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Effect of pancreatic polypeptide on the motility of the guinea-pig small intestine in vitro
Regulatory Peptides, 16 (1986) 305-314
305
Elsevier RPT 00546
Effect of pancreatic polypeptide on the motility of the guinea-pig small intestine in vitro P. Holzer 1, L. Barth61'*, I.T. Lippe 1, W. Petritsch 2 and G. Leb 2 1University Department of Experimental and Clinical Pharmacology, Universitiitsplatz 4, A-8010 Graz, and 2University Clinic of Internal Medicine, Auenbruggerplatz 15, A-8036 Graz, Austria
(Received 6 August 1986; revised manuscript received and accepted for publication 4 November 1986)
Summary The effect of porcine pancreatic polypeptide (PP) on the motor activity of the longitudinal and circular muscles of the guinea-pig isolated small intestine was investigated. PP (0.2-20 nM) inhibited cholinergic contractions of the longitudinal muscle in response to electrical field stimulation, the maximal effect being a 30% reduction of the contraction amplitude. Carbachol-induced contractions of the longitudinal muscle were not affected by PP (10 nM). PP (0.3-30 nM) also inhibited reflex contractions of the circular muscle elicited by balloon distension and recorded orally to the site of distension; the maximal effect was a 80% reduction of the reflex contraction. In contrast, carbachol-induced contractions of the circular muscle remained unaltered by PP (10 nM). It was further found that PP (10 and 100 nM) enhanced the threshold intraluminal pressure at which peristaltic waves were triggered. All these effects of PP appeared to be transient. Taken together, these data indicate that PP does not act on intestinal smooth muscle but can modulate the activity of certain enteric neurones which are involved in the regulation of intestinal motility. pancreatic polypeptide; guinea-pig small intestine; intestinal smooth muscle; enteric nervous system; enteric reflexes; intestinal peristalsis
Address correspondence to: Peter Holzer, Ph.D., University Department of Experimental and Clinical
Pharmacology, Universit/itsplatz 4, A-8010 Graz, Austria. * Permanent address: University Department of Pharmacology, Prcs, Hungary. 0167-0115/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
306
Introduction
Pancreatic polypeptide (PP) belongs to a family of structurally related peptides including polypeptide YY and neuropeptide Y [1]. PP-like immunoreactivity has been localized to endocrine cells of the pancreas and gastrointestinal tract (for reviews see Refs. 2, 3), and PP appears to be involved in the hormonal regulation of exocrine pancreatic secretion and biliary tract motility [2,4]. PP also seems to affect gastrointestinal motility, though the data reported in the literature are controversial in this respect. Both excitatory and inhibitory effects of PP on in vivo indices of gastrointestinal propulsion have been reported [4-7] but it is not clear whether differences in species, dosage, or mode of administration account for the discrepancies. In the present study the effects of PP on the longitudinal and circular muscles of the guinea-pig small intestine were analysed so as to enable a better understanding of the possible mechanisms of action of PP on intestinal motility.
Materials and Methods
Adult guinea-pigs of 350-500 g body weight and either sex were used for the experiments. The animals were killed by decapitation followed by exsanguination. A portion of the small intestine (distal jejunum and proximal ileum) was excised immediately and transferred to prewarmed and oxygenated Tyrode solution. Following dissection segments were transferred to organ baths which contained aerated Tyrode solution maintained at 37°C. The mechanical activity of the muscle was recorded with isotonic lever displacement systems (Hugo Sachs Elektronik, Freiburg, F.R.G.) and displayed on a Rikadenki pen recorder. All tissues were equilibrated for at least 20 min before starting the experiments.
Contractions of the longitudinal muscle Intact segments of 2-3 cm length were suspended vertically in 7 ml silanized glass organ baths and kept under a resting load of 0.5 g. Two electrodes made of platinum wire positioned vertically on either side of the segments were used for electrical field stimulation of the segments. Electrical square wave pulses of supramaximal voltage (40 V) and 0.1 ms duration were delivered at a frequency of 0.05 Hz. The twitches produced by these stimuli reached 35.6 + 1.7% (mean 4- S.E.M., n = 20) of the maximal contraction in response to histamine (27/~M). Contractions of the circular muscle Intact segments of 6-7 cm length were secured horizontally at the bottom of a 15-ml silanized glass organ bath by a metal bar (stainless steel, diameter 1 mm) passed through the lumen [8-10]. In addition, the two ends of the segments were also secured so as to prevent dislocation of the site of recording by longitudinal contractions. To study the enteric ascending excitatory reflex [8] an inflatable balloon made of latex rubber was introduced into the lumen of the segments. The balloon was connected, via a tubing of 1.9 mm outer diameter, to a syringe and the whole system filled with
307 Tyrode solution. With the syringe the balloon could be inflated to a diameter of 7.5 mm. The mechanical activity on the oral side of the preparation (1.5-2 cm away from the site of distension) was recorded via a frog heart clip attached to the adjacent mesentery of the segments. The preparations were kept under a resting load of 0.5 g. Before the experiments were started it was ensured, by visual inspection and, when necessary, by readjustment of the position of the clip, that the site of recording was the site where distension caused the most vigorous contraction. A similar method of eliciting and recording enteric reflexes has been used by Costa et al. [1 I].
Peristalsis Intestinal peristalsis was studied with a modification of previously described methods [12-14]. An intact segment of small intestine, about 10 cm long, was secured horizontally in a 50-ml perspex organ bath. The oral and aboral ends of the segment were connected to inflow and outflow cannulae, respectively. Tyrode solution was continuously pumped, via the inflow cannula, into the lumen of the preparation [14] at a rate of 0.5 ml/min. The aboral end of the segment was connected, via a tubing (inner diameter 4 mm), to a mercury valve [13] which was set to open at an intraluminal pressure of 4.5 mbar. The intraluminal pressure at the aboral end of the segment was monitored with a Statham pressure transducer. The fluid which passed the mercury valve was collected in a glass tube, and the amount of fluid which had passed the valve was measured by a Statham pressure transducer positioned at the lower end of the glass tube [13]. The two parameters (intraluminal pressure and fluid transport) were recorded on a pen recorder. The principle of this method of eliciting and recording intestinal peristalsis is as follows (see also Fig. 4). When fluid is pumped into the lumen of the segment intraluminal pressure increases because, at intraluminal pressures below 4.5 mbar, the mercury valve prevents escape of fluid from the system. Intraluminal pressure rises up to a threshold at which a peristaltic wave is triggered (1.21 + 0.13 mbar, mean + S.E.M., n = 12, in the present experiments). This wave of circular contraction is measured as a spike-like increase in intraluminal pressure. The pressure rise is sufficient to propel the fluid over the mercury valve and thus to empty the intestinal segment. With a pumping rate of 0.5 ml/min, peristaltic activity of a relatively low frequency (0.59 + 0.07 waves/min, mean + S.E.M., n = 12) was elicited; this probably ensured that no signs of fatigue were observed during an experimental period of up to 90 min. Pancreatic polypeptide was administered into the bath, i.e. to the serosal surface of the intestine. Drugs and solutions The following drugs were used: atropine (Merck, Darmstadt, F.R.G.), carbachol (Ebewe, Unterach, Austria) tetrodotoxin (Sigma, M/inchen, F.R.G.), histamine (Serva, Heidelberg, F.R.G.), and porcine pancreatic polypeptide (Novo, Bagsvaerd, Denmark). Pancreatic polypeptide was dissolved (1 mg/ml) in and diluted with 0.01 N acetic acid. The other drugs were dissolved (1 mg/ml) in and diluted with Tyrode solution. The Tyrode solution had the following composition (mM): NaC1 136.9, KC1 2.7, CaC12 1.8, MgCI2 1.0, NaHCO3 11.9, NaH2PO4 0.4, and glucose 5.6. Drugs were added to the organ baths in volumes less than 1% of the bath volume. Corresponding volumes of 0.01 N acetic acid had no effect on the preparations.
308
Analysis of results All results are presented as mean + S.E.M. Student's two-tailed t-test for paired data was used to analyse the results statistically.
Results
Effects of PP on the longitudinal muscle At the beginning of the experiments, the preparations were standardized with a maximally effective concentration of histamine (27/~M), and subsequent contractions were expressed as a percentage of this response to histamine. First the effect of PP on cholinergic contractions induced by electrical stimulation was investigated, concentrations of PP being tested in an ascending order. PP was left in contact with the tissue for not more than 4 min. The bath was washed repeatedly and the next dose of PP was added after the contractions had returned to pre-drug levels, but at least with 10 min in between two additions of PP. As is shown in Fig. 1A, PP depressed the cholinergic contractions in response to electrical stimu-
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Fig. !. Effect of PP on longitudinal contractions recorded from segments of the guinea-pig small intestine. (A) Effect on contractions induced by electrical field stimulation with pulses of 0.1 ms duration delivered at a frequency of 0.05 Hz. Means 4- S.E.M., n = 4. * P < 0.01 versus contractions m e a s u r ~ before addition of the respective dose of PP. (B) Effect of PP (10 nM, contact time I rain) on contractions induced by carbachol. Means 4- S.E.M. of 6 experiments.
309
lation in a concentration-dependent manner. The maximal reduction achieved by PP, however, did not exceed 30%. Moreover, the inhibitory effect of PP was only transient: Maximal reductions of the contraction amplitude were apparent within 1-2 min after addition of PP, and then the contractions started slowly to return to control values although PP had not yet been washed out. Atropine (1 #M) completely abolished the electrically induced contractions (n = 6). The effect of PP on contractions induced by carbachol was also tested. These experiments were performed in the presence of 0.3 ~tM tetrodotoxin in order to exclude any neuraUy mediated component in the action of carbachol. Carbachol concentration-response curves were recorded by adding increasing doses of carbachol at 5-min intervals; the contact time for each dose was 30 s. Ten minutes after establishing a control concentration-response curve for carbachol, a second concentrationresponse curve was recorded in the presence of PP which was added 1 min before each dose of carbachol. As is shown in Fig. 1B, contractions in response to carbachol were unaffected in the presence of 10 nM PP, a concentration which was maximally effective in depressing electrically induced contractions.
Effect of PP on the circular muscle The effect of PP was tested on the excitatory ascending enteric reflex. The reflex was elicited at 5 min intervals by balloon distension for 5 s. Increasing doses of PP were added to the bath either 1 min or 4 min before the distension stimulus. After each dose of PP, the reflex was allowed to recover to pre-drug levels before the next dose of PP was tested. Distension of the gut wall elicited a brief single contraction of the circular muscle on the oral side of the distension [see 9]. This reflex contraction amounted to 95 ± 1% (n = 10) of the response to a maximally effective concentration of carbachol (6 #M). The responses to distension were completely abolished by 0.3-1 #M tetrodotoxin (n = 6) and reduced by 93 + 5% (n =6, P < 0.01) in the presence of atropine (0.35 gM). The effect of PP (0.3-30 nM) depended on the time of exposure of the 100 c o c) t
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Fig. 2. Effect of PP on the ascending enteric reflex contraction of the circular muscle of the guinea-pig small intestine. Concentration-response relationship for the inhibitory effect of PP given either 1 rain (closed symbols) or 4 rain (open symbols) before el±citing the reflex. Means ± S.E.M., n = 6. Where no S.E.M. is shown, it is smaller than the symbols. * P < 0.05, ** P < 0.01 versus effect of the same concentration of PP given 1 min before el±citing the reflex.
310
tissue to the peptide. With a contact time of 1 min, PP inhibited the reflex contraction in a concentration-dependent manner, the maximal effect being an about 80% reduction of the contraction amplitude (Fig. 2). When the contact time was extended to 4 min, the efficacy of PP in depressing the reflex contraction was attenuated, since the maximal inhibition achieved by PP did not exceed 25-30% (Fig. 2). The effect of PP was also tested on circular muscle contractions induced by carbachol. As reported previously for bethanechol [10], carbachol produced phasic contractions of the circular muscle superimposed on a tonic contraction (see also Ref. 9). The responses to carbachol (contact time 45 s) were reproducible at 5-min intervals; they remained unaltered by tetrodotoxin [15]. PP was tested against contractions induced by 0.6-1.2 #M carbachol, i.e. approximately 10-fold threshold concentrations of carbachol; the peptide was added to the bath 1 min before carbachol. PP, in a concentration (10 nM) which suppressed the ascending reflex contraction, had no effect on the contractile responses to carbachol. In the presence of PP, the contractile responses to carbachol amounted to 103 + 2% (n = 6) of the responses measured in the absence of PP.
Effect of PP on intestinal peristalsis In these experiments only one dose of PP was added to each preparation, and peristalsis thereafter recorded for a minimum of 20 min. PP, in a concentration (10 nM) which was maximally effective in suppressing the ascending reflex contraction of the circular muscle (Fig. 2), significantly increased the threshold intraluminal pressure at which a peristaltic wave was elicited (Fig. 3). Correspondingly, the frequency of peristaltic waves decreased from 0.50 + 0.07 to 0.38 + 0.04 peristaltic waves per min (n = 6, P < 0.01). However, even a 10-fold higher concentration of PP (100 nM) did not abolish peristalsis (Figs. 3 and 4): again, only the threshold intraluminal
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Fig. 3. Time course of the effect of PP (10 and 100 n M ) on the threshold intraluminal pressure at which peristaltic waves are triggered in the isolated small intestine o f the guinea-pig. The threshold before addition of PP was 1.21 + 0.13 m b a r (n = 12), PP was added to the bath at time 0. M e a n s 4- S.E.M. o f 6 experiments with each concentration o f PP. * P < 0.05, ** P < 0.01 versus threshold before addition of PP.
311
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Fig. 4. Recording of the effect of PP (100 nM, administration to the bath indicated by an arrow) on the peristaltic reflex in the isolated small intestine of the guinea-pig.
pressure, at which a peristaltic wave was elicited, was significantly enhanced, and the frequency of peristaltic waves fell from 0.68 q- 0.12 to 0.47 + 0.07 peristaltic waves per min (n = 6, P < 0.01). Neither concentration of PP reduced the amplitude of the peristaltic waves. The effect of PP on the peristaltic reflex was transient: Maximal effects were observed within 2-4 min after addition of the peptide; thereafter the threshold intraluminal pressure tended to return to control levels although PP had not yet been washed out (Fig. 3).
Discussion The reports concerning the effect of PP on gastrointestinal motility are diverse. In the dog, low doses of bovine PP seem to inhibit whereas large doses (> 10 nmol/kg i.v.) enhance gastrointestinal motility [4]. With porcine PP, the results are conflicting because both inhibitory [7] and excitatory [6] influences on the migrating motor complexes of the canine and porcine alimentary canal have been demonstrated. In the rat, gastric emptying is increased by bovine PP [4] while the motility of the small intestine has been found either unaffected [16] or enhanced [4]. Porcine PP, on the other hand, was shown to inhibit the motor activity of the rat small intestine [5]. In man, bovine PP seems to be without effect on the migrating motor complexes [17], although both in man and dog plasma levels of endogenous PP are known to change cyclically during fasting and peaks of plasma levels are associated with maximal activity of the migrating motor complex [17-19]. The present experiments were designed to investigate the effects of porcine PP on the motor activity of the longitudinal and circular muscle of the guinea-pig small intestine and to elucidate its possible mechanism of action. The effects which were observed with PP were of an inhibitory nature: PP reduced cholinergically mediated contractions of the longitudinal muscle, suppressed the ascending reflex contraction
312
of the circular muscle, and enhanced the threshold intraluminal pressure, at which peristaltic waves were triggered. A common feature of all these effects was that PP failed to completely abolish any of the investigated motor responses and that the effects of PP were transient. It is possible that these observations reflect degradation of the peptide within the intestinal tissue and/or desensitization of the tissue to the peptide but this problem was not further studied. The inhibition of intestinal contractions caused by PP could result from an action of the peptide on enteric neurones and/or intestinal smooth muscle. PP did not alter contractile responses of the longitudinal and circular muscles to carbachol. These contractions did not involve enteric neurones since they were not affected by tetrodotoxin which blocks nerve conduction but leaves direct effects on intestinal muscle unchanged [20]. From this finding it can be inferred that PP does not act directly on the intestinal smooth muscle. Thus the effect of PP on cholinergically mediated contractions of the longitudinal and circular muscles is due to an action on enteric neurones. It is also very probable that the inhibitory action of PP on the ascending enteric reflex pathway accounts for the inhibitory action on peristalsis. The discrepancy between the maximal (80%) inhibition of the ascending reflex and the relatively modest inhibition of peristalsis caused by PP may be explained by the transient nature of the effect on the ascending reflex. Furthermore it should be borne in mind that short-lasting changes in peristaltic performance are difficult to detect in view of the slow frequency of peristaltic waves (about 0.5 waves per min) seen under the present experimental conditions. Finally the question arises as to the physiological significance of the present findings. From the localization of PP in endocrine cells of the pancreas and gut it would appear that, if PP were to regulate intestinal motility, it would do so by a hormonal action. However, the plasma levels of circulating PP in man are in the range of 0.01-0.16 nM [17,19] and thus well below the concentrations found to be active in the guinea-pig intestine. It therefore appears as if the effects of PP seen here reflect actions of neuropeptide Y (NPY) or, less likely, polypeptide YY (PYY), two peptides closely related to PP. NPY and PYY have a differential distribution in the digestive tract: while PYY occurs in endocrine cells of the mucosa, NPY is located in both sympathetic and enteric nerves [3]. The effects of PYY on intestinal motor activity have not yet been examined in detail, whereas NPY has been found to exhibit a similar spectrum of actions on enteric neurones as PP [15]. However, there are also some differences between the effects of NPY and PP. Although both NPY [15] and PP are fairly equipotent in depressing the ascending reflex contraction, half-maximal effects being produced by concentrations of 1-3 nM, the inhibition of the reflex caused by NPY is complete and sustained [15] whereas that caused by PP is incomplete and short-lasting. The transient nature of the motor effects of PP is another finding which is rather difficult to reconcile with a physiological role of this peptide in the hormonal regulation of intestinal motility.
313
Acknowledgements This work was supported by the Austrian Scientific Research Funds (grant No. 5552). The authors wish to thank Mr. W. Schluet for technical and Mrs. I. H6rzer for secretarial assistance.
References 1 Tatemoto, K., Carlqvist, M. and Mutt, V., Neuropeptide Y - a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide, Nature, 296 (1982) 659-660. 2 Schwartz, T.W., Pancreatic polypeptide: a hormone under vagal control, Gastroenterology, 85 (1983) 1411-1425. 3 O'Donohue, T.L., Chronwall, B.M., Pruss, R.M., Mezey, E., Kiss, J.Z., Eiden, L.E., Massari, V.J., Tessel, R.E., Pickel, V.M., DiMaggio, D.A., Hotchkiss, A.J., Crowley, W.R. and Zukowska-Grojec, Z., Neuropeptide Y and peptide YY neuronal and endocrine systems, Peptides, 6 (1985) 755-768. 4 Lin, T.-M., Pancreatic polypeptide: isolation, chemistry, and biological function. In G.B.J. Glass (Ed.), Gastrointestinal Hormones, Raven Press, New York, 1980, pp. 275-306. 5 Al-Saffar, A. and Tatemoto, K., Effects of peptide YY and porcine pancreatic polypeptide on migrating myoelectric complexes in the small intestine of the rat. In C. Roman (Ed.), Gastrointestinal Motility, MTP Press, Lancaster, 1984, pp. 215-222. 6 Bueno, L., Fioramonti, J., Rayner, V. and Ruckebusch, Y., Effects of motilin, somatostatin, and pancreatic polypeptide on the migrating myoelectric complex in pig and dog, Gastroenterology, 82 (1982) 1395-1402. 7 Hall, K.E., Diamant, N.E., E1-Sharkawy, T.Y. and Greenberg, G.R., Effect of pancreatic polypeptide on canine migrating motor complex and plasma motilin, Am. J. Physiol., 245 (1983) G178-G185. 8 Costa, M. and Furness, J.B., The peristaltic reflex: An analysis of the nerve pathways and their pharmacology, Naunyn-Schmiedeberg's Arch. Pharmacol., 294 (1976) 47--60. 9 Costa, M., Furness, J.B., Pullin, C.O. and Bornstein, J., Substance P enteric neurons mediate noncholinergic transmission to the circular muscle of the guinea-pig intestine, Naunyn-Schmiedeberg's Arch. Pharmacol., 328 (1985) 446-453. 10 Holzer, P., Lembeck, F. and Donnerer, J., Caerulein, substance P, serotonin and cholinomimetics induce rhythmic contractions of intestinal circular muscle, Naunyn-Schmiedeberg's Arch. Pharmacol., 312 (1980) 131-137. I 1 Costa, M., Furness, J.B. and Humphreys, C.M.S., Apamin distinguishes two types of relaxation mediated by enteric nerves in the guinea-pig gastrointestinal tract, Naunyn-Schmiedeberg's Arch. Pharmacol., 332 (1986) 79-88. 12 B/ilbring, E., Crema, A. and Saxby, O.B., A method for recording peristalsis in isolated intestine, Br. J. Pharmacol., 13 (1958) 440-443. 13 Holzer, P. and Lembeck, F., Effect of neuropeptides on the efficiency of the peristaltic reflex, Naunyn-Schmiedeberg's Arch. Pharmacol., 307 (1979) 257-264. 14 Tonini, M., Frigo, G., Lecchini, S., D'Angelo, L. and Crema, A., Hyoscine-resistant peristalsis in guinea-pig ileum, Eur. J. Pharmacol., 71 (1981) 375-381. 15 Holzer, P., Lippe, I.Th., Barthr, L. and Saria, A., Neuropeptide Y inhibits excitatory enteric neurons supplying the circular muscle of the guinea-pig small intestine, Gastroenterology, (1986) in press. 16 Gustavsson, S., Johansson, H., Lundqvist, G. and Nilsson, F., Effects of vasoactive intestinal peptide and pancreatic polypeptide on small bowel propulsion in the rat, Scand. J. Gastroenterol., 12 (1977) 993-997. 17 Janssens, J., Hellemans, J., Adrian, T.E., Bloom, S.R., Peeters, T.L., Christofides, N. and Vantrappen, G.R., Pancreatic polypeptide is not involved in the regulation of the migrating motor complex in man, Regul. Peptides, 3 (1982) 41-49. 18 Keane, F.B., DiMango, E.B., Dozois, R.R. and Go, V.L.W., Relationships among canine interdiges-
314 tive exocrine pancreatic and biliary flow, duodenal motor activity, plasma pancreatic polypeptide, and motilin, Gastroenterology, 78 (1980) 310-316. 19 Owyang, C., Achem-Karam, S.R. and Vinik, A.I., Pancreatic polypeptide and intestinal migrating motor complex in humans, Gastroenterology, 84 (1983) 10-17. 20 Gershon, M.D., Effects of tetrodotoxin on innervated smooth muscle preparations, Br. J. Pharmacol. Chemother., 29 (1967) 259-279.
305
Elsevier RPT 00546
Effect of pancreatic polypeptide on the motility of the guinea-pig small intestine in vitro P. Holzer 1, L. Barth61'*, I.T. Lippe 1, W. Petritsch 2 and G. Leb 2 1University Department of Experimental and Clinical Pharmacology, Universitiitsplatz 4, A-8010 Graz, and 2University Clinic of Internal Medicine, Auenbruggerplatz 15, A-8036 Graz, Austria
(Received 6 August 1986; revised manuscript received and accepted for publication 4 November 1986)
Summary The effect of porcine pancreatic polypeptide (PP) on the motor activity of the longitudinal and circular muscles of the guinea-pig isolated small intestine was investigated. PP (0.2-20 nM) inhibited cholinergic contractions of the longitudinal muscle in response to electrical field stimulation, the maximal effect being a 30% reduction of the contraction amplitude. Carbachol-induced contractions of the longitudinal muscle were not affected by PP (10 nM). PP (0.3-30 nM) also inhibited reflex contractions of the circular muscle elicited by balloon distension and recorded orally to the site of distension; the maximal effect was a 80% reduction of the reflex contraction. In contrast, carbachol-induced contractions of the circular muscle remained unaltered by PP (10 nM). It was further found that PP (10 and 100 nM) enhanced the threshold intraluminal pressure at which peristaltic waves were triggered. All these effects of PP appeared to be transient. Taken together, these data indicate that PP does not act on intestinal smooth muscle but can modulate the activity of certain enteric neurones which are involved in the regulation of intestinal motility. pancreatic polypeptide; guinea-pig small intestine; intestinal smooth muscle; enteric nervous system; enteric reflexes; intestinal peristalsis
Address correspondence to: Peter Holzer, Ph.D., University Department of Experimental and Clinical
Pharmacology, Universit/itsplatz 4, A-8010 Graz, Austria. * Permanent address: University Department of Pharmacology, Prcs, Hungary. 0167-0115/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
306
Introduction
Pancreatic polypeptide (PP) belongs to a family of structurally related peptides including polypeptide YY and neuropeptide Y [1]. PP-like immunoreactivity has been localized to endocrine cells of the pancreas and gastrointestinal tract (for reviews see Refs. 2, 3), and PP appears to be involved in the hormonal regulation of exocrine pancreatic secretion and biliary tract motility [2,4]. PP also seems to affect gastrointestinal motility, though the data reported in the literature are controversial in this respect. Both excitatory and inhibitory effects of PP on in vivo indices of gastrointestinal propulsion have been reported [4-7] but it is not clear whether differences in species, dosage, or mode of administration account for the discrepancies. In the present study the effects of PP on the longitudinal and circular muscles of the guinea-pig small intestine were analysed so as to enable a better understanding of the possible mechanisms of action of PP on intestinal motility.
Materials and Methods
Adult guinea-pigs of 350-500 g body weight and either sex were used for the experiments. The animals were killed by decapitation followed by exsanguination. A portion of the small intestine (distal jejunum and proximal ileum) was excised immediately and transferred to prewarmed and oxygenated Tyrode solution. Following dissection segments were transferred to organ baths which contained aerated Tyrode solution maintained at 37°C. The mechanical activity of the muscle was recorded with isotonic lever displacement systems (Hugo Sachs Elektronik, Freiburg, F.R.G.) and displayed on a Rikadenki pen recorder. All tissues were equilibrated for at least 20 min before starting the experiments.
Contractions of the longitudinal muscle Intact segments of 2-3 cm length were suspended vertically in 7 ml silanized glass organ baths and kept under a resting load of 0.5 g. Two electrodes made of platinum wire positioned vertically on either side of the segments were used for electrical field stimulation of the segments. Electrical square wave pulses of supramaximal voltage (40 V) and 0.1 ms duration were delivered at a frequency of 0.05 Hz. The twitches produced by these stimuli reached 35.6 + 1.7% (mean 4- S.E.M., n = 20) of the maximal contraction in response to histamine (27/~M). Contractions of the circular muscle Intact segments of 6-7 cm length were secured horizontally at the bottom of a 15-ml silanized glass organ bath by a metal bar (stainless steel, diameter 1 mm) passed through the lumen [8-10]. In addition, the two ends of the segments were also secured so as to prevent dislocation of the site of recording by longitudinal contractions. To study the enteric ascending excitatory reflex [8] an inflatable balloon made of latex rubber was introduced into the lumen of the segments. The balloon was connected, via a tubing of 1.9 mm outer diameter, to a syringe and the whole system filled with
307 Tyrode solution. With the syringe the balloon could be inflated to a diameter of 7.5 mm. The mechanical activity on the oral side of the preparation (1.5-2 cm away from the site of distension) was recorded via a frog heart clip attached to the adjacent mesentery of the segments. The preparations were kept under a resting load of 0.5 g. Before the experiments were started it was ensured, by visual inspection and, when necessary, by readjustment of the position of the clip, that the site of recording was the site where distension caused the most vigorous contraction. A similar method of eliciting and recording enteric reflexes has been used by Costa et al. [1 I].
Peristalsis Intestinal peristalsis was studied with a modification of previously described methods [12-14]. An intact segment of small intestine, about 10 cm long, was secured horizontally in a 50-ml perspex organ bath. The oral and aboral ends of the segment were connected to inflow and outflow cannulae, respectively. Tyrode solution was continuously pumped, via the inflow cannula, into the lumen of the preparation [14] at a rate of 0.5 ml/min. The aboral end of the segment was connected, via a tubing (inner diameter 4 mm), to a mercury valve [13] which was set to open at an intraluminal pressure of 4.5 mbar. The intraluminal pressure at the aboral end of the segment was monitored with a Statham pressure transducer. The fluid which passed the mercury valve was collected in a glass tube, and the amount of fluid which had passed the valve was measured by a Statham pressure transducer positioned at the lower end of the glass tube [13]. The two parameters (intraluminal pressure and fluid transport) were recorded on a pen recorder. The principle of this method of eliciting and recording intestinal peristalsis is as follows (see also Fig. 4). When fluid is pumped into the lumen of the segment intraluminal pressure increases because, at intraluminal pressures below 4.5 mbar, the mercury valve prevents escape of fluid from the system. Intraluminal pressure rises up to a threshold at which a peristaltic wave is triggered (1.21 + 0.13 mbar, mean + S.E.M., n = 12, in the present experiments). This wave of circular contraction is measured as a spike-like increase in intraluminal pressure. The pressure rise is sufficient to propel the fluid over the mercury valve and thus to empty the intestinal segment. With a pumping rate of 0.5 ml/min, peristaltic activity of a relatively low frequency (0.59 + 0.07 waves/min, mean + S.E.M., n = 12) was elicited; this probably ensured that no signs of fatigue were observed during an experimental period of up to 90 min. Pancreatic polypeptide was administered into the bath, i.e. to the serosal surface of the intestine. Drugs and solutions The following drugs were used: atropine (Merck, Darmstadt, F.R.G.), carbachol (Ebewe, Unterach, Austria) tetrodotoxin (Sigma, M/inchen, F.R.G.), histamine (Serva, Heidelberg, F.R.G.), and porcine pancreatic polypeptide (Novo, Bagsvaerd, Denmark). Pancreatic polypeptide was dissolved (1 mg/ml) in and diluted with 0.01 N acetic acid. The other drugs were dissolved (1 mg/ml) in and diluted with Tyrode solution. The Tyrode solution had the following composition (mM): NaC1 136.9, KC1 2.7, CaC12 1.8, MgCI2 1.0, NaHCO3 11.9, NaH2PO4 0.4, and glucose 5.6. Drugs were added to the organ baths in volumes less than 1% of the bath volume. Corresponding volumes of 0.01 N acetic acid had no effect on the preparations.
308
Analysis of results All results are presented as mean + S.E.M. Student's two-tailed t-test for paired data was used to analyse the results statistically.
Results
Effects of PP on the longitudinal muscle At the beginning of the experiments, the preparations were standardized with a maximally effective concentration of histamine (27/~M), and subsequent contractions were expressed as a percentage of this response to histamine. First the effect of PP on cholinergic contractions induced by electrical stimulation was investigated, concentrations of PP being tested in an ascending order. PP was left in contact with the tissue for not more than 4 min. The bath was washed repeatedly and the next dose of PP was added after the contractions had returned to pre-drug levels, but at least with 10 min in between two additions of PP. As is shown in Fig. 1A, PP depressed the cholinergic contractions in response to electrical stimu-
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Fig. !. Effect of PP on longitudinal contractions recorded from segments of the guinea-pig small intestine. (A) Effect on contractions induced by electrical field stimulation with pulses of 0.1 ms duration delivered at a frequency of 0.05 Hz. Means 4- S.E.M., n = 4. * P < 0.01 versus contractions m e a s u r ~ before addition of the respective dose of PP. (B) Effect of PP (10 nM, contact time I rain) on contractions induced by carbachol. Means 4- S.E.M. of 6 experiments.
309
lation in a concentration-dependent manner. The maximal reduction achieved by PP, however, did not exceed 30%. Moreover, the inhibitory effect of PP was only transient: Maximal reductions of the contraction amplitude were apparent within 1-2 min after addition of PP, and then the contractions started slowly to return to control values although PP had not yet been washed out. Atropine (1 #M) completely abolished the electrically induced contractions (n = 6). The effect of PP on contractions induced by carbachol was also tested. These experiments were performed in the presence of 0.3 ~tM tetrodotoxin in order to exclude any neuraUy mediated component in the action of carbachol. Carbachol concentration-response curves were recorded by adding increasing doses of carbachol at 5-min intervals; the contact time for each dose was 30 s. Ten minutes after establishing a control concentration-response curve for carbachol, a second concentrationresponse curve was recorded in the presence of PP which was added 1 min before each dose of carbachol. As is shown in Fig. 1B, contractions in response to carbachol were unaffected in the presence of 10 nM PP, a concentration which was maximally effective in depressing electrically induced contractions.
Effect of PP on the circular muscle The effect of PP was tested on the excitatory ascending enteric reflex. The reflex was elicited at 5 min intervals by balloon distension for 5 s. Increasing doses of PP were added to the bath either 1 min or 4 min before the distension stimulus. After each dose of PP, the reflex was allowed to recover to pre-drug levels before the next dose of PP was tested. Distension of the gut wall elicited a brief single contraction of the circular muscle on the oral side of the distension [see 9]. This reflex contraction amounted to 95 ± 1% (n = 10) of the response to a maximally effective concentration of carbachol (6 #M). The responses to distension were completely abolished by 0.3-1 #M tetrodotoxin (n = 6) and reduced by 93 + 5% (n =6, P < 0.01) in the presence of atropine (0.35 gM). The effect of PP (0.3-30 nM) depended on the time of exposure of the 100 c o c) t
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Fig. 2. Effect of PP on the ascending enteric reflex contraction of the circular muscle of the guinea-pig small intestine. Concentration-response relationship for the inhibitory effect of PP given either 1 rain (closed symbols) or 4 rain (open symbols) before el±citing the reflex. Means ± S.E.M., n = 6. Where no S.E.M. is shown, it is smaller than the symbols. * P < 0.05, ** P < 0.01 versus effect of the same concentration of PP given 1 min before el±citing the reflex.
310
tissue to the peptide. With a contact time of 1 min, PP inhibited the reflex contraction in a concentration-dependent manner, the maximal effect being an about 80% reduction of the contraction amplitude (Fig. 2). When the contact time was extended to 4 min, the efficacy of PP in depressing the reflex contraction was attenuated, since the maximal inhibition achieved by PP did not exceed 25-30% (Fig. 2). The effect of PP was also tested on circular muscle contractions induced by carbachol. As reported previously for bethanechol [10], carbachol produced phasic contractions of the circular muscle superimposed on a tonic contraction (see also Ref. 9). The responses to carbachol (contact time 45 s) were reproducible at 5-min intervals; they remained unaltered by tetrodotoxin [15]. PP was tested against contractions induced by 0.6-1.2 #M carbachol, i.e. approximately 10-fold threshold concentrations of carbachol; the peptide was added to the bath 1 min before carbachol. PP, in a concentration (10 nM) which suppressed the ascending reflex contraction, had no effect on the contractile responses to carbachol. In the presence of PP, the contractile responses to carbachol amounted to 103 + 2% (n = 6) of the responses measured in the absence of PP.
Effect of PP on intestinal peristalsis In these experiments only one dose of PP was added to each preparation, and peristalsis thereafter recorded for a minimum of 20 min. PP, in a concentration (10 nM) which was maximally effective in suppressing the ascending reflex contraction of the circular muscle (Fig. 2), significantly increased the threshold intraluminal pressure at which a peristaltic wave was elicited (Fig. 3). Correspondingly, the frequency of peristaltic waves decreased from 0.50 + 0.07 to 0.38 + 0.04 peristaltic waves per min (n = 6, P < 0.01). However, even a 10-fold higher concentration of PP (100 nM) did not abolish peristalsis (Figs. 3 and 4): again, only the threshold intraluminal
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Fig. 3. Time course of the effect of PP (10 and 100 n M ) on the threshold intraluminal pressure at which peristaltic waves are triggered in the isolated small intestine o f the guinea-pig. The threshold before addition of PP was 1.21 + 0.13 m b a r (n = 12), PP was added to the bath at time 0. M e a n s 4- S.E.M. o f 6 experiments with each concentration o f PP. * P < 0.05, ** P < 0.01 versus threshold before addition of PP.
311
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pressure, at which a peristaltic wave was elicited, was significantly enhanced, and the frequency of peristaltic waves fell from 0.68 q- 0.12 to 0.47 + 0.07 peristaltic waves per min (n = 6, P < 0.01). Neither concentration of PP reduced the amplitude of the peristaltic waves. The effect of PP on the peristaltic reflex was transient: Maximal effects were observed within 2-4 min after addition of the peptide; thereafter the threshold intraluminal pressure tended to return to control levels although PP had not yet been washed out (Fig. 3).
Discussion The reports concerning the effect of PP on gastrointestinal motility are diverse. In the dog, low doses of bovine PP seem to inhibit whereas large doses (> 10 nmol/kg i.v.) enhance gastrointestinal motility [4]. With porcine PP, the results are conflicting because both inhibitory [7] and excitatory [6] influences on the migrating motor complexes of the canine and porcine alimentary canal have been demonstrated. In the rat, gastric emptying is increased by bovine PP [4] while the motility of the small intestine has been found either unaffected [16] or enhanced [4]. Porcine PP, on the other hand, was shown to inhibit the motor activity of the rat small intestine [5]. In man, bovine PP seems to be without effect on the migrating motor complexes [17], although both in man and dog plasma levels of endogenous PP are known to change cyclically during fasting and peaks of plasma levels are associated with maximal activity of the migrating motor complex [17-19]. The present experiments were designed to investigate the effects of porcine PP on the motor activity of the longitudinal and circular muscle of the guinea-pig small intestine and to elucidate its possible mechanism of action. The effects which were observed with PP were of an inhibitory nature: PP reduced cholinergically mediated contractions of the longitudinal muscle, suppressed the ascending reflex contraction
312
of the circular muscle, and enhanced the threshold intraluminal pressure, at which peristaltic waves were triggered. A common feature of all these effects was that PP failed to completely abolish any of the investigated motor responses and that the effects of PP were transient. It is possible that these observations reflect degradation of the peptide within the intestinal tissue and/or desensitization of the tissue to the peptide but this problem was not further studied. The inhibition of intestinal contractions caused by PP could result from an action of the peptide on enteric neurones and/or intestinal smooth muscle. PP did not alter contractile responses of the longitudinal and circular muscles to carbachol. These contractions did not involve enteric neurones since they were not affected by tetrodotoxin which blocks nerve conduction but leaves direct effects on intestinal muscle unchanged [20]. From this finding it can be inferred that PP does not act directly on the intestinal smooth muscle. Thus the effect of PP on cholinergically mediated contractions of the longitudinal and circular muscles is due to an action on enteric neurones. It is also very probable that the inhibitory action of PP on the ascending enteric reflex pathway accounts for the inhibitory action on peristalsis. The discrepancy between the maximal (80%) inhibition of the ascending reflex and the relatively modest inhibition of peristalsis caused by PP may be explained by the transient nature of the effect on the ascending reflex. Furthermore it should be borne in mind that short-lasting changes in peristaltic performance are difficult to detect in view of the slow frequency of peristaltic waves (about 0.5 waves per min) seen under the present experimental conditions. Finally the question arises as to the physiological significance of the present findings. From the localization of PP in endocrine cells of the pancreas and gut it would appear that, if PP were to regulate intestinal motility, it would do so by a hormonal action. However, the plasma levels of circulating PP in man are in the range of 0.01-0.16 nM [17,19] and thus well below the concentrations found to be active in the guinea-pig intestine. It therefore appears as if the effects of PP seen here reflect actions of neuropeptide Y (NPY) or, less likely, polypeptide YY (PYY), two peptides closely related to PP. NPY and PYY have a differential distribution in the digestive tract: while PYY occurs in endocrine cells of the mucosa, NPY is located in both sympathetic and enteric nerves [3]. The effects of PYY on intestinal motor activity have not yet been examined in detail, whereas NPY has been found to exhibit a similar spectrum of actions on enteric neurones as PP [15]. However, there are also some differences between the effects of NPY and PP. Although both NPY [15] and PP are fairly equipotent in depressing the ascending reflex contraction, half-maximal effects being produced by concentrations of 1-3 nM, the inhibition of the reflex caused by NPY is complete and sustained [15] whereas that caused by PP is incomplete and short-lasting. The transient nature of the motor effects of PP is another finding which is rather difficult to reconcile with a physiological role of this peptide in the hormonal regulation of intestinal motility.
313
Acknowledgements This work was supported by the Austrian Scientific Research Funds (grant No. 5552). The authors wish to thank Mr. W. Schluet for technical and Mrs. I. H6rzer for secretarial assistance.
References 1 Tatemoto, K., Carlqvist, M. and Mutt, V., Neuropeptide Y - a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide, Nature, 296 (1982) 659-660. 2 Schwartz, T.W., Pancreatic polypeptide: a hormone under vagal control, Gastroenterology, 85 (1983) 1411-1425. 3 O'Donohue, T.L., Chronwall, B.M., Pruss, R.M., Mezey, E., Kiss, J.Z., Eiden, L.E., Massari, V.J., Tessel, R.E., Pickel, V.M., DiMaggio, D.A., Hotchkiss, A.J., Crowley, W.R. and Zukowska-Grojec, Z., Neuropeptide Y and peptide YY neuronal and endocrine systems, Peptides, 6 (1985) 755-768. 4 Lin, T.-M., Pancreatic polypeptide: isolation, chemistry, and biological function. In G.B.J. Glass (Ed.), Gastrointestinal Hormones, Raven Press, New York, 1980, pp. 275-306. 5 Al-Saffar, A. and Tatemoto, K., Effects of peptide YY and porcine pancreatic polypeptide on migrating myoelectric complexes in the small intestine of the rat. In C. Roman (Ed.), Gastrointestinal Motility, MTP Press, Lancaster, 1984, pp. 215-222. 6 Bueno, L., Fioramonti, J., Rayner, V. and Ruckebusch, Y., Effects of motilin, somatostatin, and pancreatic polypeptide on the migrating myoelectric complex in pig and dog, Gastroenterology, 82 (1982) 1395-1402. 7 Hall, K.E., Diamant, N.E., E1-Sharkawy, T.Y. and Greenberg, G.R., Effect of pancreatic polypeptide on canine migrating motor complex and plasma motilin, Am. J. Physiol., 245 (1983) G178-G185. 8 Costa, M. and Furness, J.B., The peristaltic reflex: An analysis of the nerve pathways and their pharmacology, Naunyn-Schmiedeberg's Arch. Pharmacol., 294 (1976) 47--60. 9 Costa, M., Furness, J.B., Pullin, C.O. and Bornstein, J., Substance P enteric neurons mediate noncholinergic transmission to the circular muscle of the guinea-pig intestine, Naunyn-Schmiedeberg's Arch. Pharmacol., 328 (1985) 446-453. 10 Holzer, P., Lembeck, F. and Donnerer, J., Caerulein, substance P, serotonin and cholinomimetics induce rhythmic contractions of intestinal circular muscle, Naunyn-Schmiedeberg's Arch. Pharmacol., 312 (1980) 131-137. I 1 Costa, M., Furness, J.B. and Humphreys, C.M.S., Apamin distinguishes two types of relaxation mediated by enteric nerves in the guinea-pig gastrointestinal tract, Naunyn-Schmiedeberg's Arch. Pharmacol., 332 (1986) 79-88. 12 B/ilbring, E., Crema, A. and Saxby, O.B., A method for recording peristalsis in isolated intestine, Br. J. Pharmacol., 13 (1958) 440-443. 13 Holzer, P. and Lembeck, F., Effect of neuropeptides on the efficiency of the peristaltic reflex, Naunyn-Schmiedeberg's Arch. Pharmacol., 307 (1979) 257-264. 14 Tonini, M., Frigo, G., Lecchini, S., D'Angelo, L. and Crema, A., Hyoscine-resistant peristalsis in guinea-pig ileum, Eur. J. Pharmacol., 71 (1981) 375-381. 15 Holzer, P., Lippe, I.Th., Barthr, L. and Saria, A., Neuropeptide Y inhibits excitatory enteric neurons supplying the circular muscle of the guinea-pig small intestine, Gastroenterology, (1986) in press. 16 Gustavsson, S., Johansson, H., Lundqvist, G. and Nilsson, F., Effects of vasoactive intestinal peptide and pancreatic polypeptide on small bowel propulsion in the rat, Scand. J. Gastroenterol., 12 (1977) 993-997. 17 Janssens, J., Hellemans, J., Adrian, T.E., Bloom, S.R., Peeters, T.L., Christofides, N. and Vantrappen, G.R., Pancreatic polypeptide is not involved in the regulation of the migrating motor complex in man, Regul. Peptides, 3 (1982) 41-49. 18 Keane, F.B., DiMango, E.B., Dozois, R.R. and Go, V.L.W., Relationships among canine interdiges-
314 tive exocrine pancreatic and biliary flow, duodenal motor activity, plasma pancreatic polypeptide, and motilin, Gastroenterology, 78 (1980) 310-316. 19 Owyang, C., Achem-Karam, S.R. and Vinik, A.I., Pancreatic polypeptide and intestinal migrating motor complex in humans, Gastroenterology, 84 (1983) 10-17. 20 Gershon, M.D., Effects of tetrodotoxin on innervated smooth muscle preparations, Br. J. Pharmacol. Chemother., 29 (1967) 259-279.