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. PKCϵ expression was increased in the cytosol and decreased in the membrane (P2) fraction of cerebral cortex at 10 min. At 60 min, expression of PKCϵ in the P2 fraction was increased by 42.2 ± 12%, but cytosolic levels were unchanged. In contrast, PKCγ in the P2 fraction was decreased 32.7 ± 7% at 60 min but not at 10 min post-ethanol administration. PKCγ levels in the cytosol were reduced at 10 min post-ethanol administration and unchanged at 60 min. PKCβ 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 β-chain was reduced, and phosphorylation of N-methyl-d-aspartate 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 ethanol-responsive 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 (Ca2+-dependent PKCα, β, and γ); novel nPKCs (or Ca2+-independent PKCδ and ϵ); and atypical aPKCs (Ca2+ and phorbol ester-independent ζ and λ). 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 PKCα and γ expression in rat frontal cortex (Narita et al., 2001), whereas ethanol administration for 14 days does not alter PKCγ expression in cerebral cortex (Kumar et al., 2002). Likewise, chronic ethanol exposure in PC12 cells increases PKCδ and ϵ but not α, β, or ζ 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 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 PKCγ and ϵ knock-out mice have demonstrated that ϵ and γ 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 PKCγ 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 Ca2+-dependent (β and γ) and one Ca2+-independent form (ϵ). 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 rat chow and water.
Acute Ethanol Administration. Rats (190–220 g, approximate age 10–12 weeks) were injected with ethanol (2 g/kg, 20% v/v in saline) or saline and sacrificed after 10 or 60 min. This method of ethanol administration was chosen to produce consistent blood ethanol concentrations. Trunk blood was collected, and plasma was separated by centrifugation. The brain was removed from the skull, and cerebral cortex and hippocampus were rapidly dissected over ice. Plasma and brain regions were stored at –80°C until assayed.
Chronic Liquid Diet Consumption. Rats were housed individually and administered a nutritionally complete liquid diet for the first 3 days (Dextrose diet; ICN Biomedicals, Costa Mesa, CA). Sixteen rats received ethanol (6% v/v in liquid diet) for 7 days followed by ethanol (7.5% v/v in liquid diet) for the duration of study. Control weight-matched rats (n = 8) were pair-fed the identical diet with dextrose substituted equicalorically for ethanol. Water was available ad libitum. Dietary consumption was monitored daily. The typical daily consumption was 9 to 12 g/kg. The mean body weights for the controls and pair-fed rats were similar at the termination of the experiment. This procedure reliably results in physical dependence on ethanol (Morrow et al., 1992). All animals were sacrificed between 7:00 AM and 12:00 PM. Ethanol-dependent rats had free access to ethanol diet up until the time of sacrifice. All rats were handled and habituated to saline injections and were sacrificed by decapitation.
Separate groups of rats that consumed ethanol by liquid diet (or pair-fed diet without ethanol) for 14 days were injected with a challenge dose of ethanol (2 g/kg, 20% v/v in saline) on the final day and sacrificed after 60 min. Brain regions were obtained from these rats and stored at –80°C.
Tissue and Protein Preparations. P2 membrane fractions from individual cerebral cortices or hippocampi were prepared by homogenization, low-speed centrifugation in 0.32 M sucrose, and centrifugation of the supernatant at 12,000g for 20 min. The pellet (P2 fraction) was resuspended in phosphate-buffered saline (PBS) with phosphatase inhibitor cocktail (1:100 dilution, proprietary mixture of microcystin LR, catharidin, and bromotetramisole; Sigma-Aldrich, St. Louis, MO) and stored at –80°C. The supernatant from the centrifugation described above was subjected to centrifugation at 100,000g for 45 min, and the supernatant from this centrifugation (cytosolic fraction) was collected and concentrated using Centricon Plus-20 (10,000 MW cutoff) columns (Millipore, Bedford, MA). Protein measurement was conducted using the method of Lowery et al.
Western Blot Analysis. The various subcellular fractions were analyzed by Western blot analysis under conditions of protein linearity. P2 and cytosolic fractions were subjected to SDS-PAGE using Novex Tris-glycine gels (8–16%) and transferred to polyvinylidene difluoride membranes (Invitrogen, Carlsbad, CA). The membranes with transferred proteins were probed with PKCϵ, γ, and β (BD Biosciences, San Jose, CA), NR1, and anti-phosphoserine 896 (Upstate Biotechnologies, Lake Placid, NY) antibodies. Blots were subsequently exposed to a second primary antibody directed against β-actin to verify equivalent protein loading and transfer. Bands were detected by enhanced chemiluminescence (Pierce, Rockford, IL), apposed to X-ray films under nonsaturating conditions, and analyzed by densitometric measurements using NIH Image 1.57. All comparisons were made within blots. Statistical analysis was conducted using the Student's t test.
Phosphoserine Immunoprecipitation Analysis. Receptors in the P2 fraction were immunoprecipitated with anti-phosphoserine antibody (rabbit polyclonal) as described previously (Kumar et al., 2002). In brief, P2 fraction protein (650 μg) was solubilized and denatured in radioimmunoprecipitation buffer (Sigma-Aldrich) with phenylmethylsulfonyl fluoride (1 mM), leupeptin (1 μg/μl), sodium fluoride (50 mM), sodium vanadate (200 μM), and EDTA (2 mM) to prevent protein degradation and dephosphorylation. Solubilized protein was centrifuged at 10,000g for 30 min, and supernatant (solubilized protein) was collected. Denaturation of protein in the supernatant was confirmed by SDS-PAGE analysis. Immunoprecipitation was performed using Dynal beads (Invitrogen, Carlsbad, CA). Pilot immunoprecipitation experiments were performed with various concentrations of antibody and protein to optimize the conditions for immunoprecipitation. The antiphosphoserine-specific antibody (Abcam, Cambridge, MA) was linked to magnetized Dynal beads by incubating 125 μl of Dynal beads with 100 μl of antiphosphoserine-specific antibody (0.25 μg/μl) or IgG for 1 h at room temperature. The solubilized receptors were mixed with antibody-linked beads in a final volume of 500 μl and incubated in an orbital shaker overnight at 4°C. The receptor-antibody-bead complex was washed three times with PBS. After the final wash, the receptor-antibody-bead complex was resuspended in 50 μl of SDS and boiled for 5 min. Beads were separated from the immunoprecipitate by exposure to a magnet for 2 min. Immunoprecipitates were analyzed by SDS-PAGE gel electrophoresis and Western blotting along with P2 fraction of cerebral cortex from vehicle or ethanol-treated animals to control for equivalent loading of GABA and NMDA receptor proteins. Western blots for GABAA receptors and NMDA receptors were probed using antibodies selective for GABAA receptor β chain peptides (Chemicon, Temecula, CA) and NMDA receptor NR1 subunits (Upstate Biotechnologies).
Blood Alcohol Level. Blood alcohol levels (BALs) were determined by using a commercially available kit for the Analox GL-5 (Analox Instruments, Lunenberg, MA). For each determination, 5-μl of plasma was injected, and the alcohol concentration was expressed as milligrams/deciliter.
Differential Regulation of PKC Isoform Expression following Ethanol Exposure in Naive Rats. In the present study, the expression of PKC isoforms was examined in the cytosolic and membrane fractions at two points following ethanol administration (10 and 60 min). Figure 1 shows that PKCϵ peptide expression was decreased by 18.7 ± 5% (p < 0.05, n = 8) in the P2 fraction (A) and increased by 39.04 ± 9.867 (p < 0.01) in the cytosolic fraction (B), 10 min post-ethanol administration. In contrast, PKCϵ expression was increased by 42.2 ± 12% (p < 0.01) in the P2 fraction (Fig. 1C), with no change in the cytosolic fraction 60 min following ethanol administration (Fig. 1D).
PKCγ expression was not altered in the P2 fraction but decreased by 32.73 ± 7.36% (p < 0.01) in the cytosolic fraction 10 min following ethanol administration (Fig. 2, A and B). However, at 60 min following ethanol administration, PKCγ expression was decreased by 39.29 ± 6.5% (p < 0.001) in the P2 fraction and unaltered in the cytosolic fraction (Fig. 2, C and D).
In addition, expression of PKCβ was increased by 36.06 ± 10 (p < 0.01) and 144.3 ± 52 (p < 0.05) in the P2 fraction at 10 and 60 min, respectively, following ethanol administration (Fig. 3, A and C). PKCβ levels were not altered in the cytosolic fraction at either time point (Fig. 3, B and D). Blood ethanol levels were 234.2 ± 2.168 and 199 ± 7 mg/dl at 10 and 60 min, respectively, following acute ethanol administration.
Region-Specific Regulation of PKC Expression. The distribution of PKC varies across brain regions. Because PKC-activating mechanisms following ethanol administration may vary across brain regions, we examined PKC expression in hippocampus following ethanol administration. Ethanol administration did not alter expression of PKCϵ, γ, or β in the either P2 (Fig. 4, A, C, and E) or cytosolic (Fig. 4, B, D, and F) fractions of hippocampus at 60 min post-ethanol administration.
Regulation of PKC Expression following Chronic Ethanol Administration. To determine the effect of chronic ethanol administration on the expression of PKC following acute challenge of ethanol, membrane fractions were prepared from cerebral cortices of rats that were fed a liquid diet containing ethanol for 2 weeks and challenged with an acute dose of ethanol. Because acute ethanol administration altered both expression and translocation of PKC isoforms at 60 min, we investigated the effect of ethanol challenge on PKC expression in ethanol-dependent rats at 60 min. Membrane and cytosolic fractions of cerebral cortex were analyzed by Western blot analysis and probed with PKCϵ, γ, and β antibodies. Chronic ethanol exposure did not alter the basal expression of PKCϵ (8.8 ± 11.8%), γ (–3.1 ± 6.4%), or β (4.9 ± 11.4%) in the P2 fraction of cerebral cortex compared with pair-fed controls. Furthermore, acute ethanol challenge (2 g/kg i.p.) in rats that consumed ethanol for 2 weeks did not alter expression of PKCϵ, γ, and β in the P2 fraction of cerebral cortex at 60 min (Fig. 5, A, C, and E). In addition, acute ethanol challenge following chronic ethanol administration did not alter peptide levels of PKCϵ, γ, and β in the cytosolic fraction of cerebral cortex (Fig. 5, B, D, and F). Acute systemic ethanol administration in ethanol-fed rats produced BALs of 321.15 ± 37.42 mg/dl, whereas ethanol-fed rats that received saline injections had BAL of 144.48 ± 28.24 mg/dl. The elevated BAL in ethanol-fed rats is probably due to the fact that these rats had access to ethanol in the liquid diet up to the time of the ethanol challenge.
Phosphorylation of GABAA Receptor β Chain and NMDA Receptor NR1 Subunits. The relative phosphorylation state of GABAA receptor β chain subunit and NMDA receptor NR1 subunits following acute ethanol administration was determined by immunoprecipitation of phosphorylated receptor subunits by antiphosphoserine antibody. Figure 6, A and B, demonstrates the specific labeling of phosphorylated GABAA receptor β chain and NMDA NR1 subunit peptides. Western blot analysis of the serine phosphoimmunoprecipitate showed that ethanol decreased phosphorylation of GABAA receptor β chain subunit by 34.13 ± 5.626% (p < 0.05) compared with vehicle (Fig. 6C). Acute ethanol administration also increased serine phosphorylation of NMDA receptor NR1 subunits by 60.62 ± 25.27% (p < 0.05) (Fig. 6D). To control for potential differences in total subunit expression (both phosphorylated and unphosphorylated subunits), the signal intensities of β chain or NR1 subunit labeling in vehicle and ethanol-exposed phosphoimmunoprecipitates were normalized to the respective signal intensity of total subunit labeling in the P2 fraction. There was no change in total GABAA receptor β chain subunit or NMDA receptor NR1 subunit peptide expression in P2 fraction from the same animals (Fig. 6, C and D).
The effect of ethanol on phosphorylation of the NR1 subunit was verified by Western blot analysis using an antibody against the PKC phosphorylation site serine 896. Ethanol administration increased phosphorylation of serine 896 by 40.30 ± 18% (p < 0.05) in P2 fraction compared with vehicle control (Fig. 7).
The present study demonstrates that ethanol administration selectively alters the expression and translocation of PKC isoforms in the rat brain. Acute ethanol administration altered PKC levels in cerebral cortical but not hippocampal membrane fraction in a time-dependent manner. Furthermore, acute ethanol administration decreased the serine phosphorylation of GABAA receptor β chain subunit, whereas phosphorylation of NMDA receptor NR1 subunits were increased in the P2 fraction of cerebral cortex. There was no change in expression of total β chain and NR1 subunits in P2 fraction following ethanol administration. The effect of acute ethanol challenge on PKC levels in cerebral cortex was abolished following chronic ethanol administration.
PKCϵ expression in the P2 fraction exhibited a biphasic response to ethanol, with decreased levels at 10 min and increased levels at 60 min following ethanol administration (Table 1). The reduced expression of PKCϵ in the P2 fraction at 10 min was accompanied by increased PKCϵ in the cytosolic fraction. This effect probably represents blockage of PKCϵ translocation from the cytosol to the membrane. However, at 60 min post-ethanol administration, PKCϵ peptide levels in the P2 fraction were increased without altered PKCϵ levels in the cytosol. These changes probably represent increased synthesis and translocation, because increased levels of PKCϵ in the P2 fraction would require translocation of the enzyme.
On the other hand, PKCγ expression in the P2 fraction of cortex is decreased at 60 min but not at 10 min post-ethanol administration. The early reduction (at 10 min) of PKCγ peptide levels in cytosol is probably due to reduced synthesis. However, at 60 min post-ethanol administration, PKCγ in the P2 fraction is decreased without altered cytosolic levels, suggesting a decrease in translocation of PKCγ peptide. Likewise, the expression of PKCβ is increased at both 10 and 60 min post-ethanol administration without alteration in cytosolic levels. Therefore, it is likely that increased expression of PKCβ in the P2 fraction is due to both increased synthesis and translocation or decreased proteolytic degradation of PKCβ in the membranes. Hence, ethanol differentially alters PKC expression in an isoform-specific manner.
Ethanol differentially alters the function of various neurotransmitter receptors in the brain. For example, GABAA and serotonin type 3 receptor function are increased, whereas NMDA receptor function is reduced following acute ethanol administration. Because ethanol decreased the serine phosphorylation of GABAA receptors and increased the phosphorylation of NMDA receptors, it is likely that phosphorylation contributes to the effects of ethanol on these receptors. The observation that ethanol administration decreases serine phosphorylation of GABAA receptors is consistent with other studies showing that ethanol can increase GABAA receptor responses in brain tissue (Suzdak et al., 1986; Allan and Harris, 1987; Morrow et al., 1988) and phosphorylation can decrease GABAA receptor responses in brain tissue (Brandon et al., 2000; Kumar et al., 2005).
However, not all brain areas are affected equally by ethanol in vivo. For example, ethanol depresses neural activity in medial septum without an effect on the nearby lateral septum (Givens and Breese, 1990). Likewise, acute ethanol administration potentiates GABA-mediated inhibition only in specific brain regions or cell types (Reynolds et al., 1992; Aguayo et al., 1994; Frye et al., 1994; Sapp and Yeh, 1998). The mechanism of these ethanol-mediated alterations in neurotransmitter function in specific brain areas are complex and probably involve multiple mechanisms, including heterogeneity of neurotransmitter receptor subtypes and specific PKC isoforms. Therefore, selective regulation of PKC isoforms in various brain regions following ethanol administration may contribute to the differential responses of ion channels to ethanol.
Chronic ethanol consumption results in ethanol tolerance and dependence and alters the properties of many ion channels in brain (for review see Crews et al., 1996). Because tolerance to the effects of ethanol on PKC expression and translocation are temporally related to tolerance to many behavioral effects of ethanol, ethanol effects on PKC expression and receptor phosphorylation may be related to behavioral effects of ethanol, and tolerance to this response may be related to behavioral tolerance. Previous studies in knockout mice suggest that both PKCϵ and PKCγ may contribute to the sedative effects of ethanol (Harris et al., 1995; Hodge et al., 1999; Proctor et al., 2003). Therefore, the loss of ethanol effects on both PKCϵ and PKCγ expression and membrane localization maybe related to the development of tolerance to ethanol-induced sedation. We did not detect alterations in basal PKCβ, γ, or ϵ levels following chronic ethanol administration. However, we have previously demonstrated that chronic ethanol administration alters the association of PKCγ with GABAA receptors, leading to receptor internalization (Kumar et al., 2002, 2003, 2004). Therefore, altered association of various PKC isoforms with various ion channels may contribute to changes in receptor expression and ethanol tolerance.
A growing body of evidence indicates that PKC plays an important role in development of acute functional tolerance (AFT) (Wallace et al., 2006). PKCϵ knock-out mice are more sensitive to the acute behavioral effect of ethanol than wild-type littermates. Recently, it has been shown that PKCϵ contributes to AFT to ataxic and hypnotic effects of ethanol (Wallace et al., 2006). PKCϵ has been shown to exert inhibitory control over GABAA receptor function and therefore reduce ethanol-mediated sedation (Hodge et al., 1999). Our results show that ethanol injection increases PKCϵ expression 60 min after ethanol injection. Since AFT to motor impairing effects of ethanol is reduced in PKC null mice compared with wild-type mice, it is likely that increases in PKCϵ expression following ethanol injection contributes to AFT. Increased PKCϵ expression would be expected to reduce GABAA-mediated inhibition and consequently attenuate the effects of ethanol. Similar conclusions have been made by others (Wallace et al., 2006). Likewise, PKCγ null mutant mice are innately tolerant to the effects of ethanol (Bowers et al., 2000, 2001). Therefore, the reduction of PKCγ expression 60 min following ethanol administration may also contribute to the development of acute functional tolerance to ethanol. Hence, our studies suggest that time-dependent changes in PKC isoform expression in the P2 fraction may underlie the phosphorylation of ethanol-sensitive ion channels by ethanol. PKC phosphorylation can alter receptor function directly or indirectly by altering receptor expression. Phosphorylation of β chain subunit at serine sites was decreased 60 min following ethanol administration. In contrast, NR1 subunit serine phosphorylation was increased. Since acute ethanol administration did not alter β chain and NR1 subunit expression in the cortex, it is likely that acute effect of ethanol on these channels are primarily due to direct phosphorylation. Furthermore, it is likely that phosphorylation of various ion channels by PKC are isoform-specific. Further studies investigating post-translational modification of these receptors at PKC phosphorylation sites by specific PKC isoforms will lead to a better understanding of PKC-mediated effects of ethanol on ion channels.
The molecular mechanism by which ethanol activates PKC is not known. It is likely that ethanol affects several parameters that may influence PKC activity. Both acute and prolonged ethanol exposure have been shown to affect many biochemical components required for PKC activation, translocation, and expression. For example, ethanol increases PKC activity in 30 to 60 min by increasing intracellular DAG levels in cells (Kharbanda et al., 1993). Acute ethanol alters the phosphoinositide-3-kinase signaling pathway and consequently PKC activity (Daniell and Harris, 1989; Kharbanda et al., 1993). Likewise, chronic ethanol administration alters the levels of DAG, Ca2+, and inositol triphosphate (Saito et al., 1996; Stubbs and Slater, 1999), which play a vital role in PKC activation. Therefore, altered enzymes/cofactor levels consequent to chronic ethanol administration will probably produce different effects following acute ethanol challenge in ethanol-tolerant versus naive rats.
The molecular determinants of these adaptive responses in PKC expression following ethanol challenge in ethanol tolerant rats are not known. It is likely that altered levels of inositol triphosphate receptors, DAG, and lipids in brain contribute to this response (Saito et al., 1996; Stubbs and Slater, 1999). For example, phospholipase C β1, which is important in the PKC activation pathway, is decreased by chronic but not acute ethanol administration (Pandey, 1996). Because chronic ethanol administration elicits tolerance to the ability of ethanol to alter PKC levels, these findings may have significant behavioral implications in alcohol-related diseases.
PKC isoform distribution varies across brain regions (Tanaka and Nishizuka, 1994). In the present study, different effects of ethanol on PKCϵ, γ, and β isozymes were detected in cerebral cortex but not in hippocampus following ethanol administration. This emphasizes the diversity in the regulation of PKC isozymes in different brain areas. Because ethanol is highly lipid-soluble, it is unlikely that different ethanol concentrations in various brain regions are responsible for this selective response. This raises the possibility that distinct mechanisms of PKC activation and translocation are present in different brain areas following ethanol administration. Indeed, intracellular activation and translocation of PKC depends upon fatty acid content that may differ across brain regions (Shirai et al., 1998). For example, PKCγ, but not PKCβ, is activated by micromolar concentrations of free arachidonic acid (Nishizuka, 1988). In intact platelets, sodium oleate causes a time-dependent translocation of PKCα, β-II, and δ from cytosol to membrane fractions with little effect on PKCβ I (Khan et al., 1993). Likewise, PKCγ and PKCα are much less activated than PKCβ by DAG in the presence of phosphotidylserine (Kikkawa et al., 1989). Furthermore, phorbol ester enhances the activity of the α-, β-, and γ-subspecies when added with fatty acids; however, it suppresses the activity of the ϵ-subspecies (Kasahara and Kikkawa, 1995). Hence, it is possible that ethanol differentially regulates activation of PKC isoforms by altering the fatty acid content in various brain areas. Further studies are needed to define the cell-specific activation of PKC isoforms and explain the differential response to ethanol in various brain regions.
In conclusion, the present study suggests that altered subcellular distribution of PKC isoforms may play a vital role in ethanol-mediated responses. Thus, understanding potential mechanisms for the ethanol-induced PKC levels would be extremely valuable in studying central nervous system disorders associated with alcoholism.
We thank Todd O'Buckley for technical assistance and Dr. Clyde Hodge for helpful discussions.
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.
ABBREVIATIONS: PKC, protein kinase C; NMDA, N-methyl-d-aspartate; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; AFT, acute functional tolerance; DAG, diacylglycerol.
- Received July 17, 2006.
- Accepted September 21, 2006.
- The American Society for Pharmacology and Experimental Therapeutics