Differential Effects of Systemic Ethanol Administration on Protein Kinase C , , and Isoform Expression, Membrane Translocation, and Target Phosphorylation: Reversal by Chronic Ethanol Exposure

Systemic ethanol administration alters protein kinase C (PKC) activity in brain, but the effects of ethanol on the expression and translocation of specific isoforms are unknown. Rats were administered ethanol (2 g/kg i.p.) or saline and PKC levels were measured in the cytosolic and membrane fractions by Western blot analysis. PKC expression was increased in the cytosol and decreased in the membrane (P2) fraction of cerebral cortex at 10 min. At 60 min, expression of PKC in the P2 fraction was increased by 42.2 12%, but cytosolic levels were unchanged. In contrast, PKC in the P2 fraction was decreased 32.7 7% at 60 min but not at 10 min post-ethanol administration. PKC levels in the cytosol were reduced at 10 min post-ethanol administration and unchanged at 60 min. PKC expression was increased 36 10 and 144 52% in the P2 fraction both at 10 and 60 min post-ethanol administration, whereas cytosolic levels were unchanged. Serine phosphorylation of GABAA receptor -chain was reduced, and phosphorylation of N-methyl-Daspartate receptor NR1 subunit was increased 60 min following ethanol administration. There was no effect of acute ethanol administration on PKC isoform levels in the hippocampus. Ethanol challenge did not alter PKC isoform expression in the P2 fraction of cerebral cortex following chronic ethanol administration. These findings suggest that acute ethanol administration alters PKC synthesis and translocation in an isoform and brain region specific manner that leads to alterations in serine phosphorylation of receptors. Furthermore, chronic ethanol administration prevents ethanol-induced alterations in PKC expression in the P2 fraction, where PKC interacts with ethanolresponsive ion channels. Protein kinase C is a family of phospholipid-dependent kinases that are implicated in several neuronal cell functions, including modulation of various ion channels, receptor desensitization, and synaptic plasticity. There are three major classes of PKCs: conventional cPKCs (Ca -dependent PKC , , and ); novel nPKCs (or Ca -independent PKC and ); and atypical aPKCs (Ca and phorbol ester-independent and ). These isozymes differ in their central nervous system localization, cellular targets, substrate affinities, and second messenger activators (Tanaka and Nishizuka, 1994; Newton and Johnson, 1998). Ethanol is known to alter PKC activity and expression (Stubbs and Slater, 1999). Acute ethanol administration to rats induces translocation of the conventional PKCs from the cytosol to Golgi membranes (Stubbs and Slater, 1999). In contrast, ethanol inhibits PKC translocation from the cytosol to membranes in cerebellar granule and anterior pituitary cells (Steiner et al., 1997). Ethanol administration for 5 days decreases PKC and expression in rat frontal cortex (Narita et al., 2001), whereas ethanol administration for 14 days does not alter PKC expression in cerebral cortex (Kumar et al., 2002). Likewise, chronic ethanol exposure in PC12 cells increases PKC and but not , , or isoforms (Messing et al., 1991). However, the effects of acute ethanol administration on specific PKC isoform expression, translocation, or subsequent receptor phosphorylation have not been examined in mammalian brain. The pharmacological and behavioral effects of ethanol are mediated to a great extent by ion channels in neuronal membranes that are substrates for PKC phosphorylation (for review see Kumar et al., 2004). PKC-mediated phosphorylation regulates the function of several ion channels including This work was supported by National Institutes Institute of Health Grants AA015409 (to S.K.) and AA11605 (to A.L.M.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.110890. ABBREVIATIONS: PKC, protein kinase C; NMDA, N-methyl-D-aspartate; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; AFT, acute functional tolerance; DAG, diacylglycerol. 0022-3565/06/3193-1366–1375$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 319, No. 3 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 110890/3157291 JPET 319:1366–1375, 2006 Printed in U.S.A. 1366 at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from GABAA (Poisbeau et al., 1999) and NMDA receptors (Scott et al., 2001) that contribute to effects of ethanol. For example, the anxiolytic, anticonvulsant, cognitive-impairing, and hypnotic effects of ethanol are most probably due to inhibition of NMDA and potentiation of GABAA receptor ion channels. Since ethanol changes PKC expression and ion channel function, it is likely that altered PKC expression following ethanol administration may play a role in ethanol-mediated alterations in channel function. PKC activity is regulated by the combined interaction of multiple enzyme subunits and cofactors in the cell. The accepted mechanism suggests that PKC isozymes are cytosolic in their inactive state and translocate to the cellular membrane as part of their activation process (Mochly-Rosen, 1995). The specificity of PKC isozymes is mostly attributed to their subcellular localization that brings PKCs into close proximity to their substrates (Toker, 1998; Ron and Kazanietz, 1999). There is increasing evidence that various PKC isoforms are responsible for dissimilar specialized physiological functions in the cell. Studies with PKC and knock-out mice have demonstrated that and isoforms of PKC differentially modulate the behavioral effects of ethanol and ethanol sensitivity of GABAA receptors (Hodge et al., 1999; Bowers et al., 2001; Proctor et al., 2003). Likewise, the use of specific PKC inhibitors has demonstrated that PKC is essential for ethanol withdrawal hyper-responsiveness mediated by NMDA receptors (Li et al., 2005). Therefore, the identification of altered expression of specific isozymes by ethanol is essential in understanding the mode of action of ethanol. Prolonged ethanol consumption results in the development of tolerance to many of the effects of ethanol including motor incoordination, spatial learning deficits, sedation, hypnosis and anticonvulsive activity (Grobin et al., 1998; Kalant, 1998; Morrow et al., 2001). It has been proposed that alteration of neurotransmitter receptor expression and function contributes to the development of ethanol tolerance and dependence following ethanol administration (Crews et al., 1996). Various studies provide arguments to support substantial roles for PKC in the modulation of several neurotransmitter receptors following chronic ethanol administration (Kumar et al., 2004; Newton and Messing, 2005). Because chronic ethanol administration alters the cellular levels of many enzymes and cofactors essential for PKC activation (Pandey, 1996), we postulated that alterations in PKC responses to ethanol would also be observed. The present study focused on three of the most abundant PKC isozymes implicated in ethanol-induced alterations of channel function in the central nervous system, i.e., two Ca -dependent ( and ) and one Ca -independent form ( ). We explored the effects of acute ethanol administration on PKC isoform expression in the cytosolic and membrane fractions. In addition, we explored the effects of ethanol on serine phosphorylation of both GABAA and NMDA receptors. Because chronic ethanol administration alters many biochemical pathways required for activation and translocation of PKC, we also investigated the effect of acute ethanol administration in ethanol-tolerant rats. Materials and Methods Animals. Experiments were conducted in accordance with the National Institute of Health Guidelines under Institutional Animal Care and Use Committee-approved protocols. Male Sprague-Dawley rats (150–175 g) were purchased from Harlan (Indianapolis, IN) and group-housed. Rats were maintained in standard light and dark (lights on 6:00 AM to 6:00 PM) conditions with ad libitum access to