Cultured vascular smooth muscle cells: an in vitro system for study of alpha-adrenergic receptor coupling and...

Cultured vascular smooth muscle cells derived by enzymatic dissociation of rabbit aortic media were used for study of alpha 1-adrenergic receptors (AAR) and receptor-coupled calcium flux. AAR were characterized by binding of the alpha 1-selective radioligand [3H]prazosin, and norepinephrine-stimulated 45calcium efflux was measured as an index of AAR-mediated calcium flux. The binding of [3H]prazosin to a crude cellular homogenate was to a single, saturable site of high affinity (Kd = 0.15 nM, Bmax = 75-125 fmol/mg protein), which was stereo-specific and exhibited the appropriate potency order for competition by agonists. Prazosin (Kd = 0.07 nM) was approximately 3000-fold more potent than the alpha 2-selective antagonist yohimbine (Kd = 222 nM), both of which exhibited Hill coefficients of one, indicative of binding to a single receptor type. In intact cells, norepinephrine caused a concentration-related (EC50 = 100 nM) increase in 45calcium efflux which was blocked by low concentrations of prazosin (IC50 approximately equal to 0.1 nM), but not yohimbine (IC50 greater than 100 nM). An alpha 1-selective concentration of prazosin (100 nM) fully blocked norepinephrine-stimulated 45calcium efflux, and therefore, it appears that this effect does not involve alpha 2-adrenergic receptors. Treatment of cultured cells with 1-norepinephrine for 48 h caused a concentration-related decrease in both AAR density and maximum norepinephrine-stimulated 45calcium efflux. This cultured vascular smooth muscle cell system provides several advantages for the study of alpha-adrenergic receptor coupling and regulation.