Abstract

N,N'-bis (2-diethylaminoethyl) oxamide bis-2-chlorobenzyl chloride (WIN 8077) and the corresponding bis-2-methoxybenzyl chloride salt (WIN 8078) are reversible, selective inhibitors of acetylcholinesterase (AChE), which have been shown previously to produce cholinomimetic effects in doses (0.0005 to 0.02 micromol./kgm.) far below those which cause measurable AChE-inhibition in vivo. In the present study, their pI₅₀'s for inhibition of the AChE of cat brain homogenate were found to be 7.6 and 5.6 respectively. When the same homogenate was incubated with varying concentrations of the two inhibitors, over ranges producing twenty to nearly one hundred per cent inhibition, the equivalent percentages were protected against a concentration (10^-4_M) of diisopropylfluorophosphate (DFP) which produced complete inactivation of the enzyme in the absence of the reversible inhibitors. The protective effect against DFP was also demonstrated histochemically: sections of ciliary and stellate ganglia which were incubated with WIN 8077 or WIN 8078 plus DFP, then washed and stained for AChE, showed the presence of increasing enzymatic activity with increasing concentrations of the two reversible inhibitors, whereas no activity was present after DFP alone. This principle was then employed to determine the sites of AChE-inhibition produced by various doses of WIN 8077 and WIN 8078 in vivo. Anesthetized, atropinized cats were given intravenous injections of WIN 8077 (0.0005 to 0.5 micromol./kgm.) or WIN 8078 (0.005 to 0.5 micromol./kgm.), and five minutes later received 20.0 micromol. of DFP/kgm., i.v.; thirty to forty minutes after DFP, they were sacrificed. The stellate and ciliary ganglia and a portion of intercostal muscle were removed, and the tissues were sectioned and stained for AChE-activity. Sections of ganglia from cats which had received only DFP were completely blank; motor endplates were stained faintly. The ciliary and stellate ganglia of one of the two cats which had received 0.0005 micromol. of WIN 8077/kgm. showed faint staining. Both ganglia of all cats which had received 0.005 micromol. of WIN 8077, or 0.05 micromol. of WIN 8078, or more were stained, with increasing intensity at higher doses or longer periods of incubation. Staining was most marked at the borders of the neurons of the ciliary ganglia, and in the intercellular plexuses of the stellate ganglia; the cytoplasm of the ciliary ganglion cells, which stains intensely in untreated controls, remained blank. Motor endplates showed evidence of AChE-inhibition over the same dosage ranges of WIN 8077 and WIN 8078. Results are discussed with respect to correlation between AChE-inhibition and pharmacological action, the concept of "functional" and "reserve" AChE, and possible physiological roles of AChE other than enzymatic.