Inhibition of Ca2+ channels via alpha 2-adrenergic and muscarinic receptors in pheochromocytoma (PC-12) cells.

ABSTRACT

Biochemical studies have suggested a voltage-dependent dihydropyridine-sensitive catecholamine release in adrenal chromaffin cells. This release is inhibited by activation of alpha 2-adrenergic and muscarinic receptors; the underlying molecular mechanism is not known. We used undifferentiated PC-12 cells to study the effect of epinephrine and carbachol on transmembranous currents. Applying the patch-clamp technique in the whole cell configuration and using Ba2+ as charge carrier, we identified a high voltage-activated Ca2+ channel current. Both epinephrine (10 microM, in the presence of 1 microM propranolol) and carbachol (10 microM) reversibly inhibited the Ca2+ channel current by 30-40%. Yohimbine abolished and clonidine mimicked the effect of epinephrine. Phenylephrine failed to inhibit the Ca2+ channel current. The effect of carbachol was abolished by atropine. Epinephrine and carbachol did not affect the Ca2+ channel current reduced by the dihydropyridine, PN 200-110 (1 microM), suggesting a selective inhibition of dihydropyridine-sensitive Ca2+ channels. The Ca2+ channel current and its inhibition by receptor agonists were not influenced by intracellularly applied adenosine 3',5'-cyclic monophosphate (cAMP; 100 microM). Pretreatment of cells with pertussis toxin or intracellular infusion of the GDP analogue guanosine-5'-O-(2-thiodiphosphate) was without effects on the control Ca2+ channel current but abolished its hormonal inhibition. Four pertussis toxin-sensitive G proteins were identified in membranes of PC-12 cells two members of the Gi family, Gi1 and Gi2, and two members of the Go family, Go2 and another Go subtype (possibly Go1). The present data indicate that activated alpha 2-adrenergic and muscarinic receptors inhibit dihydropyridine-sensitive Ca2+ channels via pertussis toxin-sensitive G proteins without the involvement of a cAMP-dependent intermediate step.