Changes in activity and regulation of the cardiac Ca2+ channel (L-type) by protein kinase C in chronic alcohol-exposed rats.
BACKGROUND
It has been reported recently that long-term alcohol exposure in rats increases the number of dihydropyridine binding sites in cardiac membrane preparations. We fed Sprague Dawley rats a liquid diet that contained ethanol as 36% of total calories for 4 to 6 months and studied how alcohol exposure affected the activity and regulation of the cardiac Ca2+ channel.


METHODS
Dihydropyridine-sensitive cardiac Ca2+ channel activity was measured as the rate of Mn2+ quench of the cytosolic fura-2 signal in electrically stimulated myocytes.


RESULTS
In control rat myocytes, pretreatment with phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), reduced the rate of Mn2+ quench to 68% of the untreated cell response. Pretreatment with GF109203X, a protein kinase C inhibitor, enhanced the rate of influx by 56%, whereas Gö6976, an inhibitor of PKC alpha, beta, and gamma, did not affect the rate of influx. By contrast, PMA did not affect the rate of Mn2+ quench in alcoholic myocytes; however, the PKC inhibitor GF109203X still enhanced the rate of Mn2+ quench by 33%. Similar to control myocytes, no effect was observed after pretreatment with Gö6976 in the alcoholic cells. In both Western blot and immunoprecipitation experiments, PKC epsilon expression in alcohol-exposed myocytes was reduced to 68% of the control. However, the ratio of membrane/ cytosolic distribution of PKC epsilon in alcoholic myocytes was increased from 1.6 to 2.6. No change was detected in the expression of PKC alpha and PKC delta. PKC activity, measured in the presence of Gö6976, which inhibits PKC alpha, beta, and gamma, was reduced in alcoholic myocytes to 57% of the control, but the proportion of PKC activity in the particulate fraction was increased from 26% in the control myocytes to 36% in the alcoholic myocytes.


CONCLUSIONS
Altered expression and activity of PKC may be associated with changes in the regulation of the cardiac Ca2+ channel found in the hearts of rats chronically exposed to alcohol. Specifically, we found that the novel class of PKC isozymes is responsible for regulating the cardiac Ca2+ channel in control cardiomyocytes, and that the loss of PMA modulation found in the alcoholic cells may be due, in part, to reduced expression and altered distribution of PKC epsilon.
Solem
M
M
Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA. Michele.Solem@mail.tju.edu
Almas
J
J
Rubin
E
E
Thomas
A
A
D000437:Alcoholism / Q000201:enzymology*; D000818:Animals; D020746:Calcium Channels, L-Type / Q000502:physiology*; D004558:Electric Stimulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000431:Ethanol / Q000008:administration & dosage*; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000032:analysis / Q000037:antagonists & inhibitors / Q000502:physiology; D008301:Maleimides / Q000494:pharmacology; D008345:Manganese / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology*; D011493:Protein Kinase C / Q000032:analysis / Q000037:antagonists & inhibitors / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D020746:Calcium Channels, L-Type; D004791:Enzyme Inhibitors; D007211:Indoles; D007527:Isoenzymes; D008301:Maleimides; D000431:Ethanol; D008345:Manganese; D011493:Protein Kinase C; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate