Phorbol Diester Modulates cr Adrenergic Receptor-Coupled Calcium Efflux and a-Adrenergic Receptor Number in Cultured Vascular Smooth Muscle Cells

The phorbol ester 12-O-tetradecanoyl phorbol-13-acetate was used to probe the role of protein kinase-C in the modulation of a-adrenergic receptor-coupled calcium efflux in cultured vascular smooth muscle cells derived from rabbit aorta. Exposure of cells to 12-O-tetradecanoyl phorbol-13-acetate for 6 minutes caused a concentration-related (maximum effect at >10 tat) increase in calcium-45 efflux from preloaded cells. Maximum calcium-45 efflux stimulated by 12O-tetradecanoyl phorbol-13-acetate was equivalent to maximum norepinephrine-stimulated calcium-45 efflux, and maximally effective concentrations of 12-O-tetradecanoyl phorbol-13-acetate and norepinephrine together were no more potent than either drug alone. Exposure of cells to 12-O-tetradecanoyl phorbol-13-acetate for periods of 1-24 hours resulted in complete loss of norepinephrine-stimulated calcium-45 efflux and a much slower, concentration-related reduction in a-adrenergic receptor number, with a maximum reduction of 50-60% at 12-O-tetradecanoyl phorbol-13-acetate concentrations >10 nM. Twenty-four hours of exposure to 12-O-tetradecanoyl phorbol-13-acetate (10 nM) and norepinephrine (10 fiM) together caused no greater reduction in [H]prazosin binding than did norepinephrine alone. 12-O-tetradecanoyl phorbol-13-acetate had no effect on [H]prazosin-binding affinity. These data suggest an important role for protein kihaseC in both the acute excitation-contraction coupling of vascular smooth muscle a-adrenergic receptors, and in the long-term modulation of vascular a-adrenergic receptor responsiveness. (Circ Res 58: 393-398, 1986)