Phorbol Diester Modulates α-Adrenergic Receptor-Coupled Calcium Efflux and α-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 α-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 nM) increase in calcium-45 efflux from preloaded cells. Maximum calcium-45 efflux stimulated by 12-O-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 μM) 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 kinase-C in both the acute excitation-contraction coupling of vascular smooth muscle α-adrenergic receptors, and in the long-term modulation of vascular α-adrenergic receptor responsiveness.