Different affinity states of alpha-1 adrenergic receptors defined by agonists and antagonists in bovine aorta plasma membranes

Evidence for a nonlinear relationship between alpha-1 adrenergic receptor occupancy and tissue responses, together with the finding of different affinity states for agonist binding, has raised the possibility of functional heterogeneity of alpha-1 adrenergic receptors. We have conducted studies to examine: 1) binding characteristics of [3H]prazosin, 2) competition of antagonists at these sites and 3) different affinity states of the receptor for agonists and modulation of these states by 5'-guanylylimidodiphosphate [Gpp(NH)p]. A plasma membrane-enriched vesicular fraction (F2; 15%/33% sucrose interphase) was prepared from the muscular medial layer of bovine thoracic aorta. [3H]Prazosin binding was characterized by a monophasic saturation isotherm (K(D) = 0.116 nM, B(max) = 112 fmol/mg of protein). Antagonist displacement studies yielded a relative potency order of prazosin ≥ WB4104 >> phentolamine > corynanthine > yohimbine ≥ idazoxan > rauwolscine. Competition curves for unlabeled prazosin, WB4101 (2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4 benzodioxane) and phentolamine were shallow and were best modeled to two binding sites with picomolar and nanomolar K(D) values. Gpp(NH)p was without effect on antagonist affinity. Agonist (epinephrine, norepinephrine and phenylephrine) competition with [3H]prazosin binding was biphasic with pseudo-Hill slopes less than 1.0. Binding was best described by a two-site model in which the average contribution of high affinity sites was 23% of total binding. K(D) values for the high affinity site ranged from 2.9 to 18 nM, and 3.9 to 5.0 μM for the low affinity site. In the presence of Gpp(NH)p, only a single low affinity state was found with K(D) of 2.8 to 4.3 μM equal to the lower affinity state in the absence of Gpp(NH)p. These results provide evidence for involvement of a GTP-binding protein in arterial alpha-1 adrenergic receptor coupling events. Both high and low affinity receptor states may contribute to contraction with the low affinity state providing a less efficacious response by a GTP-independent pathway. This may lead to the markedly nonlinear dose-response relationship observed by others in arterial preparations.