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-p erforated patch-clamp technique. The free cytoplasmic calcium concentr ation ([Ca2+](i)) was estimated using emission from the dye Fura-2. Ac tivation of alpha(2)-adrenoceptors by clonidine (an alpha(2)-adrenocep tor agonist) or noradrenaline (a non-selective alpha(2)-adrenoceptor a gonist), both in the presence of 0.1 mu M prazosin to block alpha(1)-a drenoceptors, caused a slow and sustained increase in [Ca2+](i) which was inhibited by 0.1 mu M rauwolscine (an alpha(2)-adrenoceptor antago nist). A similar Ca2+ response was obtained with oxymetazoline (a sele ctive alpha(2A)-adrenoceptor agonist) suggesting that the increase in [Ca2+](i) resulted from activation of the alpha(2A)-adrenoceptor subty pe. The increase in [Ca2+](i) did not occur in calcium-free solution o r in the presence of oxodipine (a voltage-dependent calcium channel bl ocker), indicating that it depended on a calcium influx. The alpha(2A) -adrenoceptor-activated calcium influx was unchanged after complete re lease of the stored calcium induced by applications of ryanodine and c affeine. Ln addition, no accumulation of inositol trisphosphate was de tected in the presence of 0.1 mu M prazosin. Taken together, these res ults indicate that alpha(2A)-adrenoceptor activation does not stimulat e phosphoinositide turnover and subsequent calcium release from intrac ellular stores. Whole-cell patch-clamp experiments showed that alpha(2 A)-adrenoceptor activation promoted calcium influx through voltage-dep endent L-type channels. Concomitant with calcium influx, alpha(2A)-adr enoceptor activation induced a 10- to 15-mV depolarization. Similar ef fects on both calcium channel current: and [Ca2+](i) were obtained wit h mastoparan, an activator of Gi-proteins. Activation of calcium influ x by both alpha(2A)-adrenoceptors and mastoparan was reduced by treatm ent with pertussis toxin and GF 109203X (a protein kinase C inhibitor) . These data suggest that activation of protein kinase C through a tra nsduction pathway involving Gi-proteins phosphorylates voltage-activat ed L-type calcium channels and thus, increases their opening probabili ty.