Effect of Cholinesterase Inhibitors and Pam on the Release of Acetylcholine From Isolated Guineapig Ileum

Abstract

Eserine and mipafox have often been used as anticholinesterases to allow measure ment of spontaneous release of acetylcholine from the ileum (1-4). Mipafox was chosen because of its apparent inability to release acetylcholine from smooth muscle (5). It has also been reported that values for acetylcholine release in the presence of mipafox (1) were much lower than those obtained by other workers using eserine (2, 3). It, therefore, fol lows that the choice of a particular anticholinesterase may influence the release of acetyl choline from smooth muscle. In order to experimentally test this possibility, we examined the effect of four cholinesterase inhibitors&mdash;eserine, prostigmine, phosphamidon and mipa fox, on the spontaneous release of acetylcholine from isolated guineapig ileum.<BR> P AM (2-pyridyl-aldoxime methiodide) reactivates the phosphorylated (inhibited) cholinesterase (6, 7); At certain concentrations, it induces the release of acetylcholine while at others, it inhibits the acetylcholine release from rat phrenic nerve diaphragm prepara tion (8). The effect of PAM on the release of acetylcholine from smooth muscle has so far not been studied. The effect of PAM, if any, on the acetylcholine output from isolat ed guineapig ileum, in the presence of anticholinesterases, has also been examined in the present study. METHODSGuineapigs (300-400 g) were killed by a blow on the neck and bled. The ileum was taken out, emptied of its contents and divided into several equal pieces. Two pieces, 3 4 cm long, from different parts of the intestine were combined into one sample to reduce the effect of the variations in choline acetylase content (9) along the length of the intestine.The pieces were tied off at both ends to prevent the mucus in the lumen from oozing into the bath fluid. The preparation was set up in the usual way in a 10 ml organ bath con taining Tyrode solution at 37°C with a resting tension of I g. The preparation was wash ed twice at 10 minutes intervals before exposure to any drug. The preparation was next bathed in Tyrode containing the cholinesterase inhibitor for 30 minutes, the bath fluid being changed at 10 minute intervals; At the end of next 15 minutes, the bath fluid was withdrawn for the assay of acetylcholine. In another series of experiments, PAM was added in the bath 15 minutes before the collection of test sample.The biological assay for acetylcholine was made on a fresh piece of isolated guinea