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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES003085-05
Application #
3250217
Study Section
Toxicology Study Section (TOX)
Project Start
1982-04-01
Project End
1988-03-31
Budget Start
1986-04-01
Budget End
1988-03-31
Support Year
5
Fiscal Year
1986
Total Cost
Indirect Cost
Name
State University of New York at Buffalo
Department
Type
Schools of Pharmacy
DUNS #
038633251
City
Buffalo
State
NY
Country
United States
Zip Code
14260
Joshi, Anil K; Witkowski, Andrzej; Berman, Harvey A et al. (2005) Effect of modification of the length and flexibility of the acyl carrier protein-thioesterase interdomain linker on functionality of the animal fatty acid synthase. Biochemistry 44:4100-7
Luo, Zhigang David; Berman, Harvey Alan (2004) Pb2+-mediated down-regulation of dihydropyridine receptors in skeletal muscle. Toxicol Lett 152:167-73
Wong, L; Radic, Z; Bruggemann, R J et al. (2000) Mechanism of oxime reactivation of acetylcholinesterase analyzed by chirality and mutagenesis. Biochemistry 39:5750-7
Taylor, P; Hosea, N A; Tsigelny, I et al. (1997) Determining ligand orientation and transphosphonylation mechanisms on acetylcholinesterase by Rp, Sp enantiomer selectivity and site-specific mutagenesis. Enantiomer 2:249-60
Luo, Z D; Berman, H A (1997) The influence of Pb2+ on expression of acetylcholinesterase and the acetylcholine receptor. Toxicol Appl Pharmacol 145:237-45
Hosea, N A; Radic, Z; Tsigelny, I et al. (1996) Aspartate 74 as a primary determinant in acetylcholinesterase governing specificity to cationic organophosphonates. Biochemistry 35:10995-1004
Jo, S A; Higgins, D M; Berman, H A (1992) Regulation of acetylcholinesterase in avian heart. Studies on ontogeny and the influence of vagotomy. Circ Res 70:633-43
Berman, H A; Leonard, K (1992) Interaction of tetrahydroaminoacridine with acetylcholinesterase and butyrylcholinesterase. Mol Pharmacol 41:412-8
Nowak, M W; Berman, H A (1991) Fluorescence studies on the interactions of myelin basic protein in electrolyte solutions. Biochemistry 30:7642-51
Decker, M M; Berman, H A (1990) Denervation-induced alterations of acetylcholinesterase in denervated and nondenervated muscle. Exp Neurol 109:247-55

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