Binding and functional characterization of alpha-2 adrenergic receptor subtypes on pig vascular endothelium

Abstract

Alpha-2 adrenergic receptor subtypes were characterized in membranes of pig vascular endothelium using [3H]rauwolscine. Alpha-2 adrenergic receptor subtypes that mediate endothelium-dependent vascular relaxation were studied in vitro by using ring segments of pig epicardial coronary arteries. Specific [3H]rauwolscine binding in endothelial membranes was saturable and to a single class of high-affinity sites with a mean KD of 0.217 +/- 0.05 nM and Bmax of 156 +/- 28 fmol/mg of protein. Nonlinear regression analysis indicated that competition binding curves for drugs that distinguish the alpha-2A adrenergic receptor subtype from the alpha-2C adrenergic receptor subtype fit best to two-site binding models. Kl values for drugs in binding to endothelial alpha-2 adrenergic receptors correlated well with their Kl values for alpha-2A (r = .98) and alpha-2C (r = .97) adrenergic receptor subtypes identified in other tissues. Vascular endothelium contained 23% alpha-2A and 77% alpha-2C adrenergic receptors. In the presence of indomethacin, the rank order of potency for agonists that cause endothelium-dependent vascular relaxation was p-iodoclonidine > clonidine > UK-14,304 > guanabenz > epinephrine > norepinephrine. KB values for antagonist inhibition of epinephrine-induced, endothelium-dependent vascular relaxation correlated best with Kl values for antagonist binding at the alpha-2A adrenergic receptor subtype. These results suggest that the alpha-2A and alpha-2C adrenergic receptor subtypes are present on pig vascular endothelium and that the alpha-2A adrenergic receptor subtype mediates indomethacin-insensitive, endothelium-dependent relaxation of pig epicardial coronary arteries.