CONSIDERATIONS OF THE EFFERENT NERVOUS MECHA NISM OF THE VAGO-VAGAL REFLEX RELAXATION OF THE STOMACH IN THE DOG

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1 CONSIDERATIONS OF THE EFFERENT NERVOUS MECHA NISM OF THE VAGO-VAGAL REFLEX RELAXATION OF THE STOMACH IN THE DOG AKIRA OHGA, YOSHIKAZU NAKAZATO AND KOJI SAITO Department of Pharmacology, Faculty of Veterinary Medicine, University of Hokkaido, Sapporo Received for publication October 11, 1969 Harper et al. (1) and Cragg and Evans (2) showed reflex relaxation of the stomach of cats and rabbits by electrical stimulation of the central cut end of the abdominal vagus nerve. One of the present authors reported that a single shock stimulation of the central end of the abdominal vagal trunk evoked a reflex discharge which was conveyed to the abdominal vicera via the other trunk (3). The transmedullary pathways involved in this reflex may exist in the immediate superficial caudal region of the obex (4). These evidences indicate that the vagus nerve provides afferent and efferent pathways for the reflex influence on the stomach. There is a considerable amount of literature which suggests that the efferent vagus nerve to the stomach contains both excitatory and inhibitory nerve fibres (See review of McSwiney (5)). Recently, the existence of the non-adrenergic inhibitory nerve fibres in the vagus was shown by Martinson (6, 7) in cats, and by Campbell (8) in guinea-pigs. Bulbring and Gershon (9) confirmed the presence of such inhibitory fibres in the vagal pathway to the stomach in guinea-pigs and mice, and suggested that 5-HT, with acetyl choline, may be a neurotransmitter at the ganglionic synapse in the inhibitory pathway. The aim of the present investigation is to obtain information concerning the nature of the efferent pathways participating in the vago-vagal gastric relaxation of the dog. Some of the results have already been described briefly (10-12). METHODS The experiments were performed on adult dogs with a weight of between 7 to 16 kg. The animals were fasted for hours before use. Surgical procedures : Seventeen dogs were anesthetized with ether and thirty-seven with pentobarbital 30 mg/kg i.v. Under these anesthesia, the trachea was cannulated in all of the animals. In twenty-eight dogs of them, the spinal cord transection was per formed between vertebra C, and C, under ether inhalation. Immediately after the spinal transection, artificial ventilation was started by means of a Starling ideal respiratory pump. In a few experiments, in addition to the spinal transection, decerebration was also per formed at the level of the midcolliculus. The chest was opened by removing the ribs from

2 7th to 13th of left side and the dorsal vagus trunk, which receives contributory branches from the right and left vagus nerves, was exposed. At the bifurcation the branch from the left vagus nerve was cut and was used for central vagal stimulation. The other branch from the right vagus was also cut and was used for peripheral vagal stimulation (Fig. 1). Bilateral greater splan chnic nerves and thoracic sympathetic chains were cut at the level of the 10th thoracic segments. The abdominal cavity was open ed by incision of the left flank, and the adrenal gland of this side and the spleen were excised or ligated completely. In most experiments the right adrenal gland was also removed. The peripheral end of the left greater splanchnic nerve was used for sympathetic stimulation. In some cases the periarterial nerves surrounding the coeliac artery were prepared for stimulation of the postganglionic sympathetic nerve. For the close arterial injection of the drugs to the stomach, a thin polyethylene tube was inserted retrogressively into a branch of the splenic artery and its tip was led to the originating point of the left gastric artery from the coeliac artery. A poly ethylene tube was also inserted in the cephalic vein for systemic administration of drugs. Recording and stimulating method: A balloon which had a capacity of approximately 300 ml was attached to the tip of a polyethylene tube and introduced into the stomach through the mouth. After inflation with about 100 ml of warmed water, the balloon was withdrawn to the cardia. In order to keep it in position (fundus and body) the connect ing tube was fixed by ligation around the oesophagus at just above the diaphragm. After wards, the tubing was connected to the flat reservoir and a small volume of water was poured into it. Afterwards it was put on the pressure transducer. The transducer was connected successively through an electronic manometer (Nihon Koden, MP-4) and a direct coupling amplifier to an ink-writing recorder (Nihon Koden, WA-205). Thus, the changes in gastric tone and motility could be displayed on a recording paper as the volume changes. FIG. 1. Schematic illustration of the position of the transections and stimulations of the nerve. L.V. : the left and R.V. : right cervical vagus nerve ; SPL : the greater splanchnic nerve ; V.V.T.: the ventral and D.V.T. : dorsal vagus trunks ; C1 and C, : level of the 1st and 2nd vertebra ; X : location of the transection of the nerves and the spinal cord ; C : electrodes for central and P : uerinheral vaeal stimulation. The reservoir-transducer system could be moved up and down freely

3 and the intragastric pressure was kept at a constant at any desired level (between 7 and 10 cm H2O). Right femoral arterial pressure was also monitored using an electronic manometer. The vagus and the sympathetic nerves were placed on a bipolar silver electrode or were passed through platinum ring electrodes (13) and were connected to a electrotonic stimulator (Nihon Koden, MSF-3R). The nerves were stimulated with a series of square wave pulses and variable strengths of 1 millisecond duration for 30 seconds or in a few cases for 1 minute delivered through an isolated unit. Frequencies were varied from 1 to 100 pulses/sec, but usually fixed at 20 pulses/sec, unless otherwise indicated. Obser vations of the responses to vagal stimulation were started at least 2 or 3 hours after the spinal transection. Throughout the experiments the animals were immobilized by re peated doses of gallamine triethiodide (Fraxedil). The drugs used were: Atropine sulphate (Merck), dl-noradrenaline hydrochloride (Sankyo), hexamethonium (Methobromin, Yamanouchi), reserpine (Serpasil, CIBA), guanethidine sulphate (kindly supplied by CIBA) and bretylium tosylate (kindly supplied by Chugai). RESULTS 1. Gastric responses to stimulation of the central and peripheral ends of the abdominal vagus nerve Stimulation of the central and of the abdominal vagus produced mostly a distinct DG. 2. Effect of intravenous atropine on the gastric responses to stimulation of the vagus nerve. A, control responses, B, after the administration of atropine (0.2 mg/kg i.v.), Upper trace : time marker, and half blank in the trace indicate duration of stimula tion ; middle trace (G.R.) : gastric respose, i.e., gastric volume change ; lower trace (B.P.) f : femoral blood pressure ; V.P.S.: stimulation of the peripheral end of the branch rom the right vagus to the dorsal vagus trunk (peripheral vagal stimulation) ; V.C. S : stimulation of the central end of the branch from the left vagus to the dorsal vagus trunk (central vagal stimulation) ; stimulation 20 pulses/sec, 1 msec duration, for 30 seconds.

4 relaxation of the stomach with frequencies at least above 5 pulses/sec (Fig. 2 A). In addi tion to the spinal transection, sometimes further decerebration was performed. There was, however, no significant difference between the vagal reflex relaxations of the stomach before and after decerebration. In some experiments, a transient increase of tone or a slight contraction also appeared in the initial phase of stimulation. In a rare case, a slight contraction only was observed during the period of stimulation at a low strength. The response to central vagal stimulation was completely abolished by sections of bilateral cervical or ventral vagal trunk (Fig. 3 A, B). In some cases, the relaxation was reduced considerably after only contralateral vagotomy as shown in Fig. 3 B, but in others these was little reduction. Therefore, it is apparent that gastric relaxation is produced by a vago-vagal reflex through the supra-spinal centre and the efferent vagal pathways are contained in bilateral cervical vagus trunks. On the other hand, stimulation of the peri pheral end of the abdominal vagus caused a contraction which was followed by various degrees of relaxation of the stomach as has been reported by many authors (Fig. 2 A). The gastric response to central vagal stimulation did not change significantly after an intravenous injection of atropine ( mg/kg). On the contrary, after atropine, FIG. 3. Effect of vagotomy on the gastric inhibitory responses to stimulation of the vagus nerve in atropinized dogs. A, effect of the transection of the ventral vagus trunk on the responses caused by central (V.C.S.) and peripheral (V.P.S.) vagal stimulation. B, effects of contralateral (Cont. cerv.) and ipsilateral (Ipsi. cerv.) cervical vagotomy on the relaxation of the stomach caused by central vagal stimulation (V.C.S.). Dose of atropine : 0.2 mg/kg i.v. See text.

5 the contraction caused by peripheral vagal stimulation was abolished and invariably revers ed to relaxation as was reported by Martinson (14) in cats and by Campbell (8) in guinea pigs (Fig. 2 B). Usually systemic arterial pressure fell during both stimulations. The fall of blood pressure upon central vagal stimulation was followed frequently by a gradual and long lasting elevation (Fig. 2 A, B). In most of the experiments, the stomach relaxed following the administration of atro pine. Under such conditions, it became more difficult to observe the inhibitory effect induced by vagal stimulation. In these cases, a large dose of bethanechol (1-2 mg/kg i.v.) was repeatedly administered for the purpose of recovering the tone of the stomach. These doses of bethanechol resulted in the recovery of the tone up to its initial level or more, and produced a favourable condition for observing the gastric relaxation in the atropinized animal. 2. Stimulation frequency-response magnitude relation Relaxations of the stomach in response to stimulation of the vagal nerves with a series of frequencies are illustrated in Fig. 4 A, B. In order to compare the magnitude of these relaxations, the area in each relaxation was calculated. From these values the frequency response curves as shown in Fig. 5 were plotted. FiG. 4. Gastric relaxations in response to stimulation of the vagus nerve at a series of frequencies. A, central vagal stimulation in non-atropinized dogs, B, peripheral vagal stimulation in reserpine plus atropine treated animals, figures : frequencies of stimula tion, in pulse/sec. Pulse duration and intensity of stimulation are kept constant.

6 Central vagal stimulation with 1-2 pulses/sec caused only a slight relaxation of about 5 % of the maximal response. The magnitude of the relaxation augmented as the frequency increased. In most cases, this response attained a maximum at pluses/sec. Even if the frequency of stimu lation increased above 50 pulses/sec, there was no further augmentation of the magni tude of the relaxation. After treatment with atropine, there was some reduction of the FiG. 5. The relation between the frequency magnitude upon stimulation at a low fre of stimulation of the vagus nerve and quency below 7 pulses/sec. However, the the magnitude of the responses 0-0 : central vagal stimulation in unatropi stimulation frequency-response relation at nized animal ; Q---0 : peripheral the frequency mentioned above has not vagal stimulation in atropinized ani shown any significant difference from that mal ; Each point represents the mean value of 5 experiments, and vertical obtained before treatment with atropine. lines indicate the standard error ; On the other hand, peripheral vagal ordinate : magnitude of relaxation as stimulation in atropinized animals caused a a percentage of the area of relaxation caused by each vagal stimulation with more distinct relaxation at 1 pulse/sec. The 20 pulses/sec ; Abscissa : frequency of magnitude of this relaxation was about 20% stimulation, in pulses/sec (logarithmic of the maximal response. The relaxation scale). See text. increased its magnitude following the increase of the frequency of stimulation until the response reached a maximum at pulses/sec. With stimulation at frequencies above 50 pulses/sec, the magnitude of the relaxation declined. Although the species were different, these relationships were closely similar to those described by Campbell (8). 3. Time courses of relaxations Time courses of the relaxations of the stomach caused by central and peripheral vagal stimulations with a series of frequencies were estimated and are graphically shown in Fig. 6. The following descriptions were made concerning the response to stimulation at 20 pulses/sec which ordinarily caused the maximum relaxation. Relaxation in response to central vagal stimulation started after the onset of stimula tion and increased its amplitude rapidly. Although the latent period of the relaxation was not estimated accurately, there was a somewhat longer delay than that of peripheral vagal stimulation. The development of the relaxation continued after cessation of stimu lation. Usually the response reached a maximum about 30 seconds after the cessation of stimulation. Afterwards, the recovery of the tone started, and developed until it return ed to the initial level. The time required for recovery ranged rather widely from 3 to 8 minutes (Fig. 6 A). After treatment with atropine, the recovery became somewhat steeper than that observed before (Fig. 6 B). Relaxation in response to peripheral vagal stimulation in the atropinized animal

7 started and rapidly developed immediately after the onset of stimulation. In most cases, the development of the relaxation terminated at the cessation of stimulation. Consequently, the maximum relaxation was attained just at the end or at least within 10 seconds after the cessation of stimulation. The recovery of this response started ab ruptly and was completed at least within 1-2 minutes (Fig. 6C). 4. Effects of drugs on the vagal relaxation of the stomach Recently, Muryobayashi et al. (15) showed that the cervical vagus and gastric branch in dogs contained green-fluorescent materials representing catecholamines and they concluded that the vagus contained adrenergic components. We attempted to determine whether the adrenergic mecha nism contributes to the incidence of the vagal relaxation of the stomach in atro pinized animals or not. a) Effect of reserpine: The effects of reserpine on the gastric relaxations produced by the central and peripheral vagal stimula tion were observed in 10 experiments using the animal which received a total of mg/kg s.c. of reserpine. In general, the FIG. 6. The time course of the gastric re laxation in response to the stimulation of the vagus nerve with a series of frequencies. A, central vagal stimula tion in unatropinized, and B, in atro pinized animal, C, peripheral vagal stimulation in atropinized animal. Each line is traced by plotting the mean value (5 experiments) of ampli tude of the relaxation every 10 seconds during the course of the response. Figures in each trace : frequency of stimulation ; Ordinate : amplitude of relaxation, as a percentage of amplitude basal tone of the stomach in the reserpinized at the terminal point of stimulation ; Abscissa : time in second. dog was higher than that of the untreated one. Even in the dog which received the largest dose of reserpine, the stimulation of the central and peripheral vagus nerve still caused the relaxation of the stomach. There was no evidence which showed fatigue of the vagal inhibitory response in the reserpinized preparation as reported by Bulbring and Gershon (9). As shown in Fig. 7 the relation between the frequency of central vagal stimulation and the magnitude of the gastric relaxation in the reserpinized dog did not differ signifi cantly from that obtained in the untreated animal. Peripheral vagal stimulation also caused distinct relaxations in a reserpine plus atropine treated animal, but upon stimu lation at a low frequency (1-10 pulses/sec) their magnitude was rather larger than those in unreserpinized ones. b) Effect of cocaine : There are many evidences which support the view that cocaine

8 potentiates the action of catecholamine or sympathetic nerve stimulation by inhibiting the rapid inactivation of catecholamines by nerve uptake. If the sympathetic mecha nism contributes to the vagal relaxation, cocaine should be expected to augment this response. The responses to stimulation of the peripheral splanchnic or periarterial nerve were used as a control of the sympa thetically induced responses. As described in the following paper (16) the responses to the sympathetic nerve stimulation in the atropinized dogs were variable, i.e. a relaxa tion, contraction or contraction followed by relaxation. These responses were mediated by adrenergic receptors irrespective of their direction, contraction or relaxation. The tonus of the stomach and the systemic blood pressure were increased by the intrvenous injection of 3-5 mg/kg of cocaine for vary FiG. 7. The relation between the frequency of stimulation of the vagus nerve and the magnitude of the responses in a reserpinized animal. Each point re presents the mean of 5 experiments. In five dogs, two are treated with reserpine 1 mg/kg/day s.c. for four days and an other three are treated 1 mg/ kg/day s.c. for the first day and 0.5 mg/kg/day s.c. for the following three days. Observations are carried out at the fourth day. FIG.8. The effects of cocaine on the responses to stimulation of the vagus, periarterial nerve stimulation and noradrenaline in an atropinized dog. A, control response ; B, after the administration of cocaine, Note that cocaine (3 mg/kg i.v.) augmented the response produced by periarterial nerve stimulation (Peri. S) and by intragastric arterial injection of 5 pg noradrenaline (NA) without any significant modification of the vagal response.

9 ing periods and gradually declined to the normal level. After cocaine the relaxation of the stomach and rise of the blood pressure caused by both sympathetic nerve stimulation and intraarterial injection of 5 ieg of noradrenaline were augmented. On the other hand, the inhibitory responses produced by vagal stimulation were never influenced by this agent (Fig. 8). c) Effect of adrenergic neurone blocking agents : The effect of bretylium and guanethidine on the sympathetically induced responses and the inhibitory vagal responses were compared. Intravenous injection of 7-15 mg/kg of bretylium or mg/kg of guanethidine abolished or greatly reduced any types of the stomach responses and pressor response to stimulation of the sympathetic nerve. However, this dose of bretylium caused slight or no reduction in the response to central and peripheral vagal stimulation. Fig. 9 shows that 5 mg/kg FIG. 9. The effects of adrenergic neurone blocking agents on the responses to stimulation of the vagus and sympathetic nerves in atropinized dogs. Note that A : guanethidine (5 mg/kg i.v.) and B : bretylium(10 mg/kg i.v.) abolished the sympathetically induced responses without any significant reduction of the vagal responses. Sp1.P.S.: peripheral splanchnic nerve stimulation. of guanethidine or 10 mg/kg of bretylium, abolished the sympathetically induced responses without any significant reduction of the vagal responses. Occasionaly, higher doses of bretylium (17-30 mg/kg) also caused some reduction of the vagal responses. Furthermore, relaxations produced by central and peripheral vagal stimulations were not reduced after treatment with both phenoxybenzamine and pronethalol which abolished the sympathe tically induced responses, as described in the following paper (16). d) Effect of hexamethonium : The effects of hexamethonium were studied with the 10 atropinized dogs. Five to 22 mg/kg of hexamethonium was injected intravenously. The relaxation of the stomach caused by central vagal stimulation was abolished in seven of them, or significantly reduced in three by 5 mg/kg of the drug.

10 min FiG. 10. Effect of hexamethonium on the responses to stimulation of the vagus in an atropinized dog. Note that the inhibitory response produced by central vagal stimu lation (V.C.S.) was abolished and that produced by peripheral vagal stimulation was reduced but never abolished by hexamethonium (5 mg/kg i.v.). The inhibitory response produced by peripheral vagal stimulation was also reduced by these doses of hexamethonium, but was never abolished in any of the experiments (Fig. 10). DISCUSSION The evidence which has been described suggests that the vago-vagal relaxation was due to the reflex excitation of the non-adrenergic inhibitory fibre in the vagal pathway to the stomach rather than the depression of the central vagal excitatory tone by the afferent volley. The ganglion cell located in the vagal efferent pathway and the cell might mainly received cholinergic preganglionic fibres an supply the non-adrenergic inhibitory fibre to the stomach. Several investigators have described a reduction of tone or the inhibition of the motility of the fundus and body of the stomach in response to stimulation of the central end of the abdominal vagus (1, 2) or to stimulation of the supra-spinal centres (17-19). Babkin and Kite (20) also reported the inhibition of motility of the pyloric antrum upon cortical stimu lation and central stimulation of the vagus, splanchnic or other somatic nerves. They postulated that cortical and afferent vagal nerve stimulations caused the depression of the parasympathetic centres rather than the stimulation of the hypothetical vagal inhibi tory neurons or of the sympatho-adrenal system. Eliasson (17, 18) also had the same view as them, namely that the mechanism of the inhibition of the gastric motility is produced by electrical stimulation of certain cortical and subcortical structures. In this experiment, the spinal cord was transected at the highest level and the greater splanchnic nerves were cut and the adrenal gland was completely removed in bilateral. Therefore, it is evident that the sympatho-adrenal system may not be responsible for the gastric relaxation in response to stimulation of the central or peripheral vagal end as sug gested by Babkin and Kite (20). Then what mechanisms are involved in the relaxation of the stomach caused by central

11 vagal stimulation? The information provided by this experiment may be of advantage in regarding this relaxation as being due to the reflex excitation of the hypothetical vagal inhibitory neurons to the gastric muscle rather than the inhibition of the motor neurons in the supra-spinal centres. The reasons are as follows. If the relaxation of the stomach caused by central vagal stimulation is due to only the reflex inhibition of the vagal tonic motor impulses, this response should never be found after the excitatory transmission from the vagus to the stomach is blocked by atropine. However, central vagal stimulation still caused relaxation after treatment with atropine, when the stomach regained its tonicity irrespective of whether the tonus rose with a large dose of bethanechol or recovered by its own intrinsic ability. After atropine, although the amplitude of the relaxation caused by this stimulation with low frequencies was slightly reduced and the recovery from the relaxation became somewhat steeper, the maximum relaxation was attained within the same range of frequencies. Martinson (21) observed that the stimulation frequency response relation of a vagally induced relaxation of the stomach in an atropinized cat differed little, whether the basal tone rose with carbachol or not. In this experiment, the same result was obtained using bethanechol. The accurate mechanism of rising of the gastric tone with a large dose of bethanechol after atropine is obscure. However, it is improbable that this is the result of the recovery of cholinergic excitatory transmission from the vagus to the gastric smooth muscle, because throughout this experiment there was no sign of the recovery of the vagal motor effect from the blocking effect of atropine. It has been reported that the gastric tone increased following the section of all the abdominal vagus (1) or of both cervical vagus nerves (22, 23). A similar phenomenon was also noted frequently in the course of the present experiment. This may be the result of the removal of the vagal inhibitory influence (1). Therefore, tonic vagal inhibitory impulses to the stomach may be delivered from the hypothetical inhibitory neurons in the central nervous system and it is also assumed that this inhibitory system is further activated by vagal af ferent inflow. Previously, Nakazato (3) showed that a single shock stimulation of the central end of the abdominal vagus trunk evoked a reflex discharge which was conveyed to the abdo minal viscerea via the other one. The transmedullary pathways involved in this reflex may exist in the immediate superficial caudal region of the obex. In the present experi ment, the reflex relaxation of the stomach was produced after the transection of the brain at the level of the midocolliculus. From these results, it is reasonable to speculate that the vagal reflex discharge described by Nakazato (3) may indicate at least partly the reflex activation of the hypothetical vagal inhibitory neurons. This viewpoint was supported by the experiment of Ohga et al. (10). They showed that the contraction of the stomach in response to the peripheral vagal stimulation or intragastric arterial injection of ACh (5-20 ag) almost completely disappeared during the period of central vagal stimulation. This inhibitory effect was completely abolished after bilateral vagotomy. Accordingly, it is reasonable to assume that central vagal stimulation accelerates the release of certain inhibitory substances from the inhibitory nerve terminals, which antagonized the action

12 of exogenous or endogenous ACh. What is the efferent mechanism of the vago-vagal reflex which participates in the initiation of the reflex relaxation of the stomach? Langley (24), McSwiney and Robson (25) and Harrison and McSwiney (26) have shown that the vagus nerve contained in hibitory nerve fibres to the stomach. Using isolated vagus-stomach preparation Greff et al. (27) and Paton and Vane (28) have postulated that the vagally induced relaxation of the stomach was due to the stimulation of the sympathetic nerve fibres included in the vagus. In recent experiments, however, the existance of the non-adrenergic inhibitory nerve fibre in the vagal inhibitory innervation of the stomach has been strongly suggested by Martinson (6, 7) in the cat and by Campbell (8) and Bulbring and Gershon (9) in the guinea-pig. It is still, however, questionable whether the contribution of the sympathetic mechanism to the initiation of the vagal relaxation of the stomach is wholly excluded. Recently Muryobayashi et al. (15) demonstrated that the existence of the adrenergic fibre in the vagus and its gastric branch in the dog was shown by the fluorescent histochemical technique. In our previous experiments (29), whether the venous blood from the stomach during gastric relaxation by vagal stimulation contained catecholamine or not was tested by means of Vane's superfusion method (30). The experiments revealed that the venous blood did not contain more than about 5 x 10-9 g/ml of noradrenaline. The present evidence also showed that the relaxation of the stomach produced by the central or peri pheral vagal stimulation behaves quite similary after treatment with adrenergic neurone and receptor blocking agents. That is, the effect of sympathetic nerve stimulation was blocked by bretylium, guanethidine and also combined use of pronethalol and phenoxy benzamine, while the inhibitory responses induced by central or peripheral vagal stimula tion were not affected (16). Both Martinson (6) and Campbell (8) have shown that sympathetic nerve stimulation caused relaxation of the stomach in the atropinized cat and in the isolated, atropinized guinea-pig stomach. They have shown that there are also differences between the frequency-response relation and the time course of the response to stimulations of the vagal and sympathetic nerves. In the atropinized dog, however, 3 types of gastric response (relaxation, contraction and contraction following relaxation) were observed in response to sympathetic nerve stimulation. Therefore, it is impossible to make an accurate comparison between the responses to the stimulations of the vagus and to the stimulation of the sympathetic nerves. Although the magnitude of the gastric response to central vagal stimulation was small at a low frequency, the maximum response was obtained at the same range of frequency and the maximum rate of relaxation was also approximately equal to that after peripheral vagal stimulation. The relation between the frequency of the stimulation and the magni tude of the relaxation caused by peripheral vagal stimulation was similar to those described by Campbell (8), although the species were different. From these results, we agree with the view of Martinson (6, 7), Campbell (8) and Bulbring and Gershon (9) that the vagal inhibitory nerve are not adrenergic, and the fibre may participate as the efferent pathway of the vago-vagal reflex arch.

13 The central vagal stimulation also caused a falling of the systemic arterial pressure which was sometimes followed by long lasting elevation. This initial fall of blood pressure coincided with the relaxation of the stomach. These events were comparable to those observed in peripheral vagal stimulation. The later elevation which started usually after the relaxation of the stomach developed considerably. On the other hand, stimulation of the sympathetic nerve always produced a rise of blood pressure depending on the stimulus strength. These facts also may support the view that the relaxation of the stomach is not due to the reflex activation of the sympathetic system. Some workers reported that there is a cholinergic synapse in the efferent vagal in hibitory pathway of the stomach (6-9, 27, 28). The present results showed that the re sponse caused by vago-vagal reflex in the atropinized preparation was entirely sensitive, but that caused by the peripheral vagal stimulation was partially sensitive to ganglion blockade by hexamethonium. The blocking action of hexamethonium on the vago-vagal relaxation was more effective after atropine administration. Martinson (5) also showed that atropine enhanced the blocking effect of hexamethonium on the gastric relaxation induced by efferent vagal stimulation. Trendelenburg (31) reported that both nicotinic and muscarinic receptors are involved in the ganglionic transmission. It is likely that both receptors are involved in the ganglionic transmission in the vagal inhibitory pathway to the stomach. These results suggest that the afferent volley from the abdominal vagus selectively induces reflex excitation of the preganglionic elements in the efferent vagal inhibitory pathway. The ganglion cells in this pathway may receive cholinergic preganglionic fibres and supply non-adrenergic postganglionic fibres to the stomach. The most important difference between gastric relaxations caused by central and peripheral vagal stimulation is the susceptibility to hexamethonium. In accordance with the fact (32) that hexamethonium (15-30 mg/kg i.v.) had no effect on the reflex discharge evoked by the central end of the abdominal vagus trunk, it is unlikely that 5-22 mg/kg of hexamethonium abolished the vago-vagal reflex relaxation of the stomach by its blocking action on the central synapses. Accordingly, the ganglion cell which is very susceptible to hexamethonium should be located in the efferent pathway of this vago-vagal reflex. On the other hand, the relaxation of the stomach caused by the peripheral vagal stimula tion was rather resistant to the ganglion blocking action of hexamethonium. What mecha nisms are invovled in the hexamethonium resistant efferent pathway of the vagal relaxa tion? Bulbring and Gershon (9) proposed the hypothesis that 5-HT, with ACh may be a neurotransmitter acting on the same ganglion cells in the vagal inhibitory pathway to the stomach. Alternatively, it might be possible that the stimulated peripheral vagal nerve contains some of the inhibitory fibres which directly innervate to the smooth muscle of the stomach. SUMMARY The nature of the efferent pathway participated in the initiation of the vago-vagal reflex relaxation of the stomach was investigated in dogs in which bilateral splanchnic

14 nerves were transected and the adrenal glands and the spleen were removed. 1. Stimulation of the central end of an abdominal vagus branch produced mostly a relaxation of the stomach whether the animal was treated with atropine or not. The reflex relaxation of the stomach was also produced after the spinal cord transection (C1 C,) and further decerebration. Peripheral vagal stimulation invariably caused a relaxa tion of the stomach in atropinized animals. 2. When a low-frequency of stimulation (1-5 pulses/sec) was used, the peripheral vagal stimulation was more effective than the central vagal stimulation in causing the relaxation of the stomach. The maximum relaxation was attained with about the same range of frequencies (20-50 pulses/sec) in both vagal stimulations. 3. The onset and recovery of the responses caused by central vagal stimulation were slower than those by peripheral vagal stimulation. 4. Reserpine (2.5-4 mg/kg s.c. in total) and cocaine (3-5 mg/kg i.v.) did not affect any significant modification of the inhibitory vagal responses. 5. Bretylium (7-15 mg/kg i.v.) or guanethidine (2.5-5 mg/kg i.v.) abolished or greatly reduced the stomach responses and pressor response to sympathetic nerve stimula tion, whereas the gastric relaxation upon central or peripheral vagal stimulation was only slightly reduced. 6. A low dose of hexamethonium (5 mg/kg i.v.) markedly reduced or abolished the gastric relaxation cuased by central vagal stimulation. However, the inhibitory response produced by peripheral vagal stimulation was reduced but was never abolished even after a high dose of this drug (22 mg/kg i.v.). 7. It was suggested that the afferent volley from the abdominal vagus selectively induced reflex excitation of the preganglionic elements in the efferent vagal inhibitory pathway. The inhibitory ganglionic cell in this pathway may receive mainly cholinergic preganglionic fibres and supply non-adrenergic postganglionic fibres to the stomach. NOTE ADDED IN PROOF During the period of the preparation of this manuscript, Jansson (33) has reported that the vago-vagal reflex relaxation of the stomach in the cat was caused at least in part by a non-adrenergic mechanism. Acknowledgements: We wish to thank Mr. N. Sasaki for his help in the initial stage of this study and to Mr. T. Kitamura for his technical assistance. We are indebted to Dr. A. Shioya of Research Laboratories, Chugai Pharmaceutical Co. Ltd., for kindly supplying bretylium, and to CIBA products Ltd., for guanethidine. REFERENCES 1) HARPER, A.A., KIDD, C. AND SCRATCHERD, T.: J. Physiol. 148, 417 (1959) 2) CRAGG, B.C. AND EVANS, D.H.L.: Exp. Neurol. 2, 1 (1960) 3) NAKAZATO, Y.: J. Physiol. Soc. Japan. 30, 172 (1968) 4) NAKAZATO, Y.: Unpublished data 5) MCSWINEY, B.A.: Physiol. Rev. 11, 478 (1931)

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