High-affinity activation by paraoxon of a muscarinic receptor subtype in rat brain striatum

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Abstract

The mechanism of action of the anticholinesterase paraoxon on the function of a muscarinic receptor subtype in rat brain striatum was investigated. Paraoxon inhibited binding of the muscarinic agonist cis-[3H]methyldioxolane, which binds to a high-affinity population of muscarinic receptors, with K0.5 of 80 nM, compared to a K0.5 of 7 μM for parathion. The inhibition was competitive, suggesting that paraoxon and CD bind to a common site. When this muscarinic receptor (possibly an M4 subtype) is activated it inhibits cAMP synthesis. Thus, function of the paraoxon-sensitive receptor was assayed by the inhibition of the forskolin-activated [3H]cAMP synthesis. Paraoxon inhibited cAMP synthesis in a dose-dependent manner as did the muscarinic agonist carbachol, and this inhibition was completely blocked by the muscarinic antagonist atropine. When low concentrations of carbachol and paraoxon were used together, there was additive inhibition of cAMP synthesis. However, there was no further increase when both paraoxon and carbachol were present in concentrations that individually produced maximal inhibition. The data suggest that paraoxon acts like carbachol, causing activation of the muscarinic receptor subtype in brain striatum and leading to inhibition of cAMP synthesis. Considering the high affinity that this muscarinic receptor has for paraoxon, it is suggested that this direct reversible action of paraoxon on the muscarinic receptor could affects its toxicity, expecially early on before most of the acetylcholinesterase is phosphorylated.

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      Finally, benzodiazepines, administered to control AChE inhibitor-related seizures (Holstege et al., 1997), are likely to depress brainstem respiratory and circulatory centers and may thus potentiate known depressant effects of OPs or carbamates on these centers (Munro et al., 1990). In addition to inhibition of AChE, several organophosphates bind to both muscarinic and nicotinic acetylcholine receptors acting as receptor agonists (Jett et al., 1991) or antagonists depending on the organophosphate, receptor and the cell type (Oortgiesen et al., 1996; Van den Beukel et al., 1996). It has been demonstrated that nicotinic acetylcholine receptors (nAChRs) identified in mammalian brain, form pentameric heteromeric or homomeric ion-channels that are composed of different (α2–α7, α9–α10, and β2–β4) transmembrane subunits (Millar, 2003).

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      Namely, competition studies with the muscarinic antagonists, [3H]quinuclidinyl benzilate ([3H]QNB) and [3H]pirenzepine ([3H]PZ), did not show any evidence of binding of soman, sarin and tabun to rat cortical membranes, VX and echothiophate displayed competitive binding with Ki values in the μM range (Bakry et al., 1988) and paraoxon demonstrated non-competitive binding with 20% displacement (Katz and Marquis, 1989). On the other hand, soman, sarin, tabun, VX, echothiophate, and paraoxon displaced the agonist—[3H]cis-dioxolane ([3H]CD) with Ki values of 1–800 nM (50–100% displacements) in brain (Bakry et al., 1988; Jett et al., 1991; Ward et al., 1993) and heart membranes (Silveira et al., 1990). These results do not seem to allow for speculation regarding the nature of the OP muscarinic binding site and obviously do not permit prediction of the effect of the OP binding on the putative biochemical events triggered by receptor occupancy.

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    1

    Permanent address: U.S. Fish and Wildlife Service, Patuxent Wildlife Research Center, Laurel, MD 20708.

    2

    Permanent address: Alexandria University, Faculty of Agriculture, Department of Plant protection, Pesticide Chemistry Division, Faculty of Agriculture, Alexandria, Egypt.

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