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MS 1277 Received 21 June 2000; accepted after revision 20 September 2000.
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ABSTRACT |
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INTRODUCTION |
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Bayliss & Starling (1899) were the first to note that inflation of a balloon in the lumen of the extrinsically denervated canine small intestine initiated local reflexes: an oral contraction and anal relaxation. If the inflated balloon was free to move, it was propelled down the bowel by a propagating wave of contraction, which they called peristalsis that was 'dependent upon activity in the local nervous system'.
Unfortunately, the term peristalsis has been used to describe a variety of propagating contractions in different regions of the gastrointestinal tract and purely muscular tubular organs, despite potential differences in their underlying mechanisms. In 1949, Bozler was well aware of this, and suggested that 'confusion has been introduced by the term peristalsis'. He believed that the term peristalsis in the bowel should be considered as a myenteric reflex that is is inhibited by drugs like cocaine or nicotine, which interfere with neuronal transmission. He went on to say that it is 'erroneous, however, to assume that every wave of contraction in smooth muscle is caused by the reflex. Simple waves of contraction, such as those in the stomach and ureter are due to muscular conduction, whereas peristalsis caused by a bolus is a much more complex phenomenon'.
The isolated guinea-pig small intestine has been the preparation of choice since the Trendelenberg (1917) technique was introduced for studying intestinal peristalsis. Once a segment of bowel is distended to a critical threshold during intraluminal fluid infusion, there is an abrupt contraction of the circular muscle which then propagates down the intestine (reviewed by Kosterlitz & Lees, 1963; McKenna & McKirdy, 1972; Tsuji et al. 1992; Waterman et al. 1994; Holzer, 1997; Smith & Robertson, 1998; Brookes et al. 1999). It has therefore been generally assumed that peristalsis is an 'all-or-none' response involving the whole length of the segment of small intestine (Kosterlitz & Lees, 1963). However, in their electrophysiological studies, Hirst et al. (1975) visually observed a propagating ring of contraction aboral to a localized distension stimulus. Since it is clear that localized stimuli lead to graded reflex responses in both myenteric neurons and smooth muscle (Smith & Furness, 1988; Smith et al. 1990, 1992; Smith & McCarron, 1998; Brookes et al. 1999; Spencer et al. 1999) then perhaps peristaltic waves in the small intestine can be graded according to stimulus intensity. Graded peristaltic waves was a feature that Bozler (1949) believed distinguished true peristaltic waves in the bowel, which were dependent on the intrinsic nervous system, from those 'all-or-none' waves dependent largely on the myogenic properties of the muscle, e.g. the ureter. Indeed, recently we have shown that peristaltic waves, which are blocked by tetrodotoxin, can also be elicited by mucosal stimulation in the guinea-pig small intestine, suggesting that they may be gradeable (Spencer et al. 1999).
The mechanisms underlying the propagation of peristalsis are still incompletely understood. What remains unclear is whether peristaltic waves require sequential contraction of the musculature, in order for their propagation to proceed along the bowel. Recently, Kunze et al. (1999) showed that during direct intracellular recordings from the intrinsic primary sensory neurons in the guinea-pig small bowel, the activity of such neurons was dependent upon the contraction and tone of the smooth muscle. Yet, it is unclear how contraction of the smooth muscles influences the propagation of peristalsis. In studies by Waterman & Costa (1994) and Waterman et al. (1994), it was suggested that the maintenance of propagation of peristalsis along the guinea-pig ileum was critically dependent upon contraction of the circular muscle, so as to 'unload' sensory neurons at the contracted region of circular muscle. Also, these investigators suggested that propagation of peristalsis required distension of the bowel, caused by the propulsion of intraluminal fluid (Waterman et al. 1994).
In this study, we have investigated whether peristaltic waves in the guinea-pig jejunum can be graded according to local stimulation intensity, whether they can occur in an empty intestine, and whether muscle tone and contraction are necessary for their initiation and propagation.
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METHODS |
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Preparation of tissues
Guinea-pigs weighing 250-400 g were killed by CO2 inhalation overdose and exanguination, in accordance with the animal ethics committee of the University of Nevada School of Medicine. The abdominal cavity was opened and approximately 15-20 cm of jejunum was removed and the lumen flushed clean with a modified Krebs solution (composition below), then placed immediately into Krebs solution (at room temperature).
Partitioned peristaltic bath
We used the partitioned organ bath technique to pharmacologically isolate particular regions of bowel (see Tonini & Costa, 1990; Kadlec et al. 1991; Smith & Robertson, 1998; Smith & McCarron, 1998; Spencer et al. 2000). The peristaltic bath was divided into three chambers by two partitions: an oral chamber (length: 7·5 cm, width: 5·5 cm, volume: 
175 ml), an intermediate chamber (length: 2 cm, width: 5·5 cm, volume: 70 ml) and an anal chamber (length: 7 cm, width: 5·5 cm, volume: 150 ml) (see Fig. 1A). The isolated jejunal segment (13 cm in length) was passed through two pores (i.d. 1-2 mm) that comprised the two partitions which bordered the intermediate chamber. For these partitions, we used fine condom rubber, which was mounted between two Perspex sheets that formed each partition (see Smith & Robertson, 1998; Smith & McCarron, 1998; Spencer et al. 1999). Both the oral and anal extremities of the preparation were mounted over a plastic tube with a rubber 'O' ring that generated a tight seal between the inflow sites in the oral chamber and the anal chamber (Fig. 1).
Recordings of circular muscle mechanical activity
The mechanical activity of the CM was monitored in each of the three chambers using isometric tension transducers (Kent Scientific Corporation, Litchfield, CT, USA) connected by small clips (Micro-serrefines No. 18055-04; Fine Science Tools Inc., Foster City, CA, USA) attached to the intestinal wall as shown in Fig. 1A. To anchor the preparation in the oral and anal chambers, a small ball bearing (diameter 1·5 mm) was placed in the lumen over a button magnet mounted on a support attached to the floor of the recording chamber (Fig. 1A). Initial tensions of the CM were routinely set to 500 mg, and were re-adjusted back to this level when time or events reduced the resting tone. Preparations were bathed in oxygenated Krebs solution at 35-37°C. The fluid in each chamber was replaced approximately every 30 min with fresh oxygenated Krebs solution.
Protocol for stimulation of peristalsis by fluid infusion
After equilibration for 2 h at 35-37°C, the lumen of the preparation in the oral chamber was infused with Krebs solution (35-36°C) at a rate of approximately 0·2 ml min-1, using a peristaltic pump (Masterflex 7523-30 with cartridge 3519-85, Cole Palmer, USA). Unlike the distal colon, which readily exhibits run down and usually does not generate rhythmical peristaltic waves for prolonged periods of time (see Smith & Robertson, 1998), the small bowel typically is not particularly susceptible to fatigue and we often infused fluid into the lumen for extended periods (i.e. 1-2 h).
In some experiments, we recorded intraluminal pressure from the segment of jejunum in the oral chamber only, while also simultaneously recording CM mechanical activity in the oral, intermediate and anal chambers. Luminal pressure was recorded using a blood pressure transducer (model TSD104A; MP100, BIOPAC systems, Inc., Santa Barbara, CA, USA) that was AC coupled to avoid movement artifacts. Both luminal pressure and mechanical activity of the CM were all recorded on a PC running Acqknowledge 3.2.6 (BIOPAC Systems, Inc., Santa Barbara, CA, USA).
Dissection procedure to prevent fluid expulsion from oral to anal chambers
To investigate whether propagation of peristalsis required distension of the intestinal wall, caused by the propulsion of intraluminal fluid, we cut open the intestine along the mesenteric border that lay in the intermediate chamber (see Fig. 6A). This was performed to prevent fluid expulsion from the segment in the oral chamber to the segment of bowel that lay in the intermediate and anal chambers. A peristaltic wave was initiated raising the intraluminal pressure by infusing fluid into the oral segment; the displaced fluid associated with the wave being expelled through a cannula into the intermediate chamber (see Fig. 6A). This procedure enabled us to investigate whether the propagation of peristaltic waves required the propulsion of fluid along the bowel and whether local paralysis of CM motor activity to the intermediate chamber would terminate peristaltic propagation into the anal chamber.
Protocol for stimulation of peristalsis by localized balloon distension
In other experiments, we tested whether a peristaltic wave could be evoked in a fluid-free preparation. That is, whether or not a peristaltic wave could propagate along the bowel without luminal contents. To maintain a fluid-free preparation, the intestine was sealed with 'O' rings at the oral and anal extremities of the jejunum, after the intraluminal balloon had been inserted into the oral region. Distension stimuli (approximately 1 s in duration) were always delivered locally to the oral region of jejunum using graded infusion volumes from 100 to 600 µl. The intraluminal balloon consisted of a 3 ml arteriolar embolectomy catheter made from silicon elastomer-coated latex (Medline, Malaysia).
Drugs and solutions
The following drugs were used throughout the current study: nifedipine and tetrodotoxin, which were obtained from Sigma Chemical Co., St Louis, MO, USA. We chose the L-type Ca2+ channel blocker nifedipine to test the involvement of muscle contraction in propagation of peristalsis, since this drug has been routinely shown to paralyse CM contraction, but preserve neuromuscular (Bywater & Taylor, 1986) or neuro-neuronal transmission in the guinea-pig small intestine (Smith et al. 1992). 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. In some experiments, the composition of the Krebs solution was modified to block all synaptic transmission in the small bowel (see Bennett, 1967). This was performed by a tenfold reduction in the Ca2+ concentration and by raising the Mg2+ concentration by the same amount (Spencer et al. 2000). This solution contained (mM): NaCl, 120·35; KCl, 5·9; NaHCO3, 15·5; NaH2PO4, 1·2; MgSO4, 12·0; CaCl2, 0·25 and glucose, 11·5. The Krebs solution in each chamber was gassed continuously with a mixture containing 3 % CO2-97 % O2 (v/v), pH 7·3-7·4.
Measurements and statistics
Student's paired t tests or analysis of variance (ANOVA) with all pairwise multiple comparison procedures (Dunnet's Method) were used where appropriate. A minimum significance level of P < 0·05 was used throughout. The use of 'n' in Results refers to the number of animals on which observations were made and data are presented as means ± standard error of the mean, S.E.M.). Measurements of amplitude and half-width, intraluminal pressure, interval between peristaltic waves and propagation velocity of peristaltic waves were all made using Acqknowledge 3.2.6 (BIOPAC Systems, Inc., Santa Barbara, CA, USA). We evaluated the propagation velocity of peristaltic waves by dividing the distance between the oral and anal recording sites by the time taken for peristaltic waves to traverse from the oral to anal recording sites (measured from half-peak amplitude).
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RESULTS |
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Effects of localized balloon distension to the oral chamber
It has been suggested that propagation of peristaltic waves requires 'the continuous stimulation by the contents of the organ' (Bozler, 1949); and similarly that 'the propagation of circular muscle contraction stops when there is no longer a sufficient distension stimulus ahead' (Waterman et al. 1994). We tested whether peristaltic waves, which were sensitive to tetrodotoxin, could be evoked and propagate along an empty intestine, by applying local balloon-distension stimuli to the oral chamber and recording circular muscle (CM) motor activity of the jejunum in the oral, intermediate and anal chambers (see Fig. 1A). It was found that, low distension stimuli (balloon volume: 100-200 µl) were typically without effect on CM activity in any chamber, although these low distension volumes occasionally evoked a local CM contraction in the oral chamber that was not observed in any other chamber. However, when the balloon distension volume was increased in a graded stepwise fashion from 200 to 500 µl (maintained for 1 s), it was possible to evoke a single peristaltic wave that was initiated in the oral chamber and propagated through the intermediate region and into the anal recording chamber (Fig. 1B). The propagation of these peristaltic waves into the anal chamber clearly did not require distension of the intestinal wall caused by the propulsion of luminal contents, since propagation always occurred in an empty intestine. Overall, from 18 preparations tested (n = 17 animals), the contraction of the CM became significantly attenuated as the peristaltic wave propagated down the bowel (oral chamber: 7·4 ± 1·1 mN; intermediate chamber: 6·1 ± 0·8 mN and anal chamber: 3·6 ± 0·6 mN; 18 preparations, P = 0·01; ANOVA; with Dunnet's Method). In six of these preparations there was no notable decrease in the amplitude of the CM contraction, as the peristaltic wave propagated down the empty segment of jejunum, an example of which is shown in Fig. 1B. There was no significant difference in the half-duration of the peristaltic wave in the three recording chambers (oral chamber: 1·5 ± 0·1 s; intermediate chamber: 1·4 ± 0·1 s; anal chamber: 1·3 ± 0·1 s; P = 0·69; 18 preparations, ANOVA; Dunnet's Method). The mean propagation velocity of these peristaltic waves was 10 ± 1 cm s-1 (range: 4-20 cm s-1; 18 preparations; n = 17). To test the involvement of the enteric nervous systems in the propagation of these peristaltic waves, TTX was applied to the intermediate chamber. TTX (1-2 µM) immediately abolished wave propagation from the oral to anal chambers, when applied to this chamber (Fig. 1C; n = 5).
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A, a segment of jejunum (~13 cm) was mounted at either end of an organ bath after it was threaded through a hole in each of the two partitions. The partitions divided the organ bath into the three chambers, an oral stimulation chamber (7·5 cm) and an anal recording chamber (5·5 cm), that were separated by an intermediate chamber (2 cm) to which drugs were applied. Graded distensions (100-600 µl) were performed with an intraluminal balloon inserted into the oral end of the segment. The preparation was anchored to the organ bath by two ball bearings (bb) inserted into the lumen, which were positioned above two magnets. A pressure transducer (BP, balloon pressure) attached to the balloon inflow line was used as a stimulus monitor. Circular muscle (CM) tension was measured in each chamber using isometric force transducers (T). B, intraluminal balloon distension (volume: 500 µl) elicited a peristaltic wave which propagated from the oral chamber through the intermediate region and into the anal chamber, without any noteable decrease in amplitude. This occurred with no free fluid in the lumen. C, addition of TTX (2 µM) to the intermediate chamber abolished the propagation of peristaltic waves into the anal chamber. | ||
Effects of localized muscular paralysis on peristalsis elicited by balloon distension
We sought to investigate whether the propagation of peristaltic waves elicited by local balloon distension would be affected by localized paralysis of CM contraction in the intermediate chamber. To test this, nifedipine (200-400 nM), was added to the intermediate chamber. This antagonist blocks L-type Ca2+ channels which abolishes action potential firing in CM, without affecting neuromuscular transmission in this tissue (Bywater & Taylor, 1986). Nifedipine rapidly abolished spontaneous activity in this chamber only. It was found that with relatively low distension stimuli (< 300 µl infusions), the propagation of peristaltic waves was either abolished, or significantly attenuated, when compared with responses obtained prior to nifedipine application (Fig. 2B). However, as the distension stimulus intensity was increased (> 300 µl balloon volumes), the amplitude of CM contractions in the oral and anal chambers also became enhanced. When compared with control conditions, the abolition of CM contraction in the intermediate chamber significantly attenuated CM contraction amplitude in the anal recording chamber (cf. Fig. 2A and B) over the range of stimulus intensities (Fig. 3). At maximal distension stimuli (500 µl balloon volume) the amplitude of CM contraction in the anal chamber was significantly attenuated from control, 4·2 ± 0·6 mN (n = 7), to nifedipine, 1·9 ± 0·5 mN (P = 0·018; n = 7) (cf. Figs 2A and B, and 3). Interestingly, there was no overall significant difference in the propagation velocity of the peristaltic waves when compared before and after application of nifedipine to the intermediate chamber (control: 6·4 ± 1·0 cm s-1, nifedipine: 7·5 ± 1·0 cm s-1, P = 0·39). Thus, local muscle paralysis appears to have no appreciable effect on the conduction velocity of a peristaltic wave.
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A, graded distensions (300, 400, 500 µl) of the jejunum evoked graded increases in peristaltic wave amplitude of CM in the oral chamber. Peristaltic waves propagated into the anal recording chamber. B, application of nifedipine (200 nM) to the intermediate chamber abolished CM contraction in this region and caused a reduction in the peristaltic wave amplitude in the anal recording chamber. | ||
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Graded increases in balloon distension in the oral chamber evoked graded increases in peristaltic CM contractions in the anal recording chamber. Addition of nifedipine (200 nM) to the intermediate chamber significantly attenuated the amplitude of the CM contraction associated with each peristaltic wave in the anal recording chamber. Note that peristaltic waves were still gradeable after nifedipine. *P < 0·05. | ||
Effects of low Ca2+-high Mg2+ solution on propagation of peristalsis
It might be argued that long descending interneurons were able to transmit neuronal activity underlying peristaltic propagation from the oral chamber into the anal recording chamber, without involving a synapse in the intermediate chamber. Therefore, we tested this possibility by applying a low Ca2+-high Mg2+ solution (see Methods) to the intermediate chamber to block all synaptic transmission. In 6 out of 6 preparations tested (n = 3) we were consistently able to abolish peristaltic propagation from the oral chamber, into the intermediate and anal chambers (within 10-15 min following application), without an effect on peristaltic activity initiated in the oral chamber (Fig. 4). Following application of this solution, spontaneous background contractions were markedly attenuated, or abolished in the intermediate chamber. Within approximately 5-10 min of replacement of normal Krebs solution to the intermediate chamber, we were able to completely recover the propagation of peristaltic waves into the anal chamber (Fig. 4). Spontaneous mechanical activity of the CM in the intermediate chamber became markedly more active almost immediately upon replacement of normal Krebs solution.
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A, balloon distension (500 µl) evoked a peristaltic wave that propagated into the anal chamber, without the presence of fluid in the lumen. B, following application of a low Ca2+-high Mg2+ solution to the intermediate chamber (for ~8 min), identical distension stimuli evoked a local contraction of the CM in the oral chamber, but this did not propagate into the intermediate or anal chambers. C, 3·3 min following normal Krebs solution to the intermediate chamber, an identical distension stimulus elicited a peristaltic wave that resumed propagation into the anal recording chamber. | ||
Effects of a longitudinal incision on propagation of a peristaltic wave elicited by balloon distension
The role of slow waves, if any, in the propagation of peristalsis is unclear in the guinea-pig small intestine. A previous report on the guinea-pig small intestine had shown that slow waves were recorded in intact tubular preparations, but were not recorded from opened sheet preparations (Smith, 1989). Therefore, to test whether opening the bowel would prevent the propagation of peristaltic waves, due to a possible impairment in slow wave activity, we made a longitudinal incision in the tissue, once reproducible peristaltic waves had been initiated by balloon distension of the oral region of jejunum. Surprisingly, in 4 out of 4 animals tested, a 1 cm incision along the mesenteric attachment of the bowel in the intermediate chamber did not have any significant effect on peristaltic contraction amplitude, half-duration or propagation velocity in the anal chamber (cf. Fig. 5A and B; P > 0·05; n = 4). Interestingly, we found that immediately following longitudinal incisions, peristaltic waves could still be elicited without any differences from control, and peristaltic waves were still gradeable in amplitude in each animal tested (see Fig. 5B). Since a longitudinal incision was not found to block propagation of peristaltic waves, this lead us to pursue the next series of experiments below.
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A, control peristaltic wave elicited following a 600 µl balloon-distension stimulus that propagated into the anal region. B, in the same preparation, following a longitudinal incision (1 cm in length) to the intermediate region of bowel (between the oral and anal recording sites, see Fig. 1A) gradeable peristaltic waves could still be elicited immediately following the incision, that propagated into the anal region without any noteable decrease in amplitude. Arrows 1, 2 and 3 represent increasing distension stimuli (400, 500 and 600 µl, respectively). | ||
Peristaltic waves elicited by fluid infusion
As a further test that propagation of peristaltic waves did not require the propulsion of fluid along the bowel, nor luminal distension, peristalsis was also elicited by slow fluid infusion into the lumen in the oral chamber, but the propulsion of fluid was diverted from reaching the lumen in the intermediate and anal chambers (see Fig. 6A). Peristaltic waves were initiated by slow infusion (at a rate of 2 ml min-1) of Krebs solution into the lumen of the preparation in the oral chamber (Fig. 6A). As the bowel gradually became distended the longitudinal muscle layer became increasingly more active (described as the preparatory phase, see Kosterlitz & Lees, 1963; Waterman et al. 1994; Smith & Robertson, 1998; Hennig et al. 1999; Brookes et al. 1999). At a critical threshold level of distension, an abrupt contraction of the CM was elicitied and a peristaltic wave could be clearly visualized as a robust propagating event that swept in an aboral direction from the oral stimulation chamber (Fig. 6B). In the oral chamber, peristaltic waves of the CM exhibited the following characteristics (amplitude: 32·3 ± 4·0 mN; half-duration: 1·7 ± 0·1 s; and waves: 0·9 ± 0·1 min-1; 17 preparations, n = 15). In the intermediate and anal chambers where the preparation was not infused with fluid and where the tissue had been incised (intermediate chamber, Fig. 6A), the amplitude of the CM contractions were significantly attenuated compared with the CM contractions in the oral chamber (P < 0·01; ANOVA using Dunnet's Method; 17 preparations, n = 15). An example of this is shown in Fig. 6B. In the intermediate chamber, CM contractions had an amplitude of 5·4 ± 2·1 mN and half-duration of 3·1 ± 0·3 s, while in the anal chamber, CM contractions had an amplitude of 3·8 ± 0·6 mN and a half-duration of 2·9 ± 0·4 s (17 preparations, n = 15).
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A, peristaltic waves were elicited by fluid infusion into the oral end of the jejunum. Approximately 10 mm of the preparation lying within the intermediate chamber was slit open along the mesenteric border and pinned (mucosal side uppermost) to a Sylgard block. The fluid expelled from the oral segment was diverted via a cannula in the intermediate chamber from reaching the anal intact segment. Intraluminal pressure was measured with a pressure gauge attached to the inflow tube. Tension of the CM was recorded in all three chambers with isometric tension transducers. B, peristaltic waves (CM contraction) initiated by fluid infusion propagated into the anal chamber, despite the diverted intraluminal fluid. Each peristaltic wave was associated with a rise in intraluminal pressure. Ball bearings (bb). | ||
Effects of localized muscular paralysis on propagation of peristalsis by fluid infusion
Once reproducible peristaltic waves were elicited in a rhythmic fashion by slow fluid infusion (Fig. 6B), the effects of local CM paralysis in peristaltic propagation was investigated. To do this, we also applied nifedipine to the intermediate chamber and recorded the effects on CM motor activity in the anal recording chamber. Immediately following nifedipine application, there was a reduction in the resting tone of the CM in this region (by 3 ± 1 mN; n = 9) and essentially all contractile activity was abolished in this chamber. The effects of local paralysis of the peristaltic contraction were somewhat variable, although it was clear that the propagation of peristalsis into the anal chamber was not blocked by local blockade of the CM contraction in the intermediate chamber. From nine animals tested, there was no overall significant attenuation of the peristaltic contraction in the anal chamber (control: 3·8 ± 0·8 mN; nifedipine: 3·2 ± 0·8 mN; P = 0·59) following nifedipine (200-400 nM) application to the intermediate chamber. Although in three of these preparations there was no effect of nifedipine on peristaltic propagation into the anal chamber, in most cases (6 of the 9 animals), nifedipine decreased the amplitude of the contraction in the anal chamber, an example of which is shown in Fig. 7. There was no difference in the half-durations of peristaltic contractions of the CM in the anal chamber, before or after nifedipine addition to the intermediate chamber (control: 2·1 ± 0·1 s, nifedipine: 2·0 ± 0·1 s; P = 0·43; n = 9; Student's paired t test). Similar to balloon-distension responses, the propagation velocity of peristaltic waves evoked by fluid infusion was not significantly modified by local paralysis of CM contraction to the intermediate chamber (control: 10 ± 1 cm s-1, range: 6-18 cm s-1; nifedipine: 9 ± 2 cm s-1, range: 5-22 cm s-1; P = 0·78; n = 9; Student's paired t test).
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Peristaltic waves initiated by fluid infusion into the oral region of jejunum. At arrow, nifedipine (400 nM) was added to the intermediate chamber where it reduced the resting tone and CM contraction in this region. Note that peristaltic waves were still able to propagate from the oral chamber into the anal recording chamber; however, their amplitude was reduced by about half: compare the control peristaltic wave (vertical line A) with the response after nifedipine (vertical line B). | ||
It is particularly noteworthy to draw attention to the movement artifacts that we initially observed in this fluid-infused preparation, where local muscle paralysis was intended. Unless the preparation in the intermediate chamber was opened as a sheet and thoroughly pinned to the Sylgard base, pronounced movement artifacts could be detected. This was noted when application of nifedipine to the intermediate chamber abolished CM contraction, but the tissue in this chamber was passively pulled from the contracting CM region in the oral chamber. During the experiment, if these preparations were more thoroughly pinned, movement artifacts disappeared. Preparations in which movement artifacts were observed were discarded for analysis in this study. Movement artifacts were not apparent in preparations where balloon distension was applied to the lumen in fluid-free tissues.
Peristalsis in an intact tubular preparation
We then sought to compare peristalsis elicited by fluid infusion in 10 preparations that were not incised in the intermediate chamber. That is, where propulsion of fluid was maintained through the entire preparation. In contrast to the incised preparations described above (Fig. 6A), it was found that the CM contractile amplitudes were not significantly different between the oral and anal extremities of the tissue, when recording 20 mm from the oral and anal extremities (oral: 48·8 ± 6·2 mN; anal: 36·1 ± 4·3 mN; P = 0·19; n = 10). The propagation velocity of peristaltic waves from these 10 preparations was 6·4 ± 1·0 cm s-1 (range: 3-13 cm s-1; n = 10). Application of TTX (1-2 µM) to the intermediate chamber immediately abolished wave propagation of fluid-driven peristaltic waves, from the oral chamber into the intermediate and anal chambers (n = 9).
Effects of muscle paralysis on the initiation of peristalsis
To test whether paralysis of CM motor activity in the oral (stimulation) chamber would prevent the initiation of peristalsis by fluid infusion, nifedipine (500 nM) was applied to the oral stimulation chamber. Immediately following application of nifedipine to the oral chamber, the amplitude of peristaltic waves elicited by fluid infusion in the oral chamber became dramatically attenuated, and after approximately 3 min following application, peristaltic initiation was abolished, at which point the bowel became swollen due to the ongoing fluid infusion, and peristalsis ceased (n = 6). In seven other animals, where peristaltic waves were elicited by local balloon distension in the oral chamber, we also applied nifedipine (500 nM) to this chamber. Immediately following application, balloon-distension responses in the oral chamber became progressively attenuated in amplitude (Fig. 8). Interestingly, it was found that as the contraction in the oral chamber became reduced, this caused a corresponding reduction in CM contraction amplitudes at the intermediate and anal recording sites that were not exposed to nifedipine (Fig. 8A). When the peristaltic contraction was abolished in the oral chamber, it abolished the contraction in the other chambers not exposed to nifedipine (Fig. 8B). This suggests that the intensity of the peristaltic contraction at the site of initiation largely determines the intensity of the peristaltic contraction at more distal sites in the bowel. Similar responses were obtained when peristaltic waves were elicited by mucosal stimulation (N. J. Spencer & T. K. Smith, unpublished observations). Peristaltic responses were also blocked by TTX (1 µM) in the oral chamber.
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A, balloon distension (500 µl) applied to the oral stimulation chamber (arrow 1) evoked a peristaltic wave which propagated into the anal chamber. Nifedipine (500 nM) was applied to the oral stimulation chamber (arrow A), where it reduced the resting tone and spontaneous activity in this region. The identical stimulus was applied in nifedipine (arrows 2 and 3), which caused a progressive attenuation of the peristaltic contraction amplitude not only in the oral chamber but also in the intermediate and anal chambers. B, in the continued presence of nifedipine, balloon-distension stimuli (500 µl, arrow 4) did not evoke any contraction in the oral chamber, or the intermediate and anal chambers. | ||
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DISCUSSION |
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There are at least two major findings from the current study: (1) peristaltic waves can be graded in amplitude according to stimulus intensity; also, they do not require a preparatory phase, and (2) once initiated, peristaltic waves elicited by either fluid infusion or balloon distension can propagate through a locally paralysed region of smooth muscle. However, our results do suggest that muscle tone and contraction are important not only for initiation but also for maintenance of peristalsis.
Does propagation of peristalsis require distension of the intestinal wall?
It had been suggested that propagation of peristalsis in the guinea-pig small intestine terminates when there is no longer a sufficient distension stimulus to activate enteric nervous pathways (Waterman et al. 1994). Indeed, Bozler (1949) also suggested that 'the reflex response to local stimulation never progresses along the intestine and that such waves cannot be produced in the empty intestine'. A major observation of our study was that local balloon-distension stimuli in the oral chamber could readily elicit a gradeable peristaltic wave that was able to propagate along the entire preparation, despite the absence of intraluminal fluid. Therefore, ongoing distension of the bowel by the displacement of fluid in the lumen is not essential to stimulate the enteric plexuses and maintain propagation. In further support for this, we also show that peristaltic waves initiated in the oral chamber by slow infusion of fluid, were still able to propagate through the intermediate chamber and into the anal chamber, even when the propulsion of fluid had been diverted from entering the anal chamber. Moreover, the velocity of peristaltic waves evoked by local balloon-distension stimuli without contents in the bowel were similar to the propagation velocity of peristaltic waves that were associated with fluid propulsion. This suggests that velocity is not appreciably modified by fluid distension in the lumen, but rather may be an intrinsic property of the nervous system. However, it is important to contrast the differences in the CM contraction amplitude, as the peristaltic wave propagated with and without fluid in the bowel. In most, but not all animals, there was a significant decline in the amplitude of the CM contraction along the jejunum, when the peristaltic wave propagated without fluid in the bowel. In contrast, when fluid was propelled within the lumen of uninterrupted segments during a peristaltic wave, there was no significant decline in CM amplitude. It could be argued from this observation that a moving bolus of fluid may amplify CM contraction by creating a wave of contraction that increases the excitability in primary afferent neurons (Kunze et al. 1998) thereby raising the excitability of other neurons in peristaltic pathways as the contraction proceeds down the bowel. Similarly, in a completely fluid-filled intestine, as in the Trendelenburg system, most neurons along the intestine may be closer to threshold owing to increased activity of local stretch-sensitive sensory neurons during the preparatory phase, and hence more readily activated by propagating nervous activity associated with a peristaltic wave. A preparatory phase is not observed when peristalsis is generated by local stimuli; it appears, therefore, that the preparatory phase may be a feature of the Trendelenburg system resulting from slowly filling the whole gut against a constant back pressure.
Are slow waves necessary for conduction of peristalsis in guinea-pig ileum?
In previous studies, various investigators have shown that drugs which interfere with nerve conduction abolish peristalsis in the small bowel, when applied to the entire tissue preparation. However, this application would also have prevented the initiation of peristalsis (Brookes et al. 1999). The difference in our study was that we were able to selectively apply TTX to the intermediate chamber, so as to avoid an action of TTX on peristaltic initiation. In this experimental arrangement, it was found that following peristaltic initiation by either balloon distension or fluid infusion, addition of TTX to the intermediate chamber consistently abolished peristaltic propagation. This shows that the enteric nervous system was critical for propagation of peristalsis by either stimuli, with or without contents in the bowel. In terms of slow waves in the guinea-pig small intestine, their role if any, in peristalsis is unknown. Although slow waves are common in other species, they are rarely recorded from the guinea-pig ileum and the recent work of Brookes et al. (1999) also did not record slow waves, nor find any role for them, at least in the initiation of peristalsis in this tissue. If slow waves are important for peristaltic propagation in the guinea-pig ileum, then in our view they cannot co-ordinate propagation of peristalsis without excitation from the enteric nerves.
Gradable versus all-or-none peristaltic waves
Peristalsis has been considered to be an all-or-none response (Kosterlitz & Lees, 1963). However, we have shown that a local graded stimulus such as distension or mucosal stimulation (see Fig. 4 in Spencer et al. 1999) can elicit a graded peristaltic wave. Brookes et al. (1999) have also recently reported that in isolated sheet preparations, slow circumferential stretch of the guinea-pig ileum can also evoke gradeable mechanical responses to subthreshold levels for peristaltic initiation. The question is why does distension of the lumen with an intraluminal balloon elicit a gradeable peristaltic wave, whereas fluid infusion produces an all-or-none response? We suggest that balloon distension is a local stimulus which, like mechanical brushing of the mucosa, is likely to activate synchronous firing in specific ascending and descending nerve pathways, the intensity of motor neuronal activation being proportional to the stimulus (Smith & Furness, 1988; Smith et al. 1990, 1992). In contrast, the mechanisms underlying the all-or-none or abrupt initiation of peristalsis during slow fluid infusion are unclear. A possible explanation is that gradual distension of the whole segment would activate sensory neurons along the entire length of the bowel. These would activate both long and short nerve pathways from every point along the entire length of the distended segment; the critical threshold for peristalsis being surpassed when there is a net activation of excitatory over inhibitory motor neurons (see Waterman et al. 1994). In this tissue, distension-evoked inhibitory junction potentials (IJPs) have been shown to be more gradeable with stimulus intensity than evoked excitatory junction potentials which occur at a higher threshold than IJPs (Smith et al. 1990).
Is circular muscle contraction necessary for peristaltic propagation to occur?
It has been unclear whether sequential contraction of the CM associated with a peristaltic wave is necessary for the maintenance of propagation down the intestine. Our data show that peristaltic waves elicited by fluid infusion or balloon distension were able to propagate through the intermediate chamber, despite paralysis of CM, and presumably longitudinal muscles (LM), in this region. This suggests that the propagation of peristalsis down the bowel can occur without sequential contraction of either muscle. However, the results with graded local distension stimuli showed that addition of nifedipine to the intermediate chamber significantly attenuated the amplitude of CM contractions in the anal recording chamber, over a range of stimulus intensities, but peristaltic wave propagation was still able to occur and was gradeable. In the light of the work of Kunze et al. (1998, 1999) it would appear that local muscle paralysis reduces synaptic transmission through the paralysed region. They showed that drugs such as nicardipine and isoprenaline, which reduced longitudinal muscle tension, also suppressed the excitability of myenteric neurons, in particular stretch-sensitive primary afferent neurons. Taken together, this suggests that some degree of mechanosensory feedback from the musculature, even in empty segments of intestine, is necessary to activate local circuits or directly excite neurons in descending excitatory nerve pathways (see Hirst et al. 1974; Brookes et al. 1999; Spencer et al. 1999, 2000) to facilitate peristaltic wave propagation. In the light of the fact that during a peristaltic wave both the circular and the longitudinal muscle layers contract at the same time (Smith & Robertson, 1998; Spencer et al. 1999, 2000; Stevens et al. 2000) mechanosensory feedback may arise from either, or both muscle layers. Mechanosensory feedback could also arise from spontaneous movements of the two muscles, in particular the longitudinal muscle which is constantly active even when the intestine is flaccid (Bayliss & Starling, 1899; Melville et al. 1975; Yokoyama & North, 1983; Yokoyama & Osaki, 1990; Hennig et al. 1999; Stevens et al. 1999, 2000). Presumably, descending inhibitory pathways are less sensitive to mechanosensory feedback than descending excitatory pathways, because they are readily activated in paralysed preparations (Smith & Furness, 1988; Smith et al. 1990).
Role of descending inhibition
In agreement with previous studies (Brookes et al. 1999; Spencer et al. 1999, 2000), we found no evidence of distal relaxation preceding a propagating contraction. Therefore, the role of descending inhibition in peristalsis remains unclear. However, owing to the fact that apamin, which blocks the inhibitory junction potential in the ileum (Smith & Furness, 1988), increases the amplitude and rate of rise of anal contractions and appears to disrupt propagation (see Waterman & Costa, 1994; Spencer et al. 1999), it is likely that descending inhibitory pathways are important for co-ordinating a peristaltic wave.
Involvement of muscle tone in the initiation of peristalsis
It is clear that muscle tone and/or contraction also regulates the threshold for generating peristalsis, since nifedipine applied to the oral stimulation chamber substantially reduced, and in many cases abolished, propagation of peristaltic waves into the other chambers that were not exposed to the drug. Interestingly, as nifedipine reduced the peristaltic contraction in the oral stimulation chamber, it also produced a corresponding reduction in the amplitude of the peristaltic contraction in the intermediate and anal chambers. Perhaps, this is not surprising since activity in primary afferent neurons is dependent on muscle tone rather than stretch per se (Kunze et al. 1998, 1999). Nifedipine would gradually decrease muscle tone and therefore the excitability of these afferent neurons and cause a corresponding reduction in their output to descending excitatory nervous pathways that underlie peristalsis.
Caution should be exercised in extrapolating our results in the small intestine to propagating neurogenic contractions in the large intestine. Rhythmical colonic migrating myoelectric complexes travel spontaneously down the isolated mouse colon, even when the whole preparation is paralysed with nifedipine (Bywater et al. 1989; Spencer et al. 1998), suggesting that propagation of myoelectric complexes in the large bowel may not depend on contraction of the musculature.
Conclusions
In summary, this study demonstrates that gradeable peristaltic waves can be elicited by local graded balloon-distension stimuli of empty intestines. Also, the propagation of peristaltic waves elicited by either balloon distension or fluid infusion, does not require ongoing luminal distension by luminal contents. However, the initiation and propagation of peristaltic waves in the small intestine is dependent upon muscle tone and contraction. This could arise from spontaneous movements of the muscle or propagating contractions of the CM or LM.
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Financial support for this project was provided by the National Institute of Health (USA) (Grant No. RO1 DK45713 to T.K.S.).
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
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