longitudinal muscle relaxes although stimulation continues. If the circular

Transcription

1 J. Physiol. (1969), 200, With 13 text-ftgurem Printed in Great Britain AN ANALYSIS OF POSSIBLE NERVOUS MECHANISMS INVOLVED IN THE PERISTALTIC REFLEX BY S. R. KOTTEGODA* From the Department of Pharmacology, University of Oxford (Received 25 July 1968) SUMMARY 1. The effects of drugs on peristalsis and on the contractions of the two muscle coats of the isolated guinea-pig ileum in response to co-axial electrical stimulation have been studied. 2. Co-axial stimulation (0.1 msec pulses) never produces simultaneous contraction of both muscle coats. When one muscle contracts, the other either relaxes or remains quiescent. 3. The circular muscle contraction has two components. The first is reflex in origin and is brought about either by distension of the gut with increasing intraluminal filling or by the contraction of the longitudinal muscle in response to electrical stimulation at low frequency (1/sec), provided this raises the intraluminal pressure to the threshold for eliciting the circular muscle contraction. As the circular muscle contracts, the longitudinal muscle relaxes although stimulation continues. If the circular muscle contraction is prevented by reducing the intraluminal filling, or by adding a ganglion-blocking drug, the longitudinal muscle remains contracted until withdrawal of the stimulus. 4. In the presence of hyoscine, the reflex contraction of the circular muscle is unimpaired but, since the longitudinal muscle contraction is abolished, a higher intraluminal pressure is required to elicit the reflex. 5. The second component of the circular muscle contraction appears in response to electrical stimulation at high frequency (3-10/sec), upon withdrawal of electrical stimulation. This delay indicates the simultaneous stimulation of a dominant inhibitory innervation. 6. The excitatory nerves to the circular muscle require a higher frequency of stimulation than those to the longitudinal muscle, which respond to single shocks. 7. Cholinergic blocking agents (hyoscine, morphine, hemicholinium and * Commonwealth Medical Fellow. Present address: Department of Pharmacology, Faculty of Medicine, Colombo 8, Ceylon. 44 Phy. 200

2 688 S.R.KOTTEGODA botulinum toxin) antagonize the responses of the longitudinal muscle to co-axial stimulation without affecting those of the circular muscle, thus suggesting that the excitatory fibres to the circular muscle are not cholinergic. Prostaglandins (E1 and E2) selectively antagonize the circular muscle contractions evoked by co-axial stimulation. Tetrodotoxin blocks both longitudinal and circular muscle responses. 8. Dimethylphenylpiperazinium (DMPP) and 5-hydroxytryptamine (5-HT) stimulate ganglia but have no direct action on the smooth muscle of guinea-pig ileum. 9. During a maintained contraction of the longitudinal muscle in the presence of high concentrations of acetylcholine (2-5 x 10-7 to 10-6 g/ml.) a contraction of the circular muscle accompanied by a relaxation of the longitudinal muscle is elicited by distension of the gut, and by co-axial stimulation. Similar reciprocal responses are produced by 5-HT or by DMPP and they are finally blocked by DMPP. 10. These results are consistent with the hypothesis that in the myenteric plexus there exists an arrangement of nerves which ensures that the two muscle coats of the intestine do not contract simultaneously but are activated reciprocally so that when one muscle layer contracts the other relaxes or is prevented from contracting. INTRODUCTION The afferent and efferent components of the nervous pathway for the peristaltic reflex are all located in the intestinal wall, and the reflex is not dependent on extrinsic nerves reaching it from the central nervous system (Bayliss & Starling, 1899; Magnus, 1904; Langley & Magnus, 1905; Biilbring, Lin & Schofield, 1958). The reflex can be obtained in isolated preparations, and it is abolished by ganglion-blocking agents (Feldberg & Lin, 1949; Paton & Zaimis, 1949). As described by Trendelenburg (1917), the peristaltic reflex consists of an initial contraction of the longitudinal muscle in response to intraluminal distension, followed by a wave-like contraction of the circular muscle travelling in an aboral direction. This contraction of the circular muscle, during which the longitudinal muscle relaxes, expels the contents of the gut. The receptors sensitive to distension are located in or near the mucosa. Biilbring et al. (1958) demonstrated that nerve fibres originating from ganglion cells in the submucous plexus penetrate into the mucosa, and that application of local anaesthetics or any procedure which affected the integrity of the mucosa abolished the peristaltic reflex. Substances which stimulate sensory receptors in general, e.g. 5-HT, phenyl diguanide or

3 PERISTALTIC REFLEX 689 substance P, when applied to the mucosa, stimulate peristalsis (Btilbring & Lin, 1958; Beleslin & Varagi6, 1958). Radial stretching of the gut appears to be the stimulus for activating the reflex contraction of the longitudinal as well as the circular muscle coat of the gut (Kosterlitz & Robinson, 1959; Ginzel, 1959). According to Kosterlitz and his co-workers (see review by Kosterlitz & Lees, 1964), the contraction of the circular muscle follows that of the longitudinal muscle merely because a greater degree of distension of the lumen is required for activation of the circular muscle than of the longitudinal muscle. They state that the contraction of one muscle layer does not trigger the contraction of the other, since the individual contraction of either muscle layer could be elicited while the contraction of the other was suppressed by suitable blocking agents. Nevertheless, the sequence of events during the peristaltic reflex suggests that the activity of one muscle layer may modulate the behaviour of the other. When the longitudinal muscle contracts suddenly in response to distension of the gut, at or near the peak of this contraction, the circular muscle begins to contract. While the circular muscle contraction develops, the longitudinal muscle relaxes. The observations of Schaumann, Jochum & Schmidt (1953) suggest that this relaxation of the longitudinal muscle is an active process: when the longitudinal muscle was kept contracted by the application of acetylcholine, and the reflex was elicited in this condition by distension of the lumen, the response of the longitudinal muscle was a relaxation. The question arises: how is this relaxation initiated? From a teleological point of view, it would appear essential to have a mechanism whereby, during the contraction of one muscle layer, the other relaxes or is prevented from contracting. Simultaneous contraction of both muscle layers would defeat the purpose of the peristaltic reflex, which is the propulsion of the contents of the gut. The final excitatory nerves to the longitudinal muscle are mainly cholinergic (Paton, 1955; Paton & Zar, 1968). Further, the longitudinal muscle contracts readily in response to numerous substances, other than choline esters, even in the absence of nerves (Paton & Zar, 1968). In strong contrast is the unresponsiveness of the circular muscle to excitatory agents. Even its response to acetylcholine is weak, compared with that of the longitudinal muscle (Harry, 1963; Brownlee & Harry, 1963). On the other hand, it has been shown (Harry, 1962) that the contraction of the circular muscle, like that of the longitudinal muscle, in response to transmural stimulation with short current pulses at low frequency of 5/min is blocked by exposure to botulinum toxin, which specifically abolishes the function of all cholinergic nerves (Ambache & Lessin, 1955). The present investigation was undertaken, first, to ascertain whether, 44-2

4 690 S.R.KOTTEGODA during the peristaltic reflex, the activity of one muscle layer influences the behaviour of the other and, secondly, to obtain further information on the nature of the neuro-transmitter to the circular muscle. A preliminary account of some of the results has been communicated to the Physiological Society (Kottegoda, 1968). METHODS In all experiments the non-terminal guinea-pig ileum was used. Peristaltic contractions were recorded by the method of Biilbring, Crema & Saxby (1958), with electrodes suitably placed for co-axial stimulation (Paton, 1955). The tissue was suspended in Krebs solution bubbled with 95 % 02-5 %/ CO2 at 370 C. In some experiments the volume of fluid expelled by each peristaltic contraction was measured. The response of the circular muscle to drugs was examined using three different preparations: (a) a segment of the whole ileum was set up as for recording peristalsis and the effect of drugs was tested in the presence of tetrodotoxin which abolishes nerve-mediated responses without interfering with effects on the smooth muscle itself (Toida & Osa, 1965; Bulbring & Tomita, 1966; Gershon, 1967); (b) the circular muscle with the mucous membrane intact was set up as above after the removal of the longitudinal muscle together with the myenteric plexus (Rang, 1964); (c) a piece of the ileum was slipped over a glass rod and the gut was cut spirally so that the continuity of the longitudinal muscle was broken. A length of this spiral was set up in the bath, in the presence of tetrodotoxin; the responses of the circular muscle were recorded by using a frontal writing lever. Some experiments were performed on an everted segment of ileum. A Grass Stimulator S4 was used for electrical stimulation with square wave pulses of 0-1 msee duration. The frequency of stimulation was varied between 1 and 10/sec at V. Usually the stimuli were applied for 5 sec, at intervals of 3 min. In a few experiments, when muscles were stimulated directly, the pulse duration was 10 msec. Drugs used were acetylcholine bromide, angiotensin II, barium chloride, botulinum toxin (type D), bradykinin, cocaine hydrochloride, dimethylphenylpiperazinium iodide (DMPP), eserine sulphate, gamma-amino butyric acid (GABA), hemicholinium bromide (HC3), histamine acid phosphate, 5-hydroxytryptamine creatinine sulphate (5-HT), hyoscine hydrobromide, morphine sulphate, pentolinium tartrate, potassium chloride, phenoxybenzamine, phentolamine, propranolol hydrochloride, prostaglandins El and E2 (PGE1 and PGE2), substance P, tetrodotoxin (Sankyo, Japan). All concentrations refer, where applicable, to the salts. I wish to thank Dr E. W. Gill for samples of hemicholinium bromide and GABA, Dr Marthe Vogt, F.R.S., for substance P, Dr D. B. Hope for bradykinin, Dr W. Cook for angiotensin II, Dr W. van Heyningen for Botulinum toxin, Dr A. D. Smith for PGE1, and Professor J. R. Vane for PGE2. RESULTS Circular muscle In preparations where the submucous plexus was intact, but where the myenteric plexus had been removed together with the longitudinal muscle, there was no spontaneous activity and no response to distension. Moreover, co-axial stimulation (0-1 msec pulses) at 5 or 10/sec for 5 sec failed to evoke a contraction. This observation lends some support to the view that the submucous plexus is mainly, if not entirely, sensory (Biilbring et al.

5 PERISTALTIC REFLEX ). It also suggests that, by removing the myenteric plexus, the remaining circular muscle may have been denervated (Paton & Zar, 1965). In all preparations (either intact, or after removing myenteric plexus and longitudinal coat, or spiral strip) the only substances which contracted the circular muscle in the presence of tetrodotoxin ( x 10-7 g/ml.), were high concentrations of acetylcholine (10-5 to 2 x 10-5g/ml.) after previous eserinization (10-7 g/ml.) and potassium chloride (10-3 g/ml.). As was found by previous workers, barium chloride, bradykinin, histamine, substance P and 5-HT had no direct action on the circular muscle, nor did angiotensin II or GABA evoke a response. Direct stimulation (10 msec pulses) produced contraction of the muscles. Everted segment of ileum If the sensory receptors in the wall of the gut which initiate the peristaltic reflex are activated by radial stretch (Kosterlitz & Robinson, 1959; Ginzel, 1959) then eversion of the gut so that the mucous membrane lies on the outside should make no difference to the response of the gut to distension. Figure 1 shows that the everted gut, like the normal gut, responds to gradually rising filling pressure with regular co-ordinated peristaltic contractions. In Fig. la it is also seen that addition of 5-HT to the bath (so that it came into contact with the mucosa) caused a brief stimulation of peristalsis (Builbring & Lin, 1958). When cocaine was similarly applied (Fig. 1 b) the peristaltic reflex was arrested (Bulbring et al. 1958), presumably due to blockade of sensory receptors, since co-axial stimulation at that time still caused contraction of the two muscle layers. Later, however, probably after diffusion of cocaine into the myenteric plexus, the muscle layers ceased to respond to stimulation. Normal segment of ileum Effect of co-axial stimulation. When the intraluminal pressure was subthreshold for the peristaltic reflex, the sequence of contractions evoked by co-axial stimulation at a low frequency resembled that seen in response to distension. This is shown in Fig. 2. A typical response to slight distension (a) was compared with that to co-axial stimulation at 1/sec (b). Both records show, first, a powerful contraction of the longitudinal muscle. As this reached its peak, the circular muscle contracted. This contraction was associated with a dramatic relaxation of the longitudinal coat which, in Fig. 2 b, took place while stimulation continued. At the end of stimulation the circular muscle relaxed and the longitudinal muscle contraction partially recovered. It may be seen that, as the longitudinal muscle contracted, the intraluminal pressure rose slightly and the circular muscle contracted in b at the same threshold at which it had contracted in a. This suggested

6 692 S. R. KOTTEGODA that it might be a reflex contraction brought about by the strong longitudinal muscle contraction. Evidence for this interpretation is shown in Fig. 3, in which stimulation was applied at two different frequencies. At 3/sec, a, first the longitudinal and then the circular muscle contracted, upon which the longitudinal a mm; H HT sec Cocaine S S S 10-4 Fig. 1. Everted guinea-pig ileum. Records of peristaltic activity elicited by slowly rising intraluminal pressure (upper tracing), showing contractions of the circular muscle. Lower tracing: contraction of the longitudinal muscle. (a) Effect of 5-HT (105 g/ml.); (b) effect of cocaine (104g/ml.). S indicates co-axial stimulation. For description see text. Time = 10 sec.

7 PERISTALPIC REFLEX 693 muscle relaxed though stimulatipn continued. In b, however, after the intraluminal pressure had been much reduced, the longitudinal muscle contraction, though of the same magnitude as in a, failed to cause the threshold distension and thus failed to evoke the contraction of the circular muscle. Moreover, the longitudinal muscle remained contracted until stimulation stopped. When a higher rate of stimulation was used, an mm H a h 10 - o 0 Circular Longitudinal 1 5 sec Fig. 2. Guinea-pig ileum. In this and subsequent tracings the records are the same as in Fig. 1. (a) Typical peristaltic reflex response. The longitudinal muscle contracted first; it relaxed abruptly as the circular muscle contracted. (b) At sub. threshold intraluminal pressure, co-axial stimulation (1/sec, white block) evoked a longitudinal muscle contraction which was followed by a circular muscle contraction. Note that the longitudinal muscle relaxed as the circular muscle contracted although stimulation continued.

8 694 S.R.KOTTEGODA additional phenomenon was observed. In Fig. 3c stimulation at 7/sec elicited a biphasic response of the circular muscle, one component during stimulation and a second component after withdrawal of stimulation. In Fig. 3d, after the intraluminal pressure had been lowered, the first component of the circular muscle contraction did not appear and, as in b, b c d _.J 5 sec 5 sec Fig. 3. Effect of lowering the intraluminal pressure on the response to co-axial stimulation. (a) At a frequency of 3/sec, the responses of the two muscle coats resembled those in Fig. 1. Between (a) and (b) the intraluminal pressure was reduced. In (b), in the absence of the contraction of the circular muscle, the longitudinal muscle remained contracted until stimulation was withdrawn. In (c) stimulation at 7/sec produced a biphasic response of the circular muscle. Between (c) and (d) the intraluminal pressure was lowered. In (d) the initial (reflex) response of the circular muscle was absent, but the second component, which appeared with the relaxation of the longitudinal muscle at withdrawal of stimulation, remained. the longitudinal muscle remained contracted throughout the period of stimulation. The second component of the circular muscle contraction appeared on withdrawal of the stimulus, while the longitudinal muscle relaxed. The question now arose: in which way did the mechanism causing con-

9 PERISTALTIC REFLEX 695 traction of the circular muscle during stimulation differ from that causing contraction at the end of stimulation? The first component seemed to be triggered reflexly by the longitudinal muscle contraction, which increased the intraluminal pressure to the threshold level. In turn, the relaxation of the longitudinal muscle coat seemed to be the consequence of the contraction of the circular layer since, if this was absent, the longitudinal muscle did not relax. Effect of cocaine. If the initial response of the circular muscle to co-axial stimulation was reflex, it should be abolished by mucosal application of cocaine. This was found to be so. When cocaine was introduced into the fluid passing through the lumen it only blocked the first (reflex) response of the circular muscle to stimulation at 7/sec and the longitudinal muscle remained contracted throughout the period of stimulation. The circular muscle still contracted on withdrawing stimulation. Effect of ganglion-blocking agents. If the first component of the circular muscle response to electrical stimulation is not due to the stimulation of its excitatory nerves, but to a reflex activation, then this component should be abolished by ganglion-blocking drugs. The observations in Fig. 4 show that this is so. During stimulation at low frequency (a and b), in spite of a very high intraluminal pressure, DMPP abolished the circular muscle contraction and, consequently, the relaxation of the longitudinal coat, indicating that this is also reflex. During stimulation at higher frequency (c and d), both reflex responses were abolished by pentolinium, but the circular muscle contraction which followed after the end of stimulation was unaffected by ganglion block. These observations suggest that the second component may be due to stimulation of post-ganglionic excitatory fibres, but may be masked, during stimulation, by the simultaneous activation of post-ganglionic inhibitory fibres to the circular muscle layer. In the experiment shown in Fig. 5, the contractions of both muscle layers increased with increasing frequency of stimulation (a, b, c). The reflex contraction of the circular muscle (first component) was clearly separated from the second contraction by a transient relaxation, which may be interpreted as the result of simultaneous stimulation of inhibitory fibres. In the presence of pentolinium (d, e, f), the reflex component of the circular muscle response was abolished. The response to stimulation of its excitatory fibres, presumably having been prevented during stimulation, only broke through at the withdrawal of the stimulus which terminated the inhibition. Effect of hyoscine. If the contraction of the circular muscle during low frequency of stimulation is merely a reflex response triggered by the contraction of the longitudinal muscle, any drug which abolished this longitudinal muscle contraction should also abolish the reflex circular muscle

10 696 S. R. KOTTEGODA contraction. Hyoscine was used to block post-ganglionic cholinergic excitation (Paton & Vane, 1963). Figure 6a shows the normal peristaltic reflex contractions caused by slow distension. In the presence of hyoscine (b) the longitudinal muscle contractions were abolished but the circular muscle continued to respond though, in the absence of the longitudinal contraction, a much higher intraluminal pressure was required to trigger the reflex. In Fig. 6c the contractions were elicited by electrical stimulation a b c d I 1 1 ~~~~~~5 sec1 ~ ~ ~~~~7 7 5e5 sec DMPP Pentolinium Fig. 4. Effect of ganglion block. (a) Responses of the muscle coats to co-axial stimulation at a frequency of I/sec; (b) in the presence of DMPP (10-5 g/ml.) the response of the circular muscle was abolished and the longitudinal muscle remained contracted until stimulation ceased; (c) response to 7/sec; (d) in the presence of pentolinium (10-4 g/ml.) the reflex component of the circular muscle response was abolished, leaving the second component of the response unaffected (cf. Fig. 3d). When the initial circular muscle contraction was absent the longitudinal muscle did not relax until cessation of stimulation.*

11 PERISTALTIC REFLEX 697 at 1/sec. In the presence of hyoscine (d) when the same stimulus was repeated, neither muscle contracted. Since hyoscine blocked the longitudinal muscle, the circular muscle response was also suppressed. However, when the frequency was increased to 7/sec the circular muscle contracted with the withdrawal of the stimulus. This observation indicated that the post-ganglionic excitatory nerve fibres to the circular muscle may not be cholinergic. a b c d e f sec Pentolinium N Fig. 5. Effect of pentolinium on the responses to different frequencies of stimulation (1/sec, 3/sec, 7/sec). Figures in the lower row: volume of fluid expelled (ml.). In this preparation the response of the circular muscle to 1/sec was also biphasic (a, b, c): controls; (d, e,f): in the presence of pentolinium (1(h4 g/ml.), at all frequencies, the initial reflex contraction of the circular muscle was absent, and relaxation of the longitudinal muscle did not occur until stimulation was discontinued. In some preparations, in the presence of hyoscine, there was a relaxation of the longitudinal muscle during co-axial stimulation at 1/sec. With increasing frequencies this relaxation became less and a small contraction appeared which may have been due to activation of non-cholinergic excitatory fibres to the longitudinal muscle (Paton & Zar, 1966). Effect of morphine. Paton (1957) has shown that morphine reduced the increase in the output ofacetylcholine produced by co-axial electrical stimulation and hence reduced the longitudinal muscle contraction of the guineapig ileum. The effect ofmorphine on the response ofthe two muscle layers to co-axial stimulation is shown in Fig. 7. Before the application of morphine

12 698 S. R. KOTTEGODA (a), stimulation (5/sec for 5 sec at 50 V) produced the typical sequence of responses: the longitudinal muscle contracted, a small circular muscle contraction followed, associated with some relaxation of the longitudinal mm a b H m Hyoscine qe~~ 1L1 - - Ssec I 1 7 Hyoscine 5X10-7 Fig. 6. Effect of hyoscine. (a) Peristaltic response to slow distension. (b) Hyoscine (10-7 g/ml.) abolished the reflex response of the longitudinal muscle; the responses of the circular muscle continued, but a higher intraluminal pressure was necessary for triggering the contractions. (c) Different preparation; responses to stimulation at I1/sec; (d) these were abolished by hyoscine (5 x 10-7g/ml.), but (e) the response of the circular muscle to a stimulus frequency of 7/sec was not suppressed.

13 PERISTALTIC REFLEX 699 coat. On stopping the stimulation, the circular muscle contracted further, while the longitudinal muscle relaxed. In the presence of morphine (b), the longitudinal muscle contraction was smaller and insufficient to distend the Morphine --D a b c d Morphine C a Fig. 7. Effect of morphine on the responses to co-axial stimulation for different durations as indicated under each record. (a--e) are from the same preparation and the frequency of stimulation was 5/sec. In (a), before application of morphine, a typical biphasic response of the circular muscle was seen. In the presence of morphine, (b-d), only the second component of the circular muscle contraction was seen, but after stimulation for 60 sec (e), it was absent. (f) is from a different preparation (stimulation frequency 7/sec). Reciprocal waves of contraction and relaxation of the two muscle coats were observed during stimulation.

14 700 S. R. KOTTEGODA gut to the degree required for the reflex contraction of the circular muscle. The longitudinal muscle now remained contracted and the circular muscle remained quiescent throughout the period of stimulation. However, as before, on withdrawing the stimulation, the longitudinal muscle relaxed and, simultaneously, the circular muscle contracted. This contraction of the circular muscle was as large as or larger than that in the absence of morphine; it was a co-ordinated wave travelling in the aboral direction, expelling the contents of the segment of gut. Prolonging the period of a b c d e f 9 h S5 10 ~~~~~~~~~~~~~~~~~05 10 sec HC3 - Tetrodotoxin Fig. 8. Effect of hemicholinium. Responses to co-axial stimulation at frequencies of 1, 5, and 10/sec before (a-c), and after exposure to HC3 (80,g/ml. for 2 hr) (d-f). While the responses of the longitudinal muscle were greatly reduced, and the reflex component of the circular muscle contraction was abolished, the second component was unaffected. Tetrodotoxin (2 x 10-7 g/ml.) abolished the remaining responses of the two muscle coats to nerve stimulation (01 msec pulses, g), but not to direct stimulation (10 msec pulses, h). stimulation (c, d) did not alter this pattern. The volume of fluid expelled diminished slightly with increasing the period of stimulation, and eventually, after 1 min stimulation (e), the withdrawal of the stimulus failed to evoke the circular muscle contraction. The record in Fig. 7f, taken from a different preparation, shows reciprocal waves of contraction and relaxation of the two muscles during stimulation, and the typical response after stimulation stopped. Effect of hemicholinium. Hemicholinium (HC3) has been shown to interfere^with the synthesis of acetylcholine (MacIntosh, Birks & Sastry, 1956) and thereby to abolish cholinergic nervous transmission (Wong & Long,

15 PERISTALTIC REFLEX ). In the experiment shown in Fig. 8, three different frequencies of co-axial stimulation were used. With low frequency (1/sec) only the first component of the circular muscle contraction appeared during stimulation (a). With higher frequencies (b, c) the second contraction appeared also after the end of stimulation. The size increased with higher frequencies of stimulation. After exposure to hemicholinium, 80 jctg/ml. for 2 hr (d, e,f), the longitudinal muscle tone showed irregular fluctuations, and the response to electrical stimulation was small. Consequently, there was no reflex contraction of the circular muscle during stimulation. The contraction of the circular muscle, however, which occurred upon discontinuing the stimulation was not affected by HC3. It was, however, of nervous origin, since it was abolished by tetrodotoxin (g). When pulses of 10 msec duration were used to stimulate the muscle directly, the muscle contracted (h). Effect of botulinum toxin. It has been shown above that the peristaltic reflex contraction of the circular muscle evoked by radial distension as well as the contraction evoked by nerve stimulation are both resistant to hyoscine. Ambache & Robertson (1953) showed that nicotine-induced contractions of the rabbit ileum which are not blocked by atropine are abolished after exposure to botulinum toxin. Harry (1962) found that the contractions of the circular and the longitudinal muscle in response to transmural stimulation with pulses of 0 3 msec duration at a frequency of 5/min were both abolished after exposure to botulinum toxin. Hence Botulinum toxin type D (Ambache & Lessin, 1955) was used in the further analysis of the circular muscle response to nerve stimulation. Figure 9 shows that exposure to botulinum toxin (106 lethal dose (LD) mouse units/ml. for 1 hr) abolished the response of the longitudinal muscle, but failed to abolish the contraction of the circular muscle produced by co-axial stimulation (0.1 msec 100 V) at frequencies of 1, 3 and 10/sec. The circular muscle contraction started after the end of stimulation, and it was abolished by tetrodotoxin, indicating that it was caused by nerve stimulation. The pattern of this circular muscle response to nerve stimulation was the same in the presence of ganglion-blockers (Figs. 4, 5), or hyoscine (Fig. 6) or morphine (Fig. 7) or hemicholinium (Fig. 8), or botulinum toxin (Fig. 9). Since the contraction is prevented during stimulation, it might appear that the inhibitory nerves to the circular muscle are, like the excitatory nerves, resistant to the four cholinergic blocking agents used (hyoscine, morphine, hemicholinium and botulinum toxin) and that both excitatory and inhibitory nerves to the circular muscle are non-cholinergic. They require a higher frequency of stimulation for their activation than the nerves to the longitudinal muscle. The threshold frequency to which some circular muscles did not respond was about 1/sec (see Fig. 6). This may

16 702 S. R. KOTTEGODA explain the apparent disagreement between the observations with Botulinum toxin shown here and those of Harry (1962), who used a very low frequency of stimulation of 5/min. The diagram in Fig. 10 might be one way of representing the nervous pathways from the sensory structures to the two muscle layers, which may be envisaged. If, for example, the inhibitory fibres to the circular muscle were linked in some part of their course with the excitatorv fibres to the a b c d e f g Ssec Tetrodotoxin--. Botulinum toxin Fig. 9. Effect of Botulinum toxin. (a-c) Responses to co-axial stimulation at 1, 3 and 10/sec. (d-f) After exposure to 106 LD mouse units/ml. Botulinum toxin, type D, for 1 hr. The longitudinal muscle contraction was abolished. The circular muscle contraction appeared on withdrawal of stimulation, suggesting simultaneous activation of inhibitory fibres. Note shorter latency with increasing frequency of stimulation. (g) Tetrodotoxin (2 x 10-7 g/ml.) abolished the response. longitudinal muscle (pathway A), then the circular muscle would be prevented from contracting while the excitatory pathway to the longitudinal muscle was activated. The obverse of such an arrangement would be a pathway common to the excitatory nerves to the circular and the inhibitory nerves to the longitudinal muscle (pathway B), as a result of which the longitudinal muscle would relax while the circular muscle contracted. Co-axial stimulation activates the nerves to both muscle layers. Since the nerves to the circular muscle have a higher frequencythreshold than those to the longitudinal layer, they are not activated when electrical stimulation is applied at low frequency; and the

17 PERISTALTIC REFLEX 703 circular muscle does not contract until the longitudinal muscle contraction has raised the intraluminal pressure to threshold, thereby presumably causing high-frequency firing from sensory receptors. This is the mechanism which operates when the peristaltic reflex is elicited by luminal distension. On the other hand, when high-frequency stimulation is applied, all nerves are activated simultaneously. Therefore, not only the reflex contraction of the circular muscle appears but also that in response to electrical Submucous Circular Myenteric Longitudinal Mucosa plexus muscle plexus muscle + Fig. 10. Diagram of envisaged arrangement of sensory, motor (+) and inhibitory (-) nerves in the intrinsic nerve plexus which may cause the reciprocal activation of the two muscle coats. For description see text. stimulation. The latter, however, is suppressed, during the stimulus, by the simultaneous activation of inhibitory nerves and thus the contraction occurs only at the end of stimulation. The peristaltic reflex never shows this pattern, since the difference in threshold for excitation of the two muscle layers staggers their activation. In addition, feed-back connexions must exist. They have not been inserted, however, to avoid complicating the diagram. Effect of high concentrations of acetylcholine. One method of testing the hypothesis of such a reciprocal nervous arrangement would be to block one of the two reflex arcs. For this purpose the observations of Schaumann et al. (1953) were used. When the gut was kept contracted during peristalsis by the addition of high doses of acetylcholine, distension of the 45 Phy. 200

18 704 S. R. KOTTEGODA lumen still evoked a contractile response in the circular muscle while the longitudinal muscle relaxed. The observations of Schauman et al. were confirmed. Figure 11 a shows the reflex contractions of both muscle coats in response to slowly rising intraluminal pressure. After the addition of acetylcholine, which caused a maintained contraction of the longitudinal muscle, the circular muscle r mm H20 b d 40 30~~ 20 0 IA 20 sec I A I 5sc A P A Ssec Fig. 11. The effect of acetylcholine. In (a) first, peristaltic reflex response to slow luminal distension. At arrow acetylcholine (A) (2.5 x 10-7 g/ml.). Distension now produced a contraction of the circular muscle with simultaneous relaxation of the longitudinal muscle. (Note that the pressure required for eliciting the reflex contraction of the circular muscle was now higher.) (b) From a different preparation, response to co-axial stimulation at I/sec. (c) Reciprocal response to stimulation in the presence of acetylcholine (10-6 g/ml.). (d and e) From a different preparation, in the presence of acetylcholine (2.5 x 10-7 g./ml.). Addition of a second dose of acetylcholine (d) produced reciprocal response of the two muscle coats. This response was abolished by pentolinium (10-4 g/ml.) (e). contractions continued, expelling the same volume of fluid as before, while the longitudinal muscle relaxed. This experiment showed that, as in the presence of hyoscine (Fig. 6), a preceding sudden contraction of the longitudinal muscle was not necessary for the co-ordinated propulsive contraction of the circular muscle, although a higher degree of distension was required. Co-axial stimulation in the presence of acetylcholine produced similar reciprocal responses of the two muscle coats (Fig. 11, c). It should be noted that, in this condition, the circular muscle contraction was not delayed but appeared immediately at the start of stimulation, indicating exclusion of pathway A (Fig. 10). The reflex response to distension in the presence of acetylcholine, like the peristaltic reflex in the absence of drugs, and the response elicited by co-axial stimulation, was abolished by tetrodotoxin.

19 PERISTALTIC REFLEX 705 Ambache (1951) suggested that the relaxant action of nicotine in the cat intestine after exposure to Botulinum toxin could be due to stimulation of peripheral adrenergic ganglia by nicotine. In the experiments described here, the relaxation of the longitudinal muscle could not have been due to activation of intra-mural adrenergic neurones since bethanidine, propranolol, phenoxybenzamine or phentolamine did not prevent a a_c --_d ~~~bc e HT 5 sec 5-HT DMPP ACh e Botulinum toxin Fig. 12. Response to co-axial stimulation at a frequency of 7/sec (a) before and (b) in the presence of acetylcholine (10-6 g/ml.). (c) Similar response to 5-HT (10-6 g/ ml.). (d) and (e) From a different preparation: responses (d) to 5-HT (10-5glml.) and (e) to DMPP (10-5 g/ml.) after exposure to botulinum toxin (106 LD mouse units/ml.) for 1 hr. the relaxation. Kosterlitz (1967) mentioned a similar observation by Watts regarding the relaxation of the longitudinal muscle seen under the experimental conditions of Schaumann et al. (1953). When the addition of acetylcholine to the gut had revealed the reciprocal reflex response to distension, it was sometimes found that further addition of similar doses of acetylcholine produced a similar reciprocal response (Fig. lid) which eventually disappeared and was replaced by a small contraction of the longitudinal muscle. The latter change was also produced by pentolinium (Fig. l1 e). This observation may be interpreted as a nicotinic action of acetylcholine on autonomic ganglia in the reflex pathway B (d) and, after ganglion block, a small direct action on the longitudinal muscle (e). Effect of 5-HT and DMPP. If the reciprocal response to acetylcholine was a nicotinic ganglion-stimulating action, other ganglion stimulants 45-2

20 706 S.R.KOTTEGODA should produce a similar effect. The ganglion-stimulating action of 5-HT is well known (Trendelenburg, 1956a, b). Figure 12 (a-c) shows that the response to 5-HT in the presence of a high concentration of acetylcholine resembled that to co-axial stimulation and was similar to the reflex response to distension shown in Fig. 11 a. DMPP had the same effect. The action of 5-HT under these conditions was blocked by DMPP. The action of DMPP, however, was not blocked by 5-HT. On the other hand, repeated application of 5-HT blocked its own action. P 5 sec Fig. 13. Effect of prostaglandin. (a) Response to co-axial stimulation 5/sec; (b) 6 min after addition of PGE1 (2 xlo-7 g/ml.) at arrow (P). (c, d, e) Responses obtained at 3 min intervals after washing out prostaglandin, showing recovery. Repeated application of DMPP also blocked its own action. It abolished the circular muscle contraction first and, eventually, also the relaxation of the longitudinal muscle. Similarly, pentolinium abolished the circular muscle contraction evoked by DMPP in these conditions, while the longitudinal muscle relaxation remained unaffected. These observations indicate the presence of cholinergic receptors on some ganglion cells in the nervous pathway B. Nevertheless, another non-cholinergic mechanism must also participate

21 PERISTALTIC REFLEX 707 in the effects of 5-HT and DMPP, since both substances contracted the circular muscle, but not the longitudinal muscle, after treatment with Botulinum toxin (Fig. 12d, e). Thus, while 5-HT and DMPP produce their excitatory effects on the longitudinal muscle by an action through a cholinergic nervous pathway (Day & Vane, 1963; Brownlee & Johnson, 1965), the excitatory innervation of the circular muscle cannot be cholinergic. Effect of prostaglrandins. In a few experiments the effects of PGE1 and PGE2 on the responses of the muscle coats to co-axial stimulation were examined. Both substances, in a dose range between 10-i and 10-6 g/ml., caused a maintained longitudinal muscle contraction and both suppressed the contractile response of the circular muscle to electrical stimulation, without affecting the longitudinal muscle response. Figure 13 shows that PGE1 (2 x 10-1 g/ml.) raised the longitudinal muscle tone. During co-axial stimulation the muscle contracted further and, on withdrawal, relaxed. The circular muscle, however, contracted neither during nor after the end of stimulation. The effect of both prostaglandins was reversible. The first (reflex) component of the circular muscle contraction was restored earlier than the second component. The prostaglandins were the only substances used in the present investigation which antagonized the circular muscle contractions. DISCUSSION The evidence presented in this investigation -does not support the assumption that the final excitatory nervous pathway to the circular muscle of the gut, like that serving the longitudinal muscle, is cholinergic. When co-axial stimulation with short current pulses (0.1 msec) was applied, cholinergic blocking agents (hyoscine, morphine, hemicholinium and Botulinum toxin) antagonized only the longitudinal but not the circular muscle response, while tetrodotoxin, which selectively blocks nerve-mediated responses, abolished both. In the peristaltic reflex, the longitudinal muscle contraction precedes that of the circular muscle, which requires a greater degree of radial distension for the activation of its sensory receptors (Kosterlitz & Robinson, 1957, 1959). During the present investigation, this could be demonstrated by applying co-axial electrical stimulation to the ileum at subthreshold intraluminal filling pressure. The shortening of the gut by the longitudinal muscle contraction then raised the pressure sufficiently to stimulate the sensory receptors for the circular muscle. With low-frequency stimulation (1/sec), the contraction of the circular muscle was then entirely reflex in origin. While the circular muscle contracted, the longitudinal muscle relaxed

22 708 S. R. KOTTEGODA before the stimulus was withdrawn. This was also shown to be reflex in origin: if the contraction of the circular muscle was suppressed either by lowering the intraluminal pressure so that the threshold distension was never reached, or by mucosal application of cocaine, or by a ganglionblocking drug, the longitudinal muscle remained contracted throughout the period of electrical stimulation. From these observations the conclusion was drawn that excitatory nerves to the circular muscle and inhibitory nerves to the longitudinal muscle share a common pathway, which is also activated during the peristaltic reflex. The excitatory nerves to the longitudinal muscle can be activated with single shocks (Paton, 1955), but the excitatory nerves to the circular muscle require higher frequencies, the maximum effect being usually obtained with 7/sec. At frequencies between 3 and 10/sec, two components of the circular muscle contraction could be distinguished, one during the period of stimulation, the second upon withdrawal of the stimulus. The first (reflex) component could be abolished, as mentioned above, by mucosal application of a local anaesthetic, by ganglion-blocking drugs, or by lowering the intraluminal pressure, in which case the longitudinal muscle remained contracted throughout the period of stimulation. The first (reflex) circular muscle contraction could also be abolished by suppressing the longitudinal muscle contraction. Thus, in the presence of cholinergic-blocking drugs (hyoscine, morphine, hemicholinium or Botulinum toxin), only the second component of the circular muscle contraction was seen, but it did not appear until the stimulus was withdrawn. One would expect that, with the higher frequencies of stimulation, both muscle coats would be activated. However, just as in the peristaltic reflex, electrical stimulation at any frequency never evoked simultaneous contraction of both muscle layers. From this observation it was inferred that stimulation also activated inhibitory nervous mechanisms which prevented simultaneous contraction of both muscles. Evidence for the existence of an intrinsic inhibitory innervation in the gastro-intestinal tract has recently been obtained (Bennett, Burnstock & Holman, 1963, 1966; Burnstock, Campbell & Rand, 1966; Biilbring & Tomita, 1967; Bulbring & Gershon, 1967). The delayed contraction of the circular muscle might then be explained by the inhibitory transmitter being more potent but less stable than the excitatory transmitter. Evidence for the non-cholinergic nature of the excitatory as well as the inhibitory nerves to the circular muscle came from the observations that (a) the reflex contraction of the circular muscle to distension was not abolished by hyoscine and (b) both the inhibition during stimulation and the contraction after withdrawal of stimulation were resistant to all cholinergic-blocking agents, including Botulinum toxin. The observation

23 PERISTALTIC REFLEX 709 by Harry (1962) that Botulinum toxin abolished the contractions of both muscle coats may be explained by the fact that he used a very low frequency of stimulation in which the circular muscle contraction was presumably entirely reflex, evoked by the longitudinal muscle contraction. When this was abolished by Botulinum toxin, the circular muscle did not contract either. In the present work, however, circular muscle contractions were evoked by higher frequencies of stimulation and were resistant to Botulinum toxin. The diagram of possible nervous pathways in the myenteric plexus, as shown in Fig. 10, is certainly oversimplified. Perhaps the most convincing evidence suggestive of an anatomical association between the inhibitory fibres to the longitudinal muscle and the excitatory fibres to the circular muscle was obtained in experiments in which a high concentration of acetylcholine was present in the bath (Schaumann et al. 1953). This caused a maintained contraction of the longitudinal muscle. However, distension of the gut evoked a propulsive contraction of the circular muscle with simultaneous relaxation of the longitudinal muscle. A similar reciprocal response to the two muscle layers was evoked by co-axial stimulation, or by 5-HT, or by DMPP. Kosterlitz, Pirie & Robinson (1956) found that the longitudinal muscle did not respond to distension of the gut or to 5-HT in the presence of acetylcholine. This may be explained by the much higher doses (0.5 x 10-5 to 10-5 g/ml.) of acetylcholine causing a depolarizing block of the ganglia. In the present experiments repeated application of acetylcholine produced such a block. One interesting observation should be mentioned. The condition created by the presence of a high concentration of acetylcholine was the only one in which the circular muscle contracted immediately at the start of coaxial stimulation. In contrast to all other conditions there was no delay, indicating that the inhibitory innervation to the circular muscle was blocked together with the excitatory innervation of the longitudinal muscle (Fig. 10, pathway A), leaving the second reciprocal pathway (pathway B) intact. It was under these circumstances that 5-HT elicited the reciprocal response of the two muscle layers similar to the response to distension and to electrical stimulation. 5-HT has no direct action on the circular muscle of the guinea-pig ileum, its effect being abolished by tetrodotoxin. However, 5-HT may be involved as a transmitter in the intramural plexus. Such a role has also been suggested by Gershon & Ross (1966) and Bulbring & Gershon (1967). Evidence for non-adrenergic inhibitory neurones in the intestinal wall has also been put forward recently by Day & Warren (1968); it is of interest that reserpine (which depletes 5-HT stores) impaired the activation of these neurones by electrical stimulation.

24 710 S.R.KOTTEGODA While 5-HT most probably participates with acetylcholine in ganglionic transmission, neither substance could be the final excitatory transmitter to the circular muscle. This transmitter, like both inhibitory transmitters to the two muscles, remains to be identified. The only known transmitter is the main exitatory transmitter to the longitudinal muscle, acetylcholine. When this is excluded by a peripheral cholinergic blocking substance, some activation of the other three efferent pathways by co-axial stimulation can be demonstrated. In the presence of morphine, and sometimes with hyoscine, prolonged stimulation at a high frequency which maintained some tone in the longitudinal muscle, evoked waves of contraction and relaxation in the circular muscle accompanied by reciprocal waves in the longitudinal muscle. During the present investigation many substances were tested for inhibitory activity on either muscle layer. PGE1 and PGE2 were found to inhibit selectively the contractile response of the circular muscle to co-axial stimulation; both substances contracted the longitudinal muscle. Bennett, Friedmann & Vane (1967), who showed that prostaglandin E1 (PGE1) is released from the rat stomach during transmural stimulation, have suggested that prostaglandins may have a role in the control of gastric motility. Prostaglandin E2 (PGE2) has been identified in the mucosa of the human stomach by Bennett, Murray & Wyllie (1968), who observed that both PGE1 and PGE2 had an inhibitory effect on the circular muscle of the human stomach. It will be important to investigate further whether prostaglandins have a physiological role in controlling peristalsis. It is a pleasure to express my thanks to Professor Edith Builbring, F.R.S., for her encouragement and advice during this investigation which was carried out during the tenure of a Commonwealth Medical Fellowship. The work was supported by a grant from the Medical Research Council. REFERENCES AMBACHE, N. (1951). Unmasking, after cholinergic paralysis by botulinum toxin, of a reversed action of nicotine on the mammalian intestine, revealing the probable presence of local ganglion cells in the enteric plexus. Br. J. Pharmac. Chemother. 7, AMBACHE, N. & LEssIN, A. W. (1955). Classification of intestomotor drugs by means of type D botulinum toxin. J. Physiol. 128, AMBACHE, N. & ROBERTSON, P. A. (1953). The nicotine-like actions of the 3-bromo and 3:5-dibromo-phenyl esters of choline (MBF and DBF). Br. J. Pharmac. Chemother. 6, BAYLISS, W. M. & STARLING, E. H. (1899). The movements and innervation of the small intestine. J. Physiol. 24, BELESLIN, D. & VARAGI6, V. (1958). Effect of substance P on the peristaltic reflex on the isolated guinea-pig ileum. Br. J. Pharmac. Chemother. 13, BENNETT, A., FRIEDMANN, C. A. & VANE, J. R. (1967). Release of prostaglandin E1 from the rat stomach. Nature, Lond. 217, BENNETT, A., MURRAY, J. G. & WYLLIE, J. H. (1968). Occurrence of prostaglandin E2 in the human stomach, and a study of its effects on human isolated gastric muscle. Br. J. Pharmac. Chemother. 32,

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