The proposed research will examine the sites and mechanisms of organophosphate toxicity at discrete loci on the ventral surface of the medulla oblongata. Organophosphates are known to act as potent inhibitors of acetylcholinesterase, the enzyme responsible for hydrolysis of acetylcholine, and produce effects nearly equivalent with excessive cholinergic stimulation. When applied to the ventral surface, organophosphates elicit a profound, long-lasting vasodepression which is readily reversed in the presence of muscarinic antagonists (atropine) and oxime reactivators (HI-6) of the enzyme. We have identified neuroanatomically well defined cell bodies within 50 Mu of the ventral surface which appear to represent cholinergic nerve tracts that play a role in central control of cardiovascular function. We plan to continue our identification of chemosensitive sites on the ventral medulla, employing fluorescent phosphonates which serve not only to induce the vasodepression, but by virtue of their fluorescence, serve as markers for acetylcholinesterase employing fluorescence microscopy. In addition, monoclonal antibodies directed against distinct molecular forms of acetylcholinesterase will be employed, in fluorescence immunoassay, as an independent assessment of acetylcholinesterase distribution in the CNS. The proposed studies will examine whether oxime efficacy arises as a consequence of reactivation of the organophosphonyl-enzyme conjugate by measuring the capacity of these agents to reverse the hypotension elicited by enantiomeric d- and l-organophosphonates. Stereospecificity in the active site of acetylcholinesterase and the optical relationship with that of the triesterase enzymes present in mammalian sera will be investigated employing enzymatic resolution of enantiomeric phosphonates and stereospecific syntheses. The proposed studies will examine the influence of organophosphonates on acetylcholinesterase turnover, and in particular, the role of organophosphate interactions with protease enzymes as one likely mechanism by which AchE turnover is altered. Protein degradation rates will be monitored by relying on measurement of the fluorescence signal derived from a labelled population of acetylcholinesterase after treatment of primary cultures of chick embryonic skeletal muscle with fluorescent phosphonates.
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