Regulation of Guinea pig intestinal peristalsis by endogenous endothelin acting at ETB receptors

GASTROENTEROLOGY 2000;119:80–88

Regulation of Guinea Pig Intestinal Peristalsis by Endogenous Endothelin Acting at ETB Receptors ANAID SHAHBAZIAN and PETER HOLZER Department of Experimental and Clinical Pharmacology, University of Graz, Graz, Austria

Background & Aims: Endothelins are expressed in many enteric neurons of the gut. Because activation of endothelin ETA and ETB receptors is known to alter intestinal muscle activity, the effect of ETA and ETB receptor agonists and antagonists on propulsive peristalsis was examined. Methods: Repetitive peristalsis in fluid-perfused segments of the guinea pig isolated small intestine was elicited by a rise of the intraluminal pressure and recorded via the pressure changes generated by the peristaltic waves. Results: Endothelin 1 (0.3–10 nmol/L added to the organ bath) stimulated peristalsis as shown by a decrease in the pressure threshold at which peristaltic waves were triggered, whereas the endothelin analog sarafotoxin 6c (0.3–10 nmol/L) inhibited peristalsis as reflected by an increase in the pressure threshold. The ETA receptor antagonist BQ-123 (3 mmol/L) converted the properistaltic action of endothelin 1 to an antiperistaltic action, whereas the ETB receptor antagonist BQ-788 (3 mmol/L) prevented the antiperistaltic action of sarafotoxin 6c. BQ-788, but not BQ-123, facilitated peristalsis on its own. Additional experiments indicated that the properistaltic action of endothelin 1 is mediated by enteric neurons, whereas the peristaltic motor effects of sarafotoxin 6c and BQ-788 are caused by a direct action on the muscle. Conclusions: ETA receptor activation stimulates, whereas ETB receptor activation inhibits, intestinal peristalsis. The ability of BQ-788 to facilitate peristalsis per se points to a physiologic role of ETB receptors in peristaltic motor regulation.

hree isoforms of the mammalian endothelin peptide family including endothelin (ET)-1, ET-2, and ET-3 have been localized to the gastrointestinal tract,1–3 where they are expressed by at least 5 different cell types. ET-1 is found in a large number of enteric neurons originating from the myenteric and submucosal plexus of the human and rat gut in which it is frequently costored with vasoactive intestinal polypeptide.2–4 All 3 ETs have been localized to mast cells and macrophages within the lamina propria of the mucosa.5–7 Some ET-1 is also produced in endothelial cells of blood vessels2 and,

T

during development, is transiently expressed in endocrine cells of the human gastrointestinal mucosa.3 The potential role of ETs in intestinal motor regulation is borne out by the presence of both types of ET receptor, termed ETA and ETB,8 in the gastrointestinal wall and by the ability of ET receptor agonists to modify intestinal muscle activity. ETA and ETB receptors are present on neurons of the myenteric and submucosal nerve plexus, stromal cells of the mucosa, blood vessels, muscularis mucosae, and circular and longitudinal muscle layer.1,2,7,9,10 The motor actions of ETs, which have been studied mostly in the guinea pig intestine, are complex and comprise both excitatory and inhibitory effects, depending on which receptors on enteric nerve or intestinal muscle are activated.11–13 Collectively, it seems that ET-evoked muscle contraction is primarily mediated by ETA receptors, whereas ET-induced muscle relaxation is predominantly brought about by ETB receptors.11,13–16 Despite this information it is still unknown whether ETs contribute to the physiologic regulation of peristalsis, the clinically most relevant motor pattern of the gut. The first objective of this study was hence to characterize the action of ET receptor agonists on peristalsis in the guinea pig isolated small intestine, because the effects of drugs on this propagated motor pattern cannot be deduced from their influence on standing motor reflexes.17 To this end, the peristaltic motor actions of ET-1, vasoactive intestinal contractor (VIC), ET-3, and sarafotoxin 6c (STX-6c) were analyzed. VIC is the mouse and rat analogue of human ET-2,18 which, like ET-1, is a high- affinity agonist at both ETA and ETB receptors.8 In contrast, ET-3 is an ETB receptor–preferring agonist,8 and the snake venom–derived peptide STX-6c is a selective agonist at ETB receptors.19 Abbreviations used in this paper: ET, endothelin; L-NAME, NG-nitroL-arginine methyl ester; NO, nitric oxide; PPADS, pyridoxal phosphate6-azophenyl-29,49-disulfonic acid; PPT, peristaltic pressure threshold; STX-6c, sarafotoxin-6c; VIC, vasoactive intestinal contractor. r 2000 by the American Gastroenterological Association 0016-5085/00/$10.00 doi:10.1053/gast.2000.8549

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The second aim was to identify the receptor types by which ETs modify peristalsis, a goal that was addressed with the ETA receptor–selective antagonist BQ-12320 and the ETB receptor–selective antagonist BQ-788.21 The third goal was to elucidate the sites of ET action, although the complexity of the neural control and muscular effector systems of propulsive motility limits this kind of analysis. The fourth objective was to seek pharmacologic evidence for an involvement of endogenous ETs in peristaltic motor regulation. An understanding of ET function in normal peristalsis is important for defining the pathophysiologic roles that these peptides are thought to play in the intestinal motor disturbances associated with ischemia-reperfusion injury22 and pancreatitis.23

Materials and Methods Propulsive Peristalsis The small intestine of adult guinea pigs (TRIK strain, either sex, 350–450 g body wt) was isolated, flushed of luminal contents, and placed for up to 4 hours in Tyrode solution kept at room temperature and oxygenated with a mixture of 95% O2 and 5% CO2.24 The composition of the Tyrode solution was (in mmol/L) NaCl, 136.9; KCl, 2.7; CaCl2, 1.8; MgCl2, 1.0; NaHCO3, 11.9; NaH2PO4, 0.4; and glucose, 5.6. The jejunum and ileum were divided into 8 segments, each approximately 10 cm long. Four intestinal segments were set up in parallel and secured horizontally in organ baths containing 30 mL of Tyrode solution at 37°C. To elicit propulsive peristalsis, prewarmed Tyrode solution was continuously infused into the lumen of the segments at a rate of 0.5 mL/min.24 The

Figure 1. Recordings of the peristaltic motor action of (A) ET-1 and (B) STX-6c administered to the organ bath at the indicated concentrations. ET-1 stimulated peristalsis as deduced from a decrease of the PPT (dots) and an increase of the peristaltic frequency. STX-6c inhibited peristalsis as deduced from a rise of PPT; in addition, peristalsis became slightly irregular.

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intraluminal pressure at the aboral end of the segments was measured with a pressure transducer whose signal was, via an analog/digital converter, fed into a personal computer and recorded and analyzed with the software Peristal 1.0 (Heinemann Scientific Software, Graz, Austria). The fluid passing through the gut lumen was directed into a vertical outlet tubing that ended 4 cm above the fluid level in the organ bath. When fluid was infused, the intraluminal pressure rose slowly until it reached a threshold at which peristalsis was triggered (Figure 1). The aborally moving wave of peristaltic contraction resulted in a spike-like increase in the intraluminal pressure, which caused emptying of the segment if the maximal pressure of the peristaltic wave exceeded the level of 400 Pa as set by the position of the outlet tubing. The peristaltic pressure threshold (PPT) was used to quantify drug effects on peristalsis. Inhibition of peristalsis was reflected by an increase in PPT, and abolition of peristalsis manifested itself in a lack of propulsive motility despite an intraluminal pressure of 400 Pa. Although in this case PPT exceeded 400 Pa, abolition of peristalsis was expressed quantitatively by assigning PPT a value of 400 Pa to obtain numerical results suitable for further statistical evaluation. The effectiveness of peristalsis was assessed by regular inspection of the preparations and by monitoring the minimum of the intraluminal pressure that was achieved after completion of each peristaltic wave. This residual intraluminal pressure, which was 5–15 Pa, is a sensitive measure of the emptying capacity of peristaltic waves.25 The preparations were allowed to equilibrate in the organ bath for a period of 30 minutes during which they were kept in a quiescent state. Thereafter, the bath fluid was renewed and peristaltic motility initiated by intraluminal perfusion of the

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segments. After basal peristaltic activity had been recorded for a period of 30 minutes, the drugs to be tested were administered into the bath, i.e., to the serosal surface of the intestinal segments, at volumes not exceeding 1% of the bath volume. The corresponding vehicle solutions were devoid of any effect. Four sets of experiments were performed. First, the peristaltic motor effects of ET-1, VIC, ET-3, and STX-6c at concentrations of 0.3–10 nmol/L were studied, and these agonists were added in a cumulative manner at 15-minute intervals (Figures 1 and 2). Second, the susceptibility of the peristaltic motor effects of ET-1 and STX-6c (0.3–10 nmol/L) to a number of drugs was tested, and these drugs were given at appropriate time intervals before exposure to the agonists (Tables 1 and 2; Figures 3 and 4). Third, the activity of the ETB receptor antagonist BQ-788 (0.1–3 µmol/L) to lower PPT was examined in the absence and presence of various drugs (Table 2; Figure 5). Fourth, the influence of BQ-788 (3 µmol/L) on the ability of adenosine triphosphate (ATP; 30–300 µmol/L) and dynorphin-(1-13) (3–100 nmol/L) to inhibit peristalsis was investigated (Table 3). Each protocol was carried out with at least 5 segments from 5 different guinea pigs.

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Table 1. Effect of Various Drugs on the PPT

Drug Vehicle Atropine (1 µmol/L) Hexamethonium (100 µmol/L) BQ-123 (3 µmol/L) BQ-788 (3 µmol/L) L-NAME (300 µmol/L) Naloxone (0.5 µmol/L) SR-140,333 (0.1 µmol/L) SR-144,190 (0.1 µmol/L) SR-140,333 (0.1 µmol/ L) 1 SR-144,190 (0.1 µmol/L) PPADS (150 µmol/L)

PPT before PPT after Exposure drug drug time exposure exposure n (min) (Pa) (Pa)

P

24 15

30 60

52 6 4 59 6 4

54 6 5 NS 120 6 13 ,0.01

19 15 34 13 12

60 30 30 30 30

66 6 5 37 6 4 50 6 3 56 6 4 58 6 5

121 6 9 36 6 3 38 6 2 44 6 4 51 6 4

,0.01 NS ,0.01 ,0.01 NS

7

30

66 6 9

68 6 9

NS

7

30

58 6 4

70 6 10

NS

11 9

30 30

59 6 3 60 6 5

62 6 3 56 6 2

NS NS

NOTE. The values represent means 6 SEM of n experiments. The P values were determined with the Wilcoxon signed-rank test. NS, not significant.

Drugs and Solutions The sources of the drugs used here were as follows: cyclo(D-a-aspartyl-L-prolyl-D-valyl-L-leucyl-D-tryptophyl) (BQ123); dynorphin-(1–13), human ET-1, N-[N-[N-[(2,6-dimethyl-1-piperidinyl) carbonyl]-4-methyl-L-leucyl]-1-(me-

thoxycarbonyl(-D-tryptophyl]-D-norleucine (BQ-788); and rat ET-3 and rat VIC were purchased from Neosystem (Strasbourg, France). Pyridoxal phosphate-6-azophenyl-28,48-disulphonic acid (PPADS) and STX-6c were obtained from RBI (Natick, MA). ATP, atropine, hexamethonium, naloxone, and NG-nitroL-arginine methyl ester (L-NAME) were obtained from Sigma (Vienna, Austria). (R)-3-51-[2-(4-benzoyl-2-(3,4-difluorophenyl)-morpholin-2-yl)-ethyl]-4-phenylpiperidin-4-yl6-1-dimethylurea (SR-144,190) and (S)1-52-[3-(3,4-dichlorophenyl)1-(3-isopropoxyphenyl-acetyl)piperidin-3-yl]-ethyl6-4-phenyl1-azoniabicyclo[2.2.2]-octane chloride (SR-140,333) were gifts Table 2. Effect of Various Drug Pretreatments on the Actions of ET-1, STX-6c, and BQ-788 (Test Substances) to Change PPT Test substance ET-1 (10 nmol/L)

Figure 2. Concentration-response relationship for the effect of ET-1, ET-3, STX-6c, and VIC to alter PPT. The concentration-response curves were recorded in a cumulative manner, and the graph shows peak changes in PPT that occurred within 15 minutes after exposure to each agonist concentration. The values represent means 6 SEM; n $ 5. *P , 0.05, **P , 0.01 vs. PPT recorded before exposure to any drug (Wilcoxon signed-rank test).

Drug pretreatment

Vehicle Atropine (1 µmol/L) ET-1 (10 nmol/L) Vehicle SR-140,333 (0.1 µmol/L) ET-1 (10 nmol/L) Vehicle SR-144,190 (0.1 µmol/L) STX-6c (10 nmol/L) Vehicle Naloxone (0.5 µmol/L) STX-6c (10 nmol/L) Vehicle L-NAME (300 µmol/L) STX-6c (10 nmol/L) Vehicle PPADS (150 µmol/L) BQ-788 (3 µmol/L) Vehicle Atropine (1 µmol/L) BQ-788 (3 µmol/L) Vehicle Hexamethonium (100 µmol/L) BQ-788 (3 µmol/L) Vehicle SR-140,333 (0.1 µmol/L) 1 SR-144,190 (0.1 µmol/L)

PPT (% of predrug)

n

66 6 4 60 6 7 71 6 4 72 6 10 61 6 7 71 6 11 220 6 17 197 6 14 240 6 31 305 6 51 187 6 17 180 6 21 71 6 6 66 6 6 70 6 5 64 6 6 71 6 6 78 6 5

8 8 7 7 7 7 17 12 8 13 10 9 9 7 6 10 7 11

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drugs expressed as a percentage of baseline PPT. Quantitative data are presented as means 6 SEM of n experiments, n referring to the number of guinea pigs used in the test. The results were evaluated with the Mann–Whitney U test, the Wilcoxon signed-rank test, or the Kruskal–Wallis H test followed by the Mann–Whitney U test, as appropriate. In select instances, drug-induced shifts in the concentrationresponse relationships for the peristaltic motor effects of ET-1 and STX-6c were analyzed by 2-way analysis of variance. Probability values of P , 0.05 were regarded as significant.

Results Effects of ET-1, VIC, ET-3, and STX-6c on Peristalsis

Figure 3. Concentration-response relationship for the effect of (A) ET-1 and (B) STX-6c to alter the PPT in the presence of vehicle, BQ-123 (3 µmol/L), or BQ-788 (3 µmol/L), which were administered 30 minutes before the ET-1 and STX-6c concentration-response curves were recorded in a cumulative manner. The values represent means 6 SEM; n $ 7. *P , 0.05, **P , 0.01 vs. respective values recorded in the presence of vehicle (Kruskal–Wallis H test followed by Mann– Whitney U test).

The PPT at baseline ranged from 35 to 65 Pa (Table 1). Administration of ET-1 (0.3–10 nmol/L) to the organ bath lowered PPT in a concentration-related manner, an effect that was associated with an increase in the frequency of the peristaltic waves and a decrease in their amplitude (Figures 1A and 2). It should be noted that peristaltic frequency is determined by PPT and the compliance of the intestinal wall and that the decrease in the amplitude of the peristaltic waves is related to the frequency rise, given that an increase in peristaltic frequency because of doubling of the luminal perfusion rate attenuated the amplitude of the peristaltic waves to a similar extent (n 5 5; data not shown). The stimulant

of Sanofi (Montpellier, France). The drugs were dissolved with appropriate media, the concentrations given hereafter in parentheses referring to the stock solutions. Dynorphin-(1-13), ET-1 (0.1 mmol/L), and VIC (0.1 mmol/L) were dissolved in 0.01 mol/L acetic acid, BQ-788 (1 mmol/L) in ethanol, ET-3 (0.1 mmol/L) in distilled water, and SR-140,333 (1 mmol/L) and SR-144,190 (1 mmol/L) in dimethyl sulfoxide. ATP, atropine (1 mmol/L), BQ-123 (1 mmol/L), hexamethonium (10 mmol/ L), L-NAME (30 mmol/L), naloxone (1 mmol/L), and PPADS (15 mmol/L) were dissolved in Tyrode solution. If necessary, the stock solutions were diluted with Tyrode solution before use.

Data Calculation and Statistics The PPT and the residual intraluminal pressure of 3 consecutive peristaltic contractions were averaged to determine the baseline values recorded immediately before administration of a drug. The same procedure was applied to calculate the peak values of drug-induced changes in PPT and the residual intraluminal pressure, unless peristalsis was abolished, in which case PPT was assigned a value of 400 Pa. To allow for a better comparison of the data obtained in different preparations, the average baseline PPT recorded in each experimental group was set as 100% and the PPT recorded in the presence of

Figure 4. Effect of hexamethonium on the concentration-dependent action of ET-1 to decrease the PPT. Vehicle or hexamethonium (100 µmol/L) was administered 60 minutes before the concentrationresponse curve for ET-1 was recorded in a cumulative manner. The values represent means 6 SEM; n 5 8. *P , 0.05, **P , 0.01 vs. respective values recorded in the presence of vehicle (Mann–Whitney U test).

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Pa (n 5 8; P , 0.01) recorded after exposure to 3 µmol/L STX-6c. ET-3 and VIC, each tested at concentrations of 0.3–10 nmol/L, were devoid of any consistent influence on peristalsis, with the exception of 10 nmol/L ET-3, which significantly increased PPT (Figure 2) and elevated the residual intraluminal pressure from a predrug value of 13 6 2 Pa to a value of 30 6 3 Pa (n 5 8; P , 0.01). Effects of BQ-123 and BQ-788 on the Peristaltic Motor Actions of ET-1 and STX-6c

Figure 5. (A) Recording of the peristaltic motor action of BQ-788 administered to the organ bath at the indicated concentrations. BQ-788 stimulated peristalsis as deduced from a decrease of the PPT (indicated by dots) and an increase of the peristaltic frequency. (B) Cumulative concentration-response relationship for the effect of BQ-788 to lower PPT. The values represent means 6 SEM; n 5 6. *P , 0.05, **P , 0.01 vs. PPT recorded before exposure to BQ-788 (Wilcoxon signed-rank test).

action of ET-1 on peristalsis was quick in onset and sustained (Figure 1A), which made it possible to record the concentration-response relationship for ET-1 in a cumulative manner, i.e., by exposing the segments to the next agonist concentration when the preceding response had peaked (Figures 1A and 2). STX-6c (0.3–10 nmol/L) increased PPT in a concentration-dependent fashion (Figures 1B and 2) and thus altered peristalsis in a manner opposite to that of ET-1. The antiperistaltic action of STX-6c set in promptly and was of a sustained nature; in addition, peristalsis became often irregular (Figure 1B). Unlike ET-1 (0.3–10 nmol/ L), which did not significantly change the residual intraluminal pressure (n 5 5; data not shown), STX-6c caused a concentration-related increase in this parameter from a value of 14 6 2 Pa at baseline to a value of 29 6 4

To identify the receptors mediating the peristaltic motor actions of ET-1 and STX-6c, we examined whether the ETA receptor antagonist BQ-123 (3 µmol/L) and the ETB receptor antagonist BQ-788 (3 µmol/L) would change the ability of ET-1 to lower PPT and that of STX-6c to elevate PPT. BQ-123 did not modify peristalsis per se, whereas BQ-788 lowered PPT to a significant extent (Table 1). Neither substance had any significant influence on the residual intraluminal pressure (n $ 15, data not shown). Figure 3A shows that the action of ET-1 (1–10 nmol/L) to decrease PPT (i.e., to stimulate peristalsis) remained largely unaltered by BQ-788, except that the response to 3 nmol/L ET-1 was significantly attenuated by BQ-788. The concentration-response curve for ET-1 was, however, not significantly changed by BQ788, as shown by 2-way analysis of variance. In contrast, BQ-123 converted the stimulant action of ET-1 on peristalsis to an inhibitory action so that the peristaltic motor inhibition caused by ET-1 in the presence of BQ-123 (Figure 3A) resembled that induced by STX-6c in the presence of vehicle (Figure 3B). The activity of STX-6c to elevate PPT (i.e., to inhibit peristalsis) was little altered by BQ-123, except that the peristaltic motor response to 3 nmol/L STX-6c was significantly diminished (Figure 3B). However, BQ-123 failed to alter the concentration-response curve for STX-6c, as revealed by 2-way analysis of variance. BQ-788, to the contrary, abolished the inhibitory action of STX-6c on peristalsis (Figure 3B). Table 3. Effect of BQ-788 on the Actions of ATP and Dynorphin-(1-13) (Test Substances) to Increase PPT Test substance

Drug pretreatment

PPT (% of predrug)

n

ATP (300 µmol/L)

Vehicle BQ-788 (3 µmol/L) Vehicle BQ-788 (3 µmol/L)

370 6 86 345 6 94 458 6 94 412 6 121

7 8 6 6

Dynorphin-(1-13) (0.1 µmol/L)

NOTE. The values refer to maximal PPT changes caused by the test substances and represent means 6 SEM of n experiments. No significant differences between the vehicle- and drug-pretreated segments were noted (Mann–Whitney U test).

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Effects of Atropine, Hexamethonium, SR-140,333, SR-144,190, L-NAME, Naloxone, and PPADS on the Peristaltic Motor Actions of ET-1 and STX-6c To probe for an involvement of endogenous acetylcholine, substance P, or neurokinin A in the stimulant action of ET-1 on peristalsis, we investigated whether atropine (muscarinic acetylcholine receptor antagonist), hexamethonium (nicotinic acetylcholine receptor antagonist), SR-140,333 (substance P/NK1 receptor antagonist), or SR-144,190 (neurokinin A/NK2 receptor antagonist) would influence the ability of ET-1 to lower PPT. Atropine (1 µmol/L) and hexamethonium (100 µmol/L) led to a transient abolition of propulsive motility. Within a period of 1 hour, however, 14 of 22 (64%) atropinetreated preparations and 29 of 31 (94%) hexamethoniumtreated segments resumed regular peristalsis, although PPT (Table 1) and the residual baseline pressure (8 6 2 Pa before and 39 6 5 Pa after atropine, n 5 15, P , 0.01; 11 6 3 Pa before and 32 6 9 Pa after hexamethonium, n 5 19, P , 0.01) remained elevated when compared with the predrug values. In contrast, SR140,333 (0.1 µmol/L) and SR-144,190 (0.1 µmol/L) had no significant influence on PPT (Table 1) and the residual baseline pressure (n 5 7). The stimulant action of ET-1 (1, 3, and 10 nmol/L) on peristalsis was left unaffected by atropine, SR-140,333, and SR-144,190. The number of experiments and the PPT changes caused by 10 nmol/L ET-1 in the absence and presence of these drugs are detailed in Table 2. In contrast, hexamethonium converted the properistaltic effect of ET-1 to an antiperistaltic action (Figure 4). The nitric oxide (NO) synthase inhibitor L-NAME (300 µmol/L), the opioid receptor antagonist naloxone (0.5 µmol/L), and the P2X purinoceptor antagonist PPADS (150 µmol/L) were used to test whether NO, opioid peptides, and ATP participate in the antiperistaltic action of STX-6c. L-NAME lowered PPT to a significant extent, an effect that was sustained for at least 30 minutes (Table 1), whereas the initial PPT decrease caused by naloxone26 and PPADS27 waned slowly during the 30-minute incubation period (Table 1). L-NAME also raised the residual intraluminal pressure from a predrug level of 12 6 2 Pa to a level of 23 6 3 Pa (n 5 13; P , 0.01), whereas naloxone and PPADS failed to affect this parameter (n $ 8). Naloxone, L-NAME, and PPADS were unable to change the antiperistaltic action of STX-6c (1, 3, and 10 nmol/L). The number of experiments and the PPT changes caused by 10 nmol/L STX-6c in the absence and presence of these drugs are documented in Table 2.

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Effect of BQ-788 on Peristalsis in the Absence and Presence of Atropine, Hexamethonium, SR-140,333, and SR-144,190 The ETB receptor antagonist BQ-788 (0.1–3 µmol/L) stimulated peristalsis in a concentration-related manner as deduced from its ability to lower PPT and to increase the frequency of the peristaltic waves (Figure 5A and B). At a concentration of 3 µmol/L, BQ-788 decreased PPT by some 30% (Figure 5B); higher concentrations of the drug were not affordable. The stimulant action of BQ-788 on peristalsis was quick in onset and sustained (Figure 5A) as is also documented in Table 1. None of the concentrations of BQ-788 tested here altered the residual intraluminal pressure to any significant degree (n 5 6). The possible contribution of endogenous acetylcholine, substance P, or neurokinin A to the properistaltic action of BQ-788 was explored with atropine (1 µmol/L), hexamethonium (100 µmol/L), SR-140,333 (0.1 µmol/ L), and SR-144,190 (0.1 µmol/L). The effects that these drugs had per se on peristalsis are shown in Table 1. Atropine, hexamethonium, and combined administration of SR-140,333 plus SR-144,190 failed to significantly modify the stimulant action of BQ-788 (0.3, 1, and 3 µmol/L) on peristalsis. The number of experiments and the PPT changes induced by 3 µmol/L BQ-788 in the absence and presence of these drugs are detailed in Table 2. Effect of BQ-788 on the Peristaltic Motor Inhibition Caused by ATP and Dynorphin-(1-13) To check the specificity of BQ-788 as an ETB receptor antagonist, we examined the influence of BQ788 (3 µmol/L) on the peristaltic motor inhibition caused by dynorphin-(1-13) and ATP. Cumulative administration of ATP (30, 100, and 300 µmol/L) and dynorphin-(113) (10, 30, and 100 nmol/L) led to a concentrationdependent inhibition of peristalsis as was evident from an increase in PPT. Exposure of the segments to BQ-788 (3 µmol/L) failed to alter the inhibitory effect of ATP and dynorphin-(1-13) on peristalsis. The number of experiments and the PPT changes caused by 300 µmol/L ATP and 100 nmol/L dynorphin-(1-13) in the absence and presence of BQ-788 are documented in Table 3.

Discussion The data of this study show that exogenous ETs modify intestinal peristalsis and that endogenous ETs participate in propulsive motor regulation. The peristaltic motor actions of ETs were characterized with the

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ETA/ETB receptor–nonselective agonists ET-1 and VIC,8 the ETB receptor–preferring agonist ET-3,8 and the ETB receptor–selective agonist STX-6c.19 The observation that at low nanomolar concentrations, ET-1 stimulated and STX-6c inhibited peristalsis suggests that ETA receptor activation facilitates, whereas ETB receptor activation suppresses, propulsive motility. This inference is supported by the finding that the ETB receptor– preferring agonist ET-3 also tended to inhibit peristalsis, whereas the receptor-nonselective agonist VIC was without significant effect. The opposite influence of ETA and ETB receptor activation on peristalsis was borne out by the data obtained with the ETA receptor–selective antagonist BQ-12320 and the ETB receptor–selective antagonist BQ-788.21 At a concentration of 3 µmol/L, which previously has been shown to be effective and selective for ETA receptors,14,16,20 BQ-123 abolished the stimulant influence of ET-1 on propulsive motility and unmasked an inhibitory ET-1 action that was indistinguishable from that of the ETB receptor agonist STX-6c. It thus seems that, after exposure to ET-1 under normal conditions, the ETA receptor–mediated stimulation of peristalsis overrides the ETB receptor–mediated inhibition of peristalsis. The inhibitory action of STX-6c on propulsive motility, on the other hand, was prevented by BQ-788, which at 3 µmol/L is an effective and selective ETB receptor antagonist.16,21,28 The inability of BQ-788 to uncover a properistaltic effect of STX-6c attests to the selective action of this snake venom peptide at ETB receptors. As was noted in other functional tests,10,13–16,28 the peristaltic motor effect of ETA receptor activation tended to be attenuated by BQ-788 and that of ETB receptor activation diminished by BQ-123. On the basis of the current study it is not possible to decide whether this finding reflects limited selectivity of BQ-123 and BQ-788 as ETA and ETB receptor antagonists, respectively, or participation of some ETB receptors in the properistaltic action of ET-1, which is predominantly brought about by ETA receptors, and of some ETA receptors in the antiperistaltic action of STX-6c, which is primarily mediated by ETB receptors. The site of action of ET-1 in the enteric nerve/muscle circuits governing propulsive motility was analyzed with the muscarinic acetylcholine receptor antagonist atropine, the nicotinic acetylcholine receptor antagonist hexamethonium, the substance P/NK1 receptor antagonist SR-140,333, and the neurokinin A/NK2 receptor antagonist SR-144,190. These drugs were used because nicotinic acetylcholine receptors mediate excitatory transmission within the enteric nerve plexuses and the excitatory motor neurons of the gut transmit via acetyl-

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choline acting on muscarinic receptors and substance P/neurokinin A acting on NK1 and NK2 receptors.26,29,30 SR-140,333 and SR-144,190 were used at concentrations known to be effective and selective for NK1 and NK2 receptors, respectively, but devoid of any major influence on peristalsis on their own.26,31 Atropine and hexamethonium, in contrast, caused a temporary peristaltic arrest which, as previously observed,32–34 was released after some time, enabling us to test drug effects on noncholinergic propulsive motility. The findings that hexamethonium eliminated the properistaltic action of ET-1, whereas atropine, SR-140,333, and SR-144,190 were without effect, indicate that ET-1 stimulates peristalsis by an action on enteric neurons. Specifically, ET-1 seems to activate excitatory nerve pathways that involve neuroneuronal communication via nicotinic acetylcholine receptors (Figure 6). The inability of atropine, SR-140,333, and SR-144,190 to prevent the properistaltic action of ET-1 does not rule out any implication of excitatory motor neurons releasing acetylcholine, substance P, and neurokinin A, given that these transmitters synergize in neuromuscular transmission.26,29,30,33,34 The antiperistaltic action of STX-6c was analyzed with respect to the possibility that this ETB receptor agonist depresses peristalsis via activation of inhibitory motor neurons, which in the guinea pig intestine relax the muscle primarily via release of NO, ATP, vasoactive intestinal polypeptide, and/or pituitary adenylate cyclase– activating peptide.35–38 However, peristaltic motor inhibition caused by STX-6c remained unaltered by an effective concentration (300 µmol/L) of the NO synthase inhibitor L-NAME,25,39 which is in line with a lack of NO involvement in ET-induced suppression of intestinal muscle contractility.11,13 Effective concentrations of the P2X purinoceptor antagonist PPADS, which antagonizes

Figure 6. Diagram showing the putative location of endothelin ETA and ETB receptors in the enteric nerve/muscle circuits governing propulsive peristalsis in the guinea pig small intestine. 1, Excitation; –, inhibition; CM, circular muscle; LM, longitudinal muscle; M, muscarinic acetylcholine receptor; MP, myenteric plexus; MU, mucosa; N, nicotinic acetylcholine receptor; NK1, NK2, tachykinin receptor types; P2X, purinoceptor type; VIP, vasoactive intestinal polypeptide.

July 2000

the antiperistaltic action of endogenously released ATP,27,40 and of the opioid receptor antagonist naloxone26 were likewise unable to prevent STX-6c from inhibiting peristalsis. It follows that peristaltic motor inhibition caused by the ETB receptor agonist STX-6c does not involve purinergic or opioidergic neurons and is very likely caused by a direct action on intestinal smooth muscle (Figure 6). Although the possible implication of vasoactive intestinal polypeptide and pituitary adenylate cyclase– activating peptide was not tested, there is additional evidence to assume that the ETB receptors causing peristaltic motor inhibition are located on intestinal muscle. First, other functional and biochemical studies have shown that ETs can relax the intestine by a direct action on muscle cells.9,13,15 Second, the antiperistaltic effect of ET-1, which is unmasked by BQ-123 and hexamethonium, seems to be mediated by ETB receptors that are situated beyond enteric pathways involving transmission via nicotinic acetylcholine receptors (Figure 6). Third, the pharmacology of the properistaltic action of BQ-788 likewise points to a muscular location of the ETB receptors. The increase in PPT caused by ETB receptor– mediated muscle relaxation may result from the effect of relaxation to inhibit the discharge of intrinsic sensory neurons subserving peristalsis41 or from the effect of relaxation to offset the muscle’s ability to contract, as can be deduced from the rise of the residual intraluminal pressure in the presence of STX-6c. Of physiologic relevance is the discovery that the ETB receptor antagonist BQ-788 stimulates peristaltic motor activity. Because the properistaltic action of BQ-788 was strictly related to its concentration and opposite to that of the ETB receptor agonist STX-6c, we infer that BQ-788 facilitates propulsion by preventing the antiperistaltic action of endogenous ETs acting via ETB receptors. This argument is supported by the specificity of BQ-788 as an ETB receptor antagonist, which has been established in many peptide and nonpeptide receptor assays21 and which is extended by the current finding that BQ-788 spares the antiperistaltic actions of ATP and the opioid receptor agonist dynorphin-(1-13). The ability of BQ788 to lower PPT thus implies that ETs released during propulsive motility play a physiologic role in peristaltic motor regulation. The failure of atropine, hexamethonium, and SR-140,333 plus SR-144,190 to counteract BQ-788 suggests that the properistaltic action of this ETB receptor antagonist does not involve excitatory pathways within the enteric nervous system but results from the occlusion of muscular ETB receptors (Figure 6) which, when activated by endogenous ETs, inhibit peristalsis.

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In summary, the present study has shown that ET-1 and STX-6c are very potent in modifying propulsive motility in the guinea pig small intestine. The facilitatory action of ET-1 on peristalsis is predominantly mediated by ETA receptors on enteric neurons, whereas the antiperistaltic action of STX-6c arises from activation of ETB receptors on the muscle. Importantly, endogenous ETB receptor agonists are released during peristalsis and participate in the physiologic regulation of propulsive motility. Because, in addition, ETs are up-regulated in inflammatory bowel disease5 and experimental peritonitis42 and released by intestinal anaphylaxis,43 it is conceivable that a deranged ET system is a factor in intestinal motor disturbances as is the case with ischemiareperfusion injury22 and pancreatitis.23

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Received August 5, 1999. Accepted March 1, 2000. Address requests for reprints to: Peter Holzer, Ph.D., Department of Experimental and Clinical Pharmacology, University of Graz, Universita ¨tsplatz 4, A-8010 Graz, Austria. e-mail: peter.holzer@kfunigraz. ac.at; fax: (43) 316-380-9645. Supported by the Austrian Research Foundation FWF (grant ¨ AD) fellowship P11834-MED), an Austrian Exchange Programme (O (to A.S.), and a stipend of the Foundation ‘‘Fu ¨r Ausla ¨nder in ¨ sterreich’’ (to A.S.). O The authors thank Milana Jocic for her help with the graphical presentation of the data.