title: Nonlinear relationship between alpha 1-adrenergic receptor occupancy and norepinephrine-stimulated calcium flux in cultured vascular smooth muscle cells.
abstract: 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.
authors.forename: W S
authors.initials: WS
authors.lastname: Colucci
authors.forename: T A
authors.initials: TA
authors.lastname: Brock
authors.forename: M A
authors.initials: MA
authors.lastname: Gimbrone
authors.forename: R W
authors.initials: RW
authors.lastname: Alexander
mesh_terms: D000818:Animals; D001011:Aorta / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D009638:Norepinephrine / Q000378:metabolism / Q000494:pharmacology*; D010643:Phenoxybenzamine / Q000494:pharmacology; D011224:Prazosin / Q000378:metabolism; D011817:Rabbits; D011942:Receptors, Adrenergic, alpha / Q000378:metabolism*; D014316:Tritium
chemical_list: D011942:Receptors, Adrenergic, alpha; D010643:Phenoxybenzamine; D014316:Tritium; D002118:Calcium; D009638:Norepinephrine; D011224:Prazosin