Protein Kinase C Participates in Up‐Regulation of Dihydropyridine‐Sensitive Calcium Channels by Ethanol

Abstract

Exposure to ethanol for several days increases the expression of dihydropyridine-sensitive, voltage-dependent Ca2+ channels in brain and in the neural cell line PC12. Since protein phosphorylation is a major mechanism by which ion channels are regulated, we used protein kinase inhibitors to investigate whether ethanol-induced up-regulation of Ca2+ channels involves activation of a protein kinase. Sphingosine and polymixin B, which inhibit protein kinase C and calmodulin-dependent kinases, prevented the enhancement of 45Ca2+ uptake induced by exposure of PC12 cells to ethanol for 4 days. In addition, sphingosine blocked the ability of ethanol to increase the number of [3H]dihydropyridine binding sites in PC12 cell membranes. Sphingosine's effect was prevented by simultaneous exposure to phorbol 12,13-dibutyrate, a potent activator of protein kinase C. Therefore, protein kinase C appears to be involved in the up-regulation of dihydropyridine-sensitive Ca2+ channels during prolonged exposure to ethanol.

Increase in Expression of α1 and α2/δ1 Subunits of L-Type High Voltage-Gated Calcium Channels After Sustained Ethanol Exposure in Cerebral Cortical Neurons

“…The increased bindings of these radiolabeled ligands were due to the increase of its binding sites, but not changes of their K d values. Similar changes with increased binding sites and no alterations in binding affinity of radiolabeled dihydropyridines after long-term exposure to ethanol were reported in animal brain (33,34) and cultured neural cells (3,4,35). In addition, these alterations in the binding sites of labeled L-type HVCC antagonists induced by long-term exposure to ethanol are considered to explain well the increased influx of 45 …”

“…On the other hand, several investigators reported that diltiazem showed somewhat different effects on central actions of ethanol, when compared with those of dihydropyridines (14), and that there was functional interaction between diltiazem and ethanol (35). The latter property of diltiazem was different from that of dihydropyridines (37 The immunoblot analysis demonstrates that the expression of α1A and α1B subunits consisting of P / Qand N-types of HVCCs, respectively, does not alter after sustained ethanol exposure, which is in good agreement with the data showing that 45 …”

Protein Kinase Cδ Mediates Ethanol-induced Up-regulation of L-type Calcium Channels

“…Eleven PKC isozymes have been identified (␣, ␤I, ␤II, ␥, ␦, ⑀, , , , , and ), and they differ in structure and requirements for activation by diacylglycerol and calcium (26 -28). In PC12 cells, we found that up-regulation of L-type channels by ethanol is inhibited by the kinase inhibitors sphingosine and polymyxin B (29). The effect of sphingosine is reversed by phorbol esters that activate all PKC isozymes except PKC and PKC (26), suggesting that ethanol-induced up-regulation of L-type channels requires activation of a phorbol ester-sensitive PKC.…”

“…Cells were grown at 37°C in plastic tissue culture flasks in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, 10% horse serum, 50 units/ml penicillin, 50 g/ml streptomycin, and 2 mM glutamine in a humidified atmosphere of 90% air and 10% CO 2 . Cells were cultured with ethanol in tightly capped tissue culture flasks or in six-well plates wrapped in Parafilm, and the medium was changed daily as described previously (8,29). Parallel control samples were cultured in a similar manner without ethanol.…”

Calcium‐Activated Neutral Protease Activity Is Decreased in PC12 Cells After Ethanol Exposure

“…Fluctuations in intracellular calcium levels are in turn associated with alterations in the activities of calcium-dependent enzymes involved in signal transduction. For example, ethanol exposure results in elevations of levels of protein kinase C (PKC) isoforms whose activity is modulated by calcium levels (Messing et al, 1990).…”