Proglumide and Benzotript: Members of a Different Class of Cholecystokinin Receptor Antagonists (Pancreatic Secretagogues/Amylase Secretion/Gastrin/Caerulein) W

Proc. NatL Acad. Sci. USA Vol. 78, No. 10, pp. 6304-6308, October 1981 Cell Biology

Proglumide and benzotript: Members of a different class of cholecystokinin receptor antagonists (pancreatic secretagogues/amylase secretion/gastrin/caerulein) W. F. HAHNE, R. T. JENSEN*, G. F. LEMP, AND J. D. GARDNER Digestive Diseases Branch, National Institute ofArthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20205 Communicated by Donald S. Fredrickson, July 7, 1981

ABSTRACT In dispersed acini from.guinea pigpancreas, pro- ilar to those of proglumide (2), to alter the actions of CCK as glumide (DL-4-benzamido-N, N-dipropylglutaramic acid) and ben- well as other secretagogues on dispersed acini from guinea pig zotript (N-p-chlorobenzoyl-L-tryptophan) caused a rightward shift pancreas. In addition, we compared the actions ofproglumide in the dose-response curve forcholecystokinin-stimulated amylase and benzotript to those of Bt2cGMP, a compound that we (13, secretion but did not alter the maximal increase in amylase se- 16) and others (15-19) have found to be a specific CCK receptor cretion caused by cholecystoldnin.. At relatively low, concentrations, proglumide did not alter the stimulation ofenzyme secretion antagonist. caused by secretagogues whose effects are mediated by adenosine MATERIALS AND METHODS 3'5'-monophosphate (e.g., vasoactive intestinal peptide or secretin) and did not alter the stimulation of enzyme secretion caused Materials. Male guinea pigs (175-225 g) were obtained from by secretagogues that have a mode ofaction similar to that ofcho- the Small Animals Section, Veterinary Resources Branch, Na- lecystokinin but act through different receptors (e.g., bombesin, tional Institutes of Health. Hepes was obtained from Boehrin- physalaemin, eledoisin, and ionophore A23187). There was a close ger Mannheim; purified collagenase (type CLSPA) was from correlation between the ability ofproglumide or benzotript to in- Worthington; carbamoylcholine (carbachol), atropine sulfate, hibit binding of '25I-labeled cholecystokinin to its receptors on 8-bromoadenosine 3',5'-monophosphate (8Br-cAMP), Bt2cGMP, pancreatic acini and the abilities ofthese compounds to inhibit the and soybean trypsin inhibitor were from Sigma; 125I-labeled N- action ofcholecystokinin on enzyme secretion and on calcium out- succinimidyl-3-(4-hydroxyphenyl)propionate (1500 Ci/mmol; flux. These results indicate that proglumide and benzotript are 1 Ci = 3.7 x 10' becquerels) was from Amersham/Searle; members ofa different class ofcholecystokinin receptor antagonists. Eagle's basal amino acid medium (100 times concentrated) was from GIBCO; essential vitamin mixture (100 times concen- Proglumide (Fig. 1) is a derivative of glutaramic acid that has trated) was from Microbiological Associates (Bethesda, MD); been used for approximately 10 years in Europe and Japan to glutamine and synthetic human gastrin I-(2-17) fragment were treat patients with peptic ulcers.(1, 2). The claimed effective- from Research Plus Laboratories (Denville, NJ); bovine plasma ness of proglumide has been attributed to its ability to reduce albumin (fraction V) was from Armour Pharmaceutical (Phoenix, secretion ofgastric acid (1, 2); however, the mechanism ofaction AZ); Phadebas amylase test and Sephadex G-50 (superfine) were ofproglumide has not been established. Some studies have con- from Pharmacia; 'Ca .(12.5 mCi/mg) was from New England cluded that proglumide inhibits only gastrin-stimulated acid Nuclear; and synthetic bombesin, physalaemin, eledoisin, and secretion and does not have anticholinergic or antihistamine vasoactive intestinal peptide (VIP) were from Peninsula Labo- activity (2-5), whereas other studies report that proglumide can ratories (Belmont, CA). Ionophore A23187 was a gift from Rob- inhibit the stimulation of acid secretion caused by gastrin, his- ert Hamill (Eli Lilly). Natural porcine CCK (greater than 99% tamine, carbamoylcholine, and insulin (3, 6-8). Proglumide has pure) and natural porcine secretin were gifts from Viktor Mutt been reported to inhibit gastrin-stimulated acid secretion com- (Gastrointestinal Hormone Research Unit, Karolinska Institu- petitively (3) as well as noncompetitively (5, 9) and to inhibit tet, Stockholm, Sweden). Synthetic COOH-terminal octapep- binding of radiolabeled gastrin to a heterogeneous mixture of tide of CCK (CCK-8) was a gift from Miguel Ondetti (Squibb gastric mucosal cellst as well as to a crude preparation ofgastric Institute for Medical Research, Princeton, NJ). Caerulein was mucosal membranes (10). Proglumide has also been reported a gift from Roberto de Castiglione (Farmatalia, Milan, Italy). to alter the action ofcaerulein on gallbladder muscle and gastric Proglumide and benzotript were gifts from Rotta Laboratories pyloric muscle, gastric acid secretion, and pancreatic fluid se- (Milan, Italy). cretion (11). Unless stated otherwise, the standard incubation solution Because gastrin, cholecystokinin (CCK), and caerulein have contained 24.5 mM Hepes (pH 7.4), 98 mM NaCl, 6 mM KCl, a common COOH-terminal pentapeptide amide (12) and exert 2.5 mM NaH2PO4, 5 mM sodium pyruvate, 5 mM sodium fu- their effects on a particular target tissue by interacting with the marate, 5 mM sodium glutamate, 2 mM glutamine, 11.5 mM same class of receptors (13-15), and because proglumide has glucose, 0.5 mM CaCl2, 1.0 mM MgCl2, 5 mM theophylline, been reported to inhibit the actions ofboth gastrin and caerulein 1% albumin, 0.01% trypsin inhibitor, 1% essential amino acid (3, 5, 11), we considered the possibility that proglumide is a mixture, and 1% essential vitamin mixture. The incubation so- specific antagonist of the interaction of CCK and structurally related peptides with their cell surface receptors. To examine Abbreviations: CCK, cholecystokinin; CCK-7 and CCK-8, synthetic COOH-terminal hepta- and octapeptides of CCK; VIP, vasoactive in- this possibility we tested the abilities of proglumide and ben- testinal peptide; Bt2cGMP, N2,O'-dibutyrylguanosine 3',5'-mono- zotript (Fig. 1), a derivative oftryptophan that has actions sim- phosphate; 8Br-cAMP, 8-bromoadenosine 3',5'-monophosphate. * To whom reprint requests should be addressed. The publication costs ofthis article were defrayed in part by page charge t Magous, R. & Bali, J. P., "Proglumide-Gastrin Receptor Antago- payment. This article must therefore be hereby marked "advertise- nist," Fourth International Symposium ofthe European Congress of mnent" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Gastrointestinal Endoscopy, Hamburg, June, 1980, p; 7 (abstr.). 6304 Downloaded by guest on September 28, 2021 Cell Biology: Hahne et aL Proc. Natd Acad. Sci. USA 78 (1981) 6305

0 H I OH 0 0 CH2-C-C--OH | H I ACH2-CH2-CH3 I HOC-CH2-CH2 -C--" NH I C C I N K33N N y NHCNH C2 C2 H3 I H C O C-'O CH2 CIH2 CI Proglulmide Benzotript CH3

OSO3H / Bt2cGMP CH3 - CH3

XCH2 =- CH2 COOH CH2 CH2 H CH2 CH2 CH2 ICH2 -NHCHCO-NHCHCO-NHCHCO-NHCHCO-NHCHCO-NHCHCO-NHCHCO-NH2

- Tyr - Met - Gly - Trp - Met - Asp - Phe -NH2 SO3H CCK-7

FIG. 1. Structures of N2,02-dibutyrylguanosine 3',5'-monophosphate (Bt2cGMP), proglumide (DL-4-benzamido-NN-dipropylglutaramic acid), benzotript (N-p-chlorobenzoyl-L-tryptophan), and the COOH-terminal heptapeptide of CCK (CCK-7).

lution was equilibrated with 100% 02 and all incubations were arations of125I-CCK used in the present studies were 900-1300 performed with 100% 02 as the gas phase. Ci/mmol. Acini from the pancreas of one animal were sus- Methods. Dispersed acini from guinea pig pancreas were pended in 10 ml ofstandard incubation solution and incubated prepared by using the published procedure (20) with three with the appropriate agents plus 50 pM 125I-CCK (approxi- minor modifications: (i) The digestion solution contained col- mately 70,000 cpm/ml of cell suspension) for 30 min at 37°C. lagenase at a concentration of 0.12 mg/ml. (ii) After digestion Nonsaturable binding of '25I-CCK was determined as the solution had been injected into the pancreas, the tissue was in- amount of radioactivity associated with the acini when the incubated with 5 ml of digestion solution for four sequential 10- cubation contained 0.1 A1M CCK-8 and was always less than 20% min periods instead of one 15-min period followed by two 10- ofthe total binding. In this paper all values for bound 125I-CCK min periods. (iii) The tissue was washed with standard incu- represent saturable binding and were calculated as total binding bation solution only after the third. 10-min digestion period in- minus nonsaturable binding. stead of after each digestion period. Amylase release was measured by using the reported pro- RESULTS cedure (20, 21). Acini from the pancreas of one animal were In acini incubated with increasing concentrations of CCK-8, suspended in 150 ml of standard incubation solution and sam- amylase secretion increased, became maximal with 0.3 nM ples (1.0 ml) were incubated with the appropriate agents for 30 CCK-8, and then decreased at peptide concentrations above 0.3 min at 37°C. Amylase activity was determined by the method nM (Fig. 2). Bt~cGMP (Fig. 2 Left) and proglumide (Fig. 2 of Ceska et al. (22, 23), using the Phadebas reagent. Amylase Right) caused a rightward shift in the dose-response curve for release was calculated as the percentage ofthe amylase activity CGK-8-stimulated amylase secretion, and the magnitude ofthe in the acini at the beginning ofthe incubation that was released shift was proportional to the concentration ofBt2cGMP or pro- into the extracellular medium during the incubation. glumide. Results similar to those illustrated in Fig. 2 with Outflux of 'Ca was determined by using the described pro- Bt2cGMP or proglumide were also obtained with benzotript cedure (24). Acini from the pancreas of one animal were sus- (not shown). pended in 10 ml of standard incubation solution and, after the When acini were incubated with a fixed concentration of acini were loaded with ',Ca, they were washed and incubated CCK-8, the pattern of action of Bt2cGMP or proglumide de- with the appropriate agents for 5 min at 37C. Outflux of 45Ca pended on the concentration of CCK-8 (Fig. 3). With concen- was calculated as the percentage of the 'Ca in the acini at the trations ofCCK-8 that were submaximal or maximal for causing beginning of the 5-min incubation that was released into the amylase secretion, increasing concentrations of Bt2cGMP or extracellular-medium during the incubation. proglumide produced a progressive decrease in amylase secre- Binding of "MI-labeled CCK ('25I-CCK) to pancreatic acini tion, and each antagonist was more potent at the lower con- was determined as described (13). '25I-CCK was prepared by centration of CCK-8 (Fig. 3). With a concentration of CCK-8 using the published modifications (13) ofthe procedure ofBol- that was supramaximal for causing amylase secretion, adding ton and Hunter (25). The specific activities ofthe various prep- increasing concentrations of Bt2cGMP or proglumide caused Downloaded by guest on September 28, 2021 6306 Cell Biology: Hahne et aL Proc. Nad Acad. Sci. USA -78 (1981)

Table 1. Effect of Bt2cGMP and proglumide on the stimulation of amylase release caused by various pancreatic secrebgogues

42 0.3 mM 1 mm Amylase release, % total -2 mM 0mm,10mogM Bt2cGMP Proglumide ~306 Secretagogue Alone (1 mM) (10 mMW) ± ± a) ~~~~~~~~3.0 None 4.9 1.7 4.9 + 1.6 5.0 1.6 ± G)24- ~~~mm CCK-8 (0.3 nM) 31.9 ± 9.6 4.9 ± 0.8* 5.7 0.8* Caerulein (01 nM) 32.3 ± 10 4.9 ± 0.3* 5.4 ± 0.9* 18- Gastrin I-(=17) (1 ,uM) 35.0 ± 3.6 6.2 ± 3.6* 4.9 ± 0.6* Physalaemin (10 nM) 12.6 ± 2.6 12.7 ± 0.6 12.8 ± 0.6 <12 Eledoisin (1 pM) 12.6 ± 2.6 13.1 ± 1.1 12.2 ± 1.3 6 Bombesin (10 nM) 30.4 + 5.1 32.4 ± 5.1 29.6 ± 6.4 A23187 (5 pM) 11.2 ± 2.5 11.5 ± 1.5 12.2 ± 0.3 -11 -10 -9 -8 -7 -6 -11 -10 -9 -8 -7 -6 Carbachol (10 jAM) 25.0 ± 4.1- 25.2 ± 3.2 25.9 ± 2.1 CCK-8, log M VIP (10 nM) 23.8 ± 4.6 23.6 ± 1.7 25.3 ± 2.6 Secretin (0.3 pM) 24.1 ± 5.7 25A ± 3.8 24.3 ± 2.0 FIG. 2. Effect of Bt2cGMP (Left) and proglumide (Right) on CCK- 8Br-cAMP (1 mM) 24.6 ± 3.3 23.0 ± 2.0 23.0 ± 1.4 8-stimulated amylase release from pancreatic acini. Acini were incu- Acini were incubated for 30 min at 370C with various pancreatic bated for 30 min at 370C with various concentrations of CCK-8 plus secretagogues alone or in the presence of Bt2cGMP or proglumide. In the indicated concentrations of Bt2cGMP or proglumide. In each ex- each experiment each value was determined in duplicate and results periment each value was determined in duplicate, and the results given are means ± SD from three separate experiments. given are means from eight separate experiments. * Significantly different (P < 0.01) from the value obtained with the secretagogues alone by Student's paired t test. amylase secretion to increase, become maximal, and, in the case of Bt2cGMP, then decrease (Fig. 3). lase secretion caused by 1 nM CCK. In terms of the abilities Table 1 illustrates that Bt2cGMP and proglumide inhibited of these compounds to antagonize these actions of CCK, the the increase in enzyme secretion caused by secretagogues that potency of proglumide was similar to that of benzotript (half- interact with CCK receptors on pancreatic acini (i.e., CCK-8, maximal inhibition at approximately 500 ,uM; Fig. 4), and each caerulein, and gastrin) but did not alter basal enzyme secretion of these antagonists was approximately one-fifth as potent as or the increase in enzyme secretion caused by-secretagogues Bt2cGMP (half-maximal inhibition at approximately 100 ,(iM;, that do not interact .with CCK receptors (i.e., physalaemin, Fig. 4). eledoisin, bombesin, A23187, carbachol, VIP, secretin, or 8Br- In pancreatic acini that have been incubated with relatively cAMP). high concentrations of CCK-8, washed, and then reincubated Previously we have shown that there is a close correlation in fresh incubation solution containing no CCK-8, there is sig- between the abilities of CCK and structurally related peptides nificant residual stimulation of amylase secretion (26). Fur- to inhibit binding of 125I-CCK to pancreatic acini and their thermore, Bt2cGMP can prevent as well as reverse this CCK- abilities to cause mobilization of cellular calcium and to stim- 8-induced residual stimulation (26, 27). As illustrated in Fig. ulate enzyme secretion (13). As illustrated in Fig. 4, there was 5, we found thatproglumide and benzotript can also reverse the a close correlation between the abilities ofBt2cGMP, proglum- residual stimulation ofenzyme secretion caused by CCK-8. The ide, and benzotript to inhibit binding of 1 nM 125I-CCK and dose-response curves for the inhibition caused by proglumide their abilities to inhibit the;increase in calcium outflux and amy-

32 0.3 nM 3O'nM03M03n 28 24 iOnM

1212 8~~~~~~~~~0.05.OnM nM 2-5 -4 -3 -2 -' 4 A OnM OM Antagonist, log M -5 4 -3 -2 -5 -4 -3 -2 FIG. 4. Abilities of BticGMP, proglumide, and benzotript to in- Bt2cGMP, log M Proglumide, log M hibitbindingof"25I-CCK (*), CCK-stimulatedoutflux of 45Ca (-), and CCK-stimulated amylase secretion (A). All experiments were per- FIG. 3. Effect of Bt2cGMP (Left) and proglumide (Right) on CCK- formed with incubation mixtures that contained 1 nM CCK plus the 8-stimulated amylase release from pancreatic acini. Acini were incu- indicated concentrations ofthe antagonist. Values are expressed asthe bated for 30 min at 370C with various concentrations of Bt2cGMP or percent of the value obtained with no added antagonist. Results for proglumide plus the indicated concentrations of CCK-8. In each ex- binding of 1'2I-CCK are means of eight separate experiments, those periment each value was determined in duplicate andthe results given for outflux of 'Ca are means of five separate experiments, and those are means from four separate experiments. for amylase release are means of four separate experiments. Downloaded by guest on September 28, 2021 Cell Biology: Hahne et al Proc. Natd Acad. Sci. USA 78 (1981) 6307 DISCUSSION

80 In general, the structural requirements for occupation ofa peptide hormone receptor are quite stringent in that only other peptides will bind to the hormone receptor, and the peptides 60 Proglumide that do bind are usually fragments ofthe hormone or analogues -Benzotript with a similar chemical structure. One exception to this general 240 Bt2cGMP pattern occurs with the opiate receptors (28). These receptors can interact not only with peptides (enkephalins and other ~20- opioid peptides) but also with morphine and structurally related alkaloid compounds. A second apparent exception to the general principle that only peptides will bind to peptide hormone -5 -4 -3 -2 receptors is the finding that butyryl derivatives ofcyclic GMP Antagonist, log M will competitively inhibit the interaction of CCK and structur- related with their on acinar FIG. 5. Abilities of Bt2cGMP, proglumide, and benzotript to in- ally peptides receptors pancreatic hibit CCK-8-induced residual stimulation of amylase secretion. Acini cells (13, 16, 17) as well as other tissues (15, 19). were incubated with 10 nM CCK-8 for 10 min at 4TC, washed three The present findings indicate that proglumide, a derivative times with 50 vol of iced standard incubation solution, resuspended in ofglutaramic acid (Fig. 1), and benzotript, a derivative oftryp- fresh standard incubation solution, andreincubated with the indicated tophan (Fig. 1), are specific antagonists of the interaction of concentration of antagonist for 30 min at 37TC. Amylase release was CCK and structurally related peptides with CCK receptors on calculated as the percentage of the amylase activity in the acini at the pancreatic acinar cells. As has been reported for B4cGMP (16), beginning of the second incubation that was released into the extra- in the cellular medium during the second incubation. Values for amylase re- proglumide and benzotript cause a rightward shift lease are expressed as the percent of the stimulation caused by CCK- dose-response curve for CCK-stimulated amylase secretion but 8 with no added antagonist. In each experiment each value was de- do not alter the maximal increase in amylase secretion caused terminedintriplicate, and results given are means from eight separate by CCK. This antagonism is selective for CCK and structurally experiments. related peptides in that proglumide does not alter the actions ofsecretagogues whose effects are mediated by cyclic AMP and does not alter the actions of that have a mode of and benzotript were parallel to the curve for the inhibition secretagogues caused by Bt2cGMP (Fig. 5). action similar to that ofCCK but act through different receptors Because has been reported to have anticholiner- (29). Proglumide can cause a small reduction in carbachol-stim- proglumide ulated this inhibition is noncom- on secretion we examined the effect amylase secretion; however, gic effects gastric acid (3), occurs with concentrations of car- on carbachol-stimulated amylase secretion in petitive, only relatively high of proglumide bachol 10 and 3 and does greater when tested at a relatively high con- (above AM) proglumide (above mM), detail. Proglumide, not occur with there is a close correlation centration did not alter the increase in se- benzotript. Finally, (10 mM), amylase between the of or to inhibit bind- cretion caused by concentrations ofcarbachol up to 10 but ability proglumide benzotript jiM, of 125I-CCK to its on acini and their it caused a 25-35% decrease in the stimulation caused by car- ing receptors pancreatic abilities to inhibit the action of CCK on enzyme secretion and bachol concentrations of 30 or greater (Table 1, Fig. 6). AM on calcium outflux. Concentrations ofproglumide of3 mM or less did not alter the Previously, we found that in acini that have been incubated increase in amylase secretion caused by any concentration of without there carbachol tested (Fig. 6). Benzotript, at concentrations up to with CCK, washed, and then reincubated CCK, 1 mM, did not alter carbachol-stimulated amylase secretion (not is significant residual stimulation of enzyme secretion and that shown). this residual stimulation can be reversed rapidly by adding Bt2cGMP (26, 27). The present results illustrate that, like Bt2cGMP, proglumide and benzotript can reverse CCK-in- 36 duced residual stimulation ofamylase secretion; therefore, pro- jM glumide and benzotript have the same spectrum of actions on 32- CCK-induced changes in acinar cell function as do butyryl de- Alone 100jWM rivatives ofcyclic GMP (13, 16, 26, 27). In terms oftheir chemical structures, however, proglumide and benzotript bear no 24 - - proglu obvious resemblance to butyryl cyclic GMP (Fig. 1); therefore, rbhmide they appear to be members ofa different class ofCCK-receptor antagonists. Moreover, because proglumide and benzotript are 16- relatively inexpensive and are active after oral administration 12 to humans (1) and animals (4), these compounds may be useful 3 MM in defining the role of CCK and related peptides in various <8- physiological processes and may be oftherapeutic value in clin- 4 ical conditions in which antagonizing the actions ofCCK might be expected to produce beneficial effects.

-7 -6 -5 -4 -3 -4 -3 -2 Carbachol, log M Proglumide, log M 1. Weiss, J. (1979) in Proglumide and Other Gastrin-Receptor An- tagonists, eds. Weiss, J. & Miederer, S. E. (Excerpta Medica, FIG. 6. Effect of proglumide on carbachol-stimulated amylase re- Amsterdam), pp. 113-131. lease. Acini were incubated for 30 min at 37TC with different concen- 2. Rovati, A. L. (1976) Scand. J. GastroenteroL 11, Suppl. 42, trations of carbachol with or without 10 mM proglumide (Left) or with 113-118. different concentrations of proglumide plus the indicated concentra- 3. Bali, J. P. (1979) in Proglumide and Other Gastrin-Receptor An- tions of carbachol (Right). Results given are means of at least four tagonists, eds. Weiss, J. & Miederer, S. E. (Excerpta Medica, separate experiments. Amsterdam), pp. 15-25. Downloaded by guest on September 28, 2021 6308 Cell Biology: Hahne et aL Proc. Natd Acad. Sci. USA 78 (1981)

4. Rovati, A. L., Casula, P. L. & DaRe, G. (1967) Minerva Med. 58, 17. Robberecht, P., Deschodt-Lanckman, M., Woussen-Colle, M.- 3656-3670. C., DeNeef, P., Camus, J. C. & Christophe, J. (1980) MoL Phar- 5. Vatier, J., Lepeltier, M. & Kuan, T. S. (1977) Gastroenterol macoL 17, 268-274. Clin. Biol 1, 643-651. 18. Philpott, H. G. & Petersen, 0. H. (1979) Pflugers Arch. 382, 6. Rovati, A. L. (1968) Br. J. Physiol 34, 667P (Abst.). 263-267. 7. Danhof, I. E. (1967) Minerva Med. 58, 3670-3677. 19. Hutchison, J. B. & Dockray, G. F. (1980) Brain Res. 202, 8. Sampietro, R. (1969) Minerva Med. 60, 1032-1037. 501-505. 9. Desvigne, C., Vagne, M., Faivre, M. & Desvigne, S. (1977) Gas- 20. Peikin, S. R., Rottman, A. J., Batzri, S. & Gardner, J. D. (1978) troenterol, Clin. Biol. 1, 447-454. Am. J. PhysioL 235, E743-E749. 10. Vidal y Plana, R. R., Cifarelli, A. & Bizarri, D. (1980) Hepato- 21. Gardner, J. D. & Jackson, M. J. (1977) J. PhysioL 270, 439-454. gastroenterology 27, 41-49. 22. Ceska, M., Birath, K. & Brown, B. (1969) Clin. Chim. Acta 26, 11. Mantovani, P., Piccinin, G. L. & Ugolotti-Adorni, M. C. (1971) 437-444. Arch. Int. Pharmacodyn. Ther. 189, 319-327. 23. Ceska, M., Birath, K. & Brown, B. (1969) Clin. Chim. Acta 26, 12. Jorpes, J. E. & Mutt, V. (1973) in Secretin, Cholecystokinin, Pan- 445-453. creozymin, and Gastrin, eds. Jorpes, J. E. & Mutt, V. (Springer, 24. Gardner, J. D., Costenbader, C. L. & Uhlemann, E. R. (1979) New York), pp. 1-177. Am. J. PhysioL 236, E754-E762. 13. Jensen, R. T., Lemp, G. F. & Gardner, J. D. (1980) Proc. Natl 25. Bolton, A. E. & Hunter, W. M. (1973) Biochem.J. 133, 529-539. Acad. Sci. USA 77, 2079-2083. 26. Abdelmoumene, S. & Gardner, J. D. (1980) Am. J. PhysioL 239, 14. Takeuchi, K., Speir, G. R. & Johnson, L. R. (1979) Am. J. Phys- G272-G279. iol 237, E284-E294. 27. Collins, S. M., Abdelmoumene, S., Jensen, R. T. & Gardner, J. 15. Poitras, P., Iacino, D. & Walsh, J. H. (1980) Biochem. Biophys. D. (1981) Am. J. PhysioL 240, G466-G471. Res. Commun. 96, 476-482. 28. Snyder, S. H. (1977) N. Engi J. Med. 296, 266-271. 16. Peikin, S. R., Costenbader, C. L. & Gardner, J. D. (1979)J. Biol 29. Gardner, J. D. & Jensen, R. T. (1980) Am. J. PhysioL 238, Chem. 254, 5321-5327. G63-G66. Downloaded by guest on September 28, 2021