| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
MS 9196 Received 27 January 1999; accepted after revision 9 March 1999.
 |
ABSTRACT |
|---|
 TopAbstract IntroductionMethodsResultsDiscussionReferences |
|---|
-nitro-L-arginine (100 µM), a nitric oxide synthesis inhibitor, had no overall significant effect on the characteristics of the LM and CM contractions, although on occasion an enhancement in their peak amplitude was noted.
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Bayliss & Starling (1899, 1900) were the first to report the existence of polarized reflexes in the gastrointestinal tract. They showed that stimulation of the canine small intestine evoked a relaxation in the smooth muscle lying anal to the stimulus site, but it was noted that a contraction was evoked orally. Such observations led them to formulate their now widely accepted 'law of the intestine' which has formed the basis of our understanding of the peristaltic reflex. Since their study, other investigators, particularly those using intracellular microelectrodes from the smooth muscle layers, have reported the existence of polarized reflex pathways that appear to generate ascending excitation (depolarization) and descending inhibition (hyperpolarization) along the small intestine (Hirst & McKirdy, 1974; Hirst et al. 1975; Smith & Furness, 1988; Smith et al. 1990, 1991; Yuan et al. 1991, 1995; Johnson et al. 1998).
Despite Bayliss and Starlings' seminal observations, a number of investigators in the first half of this century have found difficulty in demonstrating the 'law of the intestine', at least in its most simplistic form, during mechanical recordings from the small bowel of many species (see chapter 3 in the review by Alvarez, 1948). For example, Alvarez & Starkweather (1919) reported that local stimulation produced contractions both above and below the stimulus site of rabbit small intestine. Similarly, Hukuhara et al. (1936) found that local stimulation of the dog small intestine produced a contraction that propagated in both directions, with no evidence of the descending relaxation reported earlier by Bayliss & Starling (1899). One difference between these studies was that Bayliss & Starling (1899) used castor oil to purge the intestinal lumen. Even in the small bowel of man, White et al. (1934) could not see any wave of inhibition that preceded spontaneous propagating contractions. Later, Röden (1937) suggested that descending inhibition in the human small bowel 'does not seem to be valid under physiological conditions'. In fact, Alvarez (1948) was of the opinion that the widening of the small bowel that is sometimes observed to precede a wave of contraction was due 'not to inhibition but to distension by the long column of intestinal contents which is forced ahead by the wave of contraction'. The presence of a contraction and absence of relaxation anal to a local stimulus has not been restricted to mammals, but has also been reported to occur following distension of the avian small bowel (Hodgkiss, 1986). However, despite difficulty in reproducing the 'law of the intestine' in the small intestine, it seems that following a local stimulus, an oral contraction and anal relaxation is more readily recorded in the large bowel (Bayliss & Starling, 1900; Crema et al. 1970; Mackenna & McKirdy, 1972; Smith & McCarron, 1998). This is quite likely to be due to regional differences in the composition of the intestinal contents, since propulsion of a solid pellet in the distal colon necessarily requires different mechanisms for propulsion than the rapid movement of fluid in the small bowel (see Crema, 1970).
Since the pioneering observations of Bayliss & Starling at the turn of the century (Bayliss & Starling, 1899, 1900), we have gained significantly more knowledge about the enteric nervous system of mammals. In particular, the guinea-pig ileum has emerged as the model preparation for understanding how enteric neurons are integrated to regulate peristalsis; use of this model has implied that the stereotypical ascending excitatory and descending inhibitory reflexes underlie peristalsis in the small intestine (Furness et al. 1994). Immunohistochemical studies in this tissue have shown the existence of polarized projections of enteric motoneurons to the circular muscle (CM) layer, since inhibitory motoneurons project anally along the ileum (Costa et al. 1992), while the excitatory motoneurons project orally, or locally, to the CM layer (Furness et al. 1994). Further support for the existence of these polarized motoneural projections to the CM comes from intracellular recording from the CM layer, where it has been shown that distension (Hirst et al. 1975; Smith et al. 1990, 1991) or mucosal stimulation of the ileum (Smith & Furness, 1988; Smith et al. 1991; Yuan et al. 1991) elicits inhibitory junction potentials (IJPs) anal to a stimulus, while excitatory junction potentials (EJPs) are elicited orally. It has therefore been assumed that distension, or muscosal stimulation, of the guinea-pig ileum may generate similar mechanical responses in the smooth muscle (i.e. contraction orally and relaxation anally), consistent with the 'law of the intestine', originally postulated by Bayliss & Starling (1899).
Recently, we showed that, in the guinea-pig distal colon, a mucosal stimulus was sufficient to elicit an anal relaxation response and oral contraction of the smooth muscle (Smith & McCarron, 1998), consistent with the 'law of the intestine' suggested by Bayliss & Starling (1899). Remarkably, however, no studies have investigated whether the guinea-pig ileum conforms to the 'law of the intestine' under control conditions. These experiments most probably have been overlooked, due to an expectation that IJPs would cause relaxation anally and EJPs would cause contraction orally. Therefore, we have developed a preparation that allows simultaneous recording of the mechanical activity of the longitudinal muscle (LM) and CM layers of the guinea-pig ileum, both oral and anal to a stimulus site, and that avoids the possibility of any mechanical interactions between the two muscle layers. This technique has been utilized to investigate (1) whether the mechanical reflex responses of the guinea-pig ileum conform to the 'law of the intestine' proposed by Bayliss & Starling (1899), and (2) whether the LM and CM layers are reciprocally innervated, as suggested by Kottegoda (1969).
|
METHODS |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Guinea-pigs weighing 250-350 g were killed by inhalation of a rising concentration of CO2, approved by the animal ethics committee of the University of Nevada School of Medicine. The abdominal cavity was opened and the terminal 15 cm of distal small bowel was removed, the mesenteric attachment trimmed away, flushed clean with Krebs solution, and then placed immediately into a modified Krebs solution (composition below). To record the simultaneous responses of the two muscle layers, the LM and myenteric plexus (MP) were dissected free of CM and mucosa at each end to create an LMMP preparation (see Smith & Robertson, 1998). To do this, a longitudinal incision (approximately 15 mm in length) was made along the oral and anal extremities. These regions were pinned mucosal side uppermost so that the mucosa and submucosa could be delicately removed from either extremity to expose the underlying CM layer. Strips of CM were then delicately removed from the exposed oral and anal regions, to reduce the possibility of any mechanical interactions between the movements of the LM and CM muscles. Therefore, preparations consisted of a flap of LM with attached myenteric plexus that remained in continuity with the oral and anal regions of ileum (Fig. 1). The free ends of the dissected region (see LMMP, in Fig. 1) were connected via thread to independent tension transducers (see below) mounted orally and anally to the stimulus. To examine the propagation of neural reflexes along the ileum, a long segment (approximately 15 cm in length) was removed from the animal and a region (20 mm) was cut open along the mesenteric border, the mucosal surface opened and pinned (facing uppermost) approximately 30 mm from the oral end of the segment (Fig. 4). The mechanical activity of the CM was monitored using small clips (Micro-serrefines No. 18055-04; Fine Science Tools Inc., Foster City, CA, USA) mounted oral (2 cm from the opened region) and anal (3 clips placed 2 cm apart) from the opened stimulating region. These were attached via the serosal surface to the underlying CM of the intact ileum (Figs 1, 4 and 5). Tension in the LM and CM was recorded using Grass (Quincy, MA, USA) FT03 tension transducers and recorded on a 4-channel Gilson Medical Electronics 5/6H Recorder (Middleton, WI, USA). Initial tensions of the LM and CM were routinely set to 1 g, so that we could directly compare the reflex responses of the guinea-pig ileum with those we observed in the guinea-pig distal colon, where we used identical stimulating and recording methods and reflex-evoked relaxations of the LM and CM were shown to occur (Smith & McCarron, 1998). Preparations were bathed in oxygenated Krebs solution at 37°C.
Protocol for stimulation of the ileum
Oral and anal reflexes were elicited by distension (radial stretch) or by mechanical stimulation of the mucosa; the mucosa was stimulated with an artist's brush and the gut distended using a plastic plate (12 mm × 5 mm) under the serosa of the opened pinned segment (Fig. 1). This enabled mechanoreceptors to be stimulated, without distortion of the mucosa, and therefore prevent mucosal stimulation. The plate was connected via strings that pierced the lumen to a pulley system to which weights (5-30 g) were added.
Drugs and solutions
The following drugs were used in the current study: atropine sulphate, apamin, hexamethonium bromide, histamine, N-nitro-L-arginine (L-NA), sodium nitroprusside (SNP) and tetrodotoxin (TTX), all obtained from Sigma. Stock solutions were prepared in distilled water.
The composition of the modified Krebs solution was (mM): NaCl, 120·35; KCl, 5·9; NaHCO3, 15·5; NaH2PO4, 1·2; MgSO4, 1·2; CaCl2, 2·5; and glucose, 11·5. The solution was gassed continuously with a mixture containing 3 % CO2-97 % O2 (v/v), pH 7·3-7·4.
Measurements and statistics
Mann-Whitney U test and Student's (paired or unpaired) t tests were used where appropriate. A minimum significance level of P < 0·05 was used throughout. The use of n in the results section refers to the number of animals on which observations were made and data are presented as means ± S.E.M. (standard error of the mean). Measurements of the rate-of-rise of contractions were performed by dividing the peak contractile amplitude by the time-to-peak. The latter was derived by the time taken for contractions to reach peak amplitude, taken from 10 % of peak amplitude (on the rising phase).
|
RESULTS |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Mechanical responses of the longitudinal and circular muscle to mucosal stroking and radial distension
We investigated whether the mechanical reflex responses in the guinea-pig ileum conform to the 'law of the intestine' postulated by Bayliss & Starling (1899). We found that, in 73 out of 80 trials (n = 8), brush stroking the mucosa (see Methods) induced a synchronous contraction in the LM and CM, both orally and anally to the stimulus (Fig. 1B). In the remaining seven trials, the anal contractions of the LM or CM were faint, or within the level of the recording noise. The amplitude and rate-of-rise of the CM contraction oral to the stimulus were significantly enhanced compared with the CM contraction anal to the stimulus (the oral CM amplitude was 13·2 ± 3·6 mN and rate-of-rise, 6·6 mN s-1 compared with an anal CM amplitude of 4·5 ± 0·5 mN and rate-of-rise of 2·3 ± 0·4 mN s-1). There was, however, no significant difference between the amplitude and rate-of-rise of the LM contraction oral (amplitude, 7·0 ± 0·2 mN; rate-of-rise, 4·0 ± 0·6 mN s-1) or anal (amplitude, 6·9 ± 2·2 mN; rate-of-rise, 3·7 ± 1·1 mN s-1; P > 0·05; n = 8) to the stimulus, when recorded under control conditions. Under no circumstances were any relaxations recorded in either muscle layer, orally or anally to the mucosal stimulus. A maximal contractile amplitude was typically obtained with two to three brush strokes, delivered at a frequency of approximately 1-2 Hz. To examine whether distension of the ileum also elicited similar mechanical responses to those elicited by mucosal stroking, we radially distended the ileum underneath the serosal surface to avoid stimulation of the mucosa (see Methods). We found that distension also consistently evoked synchronous contractions in the LM and CM, orally and anally to the stimulus (Fig. 1C). A consistent observation was that grading the number of brush strokes (1-5 strokes) and radial distensions (5-30 g) consistently produced graded changes in the amplitude of the contractions in both the LM and CM orally and anally to the stimulus.
 |
View larger version [in this window] [in a new window] |
|
|
A, diagrammatic representation of the preparation used for simultaneous recording of the tension in the longitudinal (LM) and circular muscle (CM) oral and anal to a stimulus site. The CM at the oral and anal extremity of the tissue was dissected away, so that a flap of LM remained in continuity with the myenteric plexus (LMMP). TLM and TCM refer to isometric tension transducers for the LM and CM, respectively; g is applied radial stretch in grams. B and C, control responses of the LM and CM oral and anal to a mucosal stimulus (B; 5 brush strokes, arrow) and radial distension (C; 30 g, arrow). Note that the LM and CM contracted synchronously above and below the stimulus. | ||
We examined the possibility that inhibitory responses (relaxations) may only occur within short distances (< 2 mm) anal to a stimulus. We found that, in 24 out of 24 trials of mucosal brush stroking (n = 4), each stimulus always generated a powerful contraction of the CM, 2 mm anal to the stroking site, with no sign of muscular relaxation.
It might have been argued that the absence of muscular relaxations in the LM and CM anal to the stimulus was due to insufficient resting tone applied to the muscles. Therefore, we delivered a mucosal stimulus, after progressively applying increased levels of resting tension (1, 2 and 4 g) to the LM and CM (Fig. 2A). Regardless of the level of applied resting tone, we consistently recorded a powerful contraction in the LM and CM anal to each mucosal stimulus (n = 3; Fig. 2A).
 |
View larger version [in this window] [in a new window] |
|
|
A, effects of increasing the resting tone on the LM and CM anal to a mucosal stimulus. Progressive increases in the resting tone applied to the LM and CM (1, 2 and 4 g) consistently elicited powerful contractions in the LM and CM following mucosal stimulation (3 strokes; arrow). B, effects of pre-contracting the ileum with histamine on anal reflex responses of the LM and CM. Brush stroking the mucosa (3 strokes; arrow) elicited a synchronous contraction in the LM and CM. Subsequent addition of histamine (3 µM) caused a rapid increase in the resting tone of the LM and CM. In the presence of histamine, mucosal stimuli delivered on 3 occasions blocked contractions in the LM. In the CM, faint contractions were still elicited in the presence of histamine. No relaxations were recorded, despite enhancement of the resting tone. C, application of 3 brush strokes to the mucosa (arrow) elicited a synchronous contraction in the LM and CM anal to the stimulus. Addition of atropine (1 µM) reduced the resting tone of both the LM and CM, suggesting the presence of ongoing cholinergic tone. Contractions to mucosal stroking were abolished in the LM, and in the CM, faint non-cholinergic contractions persisted. | ||
We then examined whether pharmacological pre-contraction of the ileum with histamine (1-6 µM) would induce relaxations in the LM and CM anal to a mucosal stimulus (see Costa et al. 1986). From 11 animals tested, we found that addition of histamine caused an immediate increase in the resting tone (> 10 mN) of both the LM and CM (Fig. 2B). In eight of these animals, contractions of the LM and CM were essentially blocked with no sign of relaxation (Fig. 2B). In one preparation, we did record a faint relaxation in the CM, and surprisingly also in the LM (as the LM does not receive an intrinsic inhibitory innervation (see Hirst et al. 1975; Bywater & Taylor, 1986), relaxations in both the LM and CM of this particular animal were resistant to atropine (2 µM), but blocked by apamin (300 nM)). In the remaining two animals, the anal contractions of the LM and CM were not blocked, but were reduced in amplitude by 83 and 73 %, respectively, by histamine. To test that the muscles were capable of relaxation, we examined the effects of the nitric oxide donor SNP on the tone of the LM and CM. SNP (10 µM) was found to induce an immediate and powerful relaxation of both the LM (by 5·4 ± 1·1 mN; 4 preparations, n = 2) and CM (by 2·3 ± 0·6 mN; 4 preparations, n = 2) of the ileum, as it does in the guinea-pig distal colon (Smith & McCarron, 1998).
Effects of blockade of inhibitory neurotransmission on reflex responses of the longitudinal and circular muscle
The distension- and mucosal stimulus-evoked IJP in the CM layer of the guinea-pig ileum is abolished by the bee venom apamin (Smith & Furness, 1988; Smith et al. 1990). We examined the possibility that apamin-sensitive inhibitory neurotransmission may modulate the characteristics of the contractions of the LM and CM, oral and anal to a mucosal stimulus. Addition of apamin (300 nM) enhanced small spontaneous background contractions of the LM and CM and in half of the animals tested (4 out of 8) induced spontaneous motor complexes (see below). Apamin (300 nM) significantly increased the rate-of-rise of the contractions in the LM (3·7 ± 1·1 to 5·7 ± 1·0 mN s-1; P < 0·05; n = 8) and CM (3·2 ± 0·4 to 5·5 ± 1·1 mN s-1; P < 0·05; n = 8) anal to the mucosal stimulus, and in the LM oral to the stimulus (Fig. 3B). While in the presence of apamin (300 nM), no significant difference was found in the rate-of-rise of the oral CM contraction (Figs 3B and 4B). Further addition of L-NA (100 µM) had no significant (P > 0·05; n = 7) effect on the amplitude or rate-of-rise of the contractions oral and anal to the stimulus, although on occasion an increase in the amplitude of the LM and CM contractions was noted. To examine the involvement of nicotinic neurotransmission in these ascending and descending reflexes, hexamethonium (300 µM) was applied to the ileum. Addition of hexamethonium (300 µM) significantly reduced the amplitude of the oral contractions of the LM (by 80·0 ± 11·7 %; P < 0·05; n = 4) and CM (by 86·2 ± 9·6 %; P < 0·05; n = 4). However, overall the amplitudes of the anal contractions of the LM and CM were not reduced significantly (by 45·3 ± 22·5 and 53·0 ± 21·2 %, respectively) although in one preparation contractions were abolished. Atropine (1-2 µM) reduced the resting tone of the LM (by 2·3 ± 0·3 mN; n = 5) and CM (by 3·6 ± 1·4 mN; n = 5) (Fig. 2C) and consistently blocked contractions in the LM (n = 5) and in four out of five animals abolished the contraction in the CM oral to, and anal of, the mucosal stimulus. In one animal, contractions of the CM were reduced oral (by 60 %) and anal (by 92 %) by atropine.
 |
View larger version [in this window] [in a new window] |
|
|
A, control responses of the LM and CM layers, oral and anal to a mucosal stimulus. Brush stroking the mucosa (3 brush strokes; arrow) elicited a synchronous contraction in both the LM and CM, oral and anal to the stimulus. B, addition of apamin (300 nM) increased the amplitude of the LM contraction oral and anal to the stimulus, and the CM contraction anal, but not oral, to the stimulus. | ||
To examine whether mucosal stimuli elicited only local contractions, or responses that propagated along the ileum, multiple CM recording sites were located (2 cm apart) anal to the mucosal stimulus (see Fig. 4). In these preparations, local brush stroking of the mucosa was sufficient to elicit a wave of contraction that propagated aborally, despite the absence of fluid in the lumen. These waves exhibited a propagation velocity of 28·7 ± 9·6 mm s-1 (n = 4), consistent with the measurements for peristaltic waves reported by Tsuji et al. (1992) in this tissue. These descending waves readily conducted over 13 cm anal to the local stimulus, often with no decrement in amplitude (Fig. 4A). These evoked peristaltic waves did not occur in the presence of TTX (1·6 µM; n = 3), suggesting they were dependent upon activity within the enteric nervous system. In six out of seven animals tested, apamin (300 nM) significantly increased the amplitude (site ii: 6·0 ± 1·1 to 15·1 ± 5·1 mN; site iii: 8·6 ± 2·2 to 13·5 ± 4·5 mN; site iv: 5·8 ± 1·1 to 16·3 ± 4·4 mN) and rate-of-rise (site ii: 1·8 ± 0·3 to 5·8 ± 1·5 mN s-1; site iii: 2·2 ± 0·4 to 4·7 ± 1·0 mN s-1; site iv: 1·0 ± 0·2 to 4·2 ± 1·0 mN s-1) of the CM contractions recorded at three sites anal to the stimulus (Fig. 4B). In contrast, apamin (300 nM) had no effect on the activity of the CM oral (site i) to the stimulus (P > 0·05; n = 7). In one preparation, apamin (300 nM) did not appear to alter the characteristics of the anal or oral contractions of the CM. We also noted that apamin unco-ordinated evoked peristaltic waves, and contractions often occurred almost synchronously at all sites (Fig. 4B), leading to infinite apparent conduction velocities. Also, four out of eight preparations showed spontaneous motor complexes in apamin (300 nM), which did not appear to have any consistent preferential site of origin or direction, often originating from oral or anal regions of ileum. On other occasions, it appeared that spontaneous contractions almost occurred simultaneously at all sites in the ileum (Fig. 5). In two animals, these motor complexes were recorded in control solution (i.e. in the absence of apamin). These events appear to be similar to the intracellularly recorded myoelectric complexes that also propagate spontaneously along the isolated mouse colon (see Bywater et al. 1989; Spencer et al. 1998).
 |
View larger version [in this window] [in a new window] |
|
|
Mechanical recording of CM activity oral (i) and anal (ii, iii and iv) to a local mucosal stimulus. A, mucosal stimulation (3 strokes; arrow) elicited a contraction that propagated at least 60 mm anally despite the absence of fluid in the lumen. The rate-of-rise of the descending contraction was shown to decrease, and the time-to-peak to increase, the further the recording site was from the site of stimulation. The time-to-peak of the response increased with distance giving rise to an effective propagation of the response. B, reflex responses of the CM after addition of apamin (300 nM). Apamin increased the amplitude of the responses of CM anal, but not oral, to the stimulus and disrupted the apparent propagation of the peristaltic wave (dashed line). | ||
 |
View larger version [in this window] [in a new window] |
|
|
Mechanical recording of CM activity at 4 sites (i-iv) along the ileum in the presence of apamin (300 nM). Spontaneous contractions appeared to have reached peak tension at the same time along the length of the bowel. | ||
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
A primary finding of this study is the demonstration that local stimulation of the guinea-pig ileum (via radial distension or mucosal distortion) elicits a contraction in both the LM and CM layers, both orally and anally to a stimulus. No inhibitory responses were recorded in either muscle layer anal to a stimulus site (despite the presence of ongoing cholinergic tone in both muscle layers), suggesting that the guinea-pig ileum does not conform to the 'law of the intestine' as originally postulated by Bayliss & Starling (1899). Moreover, the LM and CM layers are not reciprocally innervated, as suggested by Kottegoda (1969), but rather contract synchronously during reflex stimulation.
We have shown that two different stimuli applied to the ileum elicit the same mechanical response in both muscle layers oral and anal to the stimulus. In the current study, we radially distended the muscle layers underneath the serosal surface, rather than using a balloon, to avoid direct distortion of the mucosa. This enabled us to selectively stimulate stretch-sensitive mechanoreceptors in the muscle, and avoid inadvertent stimulation of the mucosa (which occurs with balloon distension). Conversely, brush stroking the mucosa selectively stimulated 'mucosal' sensory neurons and avoided activation of stretch receptors. The observation that both stimuli generated the same mechanical response implies that both types of sensory neuron may converge onto the same population of motoneurons to the LM and CM (see Smith et al. 1992).
In the guinea-pig ileum, the characteristics of the oral mechanical reflex responses (ascending excitatory reflex) of the CM have been examined (see Tonini & Costa, 1990). However, the only study of mechanical reflex responses anal to a stimulus in the guinea-pig ileum that we are aware of was by Costa et al. (1986). These investigators reported that the CM relaxed anal to a distension stimulus. However, relaxations were only reported to occur when the muscle was pre-contracted with histamine. In the current study, we found that identical concentrations of histamine also pre-contracted the ileum, but abolished, or markedly attenuated, the amplitude of the descending contractions recorded in control solution, revealing faint relaxation in only one out of 11 animals. This suggests that the pharmacological intervention required to induce such relaxations is not consistent with any view that such inhibitory responses are physiologically significant. There is evidence that histamine can exert indirect actions on the enteric nervous system, by either presynaptically inhibiting the release of excitatory neurotransmitters, or postsynaptically stimulating the muscle (Daniel, 1982; Tamura et al. 1987).
In the guinea-pig small intestine, the presence of IJPs anal and EJPs oral to a local stimulus is well documented (Smith & Furness, 1988; Smith et al. 1990, 1991; Yuan et al. 1991, 1995; Johnson et al. 1998). This is due, in large part, to the fact that inhibitory motoneurons project anally to the CM layer along the guinea-pig ileum (Costa et al. 1992) and excitatory motoneurons project orally, or locally, to the CM (Furness et al. 1994). It has been assumed that stimulation of the inhibitory motoneurons would generate relaxation of the CM, especially since IJPs have been shown to occur at least up to 40 mm anal to a local distension or mucosal stimulus site (Smith & Furness, 1988). In direct contrast to the implications of immunohistochemical and electrophysiological studies on the guinea-pig ileum, the findings of the current study have shown an absence of polarized mechanical reflex responses in the LM and CM. Rather, we report the presence of ascending and descending excitation in the ileum. Descending excitation is consistent with the studies of Hirst et al. (1975) on the guinea-pig ileum, where it was reported that anal to a distension site, intracellular recording from the CM revealed an IJP which was truncated by a cholinergic EJP and if this 'reached threshold a muscle action potential was initiated.'
A major observation of this study was the ability to elicit a wave of contraction from any site along the intestine that readily conducted, often without decrement, over 13 cm anal to a mucosal stimulus. These waves were not accompanied by descending relaxation of the CM (Fig. 4A), despite the presence of ongoing cholinergic tone in this muscle layer. It is particularly interesting that the apparent conduction velocity of these waves was 25-30 mm s-1, which is similar to the values reported for peristaltic waves evoked in this tissue by fluid distension using the classical Trendelenburg technique (Tsuji et al. 1992). Our results suggest that distension caused by the presence of fluid in the lumen is not essential for the initiation and maintenance of propagation of a peristaltic wave. It is quite likely, however, that the local stimulation (stretch or mucosal) produced by a moving fluid may modify propulsive activity. Our findings appear to differ somewhat from the mechanism proposed by Waterman et al. (1994), where it was suggested that 'ascending excitatory pathways, activated by distension at each point along the intestine, are necessary for the peristaltic contraction to propagate. Descending excitatory pathways, if involved, are not sufficient to mediate the peristaltic contraction.' Our results suggest that descending excitatory pathways are important in peristalsis and that apamin-sensitive descending inhibitory pathways modulate descending excitation, most probably to co-ordinate distal transit in the small bowel. This was shown by the effects of apamin, which preferentially enhanced the amplitude of anal contractions and disrupted the phase lag between their onset at distal sites. This is consistent with the work of Waterman & Costa (1994), where it was noted that, in the presence of apamin, 'the circular muscle appeared to contract almost simultaneously along the length of intestine'.
Are the longitudinal and circular muscle layers reciprocally innervated?
There has been some controversy in the literature as to whether the LM and CM layers of the gastrointestinal tract are reciprocally innervated. This is due in part to the hypothesis put forward by Kottegoda (1969), where it was argued that as the CM contracted, the LM layer relaxed, in the guinea-pig ileum. Gregory & Bentley (1968) first drew attention to the possibility of passive mechanical interactions between the two muscle layers that can lead to false interpretations of the relative movements of the two muscles. In this study of the ileum, we paid particular attention to the mechanical isolation of the muscles by using similar dissection techniques to those we had developed for the guinea-pig colon (Smith & Robertson, 1998; Smith & McCarron, 1998). We found that, in the small intestine, the longitudinal and circular muscles contract synchronously following local stimulation. This strongly suggests that the local excitatory LM and CM motoneurons must receive a near-synchronous burst of fast excitatory postsynaptic potentials (from ascending and descending interneurons) (Smith et al. 1999), to generate synchronous contractions in both muscle layers oral and anal to a local stimulus. Under in vivo conditions, propulsion of the chyme in the ileum is most probably mediated by an imbalance in the rate-of-rise of the oral and anal CM contractions, since it was noted that the mean rate-of-rise of the CM contraction oral to the stimulus was significantly more rapid than that of contractions of the CM or LM recorded anally. The results of the current study suggest that this is likely to be mediated by the apamin-sensitive inhibitory neurotransmitter(s) anal to a local stimulus, to delay the rate-of-rise of the anal muscle contractions, facilitating aboral transit of liquid chyme. This is supported by the observations that apamin significantly enhanced the rate-of-rise of the LM and CM contractions anal, but not the CM contraction oral, to a stimulus (Figs 3 and 4). In contrast to the CM, the LM layer of the guinea-pig ileum does not receive an inhibitory intrinsic innervation following distension (Hirst et al. 1975), or transmural nerve stimulation (Bywater & Taylor, 1986). Therefore, the enhancement of the oral and anal contractions of the LM in the current study by apamin remains unclear. One possibility is that apamin may facilitate the release of acetylcholine, by increasing the excitability of the LM motoneurons (Smith et al. 1999).
We found insignificant evidence that nitric oxide modulated the characteristics of the reflex-evoked contractions of the LM and CM, although, on some occasions, we did observe an enhancement in their amplitude. This overall insignificant effect may be due to the fact that the 'slow' IJP, which has been suggested to be due to nitric oxide, is not revealed until the membrane potential actions of the known excitatory neurotransmitters (acetylcholine and substance P) have been prevented (Lyster et al. 1992; Crist et al. 1992).
Physiological significance of ascending and descending contractions
Recently, we showed that, in the guinea-pig distal colon, addition of atropine or nitric oxide donors reduced the resting tone of both the LM and CM (Smith & Robertson, 1998; Smith & McCarron, 1998). This is consistent with the findings of the current study, in that both muscle layers of the ileum also appear to be under tone, due to cholinergic drive, since they relaxed in the presence of atropine (Fig. 2C) or SNP. The presence of ongoing cholinergic tone in the ileum is supported by direct intracellular recordings from LM and short CM motoneurons from this tissue, which have shown that these neurons appear to be highly excitable, often discharging spontaneous action potentials (Smith et al. 1999). Despite the presence of tone in both muscles of the ileum and distal colon, the identical stimulus and recording conditions applied to both tissues elicited opposite responses. In the distal colon, we showed the presence of a relaxation in both the LM and CM anal to a mucosal stimulus and a contraction orally (Smith & McCarron, 1998). We believe these responses are consistent with the slow aboral transit of solid faecal pellets into the accommodating region (Smith & McCarron, 1998). In the ileum, however, we found that mucosal stroking elicited contractions both orally and anally to the stimulus. The absence of similar anal reflex-evoked relaxations in the ileum (as those recorded from the distal colon) is unlikely to be due to insufficient tone in the muscle. This is because the smooth muscle in both regions of the intestine exhibited a similar level of ongoing cholinergic tone and, furthermore, pharmacological enhancement of the tone (with histamine) usually abolished contractions, and rarely revealed relaxation. Moreover, we have shown that when an identical stimulus and recording conditions were applied to the distal colon, relaxations anal to the stimulus were clearly observed (Smith & McCarron, 1998). We suggest that the lack of a mechanical relaxation in the ileum is likely to be due to the fact that the contents of the small bowel are in liquid state and muscular relaxation is unnecessary for propulsion of a fluid.
In conclusion, the guinea-pig small intestine does not conform to the 'law of the intestine' as postulated by Bayliss & Starling at the turn of the century (Bayliss & Starling, 1899, 1900). Rather, physiological stimulation of the guinea-pig ileum elicits a contraction both orally and anally to a stimulus, and contractions occur synchronously in the LM and CM layers. That is, the two muscle layers are not reciprocally innervated as suggested by Kottegoda (1969). Furthermore, apamin-sensitive inhibitory neurotransmission facilitates co-ordinated distal transit, by modulating the amplitude and rate-of-rise of the CM contraction anal to a stimulus.
|
REFERENCES |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
| Alvarez, W. C. (1948). An Introduction to Gastroenterology, 4th edn, pp. 37-48. Paul B. Hoeber, New York. | |
| Alvarez, W. C. & Starkweather, E. (1919). Conduction in the small intestine. American Journal of Physiology 50, 252-265. | |
| Bayliss, W. M. & Starling, E. H. (1899). The movements and innervation of the small intestine. The Journal of Physiology 24, 99-143. | |
| Bayliss, W. M. & Starling, E. H. (1900). The movements and innervation of the large intestine. The Journal of Physiology 26, 107-118. | |
| Bywater, R. A. R., Small, G. S. & Taylor, G. S. (1989). Neurogenic slow depolarizations and rapid oscillations in the membrane potential of circular muscle of mouse colon. The Journal of Physiology 413, 505-519 | [Abstract] |
| Bywater, R. A. R. & Taylor, G. S. (1986). Non-cholinergic excitatory and inhibitory junction potentials in the circular muscle layer of the guinea-pig ileum. The Journal of Physiology 374, 153-164 | [Abstract] |
| Costa, M., Furness, J. B. & Humphreys, C. M. S. (1986). Apamin distinguishes two types of relaxation mediated by enteric nerves in the guinea-pig gastrointestinal tract. Naunyn-Schmiedeberg's Archives of Pharmacology 332, 79-88. | [Medline] |
| Costa, M., Furness, J. B., Pompolo, S., Brookes, S. J., Bornstein, J. C., Bredt, D. S. & Snyder, S. H. (1992). Projections and chemical coding of neurons with immunoreactivity for nitric oxide synthase in the guinea-pig small intestine. Neuroscience Letters 148, 121-125 | [Medline] |
| Crema, A. (1970). On the polarity of the peristaltic reflex in the colon. In Smooth Muscle, chap. 18, ed. Bülbring, E., Brading, A. F., Jones, A. W. & Tomita, T., pp. 542-548. Edward Arnold Ltd, London. | |
| Crema, A., Frigo, G. M. & Lecchini, S. (1970). A pharmacological analysis of the peristaltic reflex in the isolated colon of the guinea-pig or cat. British Journal of Pharmacology 39, 334-345 | [Medline] |
| Crist, J. R., He, X. D. & Goyal, R. K. (1992). Both ATP and the peptide VIP are inhibitory neurotransmitters in guinea-pig ileum circular muscle. The Journal of Physiology 447, 119-131 | [Abstract] |
| Daniel, E. E. (1982). Pharmacology of adrenergic, cholinergic and drugs acting on other receptors in gastrointestinal muscle. In Mediators and Drugs in Gastrointestinal Motility II, ed. Bertaccini, G., pp. 249-322. Spinger-Verlag, New York. | |
| Furness, J. B., Bornstein, J. C., Pompolo, S., Young, H. M., Kunze, W. A. A. & Kelly, H. (1994). The circuitry of enteric nervous system. Neurogastroenterology and Motility 6, 241-253. | |
| Gregory, J. E. & Bentley, G. A. (1968). The peristaltic reflex in the isolated guinea-pig ileum during drug-induced spasm of the longitudinal muscle. Australian Journal of Experimental Biology and Medical Science 46, 1-16 | [Medline] |
| Hirst, G. D. S., Holman, M. E. & McKirdy, H. C. (1975). Two descending nerve pathways activated by distension of guinea-pig small intestine. The Journal of Physiology 244, 113-127 | [Abstract] |
| Hirst, G. D. S. & McKirdy, H. C. (1974). A nervous mechanism for descending inhibition in the guinea-pig small intestine. The Journal of Physiology 238, 129-143 | [Medline] |
| Hodgkiss, J. P. (1986). Intrinsic reflexes underlying peristalsis in the small intestine of the domestic fowl. The Journal of Physiology 380, 311-328 | [Abstract] |
| Hukuhara, T., Masuda, K. & Kinose, S. (1936). Ueber das 'Gesetz des Darmes'. Pflügers Archiv für die Gesamte Physiologie des Menschen und der Tiere 237, 619-630. Julius Springer, Berlin. | |
| Johnson, P. J., Bornstein, J. C. & Burcher, E. (1998). Roles of neuronal NK1 and NK3 receptors in synaptic transmission during motility reflexes in the guinea-pig ileum. British Journal of Pharmacology 124, 1375-1384 | [Medline] |
| Kottegoda, S. R. (1969). An analysis of the possible nervous mechanisms involved in the peristaltic reflex. The Journal of Physiology 200, 687-712 | [Medline] |
| Lyster, D. J. K., Bywater, R. A. R., Taylor, G. S. & Watson, M. J. (1992). Effects of a nitric oxide synthase inhibitor on non-cholinergic junction potentials in the circular muscle layer of guinea-pig ileum. Journal of the Autonomic Nervous System 41, 187-196 | [Medline] |
| Mackenna, B. R. & McKirdy, H. C. (1972). Peristalsis in the rabbit distal colon. The Journal of Physiology 220, 33-54 | [Medline] |
| Röden, S. H. (1937). An experimental study on intestinal movements; particularly with regard to ileus conditions in cases of trauma and peritonitis. Acta Chirurgica Scandinavica 80, 1-146. | |
| Smith, T. K., Bornstein, J. C. & Furness, J. B. (1990). Distension-evoked ascending and descending reflexes in the circular muscle of the guinea-pig ileum: an intracellular study. Journal of the Autonomic Nervous System 29, 203-217 | [Medline] |
| Smith, T. K., Bornstein, J. C. & Furness, J. B. (1991). Interaction between reflexes evoked by distension and by stimulation of the mucosa of the guinea-pig ileum. Journal of the Autonomic Nervous System 34, 69-76 | [Medline] |
| Smith, T. K., Bornstein, J. C. & Furness, J. B. (1992). Convergence of reflex pathways excited by distension and by stimulation of the mucosa of the guinea-pig ileum. Journal of Neuroscience 12, 1502-1510 | [Abstract] |
| Smith, T. K., Burke, E. P. & Shuttleworth, C. W. (1999). Topographical and electrophysiological characteristics of highly excitable S neurones in the myenteric plexus of the guinea-pig ileum. The Journal of Physiology 517, 817-830. | [Abstract/Full Text] |
| Smith, T. K. & Furness, J. B. (1988). Reflex changes in circular muscle activity elicited by stroking the mucosa: an electrophysiological analysis in the isolated guinea-pig ileum. Journal of the Autonomic Nervous System 25, 205-218 | [Medline] |
| Smith, T. K. & McCarron, S. L. (1998). Nitric oxide modulates cholinergic reflex pathways to the longitudinal and circular muscle in the isolated guinea-pig distal colon. The Journal of Physiology 512, 893-906 | [Abstract/Full Text] |
| Smith, T. K. & Robertson, W. J. (1998). Synchronous movements of the longitudinal and circular muscle during peristalsis in the isolated guinea-pig distal colon. The Journal of Physiology 506, 563-577 | [Abstract/Full Text] |
| Spencer, N. J., Bywater, R. A. R. & Taylor, G. S. (1998). Disinhibition during myoelectric complexes in the mouse colon. Journal of the Autonomic Nervous System 71, 37-47 | [Medline] |
| Tamura, K., Palmer, J. M. & Wood, J. D. (1987). Presynaptic inhibition produced by histamine at nicotinic synapses in enteric ganglia. Neuroscience 25, 171-179. | |
| Tonini, M. & Costa, M. (1990). A pharmacological analysis of the neuronal circuitry involved in distension-evoked enteric excitatory reflex. Neuroscience 38, 787-795 | [Medline] |
| Tsuji, S., Anglade, P., Ozaki, T., Sazi, T. & Yokoyama, S. (1992). Peristaltic movement evoked in intestinal tube devoid of mucosa and submucosa. Japanese The Journal of Physiology 42, 363-375 | [Medline] |
| Waterman, S. A. & Costa, M. (1994). The role of enteric inhibitory motoneurons in peristalsis in the isolated guinea-pig small intestine. The Journal of Physiology 477, 459-468 | [Abstract] |
| Waterman, S. A., Tonini, M. & Costa, M. (1994). The role of ascending excitatory and descending inhibitory pathways in peristalsis in the isolated guinea-pig small intestine. The Journal of Physiology 481, 223-232 | [Abstract] |
| White, H. L., Rainey, W. R., Monaghan, B. & Harris, A. S. (1934). Observations on the nervous control of the ileocaecal sphincter and on intestinal movements in an unanesthetized human subject. American Journal of Physiology 108, 449-457. | |
| Yuan, S. Y., Bornstein, J. C. & Furness, J. B. (1995). Pharmacological evidence that nitric oxide may be a retrograde messenger in the enteric nervous system. British Journal of Pharmacology 114, 428-432 | [Medline] |
| Yuan, S. Y., Bornstein, J. C., Furness, J. B. & Smith, T. K. (1991). Mucosal distortion by compression elicits polarized reflexes and enhances responses of the circular muscle to distension in the small intestine. Journal of the Autonomic Nervous System 35, 219-226 | [Medline] |
Michelle Walsh is a visiting research scholar from the University of Ulster (Coleraine, UK). We wish to acknowledge Professor Kenton Sanders and Dr Fivos Vogalis for helpful suggestions and Dr Kirk Hillsley for suggestions with data analysis. The National Institutes of Health (USA) provided financial support of the project (grant no. RO1 DK45713).
Corresponding author
T. K. Smith: Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA.
Email: tks{at}physio.unr.edu
This article has been cited by other articles:
|
|
 |
|
  J. D. Huizinga and W. J. E. P. Lammers Gut peristalsis is governed by a multitude of cooperating mechanisms Am J Physiol Gastrointest Liver Physiol, January 1, 2009; 296(1): G1 - G8. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. W. M. Janssen, R. G. Lentle, P. Asvarujanon, P. Chambers, K. J. Stafford, and Y. Hemar Characterization of flow and mixing regimes within the ileum of the brushtail possum using residence time distribution analysis with simultaneous spatio-temporal mapping J. Physiol., August 1, 2007; 582(3): 1239 - 1248. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. Gwynne and J. C. Bornstein Mechanisms underlying nutrient-induced segmentation in isolated guinea pig small intestine Am J Physiol Gastrointest Liver Physiol, April 1, 2007; 292(4): G1162 - G1172. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. Huizinga, C. M. McKay, E. J. White, W. J. E. P. Lammers, T. C. Seerden, J. G. De Man, B. Y. De Winter, and P. A. Pelckmans The Many Facets of Intestinal Peristalsis Am J Physiol Gastrointest Liver Physiol, June 1, 2006; 290(6): G1347 - G1349. [Full Text] [PDF]
|
|
|
|
|
|
M. Pimentel, H. C. Lin, P. Enayati, B. van den Burg, H.-R. Lee, J. H. Chen, S. Park, Y. Kong, and J. Conklin Methane, a gas produced by enteric bacteria, slows intestinal transit and augments small intestinal contractile activity Am J Physiol Gastrointest Liver Physiol, June 1, 2006; 290(6): G1089 - G1095. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Lynn, V. Zagorodnyuk, G. Hennig, M. Costa, and S. Brookes Mechanical activation of rectal intraganglionic laminar endings in the guinea pig distal gut J. Physiol., April 15, 2005; 564(2): 589 - 601. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-C. Bian, L. F. Heffer, R. M. Gwynne, J. C. Bornstein, and P. P. Bertrand Synaptic transmission in simple motility reflex pathways excited by distension in guinea pig distal colon Am J Physiol Gastrointest Liver Physiol, November 1, 2004; 287(5): G1017 - G1027. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. Gwynne, E. A. Thomas, S. M. Goh, H. Sjovall, and J. C. Bornstein Segmentation induced by intraluminal fatty acid in isolated guinea-pig duodenum and jejunum J. Physiol., April 15, 2004; 556(2): 557 - 569. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Bisschops, P. Vanden Berghe, E. Bellon, J. Janssens, and J. Tack Electrical stimulation reveals complex neuronal input and activation patterns in single myenteric guinea pig ganglia Am J Physiol Gastrointest Liver Physiol, June 1, 2003; 284(6): G1084 - G1092. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. J. Spencer, G. W. Hennig, and T. K. Smith Stretch-activated neuronal pathways to longitudinal and circular muscle in guinea pig distal colon Am J Physiol Gastrointest Liver Physiol, February 1, 2003; 284(2): G231 - G241. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. J. E. P. Lammers, B. Stephen, and J. R. Slack Similarities and differences in the propagation of slow waves and peristaltic waves Am J Physiol Gastrointest Liver Physiol, September 1, 2002; 283(3): G778 - G786. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. J. Spencer, G. W. Hennig, and T. K. Smith Electrical rhythmicity and spread of action potentials in longitudinal muscle of guinea pig distal colon Am J Physiol Gastrointest Liver Physiol, May 1, 2002; 282(5): G904 - G917. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Vogalis, K. Hillsley, and T. K. Smith Diverse Ionic Currents and Electrical Activity of Cultured Myenteric Neurons From the Guinea Pig Proximal Colon J Neurophysiol, March 1, 2000; 83(3): 1253 - 1263. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. D. J. Thornton and J. C. Bornstein Slow excitatory synaptic potentials evoked by distension in myenteric descending interneurones of guinea-pig ileum J. Physiol., March 1, 2002; 539(2): 589 - 602. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
