Irreversible organophosphate acetylcholinesterase (AchE) inhibitors are used both clinically and as insecticides. We propose to compare and contrast the effects of a homologous series of echothiophate (217MI) derivatives (217AO and Tetram) on the neuromuscular postsynaptic membrane with their ability to directly inhibit AchE. Numerous agents interact with the acetylcholine receptor-ionophore (AchR/I) complex to speed the decay (yield) of endplate currents (e.p.c.s.) and interrupt the normal voltage dependence of yield (22). Preliminary data with these three agents (10-9 to 10-6M) demonstrate a time dependent decrease of the voltage dependence of yield while e.p.c.s. and miniature e.p.c.s. are prolonged (21). We propose to determine whether this effect is due to an inhibition of AchE or to a direct effect on the AchR/I, using electrophysiological and biochemical techniques. We propose to determine association constants with the AchE for each of the agents by measuring the rates of inhibition of AchE from cutaneous pectoris muscles of the frog (Rana Pipiens) and solubilized microsacs from Torpedo nobiliana electroplax: and to compare these constants with apparent association constants derived from their time dependent effects on yield and the voltage sensitivity of yield. In addition, we plan to directly measure the affinity constant of 3H-217MI by measuring its rate of interaction with AchE: and to determine the direct effects on the AchR/I by measuring mean single channel open times under conditions where AchE does not play a significant role, i.e., iontophoretic noise analysis and single channel currents in embryonic chick myotubes. The hypothesis that we wish to test is that voltage dependence of yield is due to the combined effects of AchE inhibition and diffusion of Ach from the synaptic cleft and that when AchE is inhibited the rate limiting step is diffusion of Ach from the synaptic cleft which should be voltage independent. It is anticipated that the results will allow us to clearly determine whether the increase in yield and interruption of its voltage dependence are due secondarily to the inhibition of AchE or to a direct effect on the AchR/I.
| Adam, L P; Henderson, E G (1990) Calcium channel effectors are potent non-competitive blockers of acetylcholine receptors. Pflugers Arch 416:586-93 |
| Henderson, E G; Post-Munson, D J; Reynolds, L S et al. (1989) Echothiophate and cogeners decrease the voltage dependence of end-plate current decay in frog skeletal muscle. J Pharmacol Exp Ther 251:810-6 |
