTA of young rats.

Section snippets

Isolation of neurons

The care and use of animals and the experimental protocol of this study were approved by the Institutional Animal Care and Use Committee of the University of Medicine and Dentistry of New Jersey (Protocol no. 00074). Sprague–Dawley rats (6–17 day-old) were decapitated as described earlier (Ye et al., 2001a). The brain was quickly excised, placed into ice-cold artificial cerebral-spinal fluid (ACSF) bubbled with 95% O2–5% CO2 mixture, glued to the chilled stage of a vibratome (Campden

Effects of ATP-γ-S on IGly and on ethanol potentiation of IGly

In agreement with our previous findings (Ye et al., 2001a), ethanol enhanced IGly in about 35% of VTA neurons. As illustrated in Fig. 1(A), ethanol (10 mM) alone did not induce a current; however, it significantly potentiated the current induced by 10 μM glycine. Fig. 1(B) shows the distribution of ethanol effects on 160 VTA neurons. Ethanol effects across cells varied greatly. Ethanol potentiation of peak IGly was from 10% to >300%. However, under control condition, ethanol potentiation of IGly

Discussion

We report here that PKC inhibitors, GF109203X, CLT and PKCϵ-TIP attenuated the potentiation effect of ethanol on IGly in VTA neurons of young rats. Since PKCϵ-TIP is a specific inhibitor of PKCϵ isoform, our results suggest that PKCϵ isoform is involved in potentiation of the function of GlyRs in a part of the VTA neurons. As an extension of previous works studying ethanol–GlyR interaction, this is the first electrophysiological evidence of an association of PKCϵ with the acute effect of

Acknowledgements

This work was supported by National Institute of Alcohol Abuse and Alcoholism, National Institute of Health Grant AA-11989 to JHY. The authors appreciate Shalini Varma for her read-through of the manuscript.

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    Present address: Department of Physiology, Institute of Nautical Medicine, Nantong Medical College, Nantong, Jiangsu, People’s Republic of China.

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