Alpha-1 adrenergic receptor binding and contraction of rat caudal artery

Alpha-1 adrenergic receptors were examined in rat caudal artery using radioligand binding of [¹²⁵I]labeled BE 2254 (¹²⁵IBE) and in vivo contraction measurements. ¹²⁵IBE bound rapidly and reversibly to a single class of high affinity binding sites in membrane preparations of caudal artery. Scatchard analysis gave an equilibrium dissociation constant (K(D)) of 110 pM and a density of binding sites of 115 fmol/mg of protein. Antagonists inhibited ¹²⁵IBE binding and phenylephrine-induced contractions competitively, with an order of potency of prazosin > ARC 239 > phentolamine > yohimbine. pA₂ values for inhibition of phenylephrine-induced contraction correlated well with K(D) values for inhibition of specific ¹²⁵IBE binding. A number of other full and partial agonists also caused contraction of caudal arteries with an order of potency of epinephrine > norepinephrine > phenylephrine > methoxamine. The order of potency of agonists and the potencies of antagonists suggests that the contractile responses of rat caudal artery were mediated by alpha-1 adrenergic receptors. The EC₅₀ values of partial agonists in causing contraction correlated well with their K(D) values for inhibition of specific ¹²⁵IBE binding. However, the EC₅₀ values for full agonists were 30 to 200 times lower than their K(D) values. Treatment of caudal arteries in vitro with 0.1 μM phenoxybenzamine for 10 min to inactivate alpha adrenergic receptors decreased both the potency of full agonists in causing contraction and the maximal contractile response. Functional equilibrium dissociation constants calculated from contraction experiments using phenoxybenzamine agreed well with K(D)'s determined from binding studies; however, phenoxybenzamine reduced ¹²⁵IBE binding sites by 50%, whereas the theoretical reduction in functional alpha adrenergic receptors averaged 96%. These data suggest that ¹²⁵IBE labels the alpha-1 adrenergic receptors mediating contraction of rat caudal artery. When receptor density is reduced, the potencies of agonists in activating the receptors agree well with their potencies in binding to the receptors, suggesting that there is a pool of 'spare' alpha-1 adrenergic receptors in this tissue.