Migrating motor complex recorded spontaneously and induced by motilin and erythromycin in an ex vivo rabbit intestinal preparation

Peptides, Vol. 15, No. 6, pp. 1067-1077, 1994 Copyright© 1994ElsevierScienceLtd Printedin the USA.All rightsreserved 0196-9781/94 $6.00+ .00

Pergamon 0196-9781(94)E0035-4

Migrating Motor Complex Recorded Spontaneously and Induced by Motilin and Erythromycin in an Ex Vivo Rabbit Intestinal Preparation L E O N A R D O MARZIO, 1 L A U R I N O GROSSI, L U A N A MARTELLI, M A R I A S S U N T A FALCUCCI AND DOMENICO LAPENNA

Istituto di Fisiopatologia Medica, G.D'Annunzio University, Chieti, Italy Received 6 O c t o b e r 1993 MARZIO, L., L. GROSSI, L. MARTELLI, M. FALCUCCIAND D. LAPENNA. Migrating motor complex recordedspontaneously and induced by motilin and erythromycin in an ex vivo rabbit intestinal preparation. PEPTIDES 15(6) 1067-1077, 1994.--We investigatedbasal motility and the motor effects of motilin, erythromycin, and prostigmine on segments of rabbit gastrointestinal tract removed from extrinsic neural and vascular pathway and immersed in an oxygenated organ bath. Motility was recorded by means of four strain gaugessutured on the serosal surfaceof the segment. During basal recording,clustersof duodenal contractions that propagated distally, resemblingphase III activity of migrating motor complex, were seen. Motilin ( 10-6 M) and erythromycin (10-6 M) induced a propagated cluster of contractions similar to the phase III recorded during the basal period. Prostigmine (10-4 M) induced a simultaneous increase in gastric and small intestinal motility. Atropine (10-5 M) prevented the motor effect of motilin, erythromycin, and prostigmine. Thus, MMCs do not appear to require central input for initiation and propagation. Motilin and erythromycin stimulate MMCs through an enteric cholinergicmechanism; therefore, the previously reported smooth muscle receptors for both substances were not apparent in the ex vivo preparation. Motility

MMC

Erythromycin

Motilin

THE migrating motor complex (MMC) is a distally propagating cyclic activity that is present in the lower esophageal sphincter, the stomach, and the small intestine (6,25). This pattern has been defined as consisting of four phases (4): phase I represents a quiescent phase with few or no contractions; phase II consists of irregular contractions; phase III is characterized by phasic contractions at maximum frequency; phase IV, which may not be always present, has intermittent contraction of short duration. One of the most prominent characteristics of the MMC is that phase III, which may start in the stomach or in the proximal duodenum, migrates downwards into the intestine, sometimes reaching the terminal ileum. Extrinsic innervation does not seem to be necessary for initiation and propagation of MMC (2,27). The studies performed to date to clarify this issue have used animal models with intestinal segments extrinsically denervated but in continuity with the systemic circulation; therefore, a hormonal control could not be excluded. The gastrointestinal peptide motilin is a substance that when secreted in the circulatory system may initiate a phase III of MMC cycle. Motilin plasma level rises with the appearance of

phase III; moreover, an IV infusion of motilin can initiate a premature phase III in the stomach (32,33). When muscle strips of rabbit stomach and small bowel are studied in vitro, motilin acts through the excitation of specific receptors on the smooth muscle (1). However, in humans, dog, opossum, and pig, motilin receptors can only be identified functionally on nerves, and release ofacetylcholine and other transmitters has been implicated in the action of motilin (11,34). Recently, erythromycin (EM), a macrolide antibiotic, has been shown to mimic the effect of motilin in the gastrointestinal tract (15,30) and to exert its action on specific receptors for motilin (22). The first aim of the present study was to investigate whether a spontaneous cyclic activity could be recorded in an intestinal segment completely excluded from any central connection (nervous and humoral). The second aim was to analyze the effect of EM and motilin in this preparation. The third aim was to determine the site of actions of these drugs. To test these hypotheses, we used an in vitro preparation consisting of the rabbit esophagus, stomach, and small intestine,

Requests for reprints should be addressed to Prof. Leonardo Marzio, Patologia Medica, Ospedale SS.Annunziata, 66100 Chieti, Italy.

1067

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MARZIO ET A L

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FIG. 1. Schematic representation of the experimental setup. Sixty centimeters of rabbit intestine are placed in a heated (37°C) and oxygenated organ bath with Kreb's solution. Serosal strain gauges are sutured in distal antrum, and in the small intestine 15 cm apart. Strain gauges are connected to a computer for data storage and analysis. Bottom: basal motor activity of the intestinal segment during the first 60 rain of recording. Cluster of contractions that start in the proximal duodenum are seen to propagate towards the distal part of the segment.

placed in an oxygenated organ bath and therefore completely isolated from the extrinsic nerves and blood circulation. In vivo the rabbit intestine has been shown to produce a MMC pattern that starts in the proximal jejunum either during fasting and or in the fed state (23). METHOD

Experiments were done on 15 rabbits of either sex, weighing 2.5_+0.5 kg (mean +_ SD). Animals were sacrificed by stunning and cervical dislocation. A segment including the esophagus, stomach, duodenum, and part of jejunum was removed and placed in oxygenated (95% 02/5% CO2) Kreb's solution (composition in raM: NaCI 120.9; NaHCO3 15.5; KC1 5.9; CaC12; MgC12 1.2; glucose ! 1.1) and kept at a constant temperature of 37°C. Experiments were performed on the duodenum and jejunum without the stomach in three rabbits; in four rabbits only the distal esophagus and stomach were isolated, and in three rabbits the activity of the ileum alone was recorded as well. Four strain gauges (RB products, Madison, WI) were sutured on the serosal surface: one on the distal antrum, one on the proximal duodenum, and the last two 15 and 30 cm distal to the proximal duodenal strain gauge, respectively (Fig. 1). Strain gauges were placed so that circular contraction could be recorded. In the experiments with the esophagus and stomach, the strain gauges were sutured in the gastric fundus, and proximal and distal antrum 3 cm apart. After positioning the strain gauges, the preparation was placed into a single-chamber organ bath (Ugo Basile, Varese, Italy) containing 1 1 of oxygenated Kxeb's solution. The two ends of the loop were clamped to tubes attached to the side walls of the bath, allowing movement and avoiding any stretching of the intestine (Fig. 1).

The strain gauges were connected to an eight-channel Dynograph Recorder R611 (SensorMedics, Milan, Italy) and balanced at a sensitivity of I MV/mm. The polygraph was connected to a computer (HP Vectra® 386, Hewlett Packard, Milan, Italy) for immediate data storage. For each channel, samples of the motility signal were collected at a rate of 5/s and subsequently analyzed with a motility program (SN Reddy, Torrance, CA USA). After a basal period of 30 rain, allowed for adaptation, a recording of 90 min was obtained for spontaneous activity. At the end of this period, stimulations were performed by the addition to the bath of erythromycin lactobionate, motilin, and prostigmine at various concentrations starting from 10-I° M . Atropine (10 -5 M) was added into the bath before motilin ( 10-s M through 10-5 M), EM (10 -6 M through 10-3 M), and prostigmine (10 -6 M through 10-4 M) in three experiments. Erythromycin, motilin, prostigmine, and atropine were obtained from Sigma Chemical Company (St. Louis, MO).

Analysis of the Motility Recording A front of activity was determined visually and identified as a period of repetitive contractions, propagating from proximal to distal sites with a duration > 2-3 rain. When such a period was identified, duration, contraction frequency, propagation velocity, and interval between two consecutive fronts of activity were calculated. At the computer the recording at each lead was normalized by the motility program so that the lower point was considered zero and the maximum 100. Motility index (i.e., the area under the curve) was computed every minute in the 15 min before and after the drug administration. Two-way analyses of variance (ANOVA) for multiple observations and Student's t-test were used to compare statistically the motility index during

MOTILIN AND ERYTHROMYCIN EFFECTS ON INTESTINAL MOTILITY

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2 3 4 5 6 7 8 9 10 1112131415161718192021222324252627282930 minute• FIG. 2. Motility index (mean _. SE) computed each minute for 15 rain before and after the administration of mofilin ( 10-4 M) (six trials), EM ( l 0 -6 M) (six trials), and prostigmine ( l0 -~ 3,/) (three trials). Horizontal bar and asterisks indicate statistical significance (p < 0.01 ) vs. control values taken l and 2 rain before drug administration.

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M A R Z I O ET AL.

TABLE 1 CHARACTERISTICS OF THE PHASE lIl SPONTANEOUSLY RECORDED AND INDUCED BY MOTILIN AND EM IN VARIOUS PREPARATIONS OF RABBIT INTESTINAL SEGMENTS IN VITRO Spontaneous

Contraction Frequency (cycles/min) Stomach Duodenum Jejunum Propagation velocity (cm/min) Stomach-proximal duodenum Proximal-distal duodenum Duodenum-jejunum Duration of the activity front (min) Stomach Duodenum Jejunum

Motilin (10-6M)

Preparation W i t h Esophagus, Stomach, Duodenum, and Jejunum

PreparationWith Esophagus,Stomach, Duodenum,and Jejunum

14.1 + 0.5* 10.1 +_ 0.8

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Erythromycin (10 6M)

PreparationW i t h Stomach Only

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Preparation With Esophagus, Stomach, Duodenum,and Jejunum

Preparation With Stomach Only

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* p < 0.05 vs. jejunum. f p < 0.05 vs. duodenum-jejunum. p < 0.01 vs. preparation with stomach, duodenum, and jejunum. basal state and after drug administration. Data are expressed as mean _+ SE. RESULTS

m~lin (tO~lVt)

Basal Recording During the basal period, clusters of duodenal contractions propagating to the distal part of the segment were recorded in 65% of the preparations (Fig. 2). They were similar to a phase III activity of M M C s recorded in vivo in rabbits and other species. Propagation velocity and contraction frequency (Table 1) were similar to the phase III described in vivo by other authors (11). The phase III occurred with a period of 16 + 4 min, which was clearly shorter than the one recorded in vivo (11). N o such contractions appeared to start in the stomach. The phase III activity was followed by a period of complete m o t o r quiescence of 3-4 min. Cyclic activity could be recorded for 45-60 min, then it progressively faded and irregular contraction not propagated distally were subsequently seen. In preparations without the stomach, the basal motility pattern was unchanged and phase III activity was recorded in the proximal and distal duodenum. The motility index of the 15 min preceding the drug administration was constant and did not show significant variations (Fig. 3).

Effect of Motilin Motilin at a concentration of 10-1° and 10 -9 M was not effective. At 10-s and 10 -6 M m o t i l i n stimulated a phase III activity that started almost simultaneously in the gastric a n t r u m and in the proximal duodenum. The phase III appeared to propagate to the middle and distal part of the intestinal preparation (Fig. 3). The phase III activity was characterized by an increase in basal tone, superimposed by phasic waves. Duration and propagation velocity of the motilin-induced activity front is summarized in Table 1, and was not different from the spontaneously occurring activity fronts. The contraction frequency decreased significantly from d u o d e n u m to j e j u n u m (Table 1). Higher concentration of motilin up to 10 -5 M did not change the motility

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pattern. The analysis of the motility index calculated every minute shows a significant increase in basal values after motilin (Fig. 2). This increase starts in the same minute in stomach and proximal duodenum and subsequently in the distal duodenum and

proximal jejunum, respectively (Fig. 2). At a magnification of the recording trace obtained with the computerized analysis, it appeared that in 60% of the trials the effect of motilin started first in the proximal duodenum and after 10-15 s in the stomach,

1072

MARZIO ET AL. erythromydn(104 M)

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In the preparations with the stomach only, EM and motilin induced a simultaneous increase in phasic motor activity in all the gastric recording sites (Figs. 7,8). The contraction frequency of the motor waves was similar to the one computed in the preparation where the stomach was in continuity with duodenum and jejunum (Table 1). The duration of motilin and EM-induced motor activity was significantly longer than the one noted in the preparation when the stomach was in continuity with the duodenum and jejunum (Table l). In preparations without the stomach, the effect of motilin and EM was identical for contraction frequency, propagation velocity, and duration of the activity front to the effect observed with the stomach in situ. Motilin and EM failed to generate a propulsive activity in preparations obtained, using segments of only distal ileum.

Effect of Prostigmine Prostigmine (10 -6 M) induced an increase in phasic and tonic activity for 7-8 rain that initiated simultaneously at all recording sites (Fig. 9).

Effect of Atropine p¢oximalduodenum

The effects of motilin, EM, and prostigmine were antagonized by a previous administration of atropine (10 -5 M) at a concentration of l0 -s to l0 -5 M for motilin, l0 -6 to l0 -3 M for EM a n d l0 -6 M for prostigmine (Fig. 10).

distal duodenum

erythmmycia(IO'3M)

Q FIG. 5. Experimental setup (as in >Fig. 1). Erythromycin (10 -6 M) induces a cluster of repetitive motor waves that starts in the stomach and migrates towards the distal recording sites. with subsequent activation of the distal portions of the segment [Fig. 4(A)]. In the rest of the cases a simultaneous activation of stomach and proximal duodenum was recorded [Fig. 4(B)]. Propagation velocity of the motilin-induced phase III significantly decreased, proceeding from proximal duodenum to jejunum similar to the spontaneously occurring activity fronts (Table l).

Effect of Erythromycin Erythromycin at a concentration of 10-6 M generated a front of activity in the stomach and duodenum that propagated to the distal part of the preparation, as shown in Fig. 5. This aspect was similar to the spontaneously occurring and the motilin-induced motility pattern with respect to propagation velocity and duration at each recording site (Table 1). Contraction frequency of the EM-induced activity front decreased, also proceeding from duodenum to jejunum (Table 1). Motility index after EM increased significantly in comparison to the basal state (Fig. 2). This increase was computed in the stomach and proximal jejunum in the same minute, and 1 and 2 min later in distal duodenum and jejunum, respectively (Fig. 2). At the highest concentration (10 -3 M), EM was effective only in the stomach, with a series of phasic motor waves that lasted for 5-6 min, and did not propagate distally (Fig. 6).

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FIG. 6. Experimental setup (as in Fig. 1). Erythromycin 00 -3 M) induces a motor activity that starts in the stomach whereas no effect is seen in the duodenal and jejunal recording sites.

MOTILIN AND ERYTHROMYCIN

EFFECTS ON INTESTINAL MOTILITY

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FIG. 8. Preparation with rabbit stomach. Motilin (10 -6 M) induces a simultaneous increase in gastric motor activity in all stomach leads.

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MARZIO ET AL.

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Addition of drugs to the proximal or distal ends of the bath did not influence the propagation polarity of the activity front that was always in the oral-aboral direction. DISCUSSION

Our findings show that rabbit gastrointestinal segments, including esophagus, stomach, and proximal small intestine, when completely separated from extrinsic nerves and systemic circulation and placed in an organ bath, show spontaneous phase III activity of the MMC at a higher frequency than the ones already described in vivo. Motilin and EM in this preparation induced a motility pattern characterized by a phase III of MMC that was similar to the spontaneous occurring one. This pattern was completely prevented by a pretreatment with atropine. Initiation and propagation of MMC has already been shown to be independent from the integrity of extrinsic nerves because they can be recorded after vagotomy, splanchnectomy, or in a Thirty-Vella loop (25). The MMC period may be reduced after extrinsic denervation, suggesting a modulatory role for extrinsic nerves on MMC frequency (28). Distal propagation of MMC is not modified after denervation, but may be blocked by the local administration of atropine and hexamentonium (27), suggesting that cholinergic intrinsic nerves are essential for this process. Our study shows that phase III of MMC may be recorded in a segment of rabbit intestine placed in an organ bath. Phase Ill period and duration recorded in our preparation are shorter than the ones described in vivo (23). These results rule out an involvement of the extrinsic nerves in the process of initiation and propagation of MMC and emphasize that ex-

trinsic innervation may be important in the regulation of the duration of various phases of MMC. Because our preparation was completely separated not only from the extrinsic nerves but also the from systemic circulation, an hormonal involvement for initiation and propagation of duodenal MMC may also be excluded. We did not record any spontaneous phase III arising from the stomach in any of the preparations studied. Phase III activity could be recorded in the small intestine in preparations either with or without the stomach. This suggests that the stomach is not determinant for generation of MMC and that stomach MMC is driven by extrinsic nerves or, as suggested by others, by hormonal (motilin) release (31) from the proximal duodenum (29). Motilin released from endocrine cells in the duodenal wall and activated by duodenal phase III should reach the stomach through the systemic circulation and in turn stimulate a stomach phase III activity. By the analysis of the motility index we found that the activity front induced by motilin and EM starts in the same minute in the stomach and proximal duodenum and subsequently in the proximal duodenum and jejunum. On closer inspection of the recording, however, it appeared that the activation of the stomach and proximal duodenum in most of the cases was backwards. A simultaneous activation of stomach, pylorus, and descendent duodenum was seen in dogs in vivo after 300 mg IV infusion of EM (14). These authors were not able to highlight a propagation of the activity front induced by EM, probably because they studied a too short se~aent ofintestine. Boeause the activity front induced by motilin and EM has a propal~tion velocity of 20-22 cm/min betwoen proximal and distal duodenum and of 10-11 cm/min between distal duodenum-jejunum, in this area

MOTILIN AND ERYTHROMYCIN EFFECTS ON INTESTINAL MOTILITY

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FIG. 10. Motility index ( m e a n + SE) performed each m i n u t e for 15 m i n before a n d 15 m i n atter atropine plus motilin, EM, a n d prostigmine. Atropine prevents the effects o f motilin (six trials), E M (six trials), and prostigmine (three trials).

two recording sites need to be at least 20 cm apart to detect a delay of I min in the appearance of phase IlL The duration of the activity front induced by EM and motilin in the preparation with the stomach only was longer than the one seen in the preparation with the stomach in continuity with the

small intestine. This difference may be due to the lack of inhibition of proximal motility induced by phase III activity when it reaches the distal small intestine. In fact, it has been postdated that phase III activity, while migrating through the distal small intestine, may inhibit contractions in the proximal sites (17).

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MARZIO ET AL.

Motilin and EM were effective either in preparations with the stomach or in those with duodenum and jejunum. No effect was recorded in the ileum. These results support the evidence that motilin receptors are mainly located in the stomach and duodenum, and that their density decreases proceeding towards the distal part of the intestine. In fact, EM contracts rabbit duodenal muscle strips in a dose-dependant manner, with poor effect on strips from ileum and colon (1), and in dogs motilin is ineffective in initiating premature phase III in the surgically isolated ileum (18). Erythromycin has been shown to produce multiple motor effects other than initiate premature MMCs, such as stimulation of giant migrating contractions and amyogenesia (21). We were not able to record the above-cited motor pattern; however, at highest concentrations EM started high and repetitive gastric contractions with no effect on small intestine. In dogs, at high dosages, EM prolongs MMC cycle length and stimulates gastroduodenal coordination (3). In humans, a prevalent antral stimulation with reduction in duodenal activity is seen (16,26). Therefore, it seems that EM has, at high dosages, a different effect from motilin; this suggests that other mechanisms in addition to the activation of specific receptors for motilin may be involved. Many studies suggest that the effects of EM and motilin on rabbit gastrointestinal tract are mediated by receptors at the smooth muscle because the contraction produced cannot be blocked by atropine and tetrodotoxin. All these studies

were performed on isolated smooth muscle strips in vitro. There are no studies on the mechanism of action of motilin in rabbit ex vivo. In contrast, in dogs neural cholinergic receptors for motilin have been identified functionally in the stomach and upper small intestine (10). In the present study, atropine completely prevented the effects of motilin and EM at all tested dosages. Nevertheless, prostigmine, when added into the organ bath, failed to mimic the effect of both drugs and induced a simultaneous increase in motor activity at all recording sites. This suggests that specific motilin receptors, if present, are at cholinergic sites and modulate the release of acetylcholine. A discrepancy between the in vivo and in vitro results of peptide actions has already been shown by others (9). Our study emphasizes that the discrepancy is not between in vivo and in vitro preparation, but between muscle strips and whole intestinal segment. Whole segments provide an integrated network that may be necessary for the expression of neural receptors and potential downregulation of smooth muscle receptors. It may be that isolation of the muscle from the integrated network allows the upregulation of muscle receptors in the in vitro situation similar to that reported for gastrin in the dog stomach (7). In summary this study shows that the capacity to generate and to propagate phase III activity of MMC is an intrinsic property of the rabbit small intestine and that motilin and eryrthromycin act at peripheral nervous sites through the release of acetylcholine.

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