The aim of this proposed research project is to elucidate how protein kinase C (PKC) modulates ethanol (EtOH) inhibition of the N-methyl-D-aspartate receptor (NMDAR). The NMDAR is believed to be involved in neuronal processes that mediate learning and memory, excitotoxicity and cell death, as well as many neural and behavioral changes attributed to the abuse of EtOH. Our preliminary results suggest PKC may be involved in EtOH inhibition of NMDARs expressed in primary cultured cerebellar granule cells (CGC)s 6-14 days in vitro (DIV). Using both whole-cell and perforated patch-clamp electrophysiological techniques we have observed significant attenuation of EtOH inhibition of NMDA-induced steady-state currents by an eight minute exposure to 100 nM calphostin C, a PKC inhibitor. This reversal of inhibition was significant for three concentrations of EtOH: 10, 50 and 100 mM. Immunoblot results indicate that PKCs alpha, betaI, betaII, epsilon, gamma, delta, mu, theta, lambda/iota and zeta are contained in our cultures between 6-14 DIV. These preliminary data support the hypothesis that acute EtOH exposure activates one (or more) of these PKC isoforms. Activation results in a translocation of the enzyme to the location of its substrate where a phosphorylation-mediated event occurs. This phosphorylation-mediated event modulates EtOH inhibition of native NMDARs. This hypothesis will be tested in the following experiments. CGCs will be exposed to EtOH and activation of PKC will be confirmed by measuring increases in enzyme activity from the cytosol to the membrane by kinase activity assays. Immunoblot analysis and PKC isoform-specific antibodies will be used to identify which isoforms have translocated to the membrane fraction. To determine if EtOH-mediated activation of PKC results in the phosphorylation of the NMDAR NRI subunit, immunoprecipitation studies will be performed with an NR1 antibody followed by immunoblotting with a phosphorylation site-specific antibody that recognizes PKC-mediated phosphorylation of the NRI. The translocation, kinase activity and phosphorylation experiments will be conducted under various physiological conditions to determine if PKC is directly or indirectly activated by EtOH. Electrophysiological experiments will establish that our observed calphostin C attenuation of EtOH inhibition of NMDARs is mediated by PKC, by assessing the EtOH sensitivity of NMDARs after exposure to PKC activators and catalytically active PKC in the presence and absence of PKC inhibitors. PKC isoform-specific inhibitors will identify which PKC isoform is modulating EtOH inhibition of NMDAR function. Additional electrophysiological experiments will examine the role of PKC with other intracellular signaling cascades and how the convergence of these cascades alters the EtOH sensitivity of the NMDAR. Overall, these studies will provide insights into the molecular mechanisms by which acute EtOH exposure alters NMDAR function. Thus, facilitating the future design of novel therapeutic strategies to be used in the treatment of alcoholism and alcohol abuse.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
5R01AA013436-03
Application #
6711823
Study Section
Alcohol and Toxicology Subcommittee 4 (ALTX)
Program Officer
Sorensen, Roger
Project Start
2002-03-01
Project End
2007-02-28
Budget Start
2004-03-01
Budget End
2005-02-28
Support Year
3
Fiscal Year
2004
Total Cost
$221,086
Indirect Cost
Name
Texas Tech University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
609980727
City
Lubbock
State
TX
Country
United States
Zip Code
79430
Nath, Vikas; Reneau, Jason C; Dertien, Janet S et al. (2012) An in vitro model for studying the effects of continuous ethanol exposure on N-methyl-D-aspartate receptor function. Alcohol 46:3-16
Reneau, Jason; Reyland, Mary E; Popp, R Lisa (2011) Acute ethanol exposure prevents PMA-mediated augmentation of N-methyl-D-aspartate receptor function in primary cultured cerebellar granule cells. Alcohol 45:595-605
Reneau, Jason C; Reyland, Mary E; Phillips, Jonathan et al. (2009) Phorbol 12-myristate 13-acetate potentiation of N-methyl-D-aspartate-induced currents in primary cultured cerebellar granule cells is mediated by protein kinase C alpha. J Pharmacol Exp Ther 330:641-9
Popp, R Lisa; Reneau, Jason C; Brotherton, B Jason (2008) Activation of protein kinase C enhances NMDA-induced currents in primary cultured cerebellar granule cells: Effect of temperature and NMDA NR2 subunit composition. Eur J Pharmacol 599:1-10
Popp, R Lisa; Reneau, Jason C; Dertien, Janet S (2008) Cerebellar granule cells cultured from adolescent rats express functional NMDA receptors: an in vitro model for studying the developing cerebellum. J Neurochem 106:900-11
Popp, R Lisa; Dertien, Janet S (2008) Actin depolymerization contributes to ethanol inhibition of NMDA receptors in primary cultured cerebellar granule cells. Alcohol 42:525-39
Popp, R Lisa; Velasquez, Oscar; Bland, Jason et al. (2006) Characterization of protein kinase C isoforms in primary cultured cerebellar granule cells. Brain Res 1083:70-84