higher bicarbonate and protein outputs than those induced by VIP or

J. Phyeiol. (1976), 255, pp. 497-509 497 With 9 text-ftgureg Printed in Great Britain COMPARSON OF VASOACTV NTSTNAL PPTD AND SCRTN N STMULATON OF PANCRATC SCRTON BY S. J. KONTURK, ANNA PUCHR AND TADUSZ RADCK From the nstitute of Physiology Medical Academy, Cracouw, Poland (Received 1 July 1975) SUMMARY 1. Pancreatic volume flow as well as bicarbonate and protein secretion have been measured in chronic pancreatic fistula cats and dogs in response to i.v. infusion of VP and secretin or duodenal perfusion of sodium oleate and HCl solution. 2. VP and secretin infused i.v. in cats produced superimposable pancreatic dose-response curves for volume flow and bicarbonate secretion, reaching almost identical observed and maximal calculated outputs with both peptides. n dogs, VP was shown previously to be a much less effective stimulant of pancreatic secretion than secretin and the maximal observed bicarbonate output in response to VP was only about 17 % of that to secretin (Konturek, Thor, Dembinski & Krol, 1975). t is concluded that VP in cats is a secretin-like full agonist, whereas in dogs it is a partial agonist of pancreatic bicarbonate secretion. 3. n cats, secretin and VP showed equal efficacy and their combination exhibited an augmentatory action on pancreatic bicarbonate secretion with additive kinetics, whereas in dogs, VP was found to have a lower efficacy than secretin and to inhibit competitively secretin-induced pancreatic secretion. These results might be explained by the interaction of VP and secretin, two chemically related peptides, on a common receptor site of the exocrine pancreas. 4. Caerulein, an analogue of CCK-P,, infused i.v. in cats and dogs caused a negligible pancreatic bicarbonate secretion and a potent dosedependent protein secretion. The combination of graded doses of VP or secretin with a background dose of caerulein resulted in significantly higher bicarbonate and protein outputs than those induced by VP or secretin alone. 5. Duodenal perfusion of sodium oleate soap in cats and dogs produced pancreatic dose-response curves for volume flow and bicarbonate output similar to those evoked by VP in these species. Pancreatic protein secretion in response to luminal oleate was slightly higher than could be

498 S. J. KONTURK AND OTHRS accounted for by the action of VP alone. This might be attributed to the release by oleate not only of endogenous VP but also CCK-P. or to the vago-vagal reflexes from gut to pancreas. The results of our combined study on cats and dogs suggest the possibility that oleate releases VP from the gut and that this peptide may play a physiological role in the stimulation of pancreatic secretion. NTRODUCTON Vasoactive intestinal peptide (VP) isolated from the small intestine of the hog by Said & Mutt (1972) and recently synthetized by Bodanszky, Klausner & Said (1973) resembles secretin and glucagon in its chemical structure. t exhibits multiple biological actions, some of which are comparable with those of these two peptides, while others are different. t has been reported that VP, like secretin, stimulates the pancreatic secretion of bicarbonate and augments the pancreatic response to cholecystokinin in the dog (Makhlouf, Said & Yan, 1974). The interaction of VP and secretin showed that VP may, as a partial agonist, at certain doses inhibit secretininduced pancreatic secretion, probably due to the competition for a common receptor site in the exocrine pancreas (Konturek et al. 1975). The physiological role of secretin is well established but the mechanism responsible for the release of VP has not yet been explored and its possible physiological significance as a hormone has been questioned on account of the marked inactivation in the liver (Said, 1974). The study now reported was an attempt to determine the release and the action of VP as compared with secretin on pancreatic secretion in chronic pancreatic fistula cats and dogs. MTHODS Six cats (2-5 kg) and four dogs (18-22 kg) were prepared with gastric and pancreatic fistulas. The cannulas used in the gastric fistulas in dogs were the type described by Thomas (1941); those in cats were the type described by mas (1960). n both the cannulas were inserted just orad to the pyloric gland area near the greater curvature. Pancreatic fistula was made by our modification of the method of Herrera, Kemp, Tsukamoto, Woodward & Dragstedt (1968). Special cannulas were prepared for dogs and for cats (Fig. 1). n both, the lateral limb of the cannula was inserted into the duodenal pouch containing the entrance of the major pancreatic duct, whereas the main limb was placed in the distal duodenum about 3 cm beyond the duodenoduodenostomy (Fig. 2). n cats, unlike dogs the common bile duct and major pancreatic duct join to form a single channel at least 1 cm before entering the duodenum through a common papilla. The common bile duct was divided and bile flow was diverted by transplanting the bile duct into the upper jejunum about 3 cm distally from the ligament of Treitz.

RLAS OF VASOACTJ V NTSTNAL PPTD 499 The secretary studies began about 3 months after surgery. Food was withheld from the cages at least 18 hr before each test. Throughout each test the gastric fistula was left open to allow drainage of gastric juice to the outside. i.,.-.> 1, 1# Fig. 1. Photograph of the pancreatic cannula and its components for the dog (above) and for the cat (below). The internal flanged end of the metal cannula is placed into the intestine and the lateral limb is put into the duodenal pouch. Additional components include a plastic protector keeping the cannula in the same position, a hollow obturator used for the perfusion of the intestine and a solid plastic plug sealing the cannula from the exterior between the secretary tests. Secretions from the pancreatic fistula were collected continuously and divided into 15 min samples. The volume was recorded to the nearest 01 ml. Bicarbonate concentration was measured by adding 0 5 ml. pancreatic juice to 1-0 ml. 041 M-HC1, boiling and back-titrating the residual HCl with 0 1 M-NaOH. Bicarbonate output was expressed as m-equiv/15 or 30 min. Protein concentration was estimated by measuring the absorbance at 280 nm in a Perkin-lmer spectrophotometer

500 S. J. KONTURK AND OTHRS (Norwalk, Conn.) with human serum albumin as standard. Protein output was expressed as mg 15 or 30 min. Several series of tests were performed on each animal. For comparison of the secretary potencies, VP or secretin (gifts from Professor V. Mutt and Dr S. Said of the Gastro-intestinal Hormone Research Unit, Karolinska nstitutet, Stockholm, Sweden) was infused i.v. in graded doses in 1-day tests. The dose level was changed every 60 min and differed by a factor of 2. Fig. 2. Scheme of a pancreatic fistula using the metal eannula shown in Fig. 1. When the cannula is closed (A), the pancreatic juice flows to the duodenum. When the cannula is opened and the hollow obturator is inserted (B), the pancreatic juice flows to the outside and can be collected into the calibrated test tubes. The interaction of VP or secretin with caerulein was studied by adding VP or secretin in graded doses to a standard dose of caerulein (a gift from Dr M. Ghione of Farmitalia, Milano, taly) infused i.v. 60 min before and throughout VP or secretin administration. The dose level of VP or secretin was changed every 60 min. The interaction of V1P with secretin was examined by injecting VP in a constant dose against varying background doses of secretin infused.v. Sodium oleate and hydrochloric acid were instilled into the duodenum as stimulants for pancreatic secretion. Sodium oleate was prepared by adding to oleic acid a sufficient amount of NaOH (about 80 % of that required for complete neutralization) to bring the final ph of the solution to ph 9*4, required for dispersion (Hofman & Small, 1967). A solution of 20 % NaCl was added as required to achieve an osmolarity of about 300 m-osmole/l. Osmolarity was measured by freezing point depression with

RLAS OF VASOACTV NTSTNAL PPTD 501 a Fiske osmometer. The test solutions were infused at graded rates into the duodenum through the hollow obturator of the main limb of the pancreatic fistula. ach test substance was studied on a separate day and each dose level was administered for 1 hr. The responses to each stimulant were compared using the t test for paired values. Differences were regarded as significant if P < 0-05. The calculated maximal response (CMR) and dose required for one half maximal response (D50) for bicarbonate secretion was calculated by the application of the linear transformation of the Michealis-Menten equation recommended by Dowd & Riggs (1965). RSULTS Comparison of pancreatic volume and bicarbonate output in response to VP and secretin. VP and secretin infused i.v. at a given dose produced the peak volume and bicarbonate secretion in the second 15 min period and these values were used to express the results. Graded doses of VP or secretin in cats evoked graded increases in pancreatic secretion attaining the highest level at the doses of 16,ug/kg. hr and 4 u./kg. hr respectively. Further increase in the dosage of VP or secretin did not result in any additional increase in the volume and bicarbonate output and these results are not presented. The mean + s.. of mean observed maximal bicarbonate outputs with VP and secretin were 1x52 + 0 09 and 156 + 0 11 m-equiv/30 min respectively. The dose-response curves with VP and secretin were superimposable. The mean volume flows in response to VP and secretin were similar (Fig. 3). Mean (±+.. of mean) CMRs for VP and secretin were 1-82 + 0'25 and 1-78 + 0*16 m-equiv/30 min respectively. The efficacy of VP based on the observed maximal bicarbonate response was 0 97 and based on CMRs was 1 0. The mean (+±.. of mean) D50 for VP and secretin was 4 95 + 1-27,#g/kg.hr and 0 95 + 0 21 u./kg.hr respectively. nteraction of VP and secretin with caerulein. VP combined with a background dose of caerulein (0-2 clg/kg. hr) produced in cats a dose-related increase in pancreatic volume flow and bicarbonate secretion reaching the highest observed output at a dose of 16,sg/kg. hr (Fig. 4). The dose-response curve for bicarbonate to the combination of VP plus caerulein was significantly above that to VP alone (P < 0.01). Similarly, the secretary response to secretin combined with caerulein (0 2 sg/kg. hr) or with VP (4 /,g/kg. hr) was significantly higher than to secretin alone, particularly at lower dose levels. n tests with a combination of VP or secretin with caerulein there was a shift in the left of the dose-response curve to VP and secretin. The highest observed bicarbonate responses to the combined agents were greater than the summed responses. Caerulein alone evoked negligible pancreatic bicarbonate secretion and these data are not presented.

502 2-0 -r20 S. J. KONTURK AND OTHRS 0 a 0 U 1-6 +16 1-2 +12 0-8 + 8 04 + 4 0 Secretin 0*25 0*5 1-0 2-0 4-0 u./kg. hr VP 1-0 2-0 4-0 8*0 16 0 ug/kg. hr Fig. 3. The pancreatic volume flow and bicarbonate outputs in response to graded doses of VP or secretin in cats. n this and subsequent Figures the vertical bars represent the s.. of the means. 2 0 r Basal 1-6 _ Secretin +caerulein C 0 U 12_- 08 _ 0-4 _- VP alone -Secretin alone Basal 0.25 20 4 05 1.0 2-0 4-0 Secretin u./kg. hr 1-0 20 40 80 160 VP ug/kg. hr Fig. 4. The pancreatic bicarbonate outputs in response to graded doses of VP or secretin alone and to graded doses of VP or secretin combined with a background dose of caerulein (0-2,g/kg. hr) as well as to graded doses of secretin combined with VP (4 #g/kg.hr) in cats. ach point is a mean (± s.:. of mean) of twelve experiments in Six cats.

RLAS OF VASOACTV NTSTNAL PPTD 503 1-6 T160 i.2 0 ~O08 04 120.80 4-0 Basal Volume Na oleate HCO3 l 0 25 0.5 1.0 2-0 40 HC m-equiv/hr 0 5 1.0 2 0 4-0 8-0 Na oleate m-mole/hr Fig. 5. The pancreatic volume flow and bicarbonate outputs in response to duodenal perfusion of sodium oleate and HCl in cats. ach point is a mean (± 8.. of mean) of twelve experiments in six cats. 6 T 60 5 C._ So 4 o U 3 50 40 C._ 30 Q CT 2 20 10 i' Basal 1*0 0-5 Naoleate 2-0 4.0 HC m-equiv/hr 8-0 1-0 2-0 4-0 Na oleate m-mole/hr -4 16-0 Fig. 6. The pancreatic volume flow and bicarbonate outputs in response to duodenal perfusion of sodium oleate and hydrochloric acid in dogs. ach point is a mean (+ s.. of mean) of eight experiments in four dogs. -4 8-0

504 S. J. KONTURK AND OTHRS Comparison of pancreatic volume and bicarbonate outputs in response to duodenal perfusion of oleate and HCl. Sodium oleate and HCl solution instilled into the duodenum in cats produced similar pancreatic dose-response curves for volume flow and bicarbonate output, which reached, respectively, about 80 and 70 % of the maximal observed response to exogenous VP or secretin (Fig. 5). Duodenal perfusion of oleate in dogs (Fig. 6) also stimulated pancreatic secretion in a dose-related manner. Bicarbonate secretion at lower loads of oleate was negligible but it started to increase at 4 0 mm oleate. The highest maximal bicarbonate response to oleate was reached at 8-0 mm oleate and it was only about 38 % of that reached with exogenous secretin. The maximal response to oleate was not attained because bicarbonate outputs continued to rise as the rate was increased to 8-0 mm and side-effects such as retching prevented the infusion of larger loads of oleate. Duodenal perfusion of HCl in dogs (Fig. 6) resulted in a stepwise increase in pancreatic volume flow and bicarbonate output. The highest 600 500 Caerulein 400 0 S ~~~~~~HC 100 Na oleate VP Secretin Basal 0-2 0 4 0-8 16 3 2 Caerulein jig/kg. hr ( J 0-5 1-0 2-0 4-0 8-0 Na oleate rn-mole/hr l 1-0 2-0 4 0 8-0 16-0 VP jug/kg. hr 0 25 0-5 1-0 2-0 40 HC m-equiv/hr 0-25 0.5 1-0 2-0 40 Secretin u./kg. hr Fig. 7. The pancreatic protein outputs in response to graded doses of caerulein, and in experiments as in Figs. 3 and 5.

RLAS OF VASOACT V NTSTNAL PPTD 505 observed pancreatic response produced by duodenal acidification was similar to that attained with exogenous secretin in the same animals. Comparison of pancreatic outputs in response to VP, secretin, oleate, HCl and caerulein. Caerulein in cats caused a potent and dose-dependent stimulation of pancreatic protein secretion. Oleate and HCl were moderate stimulants of protein secretion reaching a maximum of about 45 % of that attained by caerulein. VP and secretin did not produce significant increase in protein secretion (Fig. 7). The combination of VP or secretin with the background dose of caerulein resulted in a significantly higher protein output than VP or secretin alone (Fig. 8). 400 e300 VP+caerulein > (0O2 pg/kg) Secretin+caerulein 20 r n(0-2 pg/kg) Secretin ln 100 VP+secretin - VP alone 0 (4 pg/kg. hr) Basal 0-25 0-5 1.0 2-0 40 Secretin u./kg. hr 1-0 2*0 4*0 80 16.0 VP pg/kg. hr Fig. 8. The pancreatic protein outputs in experiments as in Fig. 4. Pancreatic protein responses to i.v. VP, secretin, caerulein, duodenal oleate and HCl in dogs are presented in Fig. 9. Caerulein was the most potent stimulus of protein secretion and resulted in a dose-dependent increase in protein output. The highest observed protein response to caerulein was reached at a dose of 0 5 #ug/kg. hr and amounted to about 800 mg protein/30 min. Oleate and hydrochloric acid were less effective stimulants of protein secretion reaching, respectively, about 87 and 63 % of that induced by caerulein. Both VP and secretin caused a slight and dose-dependent increase in protein secretion. DSCUSSON This study demonstrates that duodenal perfusion of sodium oleate in cats is as potent stimulus of pancreatic secretion as duodenal perfusion of

506 S. J. KONTURK AND OTHRS HCl and that intravenous VP in this species is as effective as secretin in the stimulation of pancreatic bicarbonate secretion. Sodium oleate soap instilled into the gut of the cat evokes a copious flow of pancreatic juice with high bicarbonate and moderate protein output. Pancreatic juice stimulated by luminal oleate very closely resembles that secreted in response to perfused HCl. With exogenous VP or secretin the pancreatic bicarbonate secretion in the cat was similar to that induced by luminal oleate or HCl, respectively. 900 700 C Caerulein 100 _ Basal 003 006 012 025 05 Caerulein,g/kg.ihr a 05 1.0 20 40 8-0 Na oleate m-mole/hr 1-0 2-0 4-0 8-0 16-0 VP ug/kg. hr 1.0 2-0 4.0 80 16.0 HC m-equiv/hr 0-25 0-5 1 0 2-0 40 Secretin u./kg. h r Fig. 9. The pancreatic protein outputs in response to graded doses of caerulein and in experiments as in Fig. 6. The stimulatory effect of fatty acids on both pancreatic bicarbonate and protein secretion was previously reported in dogs (Meyer & Jones, 1974). t was suggested that in dogs the bicarbonate-stimulating mechanism in the gut may be dissociated in part from the protein-stimulating mechanism and that fatty acids may release both secretin and CCK-P. simultaneously in different amounts depending on chain length and fatty acid loads. n our present study, increasing loads of luminal oleate in dogs were found to produce increasing outputs of pancreatic protein secretion, whereas pancreatic bicarbonate outputs were negligible at lower and started to increase

RLAS OF VASOACTV NTSTNAL PPTD 507 at higher loads. Thus our study fully confirms the previous observation and is consistent with the hypothesis (Meyer & Jones, 1974) that oleate at higher loads stimulates more bicarbonate than can be accounted for by the release of endogenous CCK-P. and that it may be attributed at least in part to the release of small amounts of secretin. However, recent study (Boden, ssa & Owen, 1975) in which secretin was measured directly by a radioimmunoassay failed to detect any change in portal or femoral venous concentration of secretin after intraduodenal infusion of oleate. Another plausible alternative might be that oleate in contact with the intestinal mucosa releases another hormone which has a secretin-like action on the pancreas but which is a less effective stimulant of pancreatic bicarbonate than secretin. f the term efficacy (of which the maximal observed or maximal calculated response is an index) is used to describe the effectiveness of an agonist-receptor complex (Goldstein, Aronow & Kalman, 1969), the hormone released by luminal oleate in the dog should have a lower efficacy than that released by duodenal acidification or than exogenous secretin. The best candidate fulfilling this requirement is VP, which in the dog is a partial, secretin-like agonist of pancreatic secretion (Makhlouf et al. 1974). The maximal observed rate of bicarbonate secretion attained with VP alone is only about 17 % of that attained with secretin. The bicarbonate response to luminal oleate slightly higher than that to exogenous VP might be explained by the concurrent release of endogenous (CK-P. by oleate. This is supported by two findings; first, the protein output in response to oleate is much higher than could be accounted for by the release of VP alone, and secondly, the pancreatic bicarbonate response to VP combined with a background dose of caerulein is close to that evoked by luminal oleate. Thus the stimulation of pancreatic bicarbonate secretion by oleate in the dog might be attributed to the release and interaction of VP and CCK-Pz. Further support for the concept that luminal oleate may release VP comes from the study on the effects of VP and oleate on pancreatic secretion in cats. The action of VP on electrolyte and water secretion in anaesthetized cats has been previously described by Said & Mutt (1972) who found that VP is a partial agonist of pancreatic bicarbonate secretion. n our present study performed on conscious cats, VP was found to be a potent stimulant of pancreatic bicarbonate secretion. The dose-response curves for bicarbonate and protein outputs to VP and secretin in conscious cats are superimposable and the CMRs to both of these peptides are almost identical. We conclude that VP in conscious cats has an efficacy as high as that of secretin, and therefore it is a full agonist for pancreatic bicarbonate secretion in this species. Because of this equal efficacy VP was not expected to inhibit secretin-induced pancreatic bicarbonate secretion in

508 S. J. KONTURK AND OTHRS cats as it did in dogs (Konturek et al. 1975). n fact it was found that the combination of VP with varying background doses of secretin in cats evoked a highly augmented pancreatic bicarbonate secretion, particularly at lower dose levels, and an unchanged maximal observed response. These results on the interaction of VP and secretin remain in full agreement with the hypothesis of digestive hormone interaction proposed by Grossman (1970). Since VP and secretin are chemically related, they are assumed to act on the same receptor sites of the bicarbonate-producing cells of the pancreas in both the cat and dog. n cats VP and secretin have equal efficacy so their combined action is augmentatory with additive kinetics. n dogs, in which the efficacy of VP and secretin is different, VP inhibits the action of secretin on bicarbonate secretion and the kinetics of this inhibition are competitive (Grossman, 1970). f oleate causes the release of VP from the gut, the pancreatic bicarbonate response to this substance should be similar to that induced by exogenous VP. As expected, the dose-response curve to luminal oleate in cats is similar to that evoked by exogenous VP or secretin. Although the bicarbonate response to luminal oleate closely resembles that to VP alone, the protein response is slightly higher than can be accounted for by the release of VP alone. This may be explained by the fact that in cats, as in dogs, luminal oleate is capable of releasing not only VP but also CCK-Pz and the pancreatic secretion results from the interaction of these two hormones. This assumption is supported by the finding of this study that caerulein is capable of augmenting the stimulation of protein secretion by VP. Another possible explanation of these data would be that luminal oleate not only releases the intestinal hormones but also activates the vago-vagal reflexes from gut to pancreas. While this is a remote theoretical possibility, the role of nervous reflex in the pancreatic response to luminal oleate cannot be excluded (Debas, Konturek & Grossman, 1975). No study has yet been made to facilitate the choice between these two possibilities, and perhaps an accurate radioimmunoassay ofthe intestinal hormones may be helpful in this matter. Our results of the combined study on cats and dogs provide strong support for the concept that oleate releases VP and that the difference in pancreatic secretion is due to the species-dependent pancreatic responsiveness to this peptide. The authors are indebted to Dr Morton. Grossman for his criticism in the preparation of the manuscript.

RLAS OF VASOACTV NTSTNAL PPTD 509 RFRNCS BODANSZKY, M., KLAusNR, Y. S. & SAD, S.. (1973). Biological activities of synthetic peptides corresponding to fragments of and to the entire sequence of the vasoactive intestinal peptide. Proc. natn. Acad. Sci. U.S.A. 70, 382-383. BODN, G., SSA, N. & OwN, 0.. (1975). ffects of intraduodenal amino acids, fatty acids and sugars on secretin concentrations. Gastroenterology 68, 722-727. DBAs, H. T., KoNTuRK, S. J. & GROSSMAN, M.. (1975). ffect of extragastric and truncal vagotomy on pancreatic secretion in the dog. Am. J. Physiol. 228, 1172-1177. DowDi, J.. & RGGS, D. S. (1965). A comparison of estimates of Michaclis-Menten kinetic constants from various linear transformations. J. biol. Chem. 240, 863-869. mas, S. (1960). Gastric secretary responses to repeated intravenous infusions of histamine and gastrin in non-ane-sthetized gastric fistula cats. Gastroenterology 39, 771-782. GOLDSTN, A., ARoNow, L. & KiAS. M. (1969). n Principles of Drug Action, p. 94. New York: Harper and Row. GROsSMS&i, M.. (1970). Gastrin, cholecystokinin and secretin act on one receptor. Lancet i, 1088-1089. HRRRA, F., KLMP, D. R., TSUKAMOTO, M., WOODWARD,. R. & DRAGSTDT, L. R. (1968). A new cannula for the study of pancreatic function. J. appi. Physiol. 25, 207-209. HoFMAN, A. F. & SMALL, C. M. (1967). Detergent properties of bile Salts: correlation with physiological function. A. Rev. Med. 18, 333-376. KONTURK, S. J., THOR, P., DMBNSK, A. & CRL, R. (1975). Comparison of secretin and vasoactive intestinal peptide on pancreatic secretion in dogs. Gastro. enterology 68, 1527-1535. M~muouF, G. M., SAD, S.. & YAN, W. M. (1974). nterplay of vasoactive intestinal peptide (VP) and synthetic VP fragments with'secwretin and octapeptide of cholecystokinin (OCTA-CCK) on pancreatic and biliary secretion. Gastro. enterology 66, 737. MYR, J. H. & JoNs, R. S. (1974). Canine pancreatic responses to intestinally perfused fat and products of fat digestion. Am J. Physiol. 226, 1178-1187. SAD, S.. (1974). Vasoactive intestinal peptide (VP). Gastroenterology 67, 735-737. SAD, S.. & MUrr, V. (1972). solation from porcine intestinal wall of a vasoactive octacosapeptide related to secretin and glucagon. ur. J. Biochem. 28, 199-204. THOMAS, J.. (1941). An improved cannula for gastric and intestinal fistulas. Proc. Soc. exp. Biol. Med. 46, 260-261.