The proposed research concerns cellular mechanisms underlying chronic toxicity of organophosphonates that arise either as a primary consequence of acetylcholinesterase (AchE) inhibition or through mechanisms independent of AchE inhibition. In vitro studies employing primary cultures of embryonic chick skeletal muscle and neural retina and in vivo studies in rat will investigate organophosphonate-induced disruption in coupling of protein synthesis and secretion; these studies will focus on the effects exerted by organophosphorus agents on protein turnover (synthesis/degradation), post-translational oligosaccharide processing, and transmembrane signaling (changes in pH and Ca++). These studies are connected by a common theme that organophosphonates can alter protein turnover through either inhibition of intracellular proteinases, thereby prolonging degradation, or through mobilization of intracellular Ca++, thereby activating proteinases and accelerating protein degradation. To distinguish between these distinct and opposit effects, the proposed studies will employ chiral and achiral, charged and uncharged, fluorescent and non-fluorescent methylphosphonates, as well as a number of readily available Ca++- and pH-selective fluorescent dyes (QUIN2, FURA2, BCECF) and calcium channel antagonists. Finally, we plan to extend our ongoing in vivo studies of organophosphonate-induced alterations in cardiovascular function by examining stereospecificity of the hypertensive response induced (in rat) by enantiomeric organophosphorus agents; examination of subsequent aging and oxime reversal will afford a means for assessing the capacity for aging to occur in vivo and the requirement for oxime efficacy to arise from direct interaction with acetylcholinesterase.
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