Extraction and partial purification of spasmogenic substances in Auerbach's plexus.

A bland procedure, conducted in ice, is described for the extraction with HCl of smooth-muscle-contracting substances from plexus-containing ileal longitudinal muscle (l.m.) sheets obtained mainly from rabbits and some guinea-pigs. The spasmogenic activity in rabbit extracts was distinguished from acetylcholine, histamine and 5-hydroxytryptamine by antagonists; and from prostaglandins, by its insolubility in ether at acid pH and by pretreatment of the animals with indomethacin. The fact that it contracts the separated l.m. of the guinea-pig ileum, whether plexus-containing or plexus-free, and in atropine distinguishes it also from methionine-enkephalin, somatostatin, 13-norleucine motilin, bombesin, and cholecystokinin octapeptide (CCK8). This activity was partially purified, first by several partitions with ether at pH 1.4-2.2 and then by treatment at pH 4.5-5 with lead acetate. The virtual absence of ATP was confirmed by the firefly bioluminescence technique. The guinea-pig-ileum-contracting component in the partially purified extracts was destroyed by pepsin, chymotrypsin and DPCC-treated trypsin, indicating its peptide nature and distinguishing it from oxytocin, vasopressin, bradykinin, etc. In parallel assays the partially purified rabbit extracts were considerably more active than Substance P on jird or rat ascending colons than on the guinea-pig l.m., suggesting the presence of a second spasmogenic component in the extracts. In guinea-pig extracts the partially purified activity was 8-16 times greater when plexus-containing than when plexus-free, pointing to Auerbach's plexus as the source of the activity.

Evidence for a strong non-adrenergic component in the motor transmission to the rabbit vas deferens.

Isolated vas deferens preparations from 16 rabbits of the New Zealand white strain were subjected to electrical and chemical excitability under physiological conditions and under the influence of drugs. Such smooth muscle fibres were strictly confined to the terminal 3 cm. segments of the distal 'urethral' portions of the vasa deferentia. Intermittent field stimulation, at 60 second intervals, was provided by a stimulator of low output impedance under constant parameters of voltage, pulse width and frequency. Results from this investigation revealed the undermentioned anomalous but distinct findings viz: (a) the presence of a minor adrenergic component which disappeared in phentolamine but remained unaffected by prolonged exposures to phenoxybenzamine; (b) the presence of a predominantly non-adrenergic component which was totally refractory to phenoxybenzamine but suffered a weak diminution in phentolamine; and (c) the picture of a phentolamine-insensitive but twitch-inhibiting effect shared by both tyramine and noradrenaline. Rabbit vasa consistently displayed a remarkable insensitivity to the motor effects of submicromolar concentrations of the putative neurotransmitter substance i.e. noradrenaline. The indirect sympathomimetic agent, tyramine failed to elicit any contractions when employed in doses as massive as 290 micrometers. i.e. 5 x 10(-5) g/ml, except in 2 out of 14 trials when a weak phentolamine-susceptible bursts of single contractions were produced. Thus, the adrenoceptors involved in the mediation of the twitch-inhibiting effects, appear not to behave towards conventional adrenoceptor antagonists in the classified manner. It seems appropriate therefore to invoke an unknown neurotransmitter for the non-adrenergic component in this motor transmission.

Adrenergic contribution to the motor transmission in the dog vas deferens.

Isolated vas deferens preparations from 9 dogs were subjected to electrical stimulation and chemical excitation under physiological conditions. The experimental smooth muscle cylinders were confined to the terminal 3 cm portions of either the distal 'urethral' segment or the proximal 'epididymal' segment. Intermittent field stimulation, at 60 sec intervals, was provided by a stimulator of low output impedance under constant parameters of frequency and voltage and an occasionally varied pulse width. Results from this examination completely confirmed the following: (i) a high degree of contractile sensitivity to minute doses of noradrenaline (0.03--0.03 micron; 10(-8)-10(-7) g/ml) and tyramine 0.58 micron (10(-7) g/ml; (ii) an apparent ease and rapidity of extinguishing the electrically-induced twitches by either small doses of phentolamine 0.25 micron (10(-7) g/ml) or phenoxybenzamine 5.8 micron (2 x 10(-6) g/ml); (iii) a complete absence of any inhibitory action by tyramine or noradrenaline on the electrically-induced twitches. The behavior of this motor transmission of the longitudinal muscle of the vas deferens to classical alpha-adrenoceptor blocking agents and the intense susceptibility to the motor actions of the putative neurotransmitter clearly fit in with a picture of an adrenergic implication in this mode of transmission.

Species of differences in postganglionic motor transmission to the retractor penis muscle.

1 Graded motor responses were elicited in isolated, desheathed, thin strips of dog, horse, pig and sheep retractor penis (RP) muscles by field stimulation with trains of 0.2 ms pulses at 10 hertz. These twitches were shown to be neurogenic in all four species, by their prompt extinction in tetrodotoxin.2 alpha-Adrenoceptor blocking drugs abolished the contractile response to noradrenaline and to tyramine in all four species.3 Motor transmission was wholly adrenergic in the horse as in the dog RP because phentolamine rapidly abolished the electrically induced twitches in both these species; but in the pig and in the sheep RP a large proportion of the motor transmission was unaffected by phentolamine given in many times the concentration required to abolish matching noradrenaline-induced contractions.4 Because of the occurrence of periodic spasms in sheep preparations, further investigation of the phentolamine-resistant transmission was confined to the pig RP. Its responses were shown to be entirely postganglionic in origin because they were unaffected by pentolinium.5 In the pig RP a considerable proportion of the phentolamine-resistant motor transmission persisted after combined blockade of alpha- and beta-adrenoceptors by phenoxybenzamine plus propranolol and was more resistant to guanethidine and bretylium than the motor transmission to the dog RP; it was not extinguished after reserpine treatment.6 The pig RP is contracted by histamine but is rather insensitive to acetylcholine, 5-hydroxytryptamine and adenosine-5'-triphosphate. The motor transmission remained unaffected after responses to these substances were blocked by the following antagonists, given alone or in combination: mepyramine, burimamide, atropine, (+)-tubocurarine, methysergide and 2-2'-pyridylisatogen tosylate.

Extraction from ox retractor penis of an inhibitory substance which mimics its atropine-resistant neurogenic relaxation.

The inhibitory post-ganglionic transmission in the retractor penis of the ox resembles that of the dog and is not cholinergic or adrenergic. Acid extracts of this tissue have yielded an unidentified, labile, inhibitory substance which mimics the effect of its inhibitory nerves.

Effects of lysergic acid diethylamide on autonomic post-ganglionic transmission.

1. Six sites of autonomic post-ganglionic transmission were examined for susceptibility to LSD. Inhibition of transmission by LSD was confined to the three sympathetic junctions. 2. Inhibition of sympathetic transmission was maximal with short trains of pulses and declined considerably as train length was increased. 3. Evidence for a presynaptic mode of action was obtained. This was the predominant effect of LSD in the rat anococcygeus and dog retractor penis because alpha-adrenoceptor-blocking properties were feeble or absent; but in dog splenic strips LSD produced marked post-synaptic alpha-blockade. 4. The presynaptic inhibitory effect of LSD was unrelated to its 5-hydroxytryptamine-blocking property because it was not shared by methysergide. Neither was it mediated by prostaglandin release because it was unaltered by indomethacin, which suppresses prostaglandin synthesis. 5. In the rat anococcygeus and dog retractor penis larger doses of LSD induced slow contractions and, as a result of the concurrent block of motor transmission, revealed relaxation responses on transmural stimulation, caused by the excitation of sacral inhibitory fibres present in these muscles. 6. The LSD contractions were due to stimulation of post-synaptic alpha-adrenoceptors because they were abolished by phentolamine or yohimbine but were present as usual in preparations taken from reserpinized animals. 7. LSD blocked presynaptic alpha-adrenoceptors in the cholinergic motor fibres to the longitudinal muscle of the guinea-pig ileum.

The rabbit rectococcygeus: a ganglion-free parasympathetically innervated preparation.

1 Isolated, desheathed preparations of the rabbit rectococcygeus muscle were relatively insensitive to spasmogens other than muscarinic drugs. Transmural stimulation with 1-50 pulses (0.2-0.4 ms at 10 Hz) elicited graded twitches which were abolished by tetrodotoxin and were therefore neurogenic; longer pulses sometimes triggered tetrodotoxin-resistant myogenic contractions.2 Twitches elicited by 0.2-0.4 ms pulses were due to post-ganglionic excitation because they were not reduced by hexamethonium, pentolinium or dimethyltubocurarine, or by ganglion-paralyzing concentrations of nicotine.3 The acetyl- and butyryl-cholinesterase activities of the rectococcygeus were determined manometrically and could be selectively inhibited by BW 284C51 (1:5-bis-(4-allyl-dimethylammonium-phenyl)-pentan-3-one dibromide) and iso-OMPA (tetramonoisopropylpyrophosphortetramide), respectively. Single-pulse twitches were greatly potentiated in amplitude and duration only when both cholinesterases were inhibited.4 The preparations could not be made to contract by nicotine (2.1-21 muM) even after cholinesterase inhibition, suggesting an absence of ganglion-cells; with nicotine (105-210 muM) small, atropine-susceptible responses were elicited, which were non-ganglionic because they were not reduced by tetrodotoxin.5 Rectococcygeus preparations that had been treated with physostigmine released acetylcholine into the bath fluid on electrical stimulation.6 The motor transmission was paralyzed by botulinum toxin (Type A) and abolished by atropine; the block of muscarinic receptors by atropine was effective against both endogenous and exogenous acetylcholine.7 Inhibitory adrenoceptors and scanty motor alpha-adrenoceptors were detected in the smooth muscle.8 Strong inhibitions of motor transmission and of rhythmic activity were produced by noradrenaline (but not by tyramine), by isoprenaline, and, after phentolamine, also by adrenaline and phenylephrine. These inhibitions were only slightly reduced by propranolol and rather more by pindolol.9 Experiments on preparations retaining their extrinsic nerve supply suggest an absence of ganglionic relays in the last 1-2 cm of the motor nerve pathway to this muscle.10 Some contrasting properties of the adjacent caudo-anal muscle, including the poor motor responses to transmural stimulation, are described.

An inhibition of post-ganglionic motor transmission in the mammalian vas deferens by D-lysergic acid diethylamide.

1. Under certain conditions D-lysergic acid diethylamide (LSD), 10(-9)-10(-6) g/ml., exerted an immediate, prolonged and slowly reversible inhibitory effect upon the post-ganglionic motor transmission in desheathed guinea-pig vas deferens preparations.2. The most critical factor influencing this action of LSD appeared to be the train length. With short trains of less than 4 or 5 pulses the twitch inhibition produced by LSD was often total. With longer trains (5-20 pulses), the degree of inhibition declined with increase in train length. These results suggest the existence of two components in the motor response to post-ganglionic stimulation, distinguished by their susceptibility to LSD.3. The inhibition of the LSD-susceptible component was related to the dose of LSD in the range 10(-9)-10(-6) g/ml., reaching a maximum at 0.5-1 x 10(-6) g/ml. The response remnants elicited by trains of more than 5 pulses under these conditions could not be reduced further by a ten- to twenty-fold increase in LSD concentration to 10(-5) g/ml. and were in fact slightly potentiated.4. The inhibition of post-ganglionic motor transmission by LSD was not explicable on the basis of an alpha-adrenoceptor blockade because it was not associated with any reduction in motor responses to noradrenaline.5. The use of propranolol excluded mediation of the LSD-inhibition by beta-adrenoceptors.6. The LSD effect was not due to a non-specific smooth muscle depression because it was not associated with any reduction in motor responses to acetylcholine, ATP or bradykinin.7. The inhibitory effect of LSD on post-ganglionic transmission resembled that of noradrenaline in that it was antagonized by phentolamine; another alpha-adrenoceptor blocking agent, phenoxybenzamine, was less effective than phentolamine in this respect.8. The LSD-inhibition was obtained in preparations taken from reserpinized guinea-pigs.9. The inhibition of motor transmission in the vas deferens by LSD was confirmed in rats, Meriones shawii and rabbits.10. The inhibition of post-ganglionic transmission by LSD was unrelated to its ability to antagonize 5-hydroxytryptamine (5-HT), to which the longitudinal muscle of the guinea-pig vas deferens is insensitive. The more potent 5-HT antagonists, methysergide and BOL 148 were either virtually inactive or considerably weaker than LSD.

Inhibition of post-ganglionic motor transmission in vas deferens by indirectly acting sympathomimetic drugs.

1. Using field stimulation with short trains of pulses (< 10 per train), the post-ganglionic motor transmission in the mammalian vas deferens has been further analysed pharmacologically.2. In preparations taken from guinea-pigs, rats and rabbits the effects of the indirectly sympathomimetic drugs, tyramine and cocaine, could be explained entirely on the basis of the actions of released, endogenous noradrenaline.3. Tyramine produced a contraction in vasa taken from normal rats but not from normal guinea-pigs. The tyramine contraction was due to release of endogenous noradrenaline because it was not seen in preparations taken from reserpinized rats and because it was abolished in normal vasa by phenoxybenzamine or phentolamine, thus denying the supposed inaccessibility, to alpha-blockers, of the motor alpha-adrenoceptors activated by endogenous noradrenaline.4. Phenoxybenzamine or phentolamine failed to block post-ganglionic motor transmission in rat and in guinea-pig vasa.5. Tyramine strongly inhibited motor transmission in vasa taken from normal but not from reserpinized guinea-pigs.6. Tyramine produced inhibition of motor transmission in phenoxybenzamine-treated preparations taken from normal but not from reserpinized rats.7. Cocaine inhibited motor transmission in guinea-pig and in rat vasa. This effect was not due to a local anaesthetic or to a smooth-muscle depressant action because it did not occur in preparations taken from reserpinized animals.8. The inhibitory effect of tyramine or cocaine was not abolished by beta-adrenoceptor blockade with propranolol.9. Whereas reserpinization abolished the tyramine- and cocaine-inhibitions, it did not affect the inhibitory actions of noradrenaline or of PGE(2).10. Indomethacin and sodium meclofenamate, which suppress prostaglandin synthesis, did not affect the twitch-inhibiting actions of noradrenaline, tyramine or cocaine.11. These results provide further support for the conclusion that post-ganglionic motor transmission to the vas deferens is non-adrenergic in these species and assign to endogenously released noradrenaline an inhibitory role upon motor transmission.