153

J. Phyeiol. (1979), 294, pp. 153-163 With 4 text-figure8 Printed in Great Britain

POTENT STIMULATION OF THE AVIAN EXOCRINE PANCREAS BY PORCINE AND CHICKEN VASOACTIVE INTESTINAL PEPTIDE

BY R. DIMALINE AND G. J. DOCKRAY From the Physiological Laboratory, University of Liverpool, Liverpool

(Received 29 January 1979) SUMMARY

1. The actions of chicken and porcine secreting and vasoactive intestinal peptides

(VIPs) were compared on the rate of flow and rate of protein secretion from the exocrine pancreas in urethane anaesthetized turkeys and rats. 2. Chicken VIP was about twice as potent as porcine VIP and 100-150 times as potent as chicken and porcine secreting in stimulating the flow of pancreatic juice in the turkey. 3. Porcine secretin was a strong stimulant of the flow of pancreatic juice in the rat, but chicken secretin and the two VIPs were only active in doses 20-50 times higher than those of porcine secretin. 4. Neither the two VIPs nor the two secreting significantly stimulated the rate of pancreatic protein secretion in the turkey or rat. 5. In the turkey i.v. infusion of graded doses of chicken VIP produced graded increases in the flow of pancreatic juice; in the presence of an infusion of a low dose of CCK8 the flow of juice secreted in response to the highest dose of chicken VIP was significantly lower compared with the infusion of VIP alone, and responses to the other doses of VIP were lower but not significantly so. The infusion of chicken secretin reduced the flow of juice in response to infusions of chicken VIP, but the differences were not significant. There was no significant difference in either the rate of flow, or rate of protein secretion from the turkey pancreas in response to an infusion of chicken secretin and CCK8, compared with CCK8 alone. 6. The results cast doubt on the importance of secretin for regulation of the avian pancreas, and suggest instead that VIP might have a physiological role in regulating the flow of pancreatic juice in birds. INTRODUCTION

In mammals, the intestinal hormone secretin strongly stimulates the flow of bicarbonate rich pancreatic juice. Secretin is related in structure to several other peptides isolated from the gut or pancreas. Two of these, glucagon and gastric inhibitory polypeptide, do not stimulate the exocrine pancreas. However, a third, vasoactive intestinal peptide (VIP), has weak secretin-like actions on the mammalian pancreas. VIP was first isolated from hog duodenum by Said & Mutt (1972) and has been shown to share nine amino acid residues with porcine secretin when the two peptides are aligned from the NH2-terminus (Table 1) (Mutt & Said, 1974). In rat, 0022-3751/79/4800-0995 $01.50 © 1979 The Physiological Society

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ACTION OF VIP ON THE AVIAN PANCREAS 155 dog and man the maximum rate of flow of pancreatic juice evoked by VIP is 15-20% that evoked by secretin (Dockray, 1973; Konturek, Thor, Dembinski & Krol, 1975; Domschke, Domschke, Rosch, Konturek, Sprugel, Mitznegg, Wunsch & Demling, 1977). In the cat, the maximum rates of flow of pancreatic juice stimulated by secretin and VIP are similar, but VIP is about twenty times less potent than secretin on a molar basis (Konturek, Pucher & Radecki, 1976). VIP also has a wide spectrum of actions on other parts of the gastrointestinal tract, as well as on the cardiovascular and respiratory systems, and on metabolism (Said & Makhlouf, 1974); nevertheless, the physiological role of this molecule remains obscure. In birds like the chicken and turkey, porcine secretin has been found to be a poor stimulant of the flow of pancreatic juice, whereas porcine VIP was a strong stimulant (Angelucci, Baldieri & Linari, 1970; Dockray, 1973, 1975). Extracts of chicken duodenum contain a strong stimulant of the avian pancreas (Dockray, 1975), and it seems possible that the active factor in these extracts might resemble VIP more closely than mammalian secretin. Recently, peptides resembling porcine VIP and secretin have been isolated from chicken doudenum (Nilsson, 1974). Chicken VIP differs from its porcine counterpart in only four of twenty-eight residues (Table 1) (Nilsson, 1975); the structure of chicken secretin has not yet been reported, but it is thought to differ significantly from porcine secretin (V. Mutt, personal communication). Little is known of the physiological role of these molecules. In the present series of experiments we have compared the actions of chicken secretin and VIP with those of their mammalian counterparts on pancreatic secretion in the turkey and in the rat. It is well established that in cat and dog the actions of secretin on the pancreas are strongly potentiated by cholecystokinin (Brown, Harper & Scratcherd, 1967; Way & Grossman, 1970; Meyer, Spingola & Grossman, 1971). In addition, therefore, we have studied the interactions of avian secretin, VIP and the active COOH-terminal octapeptide of CCK (CCK8) on the turkey pancreas. The results indicate that both chicken and porcine VIP strongly stimulate the flow of juice from the turkey pancreas, whereas both porcine and chicken secreting have low potency. These results therefore raise the possibility that in birds VIP has a physiological role in the control of the pancreas which is analogous to that of secretin in mammals. A preliminary account of some of these results has already appeared (Dockray, 1976). METHODS

Peptides Highly purified preparations (+ 95 %) of VIP and secretin isolated from chicken and porcine duodenums were generous gifts of Dr V. Mutt. Synthetic CCK8 was a gift of the Squibb Institute for Medical Research. Stock solutions (100 ,ug/ml.) of secretin and VIP were prepared in 0- 16 Macetic acid and stored at -20 0C; CCK8 was stored at -20 0C in 005 M-ammonium bicarbonate. All peptides were diluted with lightly heparinized saline before injection.

Turkeys The action of peptides on the flow of pancreatic juice was studied in anaesthetized turkeys according to published methods (Dockray, 1975). Briefly, male birds (0F6-3 4 kg) were anaesthetized with urethane (1.5 g/kg, i.P.) and a carotid artery cannulated for continuous monitoring of arterial blood pressure via a Devices physiological pressure transducer and pen

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R. DIMALINE AND G. J. DOCKRA Y

recorder. One pancreatic duct was cannulated with polypropylene tubing and the other ligated. Pancreatic juice was collected in calibrated capillary tubes and the rate of flow noted every 5 min. The protein concentration of the juice was used as an index of enzyme secretion and was determined from absorption of diluted samples of juice at 280 nm and expressed relative to a standard of bovine serum albumin. After a basal period of 30 min peptides were administered via a wing vein either by rapid injection of volumes not exceeding 0-4 ml or by continuous infusions at 3.5 ml./hr.

Rats Male rats (0-2-0-3 kg) were anaesthetized with urethane (1.5 g/kg, i.P.). The common bile and pancreatic duct was ligated proximally and the pancreatic duct was cannulated near its entry into the duodenum as previously described (Dockray, 1972). The rates of flow and protein output from the pancreas were estimated using methods similar to those employed for the turkey. Pancreatic juice was collected for a basal period of 30 min before administration of peptides. The peptides were given by rapid injection into a jugular vein in volumes not exceeding 0-2 ml.

Experimental design (1) Relative potencies of secretin and VIP. Two groups of six turkeys were studied. One group received i.v. injections of three doses each of chicken VIP (0-05, 0-2, 0-8 jug/kg), chicken secretin (0-6, 2-5, 10 gg/kg) and porcine secretin (0-6, 2-5, 10 ,ug/kg). The second group received chicken VIP (0.05, 0*2, 0*8 #sg/kg) and porcine VIP (0-05, 0-2, 0.8 jug/kg). The responses to these stimuli were defined as the highest 5 min rate of flow and highest 10 min rate of protein secretion in the twenty minute period following the injection. Two groups of rats were studied. The first group of nine animals received rapid I.v. injections of chicken VIP (1.0, 2-0, 4 0 fig/kg), porcine secretin (0-025, 0 05, 0-1 ,sg/kg) and chicken secretin (0.4, 0*8, 1-6 fig/kg). The second group of four animals received porcine secretin in the same doses as the first group and chicken and porcine VIPs (1-0, 2-0, 4 0 #sg/kg). The pancreatic responses in the rat were defined as the highest ten minute rates of flow and protein secretion. In all groups of animals the order of doses was randomized and the relative potencies were calculated from analysis of variance. (2) Interactions of secretin, VIP and CCK8. Four groups of turkeys were used to study the following interactions: secretin-VIP, secretin-CCK8, VIP-CCK8. After a basal period of 30 min the first (control) group received an infusion of chicken VIP into a wing vein in five doses that doubled every 30 min from 0 05 to 08 fug/kg. hr; saline was infused into the other wing. The second and third groups of turkeys received the same doses of chicken VIP and infusions of either chicken secretin (2.5 fig/kgg. hr) or CCK8 (0-02 fig/kg. hr) into the other wing. The infusions of chicken secretin and CCK8 were started after a basal period of 30 min and the infusion of VIP was started after a further 30 min period. The fourth group of turkeys received an infusion of CCK8 (0.1 fIug/kg) for a period of 90 min and an infusion of either saline or chicken secretin (2-5 fig/kg) for the middle 30 min of the CCK8 infusion.

RESULTS

Relative potencies of secretin and VIP in the turkey. The mean basal rate of flow of 7-4 + 0-5 isl./g gland. 5 min (n = 36, mean + S.E. of mean), and basal protein output was 0-40 + 0-06 mg/g gland. 10 min (n = 36). Rapid i.v. injection of chicken and porcine secreting and VIPs produced a prompt increase in the flow of pancreatic juice which reached a peak after 5 min and returned to basal by 20-30 min. The lowest doses of chicken and porcine VIP studied (50 ng/kg) increased the basal rate of flow 2-3 times. The peak rate of flow was related to the dose for all peptides studied (Fig. 1); dose-response lines for chicken and porcine secreting were parallel to that for chicken VIP (Fig. 1). Doseresponse lines for chicken and porcine VIPs were also parallel (not shown). On a weight

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TABLE 2. Relative potencies (by weight) of chicken VIP, chicken secretin, porcine VIP and porcine secretin in stimulating the flow of pancreatic juice in the turkey and the rat

Relative potency Peptide

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Potency ratios calculated from analysis of variance relative to chicken VIP = 100. The molecular weights of chicken and porcine VIPs and porcine secretin are similar, the potency ratios shown here approximate to the molar potency ratios; the molecular weight of chicken secretin has not been published but is probably similar to that of porcine secretin. * In the rat, the dose-response lines for chicken VIP, porcine VIP and chicken secretin were parallel to each other, but not to that of porcine secretin, that a formal potency ratio could not be calculated for the latter peptide. so

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153 J. Phyeiol. (1979), 294, pp. 153-163 With 4 text-figure8 Printed in Great Britain POTENT STIMULATION OF THE AVIAN EXOCRINE PANCREAS BY PORCINE A...
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