The contractions of the isolated guinea-pig vas deferens in response to stimulation of the sympathetic hypogastric nerve were potentiated by low concentrations and inhibited by high concentrations of the antiadrenaline agents tolazoline, yohimbine, ergotamine, phenoxybenzamine and piperoxan. Eserine potentiated the contractions of the vas deferens produced by hypogastric nerve stimulation. The cholinesterase activity of an extract of vas deferens was decreased by the antiadrenaline agents. The potentiation of responses to sympathetic stimulation by antiadrenaline drugs, which also possess anticholinesterase activity, can be explained on the basis of a cholinergic sympathetic mechanism.The effects of antiadrenaline agents on the taining 2 adjacent platinum rings. Stimulation was responses to stimulation of a sympathetic nerve do by 2 msec. pulses at a frequency of 10/sec. applied not always run parallel to their effects on responses at supramaximal strength (usually 3.0 mA.) for 10 sec.induced by noradrenaline. Thus Varagic (1956b) in every 2 min. The time cycle of stimulation was found that tolazoline potentiated the response of automatically controlled. Contractions were recorded the rabbit uterus to sympathetic nerve stimulation, by an isotonic frontal writing lever. and Hukovic (1959) reported that phenoxybenzIsolated Vas Deferens.-For experiments in which amine increased the response to sympathetic nerve nerve stimulation was not required the vas deferens stimulation in the isolated atria of the rabbit. was suspended in a 5 ml. bath. We have investigated the effects of tolazoline Cholinesterase Activity.-The vasa deferentia from and other antiadrenaline agents on the a guinea-pig were ground with Tyrode solution responses of the isolated vas deferens to (11 ml.) in a mortar. The whole extract was divided sympathetic nerve stimulation, noradrenaline and into 2 ml. aliquots to serve as source of enzyme in acetylcholine-and on the cholinesterase activity of each reaction mixture. The drugs examined for acetylcholine, and on anticholinesterase activity were mixed with the enzyme the vas deferens. 10 min. before the addition of substrate and brought METHODSto 32°. Acetylcholine as substrate was added to reach Isolated Innervated Vas Deferens.-The isolated vas a final concentration of 0.5 lxg. /ml. in a final volume deferens with intact hypogastric nerve was prepared of reaction mixture of 5 ml. At various time intervals by the method of Hukovic (personal communication). after this, the reaction mixture was agitated and 0.1 Guinea-pigs weighing about 500 g. were killed by a ml-samples were withdrawn and assayed for acetylblow on the head. The abdomen was opened in the choline on a strip of guinea-pig ileum suspended in midline and the distal colon retracted to one side. Tyrode solution in a 5 ml. bath. A dose-response The hypogastric nerves were identified and dissected curve for acetylcholine was obtained upon the ileum free. The vasa deferentia were cut from their attach-at the beginning and the end of each exp...

The adrenergic blocking agents tolazoline, phentolamine, piperoxan, yohimbine, phenoxybenzamine, bretylium and guanethidine block the excitatory actions both of cholinergic nerves and of added acetylcholine on a variety of vertebrate smooth muscle preparations. These cholinergic blocking actions often occurred with concentrations lower than those required to block the response of the guinea-pig vas deferens to stimulation of the adrenergic hypogastric nerve. The anti-acetylcholine activities of these drugs have been studied in detail, using the guinea-pig rectum and the toad bladder' as test organs. In preparations sensitive to eserine, the anticholinesterase actions of the drugs competed with their anti-acetylcholine actions, so that either potentiation or block of responses to acetylcholine and to cholinergic nerve stimulation occurred with different concentrations. The responses of the toad bladder to acetylcholine were not potentiated by eserine. This enabled the antagonism of acetylcholine by the anti-adrenergic drugs to be estimated without interference from their anticholinesterase activity. When blocking activity was assessed on guinea-pig rectum previously treated with dyflos, the results were qualitatively similar to those on the toad bladder. Phenoxybenzamine often completely blocks responses both to added acetylcholine and to cholinergic nerve stimulation in concentrations less than those required to block adrenergic nerves. Guanethidine and piperoxan also show strong cholinergic blocking activity. Bretylium, yohimbine, tolazoline and phentolamine were less potent. However, in concentrations required to block the effect on the vas deferens of hypogastric nerve stimulation, these drugs at least halved the effects of acetylcholine and often of cholinergic nerve stimulation. It is concluded that these adrenergic blocking agents cannot be used to distinguish conclusively between adrenergic and cholinergic nerves. For reliable analysis of autonomic innervation, the substances released upon nerve stimulation must be identified by specific biochemical techniques or bioassay.

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An improved binding assay involving (−)‐[3H]‐dihydroalprenolol (DHA) and KCl‐washed cardiac membranes was developed to study β‐adrenoceptors in the canine heart quantitatively. Receptor numbers varied from 3.8 to 7.1 pmol/g fresh tissue, showing a steady increase from left atrium → right atrium →right ventricle → interventricular septum → left ventricle. With one minor exception, the same pattern was found for adenylate cyclase activity and Na+, K+ ‐activated ATPase activity. The binding of DHA was inhibited in the expected manner by β‐adrenoceptor agonists and antagonists, and was stereospecific, in confirmation of previous studies. Dissociation constants determined from Scatchard analyses included DHA: 2.5 nm; (−)adrenaline: 230 nm; (−)noradrenaline: 1167 nm. Kinetic analyses of the binding of DHA yielded a KD of about 4 nm. The distribution of β‐receptors is closely related to that of blood flow and the arrival plus retention of a circulating catecholamine, but is markedly different from that of endogenous noradrenaline, and thus adrenergic nerve terminals. Most receptors thus appear not at synapses but diffusely localized where they can react with circulating adrenaline. Evidence is discussed that β‐receptors at synapses respond primarily to neural noradrenaline, less to circulating adrenaline, and hardly at all to circulating noradrenaline; responses mediate increased cardiac output during exercise. In contrast most cardiac β‐receptors appear to respond only to adrenaline, and to be used, except at times of severe circulatory stress, during psychological stress.