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 α, β, and γ, 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 ? expression in alcohol‐exposed myocytes was reduced to 68% of the control. However, the ratio of membrane/cytosolic distribution of PKC ? in alcoholic myocytes was increased from 1.6 to 2.6. No change was detected in the expression of PKC α and PKC δ. PKC activity, measured in the presence of Gö6976, which inhibits PKC α, β, and γ, 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 ?.