1 The affinity of 17 compounds for muscarine-sensitive acetylcholine receptors in atrial pacemaker cells and ileum of the guinea-pig has been measured at 290C in Ringer-Locke solution. Measurements were also made at 370C with 7 of them. 2 Some of the compounds had much higher affinity for the receptors in the ileum than for those in the atria. For the most selective compound, 4-diphenylacetoxy-N-methylpiperidine methiodide, the difference was approximately 20-fold. The receptors in the atria are therefore different in structure from those in the ileum. 3 The effects of temperature on affinity are not the same for all the compounds tested, indicating different enthalpies and entropies of adsorption and accounting for some of the difficulty experienced in predicting the affinity of new compounds.

I The dissociation constants of many phenolic amines, including benzylamines, phenethylamines, phenylethanolamines, phenylpropylamines, catecholamines, and apomorphine have been measured by potentiometric titration at 25°C. Measurements have also been made with many of their methoxy derivatives and with series of phenolic quaternary ammonium salts. Some compounds were also studied at 370C. 2 Usually at least five titrations were made with each compound and Debye-Huckel theory was applied to convert concentrations to activities but the estimates of pKa were not constant and found to increase with increasing concentration. The range studied was usually 5-15 mM and a least-squares line-fit, based on the empirical assumption that pK5 varies with (concentration)"12, has been used to calculate values for 10 mm solutions and to extrapolate to infinite dilution and to 100 mM. The dependence of pKa on concentration was much less at 370C than at 25°C. 3 At 370C the pKa values of many biologically interesting compounds in the group, dopamine, noradrenaline, adrenaline and isoprenaline, coryneine (the trimethylammonium derivative of dopamine) and apomorphine are within 1 log unit of physiological pH, indicating the presence of a significant proportion of either the zwitterion or of the uncharged phenolic amine. 4 Zwitterion constants have been estimated from the pKa values of the phenolic amines and those of their methoxy and quaternary trimethylammonium analogues. Zwitterion formation does not appear to be associated with activity at a-adrenoceptors and probably not with activity at P-receptors. The active species seems likely to.contain the unionised phenolic group but at dopamine receptors this may be in the uncharged phenolic amine rather than in the phenolic ammonium salt.

. Series of analogues of acetylcholine have been prepared in which the acetyl group was replaced by phenylacetyl, cyclohexylacetyl, diphenylacetyl, dicyclohexylacetyl, (±)‐phenylcyclohexylacetyl, benziloyl and (±)‐phenyl‐cyclohexylhydroxyacetyl groups and the trimethylammonium group was replaced by Me2EtN+, MeEt2N+, Et3N+,Further series were prepared in which the acetoxyethyl group was replaced by ethoxyethyl, phenylethoxyethyl, cyclohexylethoxyethyl, diphenylethoxyethyl, and dicyclohexylethoxyethyl groups, and by n‐pentyl, 5‐phenylpentyl, 5‐cyclohexylpentyl and 5:5‐diphenylpentyl groups. . The ethoxyethyl and n‐pentyl series contain some compounds which are agonists or partial agonists when tested on the isolated guinea‐pig ileum, but all the other compounds are antagonists. . The affinity of the compounds for the postganglionic (“muscarine‐sensitive”) acetylcholine receptors has been measured in conditions in which the antagonists have been shown to be acting competitively. There were considerable differences between their affinities, the most active (log K, 9.8) having one million times the affinity of the least active (log K, 3.7). . The changes in affinity as the onium group was modified were not entirely independent of changes in the rest of the molecule. Increasing the size of the onium group, as judged from conductivity measurements on simpler onium salts, increased affinity in the series containing one large group (phenyl or cyclohexyl) but, in the series with two large groups, affinity declined when the size was increased beyond ‐N+MeEt2. . In general, the effects of changes in the rest of the molecule on affinity were bigger than the effects of changes in the onium group and there were bigger interactions. Affinity was increased to a greater extent by introducing one phenyl and one cyclohexyl group together than by introducing either two phenyl or two cyclohexyl groups; the increment was greater than the separate contributions made by one phenyl and one cyclohexyl group. . The factors which influence the binding of molecules to receptors are discussed. There is no evidence that the separation between the onium group and the group in the receptor with which it interacts is greater in compounds with high affinity nor is there any evidence, from the study of the series which contain agonists and partial agonists, that ability to activate receptors depends upon the onium group being able to come close to this charged group in the receptors.

It has long been known that, whereas tetramethylammonium ions (Me4N+) stimulate nicotine-sensitive acetylcholine receptors, tetraethylammonium ions (Et4N+) block them. The early work is reviewed by Raventos (1937), who himself made a systematic survey of the effects of the step-wise replacement of methyl groups in Me4N+ by ethyl groups. On the frog rectus he found that activity declined eight-fold when one methyl group was replaced by ethyl, and that further replacement gave compounds which showed neither agonist nor antagonist activity in the highest concentrations tested. Marshall (1916), on the other hand, had found that the compounds dimethyldiethylammonium (Me5N+Et2), methyltriethylammonium (MeN Et3) and Et4N+ antagonized the contracture produced by tetramethylammonium on the frog sartorius muscle. This would seem to indicate that the compounds still combine with the receptors, so the loss in activity must be due to a loss of efficacy. None of the results, however, give any quantitative indication of the effect of the replacement of methyl by ethyl on the affinities of these compounds for the receptors and we have, therefore, re-examined them together with their pyrrolidine, piperidine, quinuclidine and pyridine analogues. The blocking actions of ions as similar as these are likely to be competitive, and we have measured the affinity constants of the antagonists and partial agonists for the nicotine-sensitive acetylcholine receptors in the frog rectus muscle, as well as the relative activities of those of the compounds which are agonists. METHODSThe frog rectus preparationThe rectus abdominis muscle from Rana pipiens was used in all experiments. It was mounted in frog-Ringer solution, through which air was blown, and kept at room temperature (around 15°C). Contractions were recorded with an isotonic gimbal-mounted lever, writing on a smoked drum. The load was between 0.5 and 1.0 g. The volume of the bath was approximately 5 ml., but this is not important because all drugs were made up to the desired concentration in frog-Ringer. This solution flowed from a reservoir on to the tissue at the appropriate time, determined by Opening and closing a magnetic relay, operated by a time-clock and uniselector. In all the experiments the agonist was in contact with the tissue for 41 min. It was then washed out and the preparation gently stretched (by increasing the load on the lever by about 5 g) for 15 min, during which period it was again washed twice. It was then left with the extra load removed and the next application of