In Vitro excitation of purified membrane fragments by cholinergic agonists

Summary

Excitation of membrane fragments by cholinergic agonists is measuredin vitro by a filtration technique. Membrane fragments which contain high levels of the enzyme acetylcholinesterase and presumably originate from the innervated excitable faces of electroplax are first purified from homogenates of electric organ ofElectrophorus electricus by centrifugation in a sucrose gradient. Then the fragments, which make closed vesicles or microsacs, are equilibrated overnight with a medium containing²²Na⁺. After equilibration of the inside of the microsacs with the outside medium, the suspension is diluted into a nonradioactive medium. The²²Na⁺ content of the microsacs as a function of time is then followed by rapid filtration on Millipore filters. In the presence of cholinergic agonists, the time course of²²Na⁺ release changes: the rate of²²Na⁺ release increases. This increase is blocked byd-tubocurarine and is absent with microsacs derived from the non-innervated inexcitable membrane of the electroplax. The response to cholinergic agonists is thus followed on a completely cell-free system, in a well-defined environment. The dose-response curves to cholinergic agents obtainedin vitro agree, quantitatively, with the dose-response curves recordedin vivo by electrophysiological methods. In particular, the dose-response curve to agonists is sigmoid, the antagonism betweend-tubocurarine and carbamylcholine competitive, and the antagonism between tetracaine and carbamylcholine noncompetitive. The effects of two different affinity labeling reagents on the response to agonists and on the catalytic activity of acetylcholinesterase are followed in parallel on the same microsac preparation. The effects of dithiothreitol and of gramicidin A on the microsacs are studied and are found to be similar to those observedin vivo with the isolated electroplax.