The Role of Protein Kinase A in the Ethanol-Induced Increase in Spontaneous GABA Release Onto Cerebellar Purkinje Neurons

Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7178, USA.
Journal of Neurophysiology (Impact Factor: 2.89). 11/2008; 100(6):3417-28. DOI: 10.1152/jn.90970.2008
Source: PubMed


Ethanol increases miniature inhibitory postsynaptic current frequency and decreases the paired-pulse ratio, which suggests that ethanol increases both spontaneous and evoked GABA release, respectively. We have shown previously that ethanol increases GABA release at the rat interneuron-Purkinje cell synapse and that this ethanol effect involves calcium release from internal stores; however, further exploration of the mechanism responsible for ethanol-enhanced GABA release was needed. We found that a cannabinoid receptor 1 (CB1) agonist, WIN-55212, and a GABA(B) receptor agonist, baclofen, decreased baseline spontaneous GABA release and prevented ethanol from increasing spontaneous GABA release. The CB1 receptor and GABA(B) receptor are Galpha i-linked G protein-coupled receptors with common downstream messengers that include adenylate cyclase and protein kinase A (PKA). Adenylate cyclase and PKA antagonists blocked ethanol from increasing spontaneous GABA release, whereas a PKA antagonist limited to the postsynaptic neuron did not block ethanol from increasing spontaneous GABA release. These results suggest that presynaptic PKA plays an essential role in ethanol-enhanced spontaneous GABA release. Similar to ethanol, we found that the mechanism of the cannabinoid-mediated decrease in spontaneous GABA release involves internal calcium stores and PKA. A PKA antagonist decreased baseline spontaneous GABA release. This effect was reduced after incubating the slice with a calcium chelator, BAPTA-AM, but was unaffected when BAPTA was limited to the postsynaptic neuron. This suggests that the PKA antagonist is acting through a presynaptic, calcium-dependent mechanism to decrease spontaneous GABA release. Overall, these results suggest that PKA activation is necessary for ethanol to increase spontaneous GABA release.

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Available from: George R Breese, Aug 21, 2014
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    • "However, other biochemical events activate HSF1 at normal physiological temperature and there is a consensus within the field that conditions that alter normal protein conformation (temperature, calcium, urea, pH) can also induce HSF1-DNA binding (Mosser et al. 1990). As recent studies have observed that acute ethanol can trigger the release of calcium from internal stores (Kelm et al. 2007, 2008, 2010), we speculate that ethanol may increase free intracellular calcium concentrations to alter protein conformation and activate HSF1 and the heat shock cascade. "
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    ABSTRACT: Astrocytes are critical for maintaining homeostasis in the central nervous system (CNS), and also participate in the genomic response of the brain to drugs of abuse, including alcohol. In this study, we investigated ethanol regulation of gene expression in astrocytes. A microarray screen revealed that a brief exposure of cortical astrocytes to ethanol increased the expression of a large number of genes. Among the alcohol-responsive genes (ARGs) are glial-specific immune response genes, as well as genes involved in the regulation of transcription, cell proliferation, and differentiation, and genes of the cytoskeleton and extracellular matrix. Genes involved in metabolism were also upregulated by alcohol exposure, including genes associated with oxidoreductase activity, insulin-like growth factor signaling, acetyl-CoA, and lipid metabolism. Previous microarray studies performed on ethanol-treated hepatocyte cultures and mouse liver tissue revealed the induction of almost identical classes of genes to those identified in our microarray experiments, suggesting that alcohol induces similar signaling mechanisms in the brain and liver. We found that acute ethanol exposure activated heat shock factor 1 (HSF1) in astrocytes, as demonstrated by the translocation of this transcription factor to the nucleus and the induction of a family of known HSF1-dependent genes, the heat shock proteins (Hsps). Transfection of a constitutively transcriptionally active Hsf1 construct into astrocytes induced many of the ARGs identified in our microarray study supporting the hypothesis that HSF1 transcriptional activity, as part of the heat shock cascade, may mediate the ethanol induction of these genes. These data indicate that acute ethanol exposure alters gene expression in astrocytes, in part via the activation of HSF1 and the heat shock cascade.
    Brain and Behavior 03/2013; 3(2):114-33. DOI:10.1002/brb3.125 · 2.24 Impact Factor
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    • "Lastly, the difference in ethanol and muscimol-stimulated effects may be related to ethanol or PKA effects on presynaptic GABA release. Ethanol is well documented to increase GABA release (e.g., Criswell et al., 2008), but recent work has indicated that blocking adenylyl cyclase or PKA activity prevents ethanol from increasing GABA release (Kelm et al., 2008). "
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    ABSTRACT: Protein kinases are implicated in neuronal cell functions such as modulation of ion channel function, trafficking, and synaptic excitability. Both protein kinase C (PKC) and A (PKA) are involved in regulation of γ-aminobutyric acid type A (GABA(A)) receptors through phosphorylation. However, the role of PKA in regulating GABA(A) receptors (GABA(A)-R) following acute ethanol exposure is not known. The present study investigated the role of PKA in the effects of ethanol on GABA(A)-R α1 subunit expression in rat cerebral cortical P2 synaptosomal fractions. Additionally, GABA-related behaviors were examined. Rats were administered ethanol (2.0-3.5 g/kg) or saline and PKC, PKA, and GABA(A)-R α1 subunit levels were measured by western blot analysis. Ethanol (3.5 g/kg) transiently increased GABA(A)-R α1 subunit expression and PKA RIIβ subunit expression at similar time points whereas PKA RIIα was increased at later time points. In contrast, PKC isoform expression remained unchanged. Notably, lower ethanol doses (2.0 g/kg) had no effect on GABA(A)-R α1 subunit levels, although PKA type II regulatory subunits RIIα and RIIβ were increased at 10 and 60 min when PKC isozymes are also known to be elevated. To determine if PKA activation was responsible for the ethanol-induced elevation of GABA(A)-R α1 subunits, the PKA antagonist H89 was administered to rats prior to ethanol exposure. H89 administration prevented ethanol-induced increases in GABA(A)-R α1 subunit expression. Moreover, increasing PKA activity intracerebroventricularly with Sp-cAMP prior to a hypnotic dose of ethanol increased ethanol-induced loss of righting reflex (LORR) duration. This effect appears to be mediated in part by GABA(A)-R as increasing PKA activity also increased the duration of muscimol-induced LORR. Overall, these data suggest that PKA mediates ethanol-induced GABA(A)-R expression and contributes to behavioral effects of ethanol involving GABA(A)-R.
    Frontiers in Neuroscience 04/2012; 6:44. DOI:10.3389/fnins.2012.00044 · 3.66 Impact Factor
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    • "Additionally, because the EC system mediates retrograde signaling at both glutamatergic and GABAergic synapses there is more biochemical complexity than in most classical neurotransmitter systems. The contrast between the effects of ethanol observed at hippocampal glutamate synapses (Basavarajappa et al., 2008) and at GABAergic synapses (Clarke and Adermark, 2010; Kelm et al., 2008; Roberto et al., 2010) are examples of this complexity. Lastly, the state of the system under study may have a large impact on EC signaling. "
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