Minneman K P, Abel P W
Mol Pharmacol. 1984 Jan;25(1):56-63.
The existence of "spare" alpha 1-adrenergic receptors in rat vas deferens was examined directly using radioligand binding assays and contractility measurements. Alpha 1-adrenergic receptors in homogenates of rat vas deferens were labeled with [125I]BE 2254 (125IBE). Norepinephrine and other full alpha 1-adrenergic receptor 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 inactivate alpha 1-adrenergic receptors caused a large decrease in the potency of full agonists in causing contraction of this tissue and a 23-48% decrease in the maximal contraction observed. Using those data, equilibrium constants for activation (Kact values) of the receptors by agonists were calculated. These Kact values agreed well with the equilibrium binding constants (KD values) determined from displacement of 125IBE binding. The reduction in alpha 1-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 the agonist dose-response curves before and after phenoxybenzamine treatment. Exposure to 300 nM phenoxybenzamine for 10 min resulted in a 39% decrease in 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 microM for 15 min) that completely abolished the contractile response to alpha 1-adrenergic agonists caused an 82% decrease in the density of 125IBE binding sites. Tissues exposed to 300 nM phenoxybenzamine in the presence of 100 microM phentolamine or 3 microM 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 alpha 1-adrenergic receptors at or near the receptor binding site. These experiments suggest that the potencies of agonists in activating alpha 1-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 alpha 1-adrenergic receptors in rat vas deferens, but that the decrease in receptor density is much smaller than that predicted from receptor theory.
运用放射性配体结合分析和收缩性测量方法，直接检测了大鼠输精管中“备用”α1 - 肾上腺素能受体的存在情况。大鼠输精管匀浆中的α1 - 肾上腺素能受体用[125I]BE 2254（125IBE）进行标记。去甲肾上腺素和其他完全性α1 - 肾上腺素能受体激动剂在抑制125IBE结合方面的效力，远低于其在体外使输精管收缩的效力。用300 nM酚苄明处理10分钟以不可逆地使α1 - 肾上腺素能受体失活，导致完全激动剂引起该组织收缩的效力大幅下降，且观察到的最大收缩力下降了23% - 48%。利用这些数据，计算了激动剂激活受体的平衡常数（Kact值）。这些Kact值与通过125IBE结合位移测定的平衡结合常数（KD值）吻合良好。通过对特异性125IBE结合位点的Scatchard分析，确定了酚苄明处理后α1 - 肾上腺素能受体密度的降低情况，并与根据酚苄明处理前后激动剂剂量 - 反应曲线计算出的预期降低值（q值）进行比较。暴露于300 nM酚苄明10分钟导致特异性125IBE结合位点减少39%，这与根据计算出的q值预期的93%的减少不一致。用能完全消除对α1 - 肾上腺素能激动剂收缩反应的剂量的酚苄明（10 μM，处理15分钟）处理输精管，导致125IBE结合位点密度降低82%。与对照组相比，在100 μM酚妥拉明或3 μM哌唑嗪存在的情况下，暴露于300 nM酚苄明的组织，激动剂诱导收缩的剂量 - 反应曲线或125IBE结合位点密度均未发生变化。这表明酚苄明在受体结合位点或其附近使α1 - 肾上腺素能受体发生功能性失活。这些实验表明，激动剂在激活大鼠输精管中α1 - 肾上腺素能受体方面的效力，与其与受体结合的效力吻合良好。激动剂引起收缩的更大效力可能归因于该组织中的备用受体。数据还表明，酚苄明可不可逆地使大鼠输精管中的α1 - 肾上腺素能受体失活，但受体密度的降低远小于受体理论预测的值。
