Pituitary Thyrotropin-Releasing Hormone Receptors: Local Anesthetic Effects on Binding and Responses

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Abstract
Pharmacological agents are widely used to probe the mechanism of action of TRH. A number of these drugs behave as local anesthetics at high concentrations. The effect of local anesthetics on the binding of [3H]Me-TRH to specific receptors was studied using the GH4C1 line of rat pituitary tumor cells. [3H] Me-TRH binding was inhibited by classical local anesthetics with the order of potency (IC50 values): dibucaine (0.37 mM) > tetracaine (1.2 mM) > lidocaine (3.3 mM) > procaine and benzocaine (> 10 mM). IC50 values for other drugs with local anesthetic properties that inhibited [3H]Me-TRH were: 100 .mu.M trifluoperazine, 100 .mu.M imipramine, 170 .mu.M chloropromazine, 300 .mu.M verapamil, and 700 .mu.M propranolol. Inhibition by tetracaine and verapamil increased as the pH was raised from 6 to 8.5, indicating that the free base for of the amine drugs was the inhibitory species, and local anesthetic effect was greater at 37 C than at 24 C or 0 C. [3H]Me-TRH binding at receptors in isolated membranes was inhibited to the same extent as binding to receptors on intact cells. Local anesthetics were 3- to 20-fold less potent at inhibiting [3H]Me-TRH to digitonin-solubilized receptors than binding to intact cells. In contrast, the potency of chlordiazepoxide, a putative TRH antagonist, to inhibit [3H]Me-TRH binding was equal using cells and solubilized receptors (IC50 = 10 .mu.M). Local anesthetics inhibited TRH-stimulated PRL release and also inhibited basal PRL secretion and secretion stimulated by two nonhormonal secretagogues, (Bu)2cAMP and a phorbol ester. These data suggest that local anesthetics inhibit [3H]Me-TRH binding by a nonspecific intercalation into the plasma membrane and inhibit at multiple points in the PRL secretory pathway. When drugs with local anesthetic potential are used at high concentrations, inhibition of [3H]Me-TRH binding may occur and preclude interpretation of any reduction in TRH responses based on other sites of drug action.

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