ALPHA(2)-ADRENOCEPTORS ACTIVATE DIHYDROPYRIDINE-SENSITIVE CALCIUM CHANNELS VIA GI-PROTEINS AND PROTEIN-KINASE-C IN RAT PORTAL-VEIN MYOCYTES

The presence of functional alpha(2)-adrenoceptors was investigated in isolated smooth muscle cells from rat portal vein using the nystatin-perforated patch-clamp technique. The free cytoplasmic calcium concentration ([Ca2+](i)) was estimated using emission from the dye Fura-2. Activation of alpha(2)-adrenoceptors by clonidine (an alpha(2)-adrenoceptor agonist) or noradrenaline (a non-selective alpha(2)-adrenoceptor agonist), both in the presence of 0.1 mu M prazosin to block alpha(1)-adrenoceptors, caused a slow and sustained increase in [Ca2+](i) which was inhibited by 0.1 mu M rauwolscine (an alpha(2)-adrenoceptor antagonist). A similar Ca2+ response was obtained with oxymetazoline (a selective alpha(2A)-adrenoceptor agonist) suggesting that the increase in [Ca2+](i) resulted from activation of the alpha(2A)-adrenoceptor subtype. The increase in [Ca2+](i) did not occur in calcium-free solution or in the presence of oxodipine (a voltage-dependent calcium channel blocker), indicating that it depended on a calcium influx. The alpha(2A)-adrenoceptor-activated calcium influx was unchanged after complete release of the stored calcium induced by applications of ryanodine and caffeine. Ln addition, no accumulation of inositol trisphosphate was detected in the presence of 0.1 mu M prazosin. Taken together, these results indicate that alpha(2A)-adrenoceptor activation does not stimulate phosphoinositide turnover and subsequent calcium release from intracellular stores. Whole-cell patch-clamp experiments showed that alpha(2A)-adrenoceptor activation promoted calcium influx through voltage-dependent L-type channels. Concomitant with calcium influx, alpha(2A)-adrenoceptor activation induced a 10- to 15-mV depolarization. Similar effects on both calcium channel current: and [Ca2+](i) were obtained with mastoparan, an activator of Gi-proteins. Activation of calcium influx by both alpha(2A)-adrenoceptors and mastoparan was reduced by treatment with pertussis toxin and GF 109203X (a protein kinase C inhibitor). These data suggest that activation of protein kinase C through a transduction pathway involving Gi-proteins phosphorylates voltage-activated L-type calcium channels and thus, increases their opening probability.