Acute ethanol administration rapidly increases phosphorylation of conventional protein kinase C in specific mammalian brain regions in vivo.

Protein Kinase C (PKC) is a group of Ca2+ and phospholipid-dependent serine/threonine kinases that are involved in a variety of cell signaling systems (Nishizuka, 1984). The PKC family of enzymes is divided into subgroups based on the structural and functional properties of the kinase regulatory domain: conventional (α, βI, βII, and γ), novel (δ, e, η, and θ), atypical (λ and ς), and the recently described (μ and ν) isoforms (Casabona, 1997; Dempsey et al., 2000; Newton, 2003; Nishizuka, 1984). The various isoforms of PKC also show distinct patterns of tissue expression and cellular localization (Akita, 2002; Saito et al., 1988), which suggests specificity of function. Indeed, specific members of the PKC family regulate a variety of neurobiological functions including ion channel activity, neurotransmitter release, receptor desensitization, and cell differentiation (Martelli et al., 2006; Tanaka and Nishizuka, 1994). In addition to these more general functions, specific isoforms of PKC have been shown to regulate behavioral, biochemical, and physiological responses to ethanol (Hodge et al., 1999). Growing evidence from gene knockout mice indicates that PKCγ, a member of the conventional PKC (cPKC) subgroup, mediates both acute and chronic effects of ethanol. For example, PKCγ null mice show decreased sensitivity to the acute hypnotic effects of ethanol but display normal responses to other sedative/hypnotic compounds such as pentobarbital (Harris et al., 1995). PKCγ null mice also fail to develop tolerance to ethanol hypnosis after 10 days of ethanol liquid diet exposure (Bowers et al., 1999). Ethanol-induced enhancement of γ-amino butyric acid (GABA)A receptor-mediated inhibitory postsynaptic currents (IPSCs) from hippocampal CA1 pyramidal neurons (Proctor et al., 2003) and stimulation of GABAA receptor-mediated Cl-flux from cortical membranes (Harris et al., 1995) are blunted in PKCγ null mice. In agreement with these data showing loss of ethanol sensitivity, PKCγ null mice voluntarily consume significantly more ethanol than wild-type controls (Bowers and Wehner, 2001). Ethanol also alters association of PKCγ with GABAA receptors in the cerebral cortex (Kumar et al., 2002) and mediates the effects of ethanol withdrawal on N-methyl-D-aspartate receptor currents in the spinal cord (Li et al., 2005). Moreover, ethanol prevents translocation of the βII isoform of PKC, another member cPKC subgroup (Ron et al., 2000). Together, these findings suggest that conventional cPKC isoforms may regulate response to ethanol. Members of the cPKC subfamily are activated by Ca2+ and diacylglycerol (DAG) in the presence of phosphatidylserine (Nishizuka, 1995). Ethanol has been shown to activate phospholipase C, which initiates intracellular signaling responses including formation of inositol-1,4, 5-trisphosphate, the release of intracellular Ca2+, and formation of DAG, which leads to stimulation of cPKC (Hoek and Rubin, 1990). Evidence also indicates that ethanol (100 mM) induces translocation of PKCγ from the nucleus to the cytoplasm in neonatal spinal cord motor neurons (Li et al., 2005). However, to be catalytically active, cPKC isoforms also require a series of phosphorylations (Newton, 2003). For PKCγ, the activation loop threonine (T514) of the kinase is phosphorylated first by phosphoinositide-dependent kinase-1 (PDK1), which then enables an apparent autophosphorylation on T655 in the turn loop, and subsequent phosphorylation at a T674 site in the hydrophobic loop of the c-terminus (Parekh et al., 2000). The purpose of the present study was to determine whether acute ethanol exposure alters the phosphorylation state of cPKC in the mammalian brain. To accomplish this goal, male Long–Evans rats were administered either an acute dose of ethanol or a corresponding volume of water via intragastric (IG) gavage. Animals were killed after 10 minutes and the brains were prepared for analysis of phosphorylated PKCγ (phospho T674; p-PKCγ) expression by immunohistochemistry or Western blot methods. The T674 site was chosen for analysis because phosphorylation of the hydrophobic loop is required for PKCγ to be fully active (Newton, 2003). It should be noted that peptide competition assays have shown that the p-PKCγ antibody also labels the other cPKC isoforms. For this reason, here we refer to the antibody as phosphorylation of cPKC (p-cPKC). Total PKCγ immunoreactivity was also analyzed by immunohistochemistry throughout the brain and by Western blot analysis in the nucleus accumbens to determine whether acute ethanol produced any changes in the abundance of PKCγ.