Nonlinear relationship between alpha 1-adrenergic receptor occupancy and norepinephrine-stimulated calcium flux in cultured vascular smooth muscle cells

WS Colucci, TA Brock, MA Gimbrone and RW Alexander

To determine the relationship between vascular alpha 1-adrenergic receptor occupancy and receptor-coupled calcium flux, we have studied [3H]prazosin binding and l-norepinephrine-induced 45Ca efflux in cultured vascular smooth muscle cells isolated from the rabbit aorta. In a crude cellular homogenate, [3H]prazosin bound to a single high affinity site (Kd = 0.096 nM; Bmax = 105 +/- 15 fmol/mg of protein), whereas l-norepinephrine (NE) binding was best described by a two-site model with 43 +/- 8% of sites of high affinity (KH = 92 +/- 3 nM) and 57 +/- 7% of sites of low affinity (KL = 7460 +/- 4330 nM). NE- stimulated 45Ca efflux was concentration-dependent (EC50 = 108 nM) and potently inhibited by prazosin (IC50 = 0.15 nM), but not yohimbine (no inhibition at 10 microM). For the total receptor pool identified by [3H]prazosin binding, the relationship between receptor occupancy by NE and NE-stimulated 45Ca efflux was markedly nonlinear, such that 50% of maximum NE-stimulated efflux occurred with occupancy of only approximately 7% of receptors. Likewise, following irreversible inactivation of 69 +/- 5% of receptors by phenoxybenzamine, maximal NE- stimulated 45Ca efflux was decreased by only 8 +/- 2%. These two experimental approaches provide direct evidence for the presence in cultured rabbit aortic smooth muscle cells of a sizable pool of alpha 1- adrenergic receptors in excess of those needed for maximum NE- stimulated 45Ca efflux. This evidence of "spare" receptors, together with the finding of two affinity states of agonist binding, raises the possibility of functional heterogeneity of alpha 1-adrenergic receptors in this system.

Volume 27, Issue 5, pp. 517-524, 05/01/1985
Copyright © 1985 by American Society for Pharmacology and Experimental Therapeutics

This article has been cited by other articles:

				M. Israilova, T. Tanaka, F. Suzuki, S. Morishima, and I. Muramatsu Pharmacological Characterization and Cross Talk of {alpha}1A- and {alpha}1B-Adrenoceptors Coexpressed in Human Embryonic Kidney 293 Cells J. Pharmacol. Exp. Ther., April 1, 2004; 309(1): 259 - 266. [Abstract] [Full Text]

				J. Bevan, M. Oriowo, and R. Bevan Physiological variation in alpha-adrenoceptor-mediated arterial sensitivity: relation to agonist affinity Science, October 10, 1986; 234(4773): 196 - 197. [Abstract] [PDF]