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The leech muscle is one of the oldest preparations for the bioassay of acetylcholine and has served in this capacity on many venerable occasions. The last systematic pharmacological study was carried out by Minz (1932). Minz found the contraction of the eserinized leech muscle practically specific for acetylcholine. Several other agents either were not potentiated by physostigmine or were simply inactive save in very high concentrations.No information is available concerning the effects on the leech muscle of a number of agents which are known to, or assumed to, interact with acetylcholine receptors and which have become available only within the past 20 years. We consider it of sufficient interest and importance to try and fill in these gaps. The results of these investigations indicate an even closer pharmacological similarity between the leech muscle and the endplate of vertebrate skeletal muscle than previously recognized. Succinylcholine and decamethonium were found to be agonists in the leech, but could clearly be differentiated from acetylcholine, carbachol, and nicotine.In addition, the ability of some anticholinesterase agents to potentiate the effect of acetylcholine and related agonists has been studied. These studies, too, take on an added significance in view of the close pharmacological similarity between vertebrate endplate and leech muscle. METHODSThe dorsal muscle of the leech (Hirudo medicinalis) was dissected free of all tissue adhering to its inner surface as described by MacIntosh & Perry (1950). Two strips of muscle were prepared by dividing along the midline. The paired muscles were mounted vertically in identical Lucite organ baths of 10 ml. capacity and suspended in a bicarbonate Ringer solution. The lower end was secured to a hook at the bottom of the bath and the upper end attached by a length of inelastic thread to a strain gauge tension transducer (Grass model FT 03) mounted on an adjustable rack. On isolating and mounting, the muscles tend to go into spasm. This could be overcome by perfusing the organ bath with the Ringer solution at a rate of approximately 3 ml./min.

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In a study on the effects of some agonists on the leech muscle, Flacke & Yeoh (1968) showed that two groups of agonists, acetylcholine, carbachol and nicotine on the one hand, and succinyicholine and decamethonium on the other, could be differentiated by their potencies and the sizes of the maximal responses obtainable. It was thought that these differences could be explained on the basis of different sites of action of the two groups of agonists. This possibility was tested, and the present communication describes the interaction of the two groups of agonists with each other and with antagonist agents. METHODSThe methods used have already been described (Flacke & Yeoh, 1968). The effects of different concentrations of an antagonist agent were determined after exposure of at least 30 min to each concentration. The antagonist was present throughout the test sequence-that is, during and between successive tests with an agonist. Exposure to the agonist was for a period of 30 sec. The shift in the concentration-response curves was determined with concentrations of the agonist causing 20-80% of the estimated maximum response, which was determined only at the end of the experiment by adding a supramaximal dose; this procedure was necessary because a maximum response was followed by a period of depressed sensitivity. The effect of an antagonist was measured by determining the concentration ratio of the agonist at 50% of the maximum response. DrugsThe following drugs were used: acetylcholine chloride (Merck); carbaminoylcholine chloride (Merck); nicotine hydrogen tartrate (BDH); succinylcholine chloride (Burroughs Wellcome); decamethonium bromide (Burroughs Wellcome); physostigmine sulphate (Merck); (+)-tubocurarine chloride (Abbott); gallamine triethiodide (Davis and Greck). All drug concentrations are expressed as molar concentrations of the base. RESULTSPreliminary experiments confirmed earlier observations by several investigators (Minz,

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