McHardy Smith - Modulation of muscarinic cholinergic receptor affinity for antagonists in rat heart.

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  Martin, MW, Smith, MM, Harden, TK 230 424-30-

  Abstract: Modulation of the affinity of agonists   and antagonists at muscarinic cholinergic receptors in rat heart   membranes was investigated using the radiolabeled antagonist,   [3H]quinuclidinyl benzilate ([3H]QNB), and the radiolabeled   agonist, [methyl-3H]oxotremorine acetate ([3H]OXO). Receptor   affinity for oxotremorine measured in competition binding assays   with [3H]QNB or by equilibrium binding of [3H]OXO was increased   when the incubation temperature was reduced to 4 degrees C. In   contrast, the receptor affinity for [3H]QNB was decreased at   lower incubation temperatures and a marked effect of guanine   nucleotides on the affinity for [3H]QNB was revealed. Guanine   nucleotides increased receptor affinity for [3H]QNB without   changing the total number of binding sites. The GTP-induced   increase in the affinity for [3H]QNB was reflected by an increase   in the rate constant for association of [3H]QNB. At subsaturating   ligand concentrations, guanine nucleotides increased [3H]QNB   binding and decreased [3H]OXO binding with the same order of   potency: GppNHp = GTP gamma S greater than GTP greater than   guanosine 5'-diphosphate greater than GMP. Free Mg++ ion was   required to observe guanine nucleotide effects on antagonist   binding. Pretreatment of heart membranes with N-ethyl-maleimide   increased [3H]QNB affinity and blocked the effects of guanine   nucleotides. N-Ethylmaleimide also decreased [3H]OXO binding and   increased [3H]QNB binding with a similar concentration-effect   relationship. Thus, antagonist and agonist binding to muscarinic   cholinergic receptors is modulated in a reciprocal manner by a   number of factors; this modulation appears to reflect interaction   of agonist and antagonist-occupied receptors with a guanine   nucleotide regulatory protein, Ni.

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