'Spare' alpha₁-adrenergic receptors and the potency of agonists in rat vas deferens

The existence of 'spare' alpha₁-adrenergic receptors in rat vas deferens was examined, directly using radioligand binding assays and contractility measurements. Alpha₁-adrenergic receptors in homogenates of rat vas deferens were labeled with [¹²⁵I]BE 2254 (¹²⁵IBE). Norepinephrine and other full alpha₁-adrenergic receptors agonists were much less potent in inhibiting ¹²⁵IBE binding than in contracting the vas deferens in vitro. Treatment with 300 nM phenoxybenzamine for 10 min to irreversibly inactive alpha₁-adrenergic receptors caused a large decrease in the potency of full angonists in causing contraction of this tissue and a 23-48% decrease in the maximal contraction observed. Using those data, equilibrium constants for activation (K(act) values) of the receptors by agonists were calculated. These K(act) values agreed well with the equilibrium binding constants (K(D) values) determined from displacement of ¹²⁵IBE binding. The reduction in alpha₁-adrenergic receptor density following phenoxybenzamine treatment was determined by Scatchard analysis of specific ¹²⁵IBE binding sites and compared with the expected reduction (q values) calculated from agonist dose-response curves before and after phenoxybenzamine treatment. Exposure to 300 nM phenoxybenzamine for 10 min resulted in a 39% decreased specific ¹²⁵IBE binding sites, which did not agree with the 93% decrease expected from the calculated q values. Treatment of vas deferens with a dose of phenoxybenzamine (10 μM for 15 min) that completely abolished the contractile response to alpha₁-adrenergic agonists caused an 82% decrease in the density of ¹²⁵IBE binding sites. Tissues exposed to 300 nM phenoxybenzamine in the presence of 100 μM phentolamine of 3 μM prazosin showed no change in the dose-response curves for agonist-induced contraction or in the density of ¹²⁵IBE binding sites when compared with controls. This suggests that phenoxybenzamine functionally inactivates alpha₁-adrenergic receptors at or near the receptor binding site. These experiments suggest that the potencies of agonists in activating alpha₁-adrenergic receptors in rat vas deferens agree well with their potencies in binding to the receptors. The greater potency of agonists in causing contraction may be due to spare receptors in this tissue. The data also demonstrate that phenoxybenzamine irreversibly inactivates alpha₁-adrenergic receptors in rat vas deferens, but that the decrease in receptor density is much smaller than that predicted from receptor theory.