Effects of various peptides on isolated rabbit lower esophageal sphincter

Peptides, Vol. 17,No. 6, pp. 927-931, 1996 Copyright0 1996Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/96$15.00+ .OO PI1 SO196-9781( 96) 00137-4

ELSEVIER

Effects of Various Peptides on Isolated Rabbit Lower Esophageal Sphincter ATSUSHI KOHJITANI,”

JUNJI SHIRAKAWA,”

SAEKO OKADA?

AND HIDEFUMI OBARA”

*Department of Anesthesiology, Kobe University School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650, fSaiseikai Hyogo Prefectural Hospital, Japan Received 6 December 1995 KOHJITANI, A., .I. SHIRAKAWA, S. OK ADA AND H. OBARA. E’ects of variouspeptides on isolated rabbit lower esophageal sphincter. PEPTIDES 17(6) 927-931, 1996.-Strips of lower esophageal sphincter (LES) from rabbits were tested for their responses to several peptides, and to electrical field stimulation (EFS) under the presence of some peptides. Substance P (SP), motilin, and bombesin induced contraction, and vasoactive intestinal peptide (VIP) induced relaxation. SP- and bombesin-induced contractions were antagonized by SP antagonist. VIP-induced relaxation was antagonized by phentolamine and VIP antagonist. Pretreatment with atropine, phentolamine, and diphenhydramine antagonized the motilin- and bombesin-induced contraction. Pretreatment with tetrodotoxin (TTX) attenuated the motilin- and bombesin-induced contraction, but not the SP-induced contraction and VIP-induced relaxation. EFS induced contraction, which was attenuated by ‘FIX. Calcitonin gene-related peptide and neuropeptide Y had no effect on LES; however, they attenuated EFS-induced contraction. These findings suggest some characteristic peptidergic involvement in rabbit LES smooth muscle. Substance P Vasoactive intestinal peptide Lower esophageal sphincter Neuropeptide Y

Calcitonin gene-related peptide Smooth muscle Rabbit

THE enteric nervous system predominantly controls the local modulation of intestinal motility. In recent years there has been considerable interest concerning the physiological and pharmacological properties of the lower esophageal sphincter (LES) to understand the etiology of sphincter incompetence-induced gastrointestinal disorders (48). Various neurotransmitters, hormones, and peptides are: thought to play roles in the regulation of esophageal motility and LES functions ( 11) Immunohistochemical methods (2,3,:!4,25,27,32,33) and radioligand binding studies (20,31,38) have revealed the precise topography of peptide-containing neurons and their specific receptors in the enteric nervous system. Howev’er, the roles of these neurohumoral substances and their receptors in the physiological regulation of the LES have been a subject of considerable controversy. Isolated muscle strips from dog, rat, and guinea pig are not affected by motilin. The rabbit has been useful for investigating the mechanisms of pepti’des, particularly motilin (33). This study investigated the effects of several peptides on isolated rabbit LES smooth muscle: substance P (SP) , vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) , motilin, neuropeptide Y (NPY), and bombesin. Effects of peptides on EFSinduced response were also investigated. METHOD The experimental protocol was approved by the Kobe University Animal Use Committee. Twenty-four adult male Japanese

Motilin

Bombesin

White rabbits weighing 2-3 kg were anesthetized with thiamylal sodium (50 mg/kg, IV) and killed by exsanguination. The lower part of the esophagus and stomach were immediately isolated. The sphincteric region was opened along the longitudinal axis, and the LES was excised by sharp cutting in a circular direction, making strips about 2 mm wide and 5 mm long. The mucosa was then removed by a sharp dissection to exclude the effects of autacoids derived from the mucosa, and to precisely record the contractility of the smooth muscle. The strips were suspended between a fixed hook in a holder and the force-displacement transducer (Strain Amplifier 6M82, Nihondenki-San-ei Co., Tokyo, Japan) under 1 g tension. Changes in the isometric tension of circular muscle were recorded (Recti-Horiz 8K, Nihondenki-San-ei Co., Tokyo, Japan). The strips were suspended in a thermostatically controlled (37.0 2 05°C) 5-ml organ bath containing Krebs-Ringer solution (118 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl*, 25 mM NaHC03, 1.18 mM KI-12P04, 1.19 mM MgS04, 11 mM glucase). The bath fluid was aerated with a mixture of 95% O2 and 5% CO* to keep the pH within 7.35 to 7.45. Before starting the experiments the strips were allowed to equilibrate for 60-90 min in the Krebs solution, which was replaced every 15 min. LES strips were precontracted submaximally with 30 mM KC1 before being exposed to a peptide, except for VIP. In the VIP-induced relaxation experiment, maximal relaxation was obtained by application of 10e4 M papaverine, which is an inhibitor of phosphodiesterase activity (26). The peptides and drugs were

’ Requests for reprints should be addressed to Atsushi Kohjitani. 927

928

KOHJITANI

N=4

9 8 7 6 5 4 concentration of substance P ( -log[M]

ET

AL.

I

11 conceZration if VIP (-13og[fvl]) 7

)

FIG. 1. Concentration-response curves for SP-induced mechanical contraction in the absence (0) or presence (0) of SP antagonist. The concentration-response curve is expressed as a percent of the maximum contraction induced by 30 mM KCI. Each point represents the mean from tissues from four animals; vertical lines show SEM. *Significantly different from the value in the absence of agents.

FIG. 2. Concentration-response curves laxation in the absence (0) or presence centration-response curve is expressed relaxation induced by lo-” M papaverine. from tissues from four animals; vertical different from the value in the absence

for VIP-induced mechanical re(0) of VIP antagonist. The conas a percent of the maximum Each point represents the mean lines show SEM. *Significantly of agents.

RESULTS

applied peptides

directly to the organ bath by micropipette. The effects of were expressed as percent contraction of 30 mM KCl-

induced contraction, or percent relaxation of 10e4 M papaverineinduced relaxation. EFS (100 mA, 1 Hz, 50 ms) was delivered through platinum wire electrodes placed in parallel with the strips at a distance of 1 mm from the muscle. The stimulus originated from a Grass stimulator (DPS-1 IOOD, DIA Medical System Co., Tokyo, Japan). In a series of experiments, each peptide was administered cumulatively, and a dose-response curve was obtained. All antagonists were preincubated for the given time and remained in the bath for the cumulative dose-response curve. The results are expressed as mean values +- SEM. Student’s t-tests were used to test for statistical significance between paired data. For all statistical tests, a value of p 5 0.05 was taken as significant. The following drugs were used: [ D-Pro’,~-Tip”~] substance P, VIP, [ Ac-Tyr ’ ,D-Phe*] GRF( l -29)amide (VIP antagonist), NPY, and CGRP from Peninsula Laboratories Inc. (Belmont, CA) ; SP, motilin (porcine), and bombesin from Peptide Institute Inc. (Osaka, Japan) ; potassium hydrochloride, or,-propranolol hydrochloride, diphenhydramine hydrochloride, and phentolamine hydrochloride from Nacalai Tesque (Kyoto, Japan) ; atropine sulfate, papaverine hydrochloride, and TTX from Sigma Chemical (St. Louis, MO). All agents were dissolved in distilled water and handled in siliconized glassware. All other chemicals used were of reagent grade.

TABLE EFFECTS

OF VARIOUS

ANTAGONISTS

Peptide

Control

SP (3 x lo-’ M) (N = 3) VIP (lo-’ M) (N = 3) Motilin (lo-’ M) (N = 4) Bombesin (10d7 M) (N = 4)

100 100 100 100

SP induced dose-dependent contractions in the LES strips in doses from lo-’ to 10m4 M. VIP induced dose-dependent relaxation, from 10-r’ to lo-’ M. The application of the putative SP antagonist [ o-Pro*,~-Trp’.‘] substance P (3 X 1O-6 M for 15 min) antagonized the contractile effect of SP (Fig. 1). The application of the VIP analogue, [ Ac-Tyr ’ ,o-Phe *J GRF ( l29)amide (3 X 10m6 M for 15 min), antagonized the VIP-induced relaxation (Fig. 2). The effects of various antagonists on lo-’ or 3 X lo-’ M peptide-induced contraction or relaxation are shown in Table 1. Pretreatment with atropine (3 X 10 -6 M), propranolol (3 X 1O-6 M), phentolamine (3 X 10m6 M), and diphenhydramine (3 X 10m6 M) for 10 min did not alter the SPinduced contraction. VIP-induced relaxation was antagonized by phentolamine. TTX (3 x lo-’ M for 15 min) did not alter the SP-induced contraction or the VIP-induced relaxation. Motilin caused dose-dependent contraction of the LES. Pretreatment with atropine, phentolamine, diphenhydramine, and TTX significantly attenuated the motilin-induced contraction (Table 1) . After pretreatment with all these antagonists simultaneously, a part of the contractile component still remained (Fig. 3). Bombesin induced dose-dependent contractions in the LES. Pretreatment with atropine, phentolamine, diphenhydramine, and TTX significantly attenuated the response (Table 1) When [DPro2,D-Trp7,9] substance P was pretreated, bombesin-induced contractions were antagonized (Fig. 4).

1

ON PEPTIDE-INDUCED Ampine

96.4 95.6 26.5 40.8

? % ? -c

2.2 6.4 2.3* 8.9*

CONTRACTION

Propramlol

92.1 94.7 49.9 62.6

2 2 * ?

4.9 3.9 15.1 8.3

OR RELAXATION

Phentolamine

111.1 74.2 42.2 35.2

2 11.1 2 6.8* ? 6.S* i 5.9*

Diphenhydramine

115.0 93.5 37.3 55.3

2 13.7 2 5.1 2 3.5* 2 8.2*

Contraction or relaxation which was induced by lo-’ M or 3 X IO-’ M of each peptide was taken as 100%. Values are expressed as mean 5 SEM. *Significantly different from the control values.

VARIOUS

PEPTIDES

ON RABBIT

929

LES

. .i 50. t 2 5 40.

N=5

! 5 f 30. Y zz E 20s :: z 3 lo-

FIG. 3. Concentration-response curves for motilin-induced mechanical contraction in the absence of agents (0), presence of atropine, propran0101, phentolamine and diphenhydramine simultaneously (e), or presence of TTX (0). The concentration-response curve is expressed as a percent of the maximum contraction induced by 30 mkf KCI. Each point represents the mean from tissues from six animals; vertical lines show SEM. *Significantly different from the value in the absence of agents for TTX. fsignificantly different from the value in the absence of agents for the simultaneous pretreatment of atropine, propranolol, phentolamine, and diphenhydramine.

EFS

induced

of the LES. Pretreatment with TTX the EFS-induced contraction (Fig. 5). CGRP and NPY did not affect the LES response; however, pretreatment with CGRP or NPY ( 10m7 A4 for 15 min) significantly attenuated the EFS-induced contraction (Fig. 5).

v xt

o10

9

7

8

6

concentration of bombesin( -log[Ml ) FIG. 4. Concentration-response curves for bombesin-induced mecharical contraction in the absence of agents (O), presence of SP antagonist (O), or presence of TTX (0). The concentration-response curve is expressed as a percent of the maximum contraction induced by 30 mM KCl. Each point represents the mean from tissues from five animals: vertical lines show SEM. *Significantly different from the value in the absence of agents for SP antagonist. tsignificantly different from the value in the absence of agents for TTX.

contracf.on

(3 x 10 -7 M for 15 min) attenuated

DISCUSSION The effects of various gut-brain peptides and those on EFSinduced response on isolated rabbit LES smooth muscle were investigated in the present study. SP-induced contraction was not

affected by adrenergic, cholinergic, or histaminergic antagonists or by TTX; it was antagonized by its specific antagonist. VIPinduced relaxation was antagonized by phentolamine and the VIP antagonist, but not by TTX. We suggest that SP and VIP exert excitatory and inhibitory effects on the contractility of LES predominantly by stimulating their own receptors located on the esophageal smooth muscle, with additional involvement of an LYadrenergic pathway in VIP-induced relaxation. As the motilinand bombesin-induced #contraction were attenuated by TTX, these LES contractile responses appear to have been induced partially via neural pathways. Atropine, phentolamine, and diphenhydramine attenuated the excitatory effects of motilin and bombesin. The possible inhibitory mechanisms of these antagonists are that the motilin- and bombesin-containing neurons coexist with other neurons such as cu-adrenergic, cholinergic, and histaminergic neurons, and that presynaptic intemeurons of motilin and bombesin link to those neurons. The direct stimulation of esophageal smooth muscle may mediate the antagonist-resistant contraction. EFS induced contraction, and TTX attenuated the EFS-induced contraction to about 60%. Therefore, EFS-induced contraction appears to be mediated partially by neural pathways. In in vitro studies it has been reported that EFS induced relaxation (28,35,37), contraction or relaxation (5) in opossum LES. Intrinsic neurons, which are part of the nonadrenergiclnoncholinergic (NANC) inhibitory system, mediate this stimulation-induced relaxation (10,37). The neurotransmitter of NANC innervation has not been demonstrated; recently nitric oxide (NO) or NO-containing compound has been proposed (36).

To investigate the effects of regulatory peptides on the gastrointestinal tract, various species of laboratory animals such as dog, feline, opossum, rabbit, rat, pig, and guinea pig have been examined. Some animal studies focused on the species’ own gastrointestinal characterics. For example, studies of the histological and innervational similarities to the human esophagus in opossum (5,ll) and feline ( 11) esophagus, the similarity in the pattern of peptidergic innervation to human in pig LES (2), and the high responsiveness of peristaltic activity to motilin in rabbit gastrointestinal tract ( 18) have helped to elucidate the mechanisms of intestinal motility. However, these animal models do not suffice to explain human gastrointestinal mechanisms. Specific arrangements of excitatory and inhibitory nerves mediate the intrinsic neurons of the lower esophageal sphincteric

c

[“bl

N=5

contra

NPY

CGRP

lTX

FIG. 5. EFS-induced mechanical contraction in the absence or presence of NPY, CGRP, or TTX. The response is expressed as a percent of the contraction induced by stimulation under 100 mA, 1 Hz, 50 ms condition. Each bar represents the mean from tissues from five animals; vertical lines show SEM. NPY (hatched bars), CGRP (dotted bars), and TTX (close bars) were preincubated for 15 min before stimulation. *Significantly different from the control values (open bars).

KOHJITANI

930

smooth muscle. SP has been established as an excitatory neurotransmitter throughout the gastrointestinal tract (9,22,30), and VIP has been established as an inhibitory neurotransmitter in LES (9,12) in various animal species. SP and VIP innervation is one of excitatory and inhibitory neurons in LES smooth muscle. The present findings of an increase in the tone of rabbit LES strips induced by SP and a decrease induced by VIP are in agreement with the data of other studies on LES from different species of animals under in vivo and in vitro conditions. Few studies have been performed on the effect of motilin on LES. In opossum LES, motilin induced contraction ( 13,15 ) and plasma motilin levels were related to LES pressure and migrating motor complex cycles ( 15). Motilin has also been reported to induce contraction of the duodenum, ileum and rectum, gastric antrum, and colon in rabbit ( 1,18 ) . Immunohistochemicai studies revealed that the motilin-containing cells were present especially in the duodenum of rabbit gastrointestinal tract (33 ), and in the duodenum and jejunum of rat gastrointestinal tract (32). The mechanism of the stimulating action of motilin on gastrointestinal motility seems to differ among species: in dogs by excitation of cholinergic neurons ( 17) and in rabbit by direct action on the smooth muscle cells (1,34). In our study, the excitatory effect of LES smooth muscle by motilin seems to have been mediated by some neural pathways, including cr-adrenergic, cholinergic, and histaminergic pathways. Both NPY and CGRP has been accepted to possess inhibitory effect on intestinal motility. NPY has been reported to induce biphasic response in feline LES (25), to inhibit neurally mediated contraction of guinea pig ileum (3), to inhibit the motility of cat colon (14), and to inhibit the peristaltic reflex of guinea pig small intestine ( 16). CGRP has been reported to mediate potent inhibitory transmission in opossum LES (27). The inhibitory effect of CGRP on LES is exerted both on nonadrenergic/ noncholinergic inhibitory neurons and on smooth muscle directly

ET AL.

in feline (24 ) and opossum (27). In LES, NPY immunoreactivity was highest among the gastrointestinal tract in rat, pig, and guinea pig ( 3 ), and CGRP-like immunoreactivity has been demonstrated between the circular and longitudinal muscle in feline (24). In the present study both NPY and CGRP showed no apparent change in the isometric tension of rabbit LES preparations; however, preincubation of NPY or CGRP attenuated the EFSinduced contraction. The amphibian skin peptide bombesin has been investigated in several human-model studies (6,7), and a recent study demonstrated the specific binding sites for bombesin in the gastrointestinal tract (38). In previous studies, bombesin stimulated LES smooth muscle in opossum (23) and gastric smooth muscle in dog (21) due to its direct effect on smooth muscle, and due to its indirect effect on some neural pathway (29) or on postganglionic adrenergic neurons, which release the contractile neurotransmitters in opossum LES (23). In contrast, the ability of bombesin to modulate the release of nonadrenergic/noncholinergic inhibitory neurotransmitters in cat LES has been reported ( 19). In rabbits, the interaction of bombesin-containing neurons with neurons whose neurotransmitter is acetylcholine, noradrenaline, histamine, or SP would be suggested. In conclusion, in rabbit, SP, and VIP appear to exert excitatory and inhibitory effects on the contractility of LES predominantly by stimulating their own receptors located on the esophageal smooth muscle. Both motilin and bombesin increase LES contraction via some neural pathways. An SP pathway may be involved in bombesin-induced contraction. Both NPY and CGRP inhibited EFS-induced contraction. Integration of immunohistochemistry, autoradiography, pharmacological approaches and molecular biological techniques in various species will contribute to a better understanding of the physiological role of these peptides in the regulation of LES functions.

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VARIOUS PEPTIDES ON RABBIT LES

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