Further biochemical characterization of [3H]dihydroazapetine binding to α-adrenoceptor — related proteins from the rat vas deferens

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Abstract
The binding of [3H]dihydroazapetine, a reversible blocker of the α-adrenoceptor, has been characterized in a membrane preparation from the rat vas deferens. Binding is rapid, reversible and shows evidence of saturation. Postganglionic sympathetic denervation of the tissue does not decrease binding to the subcellular fraction. Drugs known to act on the α-adrenoceptor are specific inhibitors of [3H]dihydroazapetine binding while those acting on other receptors are without effect. Scatchard plot analysis suggests that two specific binding components are present, one of which has a similar dissociation constant for [3H]dihydro-azapetine to that determined pharmacologically. [3H]Dihydroazapetine binding is lowest in the physiological pH range and tends to decrease with increasing temperature in the range of 25 to 60°. Calcium, magnesium, sodium and potassium inhibit binding of [3H]dihydroazapetine with the divalent ions being approximately 100 times more potent. Studies with group selective reagents indicate that a disulphide bridge, and sulphydryl and carbonyl groups, are at or near the binding site and are necessary for binding. The site is protein possibly with involvement of phospholipids. Specific binding of [3H]dihydroazapetine is inhibited by low concentrations of local anaesthetics suggesting that the binding site of the antagonist may be related to the ‘calcium mobilization’ site of the α-adrenoceptor.