Changes in affinity states during down‐regulation of muscarinic receptors in tracheal smooth muscle of organophosphate‐treated swine

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Abstract
1 Subacute (daily) treatment of male swine with the organophosphate acetylcholinesterase inhibitor diisopropylfluorophosphate (DFP) resulted in tolerance to the effects of DFP within 5-6 days. 2 Subacute administration of DFP resulted in a 98% inhibition of tissue cholinesterase after 7 days and in a decrease of [3H]quinuclidinyl benzilate ([3H]QNB) binding sites in homogenates of tracheal smooth muscle by 77%. The maximal density of receptors (Bmax) decreased from 1.8 .+-. 0.4 to 0.5 .+-. 0.1 pmole mg-1 protein. There was no significant change in the dissociation constant (Kd)for [3H]QNB binding. 3 Pirenzepine displacement of [3H]QNB binding was best described by a single binding site model, with a Ki of 230 .+-. 40 nM. This value was unchanged following seven days of DFP treatment (250 .+-. 30 nM). The low affinity for this M1 antagonist suggests that there is predominantly a single population of [3H]QNB binding sites of the M2 subtype in tracheal smooth muscle. 4 Carbachol displacement of [3H]QNB binding yielded data best fit by a two-binding site model. The dissociation constants were KiL = 210 .+-. 60 .mu.M (61 .+-. 1%) and KiH = 1.2 .+-. 0.4 .mu.M (39 .+-. 1%) respectively (n = 7) for the low and high affinity states. Seven-day treatment with DFP reduced the percent of high affinity receptors to 25 .+-. 4%. 5 Addition of Mg++ to the incubation medium prevented this shift in the proportion of low and high affinity receptors. Gpp(NH)p and Mg++ together decreased the proportion of the high affinity receptors when added to the incubation medium in control tissue (to 25%), but not tissue from 7-day DFP-treated swine. NEM increased the proportion of muscarinic receptors in the high affinity state both for controls and for the DFP-treated swine, in both cases yielding receptors with identical binding properties. 6 Thus, subacute administration of DFP causes not only a decrease in the number of receptors, but also a change in the affinity of the receptors for agonists which is related to the interaction of the guanine nucleotide binding protein and the muscarinic receptor.