Protein kinase A regulates regulates inhibition of N- and P/Q-type calcium channels by ethanol in PC12 cells.
Ethanol inhibits L-type Ca++ channels, but little is known about its effect on other voltage-gated Ca++ channels. To examine non-L-type channels we used nerve growth factor-differentiated PC12 cells treated with the L channel blocker nifedipine. Using selective Ca++ channel antagonists, we found that N-type and P/Q-type channels mediate most of the remaining depolarization-evoked Ca++ rise. Ethanol (10-150 mM) inhibited depolarization-induced rises in intracellular Ca++ with maximal inhibition of 46% achieved using 50 mM ethanol. Inhibition was time dependent, requiring at least 8 min to develop fully. Ethanol did not alter Ca++ mobilization, sequestration, extrusion or capacitative entry. Sp-adenosine cyclic 3',5'-phosphorothioate, a specific activator of protein kinase A (PKA), blocked inhibition by ethanol, whereas the protein kinase C activator phorbol 12-myristate, 13-acetate did not. Okadaic acid, an inhibitor of protein phosphatases type-1 and type-2A, also blocked inhibition by ethanol with an IC50 of 3 nM. This was prevented by inhibiting PKA, indicating that the action of okadaic acid was due to increased PKA-mediated phosphorylation. These results indicate that ethanol can inhibit N-type and P/Q-type channels and this is antagonized by activating PKA. The findings suggest the sensitivity of these channels to ethanol is regulated by a phosphoprotein that is a substrate for PKA and protein phosphatase type-2A.
Solem
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Ernest Gallo Clinic & Research Center, University of California, San Francisco, USA.
McMahon
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Messing
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D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D015220:Calcium Channels / Q000187:drug effects*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D000431:Ethanol / Q000494:pharmacology*; D016716:PC12 Cells; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D012964:Sodium / Q000378:metabolism
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D000431:Ethanol; D012964:Sodium; D017868:Cyclic AMP-Dependent Protein Kinases; D011188:Potassium; D001920:Bradykinin; D002118:Calcium