Regulation of alpha 1-adrenergic receptor-coupled calcium flux in cultured vascular smooth muscle cells.
To study the role of the alpha-adrenergic receptor (AAR) pathway in the regulation of catecholamine-induced vascular contraction, we developed methods for the direct evaluation of AAR and AAR-coupled calcium efflux in cultured vascular smooth muscle cells derived from the rabbit aorta by enzymatic dissociation. AAR were characterized by the binding of the alpha 1- and alpha 2-selective radioligands [3H]-prazosin and [3H]-yohimbine, respectively. Norepinephrine-stimulated efflux of 45calcium from preloaded cells was measured as an index of AAR-mediated calcium flux. The [3H]-prazosin binding was of high affinity (Kd = 0.15 nM), saturable (Bmax = 75-125 fmol/mg protein), and competed for by agonists and antagonists in the order expected for an alpha 1 receptor. There was no specific binding of [3H]-yohimbine. Norepinephrine-stimulated 45Ca efflux was concentration-dependent (EC50 congruent to 100 nM), and potently blocked by prazosin (IC50 congruent to 0.1 nM), but not yohimbine (IC50 greater than 100 nM), indicating, together with the binding data, that norepinephrine-stimulated 45Ca efflux in this system is alpha 1 mediated. Following treatment of cells with 1-norepinephrine (0.1 nM) for 24 hours, the density of AAR and maximal norepinephrine-stimulated 45Ca efflux were decreased by 72% +/- 14% and 91% +/- 9%, respectively. This cultured cell system offers several advantages for study of the mechanisms of coupling and regulation of alpha-adrenergic receptors in vascular smooth muscle.
- Copyright © 1984 by American Heart Association