PERISTALSIS AND ANTIPERISTALSIS IN THE CHICKEN CAECUM ARE MYOGENIC

Quarterly Journal of Experimental Physiology (1984) 69, 161-170 161 Printed in Great Britain PERISTALSIS AND ANTIPERISTALSIS IN THE CHICKEN CAECUM ARE MYOGENIC J. P. HODGKISS Agricultural Research Councils Poultry Research Centre, Roslin, Midlothian EH25 9PS (RECEIVED FOR PUBLICATION 30 MARCH 1983) SUMMARY Peristalsis in the chicken caecum was investigated by recording the responses of isolated segments of the caecum to either fluid distension using a modified Trendelenburg apparatus or by monitoring circular muscle activity following localized radial distension of adjacent areas. Raising the intraluminal pressure did not initiate peristalsis except in the presence of tetrodotoxin, local anaesthetics and high concentrations of phentolamine. The effect of these drugs was reversible. Localized distension generally produced either a small relaxation of the circular muscle or no response at all on both sides of the site of distension. After exposure to tetrodotoxin one, or more (usually several), rhythmic propogating contractions were initiated at the point of distension. These results suggest that intrinsic inhibitory neurones are present in the caecum and may be triggered by distention. The possible role of these inhibitory neurones in the emptying and filling of the caecae is considered. INTRODUCTION Since the classical studies of Bayliss & Starling (1899, 1900, 1901) abundant evidence has accumulated for ascending excitatory and descending inhibitory neuronal mechanisms in the mammalian intestine (see Costa & Furness, 1982). These mechanisms form the basis of the peristaltic reflex which serves to propel ingesta from oral to anal poles of the gut. Studies on isolated preparations of mammalian intestine have clearly demonstrated that peristalsis is mediated by intrinsic nerves having a fixed polarity (Frigo & Lecchini, 1970; Crema, 1970; Frigo, Torsoli, Lecchini, Falaschi & Crema, 1972; Furness & Costa, 1976; Costa & Furness, 1976, 1982). Thus, blockade of nervous conduction or ganglionic transmission effectively inhibits peristalsis (Feldberg & Lin, 1949; Paton & Zaimis, 1949; Kosterlitz, Pirie & Robinson, 1956; Kosterlitz & Robinson, 1957; MacKenna & McKirdy, 1972). Similar studies on the oesophagus and rectum of the chicken have failed to confirm these findings. There is evidence that in these regions of the avian gut peristalsis is myogenic (Bartlet, 1973; Mizuta, Ohashi, Okada & Takewaki, 1980). In this study peristalsis and reverse peristalsis (antiperistalsis) in the blind-ended chicken caecum have been investigated in an attempt to characterize the underlying mechanism in this structure. METHODS Brown Leghorn chickens of both sexes hatched and reared at the Poultry Research Centre were used in this study when they were between 12 and 24 weeks of age. The birds had free access to food and water up to the time of experimentation. They were killed by cervical dislocation and an abdominal incision was made to expose the caecae. 6 EPH 69

162 J. P. HODGKISS The caecum of the fowl is formed of three parts though only two regions can be distinguished with ease (McLelland, 1975). The proximal segment (or base of the caecum) originates from the ileocaecolic (i.c.c.) junction. It has a narrow lumen and a relatively thick wall and extends 4-6 cm from the i.c.c. junction before giving way to the distal segment. This has a much wider lumen, thinner walls and a length of 8-10 cm. Four to five centimetres of either the proximal or distal caecum were removed and placed in Krebs solution containing (mm): NaCl, 118; KCI, 4 7; CaCl2, 2-5; NaH2PO4, 1-2; MgSO4, 12; NaHCO3, 25 and glucose, 11. This solution was also used to gently flush out the contents of the caecum. The response of the caecum to increases in intraluminal pressure was studied using the apparatus described by Kosterlitz, Pirie & Robinson (1956) except that conventional pressure transducers were used instead of condenser manometers and changes in the tension of the longitudinal muscle were recorded with an isometric transducer (Dynamometer UFI). The proximal caecum was mounted with either the i.c.c. end or distal (sac) end nearest to the intraluminal pressure transducer. The preparation was suspended in a 100 ml organ bath containing Krebs solution at 38 C which was gassed with 5% CO2 in 02 and subjected to a longitudinal tension of 1-1 5 g. During peristalsis the fluid in the caecum was ejected through a 100 mm length of capillary tube of 1 mm i.d. The ejected fluid caused a rise in pressure in an air-filled glass jar (volume = 2 5 1). The response of the caecum to localized distension was studied using the technique described by Costa & Furness (1976). The required segment ofcaecum was slipped over a stainless-steel rod secured to the bottom of the organ bath. The wall of the caecum was distended over 5-7 mm of its length by tying weights (5-40 g) to a bent stainless-steel pin inserted through the wall at either of two locations (see Fig. 2, Costa & Furness, 1976). Circular muscle activity was recorded auxotonically by gripping a small area of the wall with a frog heart clip connected via a 8 g. cm-' spring to an isometric transducer. In all experiments the preparation was equilibrated in the organ bath for 1 h, and exposed to drugs for at least 10 min before data were collected. Stock solutions of the following drugs were used, guanethidine sulphate (Ciba), hexamethonium bromide (Sigma), methysergide bimaleate (Sandoz), DL-propranolol HCI (Sigma), phentolamine mesylate (Ciba), procaine HCI (Sigma), cinchocaine HCI (Sigma), naloxone HCI (Endo Laboratories) and tetrodotoxin (TTX) (Sigma) made up in distilled water. Final bath concentrations are given in terms of the salt except for tetrodotoxin. The drug solution did not alter the bath volume by more than 100. RESULTS The results were obtained from thirty-two preparations from as many animals. Twenty-one preparations were studied in the modified Trendelenburg apparatus, the other eleven were mounted in the apparatus of Costa & Furness (1976). General observations Exposure of the caecae was achieved by moving the small intestine to one side and this showed them to be highly active structures. This activity was undoubtedly the result of mechanical stimulation resulting from displacing the overlying intestines. When the wall of the proximal caecum was lightly touched the immediate response was a localized ring of contraction at the point of stimulation. From this two waves of contraction propogated for variable distances in both directions along the caecum. Often the contents of the caecum were observed being discharged into the rectum as a contraction wave arrived at the i.c.c. junction. The response to fluid distension in the modified Trendelenburg apparatus After equilibration in the organ bath seven of the Trendelenburg preparations showed spontaneous relaxations of the longitudinal muscle when subjected to a tension of 1-1 5 g. A further four preparations exhibited both spontaneous contractions and relaxations. The

PERISTALSIS IN THE CHICKEN CAECUM 163 39 mmh20 67 84 128 167 A B C D E TTX, 0 1 ug.ml- F HH J Fig. 1. Responses of the proximal caecum to raising intraluminal pressure from 0 to various levels up to 167 mmh20 before (A-E) and after (F-i) adding TTX (10-7 g.ml-1) to the bath. Each record shows the longitudinal tension (top trace, increase upward), pressure in the 2 5 1 air space (middle trace, increase downward) and intraluminal pressure (bottom trace, increase upward). Vertical calibration; 3 9 g tension, 83 mmh20 pressure in the air space and 334 mmh20 intraluminal pressure. duration of the relaxations varied from 30 to 120 s. The spontaneous relaxations were abolished by TTX (10-7 g.ml-h) (Fig. 1). In six of the preparations the response to increases in intraluminal pressure of 1-2 min duration was sometimes a single peristaltic contraction, especially with pressures in the region of 80-100 mmh2o. However, the other fifteen preparations exhibited no peristaltic activity at all to such stimuli (Figs. 1 and 2). When stimuli of longer duration (10-15 min) were used, weak peristaltic contractions occurred intermittently. Such contractions were seen to progress in both directions along the caecum. The only difference between the responsiveness of the proximal and distal segments of the caecum to distension was that the latter exhibited small rhythmic non-propulsive contractions when distended (Figs. 1 and 2). Similar results were obtained irrespective of the orientation of the caecum within the apparatus. The only observed difference between the response to distension with different orientations of the preparation was that when the i.c.c. end was nearest the intraluminal pressure transducer one or more contractions were often observed when distension was withdrawn. The general lack of responsiveness of the two segments of caecum to distension was in marked contrast to the behaviour observed when TTX (1 0-2-0 x 10-7 g. ml-1) was present in the bath. In fourteen experiments raising the intraluminal pressure to 20-50 mmh2o in the presence of TTX resulted in the appearance of regular peristaltic contractions (Figs. 1 and 5). These contractions propagated either in both directions along the caecum when the initial contraction(s) took place in the middle of the preparation (Fig. 3 A) or toward the open end of the caecum when the contraction started at the occluded end (Fig. 3 B). The former pattern of activity tended to predominate at low distending pressures, the latter at high distending pressures. When contractions started at a number of points along the length of the caecum they 120 s 6-2

164 J. P. HODGKISS A 49 mmh2o B 60 C ----r 102 140 D Procaine HCI, 375 gg mnl` 120 s Fig. 2. Responses of the distal caecum to raising intraluminal pressure from 0 to various levels up to 140 mmh2o before (A-D) and after (E-H) adding procaine (375 l,g.ml-') to the bath. Each series of records shows longitudinal tension (top trace, increase upward) pressure in the 2 5 1 air space (middle trace, increase downward) and intraluminal pressure (bottom trace, increase upward). Vertical calibration; 6 0 g tension, 207 mmh2o pressure in the air space and 512 mmh2o intraluminal pressure. A B 1-1- - 2 3 4 < 3 H.,--- 4! 5 5 k/4 6 Fig. 3. Schematic diagram of the types of peristaltic activity seen in the distended caecum after exposure to TTX or local anaesthetics. Contractions which started in the middle of the preparation (A) propagated in both directions. Those reaching the blind end of the preparation then reversed direction and began to expel fluid into the air space. This resulted in a complex pressure spike. Another type of activity consisted of a contraction which started at the blind end of the preparation (B) and progressed smoothly along the length of the preparation to give a monophasic pressure spike.

PERISTALSIS IN THE CHICKEN CAECUM 165 1-A-A 20 s Fig. 4. Changes in longitudinal tension of the proximal caecum (top trace, increase upwards), intraluminal pressure (middle trace, increase upwards) and pressure in 2 51 air space (bottom trace, increase downwards) during peristaltic activity initiated by raising intraluminal pressure to 128 mmh2o in the presence of TTX (0-1 ug.ml-'). Vertical calibration bar 0 85 g longitudinal tension, 127 mmh20 intraluminal pressure and 10-4 mmh20 pressure in air space. propagated in both directions thus producing a pressure spike with a complex wave form. The complexity of these pressure spikes made a quantitative analysis of peristalsis difficult. However, visual inspection of the records showed that, within limits, increasing intraluminal pressure resulted in an increase in the number of peristaltic contractions (Fig. 1). In those experiments where the intraluminal pressure change consisted of a single pressure spike its relationship with changes in tension in the longitudinal muscle was examined. The phase relationship between tension changes in the longitudinal muscle and changes in intraluminal pressure is shown in Fig. 4. It is clear that during the peristaltic reflex in the caecum the circular muscle starts to contract first. This is followed by a contraction of the longitudinal muscle with both reaching maximum at approximately the same time. These phasic changes in the tension of the longitudinal muscle were superimposed on an over-all fall in tension which persisted for as long as distension was maintained (Figs. 1 and 5). Similar results were obtained with segments of proximal and distal caecum. The orientation of the caecum in the apparatus did not affect the ability of the caecum to undergo peristalsis. The contractions sometimes seen when distension was withdrawn were abolished by TTX. The affect of TTX was reversible on washing with drug-free solution. In four experiments exposure of the caecum to the local anaesthetics procaine (375,pg. ml-') or cinchocaine (200,ug. ml-') also resulted in peristalsis when the intraluminal pressure was raised (Fig. 2). In this respect the action of local anaesthetics was similar to that of TTX. The action of local anaesthetics was reversible. No peristaltic activity was seen in response to raised intraluminal pressure after exposure of the preparation to the following drugs: hexamethonium, 100-500 pg. ml-m (Fig. 5 H); methysergide, 1 /sg. ml-'; naloxone, 40 ng. ml-'; guanethidine, 5 /sg. ml-'; propranolol, 5,tg. ml-'; phentolamine, 5 jug. ml-' (Fig. S C). Propranolol (5 jug. ml-') in combination with phentolamine (5,g. ml-') was also ineffective (Fig. 5E). The immediate effects of phentolamine, propranolol, hexamethonium and TTX, on the longitudinal muscle in particular, are shown in Fig. 5 B, D, G and I respectively. All four drugs caused a contraction of the longitudinal muscle. In the presence of higher concentrations of phentolamine (50-100 #ug. ml-') distension did cause regular peristaltic contractions of the caecum. The effect was reversed by washing with drug-free Krebs solution.

166 J. P. HODGKISS A pb,/-- C D E L11- v- I Phen. I Prop. I F Gl H I -r7 J fi. Hex. TTX Fig. 5. The effect of raising intraluminal pressure to 156 mmh20 on the response of the proximal caecum after exposure to C, phentolamine (phen., 5,ug. ml-'); E, propranolol (prop., 5 jug. ml-'); G, hexamethonium (hex., 500,sg ml-') and J, tetrodotoxin (TTX, 0 1 zg. ml-'). The control responses are shown in A and F. The records in B, D, G and I are included to show the immediate effects of these drugs when added to the bath. Each series of records shows longitudinal tension (top trace, increase upwards), pressure in 2-5 1 air space (middle trace, increase downwards) and intraluminal pressure (bottom trace, increase upwards). Calibration bars: 3 g longitudinal tension; 104 mmh20 pressure in air space and 200 mmh20 intraluminal pressure. 240 s The effect of localized distension on the response of an adjacent segment of circular muscle Of the eleven preparations examined for their responsiveness to localized distensions only four exhibited spontaneous activity. This took the form of either non-propagating, local, spike-like contractions at the point of recording (Fig. 7 Al and A2) or contractions which propagated from one end of the preparation to the other past the point of recording. Often these latter contractions were preceded by a small relaxation (Fig. 7A4 and Bi). When a 7 mm length of the wall of the caecum located 3-10 mm on the sac side of the point of recording was distended, usually (nine experiments) either no response was recorded (Figs. 6B and 7A2) or there was a small relaxation of the circular muscle

PERISTALSIS IN THE CHICKEN CAECUM 167 A -B TTX, 0-15,ugml- 60 s E i..c sac e 9mm*9mm4 Fig. 6. Response of the circular muscle of the proximal caecum to localized distension using the arrangement shown in E before (A and B) and after (C and D) exposure to TTX (0.1 sg. ml-'). The bars above each record indicate the time over which distension was applied at the ileocaecolic (i.c.c.) (A and C) or distal (sac) (B and D) ends of the preparation. (Fig. 7 B2). In two experiments a small maintained contraction was recorded. Distension of the circular muscle on the i.c.c. side of the site of recording produced similar results. Thus, in seven experiments no response or a small relaxation (Fig. 7Bi) was obtained, and a single phasic contraction was recorded in three experiments. The latter originated at the point of distension and propagated the length of the preparation (Fig. 6A). A small tonic contraction was seen in only one experiment (Fig. 7 Al). When TTX (0 1,ug. ml-') was added to the bath, distension at the i.c.c. end of the caecum in six experiments resulted in one or more contractions, initiated at the point of distension, which propagated past the point of recording to the other end of the preparation (Figs. 6 C and 7 A3, 7 B3). Distension at the sac end in five experiments gave similar results (Figs. 6D and 7A4, 7B4). DISCUSSION In the first series of experiments with the Trendelenburg apparatus, raising the intraluminal pressure to values up to 167 mmh2o did not initiate peristalsis in segments of either proximal or distal caecum. In the case of the proximal caecum this was so, irrespective of the orientation of the preparation in the apparatus. These results are similar to those obtained with the chicken oesophagus (Bartlet, 1973) and rectum (Mizuta et al. 1980). Peristalsis was seen, however, in the presence of TTX or local anaesthetics when the intraluminal pressure was raised to 20-50 mmh2o. These results suggest that some intrinsic

168 J. P. HODGKISS A 20g 20g 20g 20g 3 4 B 15 g 15 g 1 2 3 4 3 g 120 s Fig. 7. Response of the circular muscle of the proximal caecum in two preparations (A and B) to localized distension obtained before (1 and 2) and after (3 and 4) exposure to TTX (0 1 /sg. ml-). The same arrangement as in Fig. 6 was used, the bars above each record indicate the time over which distension was applied at the ileocaecolic (1 and 3) or distal (2 and 4) ends of the preparation. neurones in the caecum inhibit the musculature during distension and support the view that the mechanism of peristalsis in the chicken gut is quite different to that found in mammalian species. Increases in intraluminal pressure were almost invariably accompanied by a fall in tension of the longitudinal muscle of the caecum. This was most likely to be due to passive mechanical elongation (Hukuhara & Fukuda, 1965) since it was also observed in the presence of TTX and local anaesthetics. During peristalsis, contractions of the longitudinal muscle were recorded which were in phase with the intraluminal pressure spikes resulting from propagated contractions of the circular muscle. These results also differ from those obtained in the guinea-pig small intestine using essentially the same apparatus. In the guinea-pig each circular muscle contraction was associated with a relaxation of the longitudinal muscle and thus contractions of the two muscle layers were not in phase (Kosterlitz et al. 1956). Another difference is the polarity of the peristaltic reflex; in the present experiments peristalsis ofthe proximal caecum occurred irrespective ofthe orientation of the preparation. Thus, after withdrawal of neuronal inhibition, the caecum had the ability to propel luminal contents in either direction. Myogenic peristalsis in the chicken caecum can, therefore, occur in either direction. Peristalsis in the mammalian intestine on the other hand exhibits a definite polarity (Crema, 1970).

PERISTALSIS IN THE CHICKEN CAECUM The presence of intrinsic inhibitory neurones triggered by distension was also suggested by experiments in which a localized distension was applied to the wall of the caecum. Such stimuli most often produced no response or a small relaxation of the circular muscle layer on both sides of the point of distension. These responses are very similar to those recorded on the anal side of the site of distension in the guinea-pig small intestine (Costa & Furness, 1982) and suggest that distension may trigger ascending and descending inhibitory mechanisms in the caecum. Thus, in the presence of TTX distension leads to myogenic contractions of the circular muscle of the caecum. On the other hand, in the guinea-pig small and large intestine an ascending excitatory and descending inhibitory reflex have been demonstrated in response to distension. TTX blocks both reflexes but does not induce myogenic activity in the circular muscle (Costa & Furness, 1976; Furness & Costa, 1976). Preliminary studies were carried out to determine the nature of the inhibitory transmitter in the chicken. It was not a catecholamine nor apparently was it exerting an inhibitory action through opiate receptors. It was not sensitive to blockade by methysergide which suggests that the transmitter may not be 5-hydroxytryptamine (5-HT). However, the evidence is not unequivocal because, methysergide did not antagonize the actions of 5-HT on the longitudinal muscle of the chicken rectum (Bartlet, 1974) although it did so in the chicken ileum (Ahmad, Singh & Garg, 1978). The lack of effect of high concentrations of hexamethonium suggests that the intrinsic inhibitory neurone does not receive a nicotinic input. In the present experiments high concentrations of phentolamine resulted in peristalsis during distension. Phentolamine in such high concentrations has been found to antagonize the responses to adenine nucleotides and stimulation of non-adrenergic, non-cholinergic nerves in the guinea-pig intestine (Satchell, Burnstock & Dann, 1973). Furthermore, the adenine nucleotides have been found to be released from isolated avian myenteric plexus following stimulation (Burnstock, Campbell, Satchell & Smythe, 1970). Thus the possibility that an adenine nucleotide is the inhibitory transmitter to the circular muscle of the caecum merits further investigation. The mechanism of filling and emptying of the caecae has received scant attention. Radiographic studies in the chicken (Akester, Anderson, Hill & Osbaldiston, 1967) and quail (Fenna & Boag, 1974) suggest that antiperistaltic movements of the large intestine occurring at the same time as peristaltic movements of the ileum create a high pressure zone at the ileocaecolic junction thus forcing material into the caecae. The results of the present study indicate that such material could be transported to the distal caecum by the sequential removal of circular muscle inhibition along the length of the caecum (antiperistalsis). Similarly the removal of material from the caecum could be achieved by the sequential release of inhibition in the reverse direction (peristalsis). However, the experiments reported here shed no light on how intrinsic inhibitory neurones could be switched on and off in this way, although it seems likely that the intestinal nerve (of Remak) may be involved. 169 REFERENCES AHMAD, A., SINGH, R. C. P. & GARG, B. D. (1978). Evidence of non-cholinergic excitatory nervous transmission in chick ileum. Life Sciences 22, 1049-1058. AKESTER, A. R., ANDERSON, R. S., HILL, K. J. & OSBALDISTON, G. W. (1967). A radiographic study of urine flow in the domestic fowl. British Poultry Science 8, 209-212. BARTLETT, A. L. (1973). Myogenic peristalsis in isolated preparations of chicken oesophagus. British Journal of Pharmacology 48, 36-47.

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