Reflex gastric motor inhibition caused by intraperitoneal bradykinin: Antagonism by Hoe 140, a bradykinin antagonist

Peptides.Vol. 13, pp. 1073-1077, 1992

0196-9781/92 $5.00 + .00 Copyright © 1992PergamonPress Ltd.

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Reflex Gastric Motor Inhibition Caused by Intraperitoneal Bradykinin: Antagonism by Hoe 140, a Bradykinin Antagonist PETER

HOLZER

University of Graz, Department of Experimental and Clinical Pharmacology, Universitiitsplatz 4, A-8010 Graz, Austria R e c e i v e d 12 M a r c h 1992 HOLZER, P. Reflexgastricmotorinhibitioncausedby intraperitonealbradykin&:Antagon&mby Hoe 140, a bradykininantagonist. PEPT1DES 13(6) 1073-1077, 1992.--Bradykinin (BK) has been reported to have mixed excitatory/inhibitory effects on gastrointestinal motility. The present study examined the mechanism responsible for the inhibition of gastric motor activity caused by intraperitoneal administration of BK. Gastric motor activity was measured by recording the intragastric pressure (IGP) of phenobarbital-anesthetized rats via a transesophageal catheter. To facilitate the study of inhibitory influences, gastric motility was stimulated by neurokinin A (NKA), which on intravenous injection evoked reproducible gastric contractions as measured by a rise of IGP. lntraperitoneal injection of BK (0.1-10 nmol) inhibited the NKA-induced increase in IGP in a dose-dependent manner, and the effect of epigastric administration of BK was not significantly different from that of intraperitoneal administration. The inhibitory effect of intraperitoneal BK on gastric motility was due to an effect on BK2 receptors because it was blocked by prior intraperitoneal injection of the BK2 antagonist Hoe 140. The specificity of this BK antagonist was demonstrated by its inability to antagonize the effect of intraperitoneal hydrochloric acid (HC1), which, like BK, inhibited the NKA-induced gastric contractions. Because the BK- and HCl-induced inhibition of the NKA-induced rise of IGP was abolished by acute removal of the celiac-superior mesenteric ganglion complex, but left unaltered by acute bilateral subdiaphragmatic vagotomy, it is inferred that intraperitoneal BK inhibits gastric motor activity via activation of an autonomic reflex that involves prevertebral ganglia. Bradykinin

Bradykinin antagonist

Reflex inhibition of gastric motor activity

BRADYKININ (BK) is an autacoid nonapeptide that displays algesic and inflammatory properties. Apart from a direct action on gastrointestinal smooth muscle (7,14,18), BK may also influence gastrointestinal motility by way of autonomic reflexes that are triggered by the peptide formed in the course of inflammatory processes. Bradykinin is capable of stimulating visceral afferent neurons that are connected to nociceptors (6,12), which in turn can activate sympathetic nerve activity (17) leading to reflex changes in the cardiovascular system (1,16). In addition, BK administered close arterially (3) or intraperitoneally (2) causes inhibition of gastric motor activity. Since the inhibitory motor effect of BK is reduced after ablation of capsaicin-sensitive afferent neurons (2) or ganglionic blockade (3), it appears as if it is mediated by an autonomic reflex. The objective of the present study was threefold. First, we sought to develop a simple experimental model to study inhibition of gastric motor activity in anesthetized rats and found that gastric contractions induced by intravenous injection of neurokinin A (NKA) represent a gastric motor effect that is reproducibly inhibited by intraperitoneal BK. Second, we investigated whether the inhibitory effect of intraperitoneal BK on gastric motility is mediated by specific BK receptors. To this end, a newly developed, potent, and specific antagonist of BK, Hoe 140 (7,14,19), was used. Third, we examined whether the

Sympathetic nervous system

BK-induced inhibition of gastric motor activity is due to a direct effect on the stomach or whether it is mediated by an extrinsic autonomic reflex involving vagal or splanchnic pathways. METHOD

A nimal Preparation All experiments of this study were approved by the Ministry of Science and Research of the Republic of Austria. SpragueDawley rats (Strain OFA-SD, Forschungsinstitut ftir Versuchstierzucht, Himberg, Austria), of either sex and weighing 220240 g, were used. The animals were deprived of food for 20 h before experimentation but were allowed free access to tap water. After induction of anesthesia by intraperitoneal injection of phenobarbital sodium (0.92 mmol/kg) the trachea was cannulated to facilitate spontaneous breathing. The left carotid artery was cannulated and connected to a pressure transducer to monitor mean arterial blood pressure (MAP). The left jugular vein was cannulated for intravenous administration of drugs and for continuous infusion of physiological saline at a rate of 0.6 ml/ h to avoid dehydration of the animals. Intragastric pressure (IGP) was measured by a catheter (outer diameter: 1.9 mm) passed down to the stomach via the esophagus. The position of the catheter tip in the corpus region of the stomach was verified at

1073

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HOLZER

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F1O. l. Recordings of mean arterial blood pressure (MAP) and intragastric pressure (IGP) in phenobarbital-anesthetized rats showing the effects of intravenous injection of neurokinin A (NKA, 0.9 nrnol/kg) and of intraperitoneat injection of bradykinin (BK, 10 nmol) and Hoe 140 (100 nmol). The three recordings represent consecutive tracings of one experiment; the interval between the tracings is 15 rnin.

the end of each experiment. The catheter had two side holes in its tip segment and was continuously perfused with physiological saline at a rate of 0.6 ml/h. The intragastric catheter was connected to a pressure transducer to record IGP. Both MAP and IGP were displayed on a chart recorder and recorded at a speed of 1 cm/min. The temperature of the rats was kept at 36-37°C by means of a heating pad. Bilateral subdiaphragmatic vagotomy or removal of the celiac-superior mesenteric ganglion complex was performed as described previously (8,13). Control rats were sham operated.

Intraperitoneal Injection of BK or Hydrochloric Acid (HCl) Solutions of BK (1, 10, or 100 #M), HC1 (150 mM), and their vehicle (saline) were used. Because intraperitoneal insertion of a needle influenced MAP and IGP by itself, the appropriate number of needle-tip infusion catheters (one for each drug concentration and the vehicle) was placed intraperitoneally after laparotomy. The tips of the catheters were positioned within the loops of the small intestine approximately halfway between stomach and urinary bladder and were not in contact with the stomach except in a separate experiment in which BK and its vehicle were applied epigastrically, i.e., on the serosal surface of the gastric corpus. Care was taken that handling of the syringes did not distort the position of the intraperitoneal needles. All injections were made at volumes of 0.1 ml.

Evaluation of Data The gastric motor effect of NKA was quantitated by calculating the area under the curve versus baseline IGP (measured immediately before injection of NKA). This was done by using a digitizing tablet and the software SigmaScan (Jandel Scientific, Cone Madera, CA). The IGP results are presented in two different ways, depending on the question under study. I. To evaluate possible influences of vagotomy or celiac ganglionectomy on the gastric motor responses to NKA, the stimulant motor effect of NKA is presented by an absolute motor index, which is given by the arbitrary area units calculated by SigmaScan. Given the calibration of the IGP chart

recorder curve, generation of an IGP of 5 cm H20 over baseline for a period of I min gave a motor index of 2080. 2. To evaluate the inhibitory effects of intraperitoneal BK and HC1, the motor effect of NKA is presented by a relative motor index. In this instance, the motor effect of NKA measured before administration of BK or HC1 is set as 100% and the motor effect of NKA measured immediately after application of BK or HCI is expressed as a percentage of the control value. All data are presented as means + SEM. For statistical comparison of the means the Mann-Whitney U-test or the Wilcoxon test for pair differences was used as indicated. Probability values o f p < 0.05 were regarded as significant.

Substances and Solutions Atropine (Merck, Darmstadt, FRG), BK (Bachem, Bubendorf, Switzerland), and Hoe 140 (o-Arg-[Hyp3,ThiS,D TicV,OicS]bradykinin; Hoechst, Frankfurt am Main, FRG) were dissolved in saline (0.9% NaCI, wt./wt.). Neurokinin A (Cambridge Research Biochemicals, Cambridge, UK) was dissolved in 0.01 N acetic acid and diluted with saline. RESULTS

Experimental Model Following intragastric placement of the recording catheter it took about 10-15 min until IGP stabilized at a value between 2 and 3 cm H20. Intravenous bolus injections of NKA (0.9-1.8 nmol/kg) at 30-min intervals stimulated gastric motor activity. The motor effect of NKA consisted of oscillatory changes of IGP (frequency: 5-6/min) on top of a tonic increase in IGP and lasted 8-12 min (Fig. l). Except for the first two dosings, the NKA-induced contractions of the stomach were reproducible for up to 6 h (n = 6). In separate experiments it was ensured that the test doses of NKA (0.9-1.8 nmol/ks) were submaximally effective because a threefold higher dose of NKA caused a 3.4 _ 1.0 (n = 4) times larger response than the test dose. Neurokinin A also led to a brief fall of MAP, but this hyootensive response to NKA was over before the gastric motor effect came to a peak

REFLEX INHIBITION OF GASTRIC MOTILITY

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deduced from a comparison of the effects of BK ( 10 nmol) shown in Fig. 2A and B. Within individual rats, however, gastric motor inhibition caused by l0 nmol BK was reproducible on repeated administration of this BK dose (Fig. 2B). Saline (0.1 ml) given intraperitoneally 1 min before N K A had no influence on the gastric motor response to N K A (Fig. 2A). Bradykinin (10 nmol) administered epigastrically evoked a similar inhibition of the NKA-induced rise of IGP as intraperitoneal injection of the same BK dose (Fig. 2B), the degrees of inhibition being not significantly different from each other. Epigastric administration of saline (0. l ml) was without influence on the NKA-induced increase in IGP (Fig. 2B).

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(Fig. l). Atropine (0.35 #mol/kg injected intravenously 5 min before N K A ) did not influence NKA-induced stimulation of gastric motility, the motor effect after atropine injection being 104 _+ 14% (n = 1 l) of that before atropine.

Effect of BK Intraperitoneal injection of BK caused a slight fall in basal IGP and inhibited the NKA-induced increase in IGP (Fig. 1). As preliminary experiments had shown that BK administered 5 min before N K A was not as active in depressing the m o t o r stimulation caused by N K A as when BK was injected 1 min before N K A (n = 4), all experiments reported here were made by injecting BK 1 min before N K A . The inhibitory motor effect of BK was dose dependent (Fig. 2A) and short lasting because the NKA-induced increase in IGP was fully restored 30 min after administration of even the highest dose of B K (10 nmol). This enabled multiple tests of the inhibitory m o t o r effect of BK, which were performed at l-h intervals. The magnitude of the effect of intraperitoneal BK showed some variation, as can be

25 0 BKAT

BK (2) i.p.

FIG. 2. (A) Dose-dependent inhibitory effect ofintraperitoneal injection of bradykinin (BK, 0.1-10 nmol), and lack of effect of its vehicle (SAL, saline, 0.1 ml), on gastric contractions evoked by intravenous injection of neurokinin A (0.9-1.8 nmol/kg). (B) Inhibitory effect of epigastric and of repeated intraperitoneal injection of bradykinin (BK, 10 nmol; second injection ofBK 1 h after the first injection) on gastric contractions evoked by intravenous injection of neurokinin A (0.9-1.8 nmol/kg), and lack of effect of epigastric injection of the vehicle (SAL, saline, 0.1 ml). Bradykinin and SAL were injected 1 min before administration ofneurokinin A. The neurokinin A-induced rise of IGP was quantitated by measuring the area under the IGP curve, and the motor effects seen after administration of SAL and BK were expressed as a percentage of the motor response to neurokinin A measured before (control). Means + SEM, n = 6-9, *p < 0.01 versus control (Wilcoxon test for pair differences).

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FIG. 3. (A) Antagonism of the inhibitory motor effectof intrapefitoneal injection of bradykinin (BK, 10 nmol) by the bmdykinin antagonist (BKAT) Hoe 140 (100 nmo]), lack of effectof the B K A T on the inhibitory motor effectof intraperitoneal injection of hydrochloric acid (HC], 0.1 ml of a 150 ~ solution), and lack of an inhibitory motor effectof the

BKAT per se. Bradykinin and HCI were tested against gastric contractions evoked by intravenous injection of neurokinin A (0.9-1.8 nmol/kg) and were injected 1 min before neurokinin A. Bradykinin antagonist was administered 1 min before BK and HCI or, when its effect per se was examined, 1 min before neurokinin A. Means _+ SEM, n = 5-6, *p < 0.05 versus control (Wilcoxon test for pair differences), #p < 0.01 versus BK alone (Mann-Whitney U-test). (B) Influence of acute celiac ganglionectomy (CGE) and of acute bilateral subdiaphragmatic vagotomy (VGT) on the inhibitory motor effects ofintraperitoneal injection of bradykinin (10 nmol) and hydrochloric acid (HCI, 0. l ml of a 150 mM solution). Control rats were sham operated (SHAM). Bradykinin and HCl were tested against gastric contractions evoked by intravenous injection of neurokinin A (0.9-1.8 nmol/kg) and were injected 1 min before neurokinin A. The operations were carried out 2 h before the tests of bradykinin and HCI. The neurokinin A-induced rise of IGP was quantitated by measuring the area under the IGP curve, and the effects seen after the various treatments were expressed as a percentage of the motor response to neurokinin A measured before (control). Means +_ SEM, n = 8, *p < 0.05 versus SHAM (Mann-Whitney U-test).

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HOLZER

The inhibitory motor effect of BK was often associated with a decrease in MAP (see Fig. 1), but the effects of BK on MAP were variable among different rats and hence were not further evaluated.

Effect of the BK Antagonist Hoe 140 Intraperitoneal injection of 100 nmol Hoe 140 (1 m i n before BK) abolished the inhibitory action of BK (10 nmol injected intraperitoneally 1 rain before intravenous NKA) on the NKAinduced rise of IGP (Figs. 1 and 3A). The BK-induced fall of MAP was also inhibited by Hoe 140, whereas that evoked by N K A was not affected (Fig. 1). Hoe 140 per se did not change the gastric motor response to NKA (Fig. 3A) but caused a brief hypotensive response (Fig. 1). The BK-antagonistic effect of Hoe 140 on the NKA-induced contractions of the stomach was reversible within a period of 2 h (n = 6). The specificity of Hoe 140 as a BK antagonist was demonstrated by its inability to antagonize the inhibitory action of HCI (0.1 ml of a 150 m M solution injected intraperitoneally 1 rain before intravenous NKA) on the NKA-induced increase in IGP (Fig. 3A). The inhibitory motor effect of this dose of HCI amounted to an about 30% depression of the gastric motor response to NKA.

Effect of Vagotomy and Removal of the Celiac Ganglion As analyzed previously (10), acute bilateral subdiaphragmatic vagotomy (2 h before) enhanced the NKA-evoked rise of IGP, whereas acute removal of the celiac-superior mesenteric ganglion complex (2 h before) had no effect. The absolute motor index in sham-operated rats was 4411 + 550 (n = 8) compared with values of 4580 + 565 (n = 8) in ganglionectomized rats and 7312 + 980 (n = 8) in vagotomized rats (p < 0.05, M a n n - W h i t ney U-test). Vagotomy, however, failed to alter the inhibitory effects ofintraperitoneal BK and HC1 on the NKA-evoked gastric contractions (Fig. 3B). In contrast, acute removal of the celiac ganglion abolished the BK- and HCl-induced inhibition of the NKA-induced increase in IGP (Fig. 3B).

suring IGP via an transesophageal catheter was ascertained by the similarity of the NKA-evoked increase in IGP to that recorded when N K A was given close arterially to the rat stomach and IGP was measured via a transpyioric catheter (l l). The stimulant motor effect of N K A was not modified by atropine, which indicates that the action of NKA does not involve cholinergic motor neurons and hence most probably is due to a direct action on the circular muscle of the gastric corpus (l l ). Intraperitoneal injection of BK inhibited gastric motor activity in a dose-dependent manner, an effect that was mediated by specific BK receptors because it was blocked by the BK antagonist Hoe 140 (7,14,19). Since Hoe 140 is selective for BK2 receptors (7), it follows that this subtype of BK receptor is responsible for BK's inhibition of NKA-evoked gastric contractions. The observation that Hoe 140 did not alter the inhibitory motor effect of intraperitoneal HC1 underlines the specificity of Hoe 140 as BK antagonist and indicates that HC1 does not act via endogenous BK. The inhibitory action of BK on the NKA-induced increase in IGP was apparently independent of whether BK was administered intraperitoneally or epigastrically. Since administration of BK to isolated preparations of rat gastric smooth muscle induces contraction (18), it is inferred that BK-induced inhibition of gastric motility arises from a fundamentally different mechanism that involves extragastric systems. This conjecture is supported by the results of acute extrinsic denervation of the stomach. Abolition of the inhibitory motor effect of BK by acute removal of the celiac-superior mesenteric ganglion complex, but not vagotomy, demonstrates that intraperitoneal BK inhibits gastric motor activity via activation of a neural mechanism that involves prevertebral ganglia. This suggests that intraperitoneal BK, like HC1 (5,9), inhibits gastrointestinal motility via activation of inhibitory intestino-intestinal reflexes. As these reflexes are thought to participate in pathological inhibition of gastrointestinal motility (4,15), it is proposed that BK formed under conditions of peritoneal inflammation could contribute to a reflex shutdown of gastrointestinal motor activity. This unwanted action of BK can be prevented by the use of specific receptor antagonists of BK.

DISCUSSION

ACKNOWLEDGEMENTS

The present data demonstrate that the stimulant effect of intravenous N K A on IGP can be utilized to study inhibitory influences on gastric motility because both the NKA-evoked gastric contractions and the BK-induced inhibition of NKA's motor effect are reproducible for several h. The validity of mea-

This work was supported by the Austrian Scientific Research Council (grant # P7845). Hoe 140 was generously provided by the Hoechst AG, Franfurt am Main, FRG. The author thanks Dr. Ulrike Holzer-Petsche for her comments on the manuscript and Wolfgang Schluet for expert technical and graphical assistance.

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