EFFECT OF HORMONES ON PANCREATIC MACROMOLECULAR TRANSPORT

Transcription

1 GASTROENTEROLOGY 68: , 1975 Copyright 1975 by The Williams & Wilkins Co. Vol. 68, No.6 Printed in U.S.A. EFFECT OF HORMONES ON PANCREATIC MACROMOLECULAR TRANSPORT MANJIT SiNGH, M.D., F.R.C.P. (C), AND PAUL D. WEBSTER, III, M.D. Gastroenterology Research Laboratory. Forest Hills Division, Veterans Administration Hospital, and the Department of Medicine, Medical College of GeorgW., Augusta, Georgia The role of subcellular organelles in the synthesis, transport, and secretion of pancreatic digestive proteins has been well documented. This study was designed to examine effects of pentagastrin, secretin, and acute and chronic administration of cholecystokinin-pancreozymin (CCK-PZ) on pancreatic macromolecular transport and secretion. Pooled rat pancreas slices were pulse-labeled with L-p c )phenylalanine and migration of 14 C-labeled proteins studied by "chase" incubation for 15, 30, 60, and 120 min. After in vitro incubation of control and drug-treated pancreatic slices, subcellular particles were isolated by differential centrifugation. Specific activity of radioactive labeled proteins was determined in subcellular involved in transport and secretion of digestive proteins. These studies indicate that pentagastrin and acute and chronic stimulation with CCK-PZ did not alter the rate of transport of labeled proteins from ribosomes to the zymogen granules. Pentagastrin and CCK-PZ stimulated secretion of labeled proteins from the zymogen granules into the medium in the concentrations used as evidenced by significant increase in the amount of labeled proteins in the medium and significant decrease in the specific activity of the zymogen granule after 60- and 120-min periods of incubation. Secretin did not alter the rate of transport or secretion of the pulse-labeled proteins. In pancreatic exocrine cells, proteins meant for export are synthesized on ribosomes attached to endoplasmic reticulum. Nascent proteins are released from membrane-bound ribosomes into the cisternae of the rough endoplasmic reticulum. From this site they move to Golgi complex where condensation takes place leading to the formation of mature zymogen granules. Application of secretory stimuli leads to the discharge of zymogen granules into pancreatic ductules. 1-3 It has been demonstrated that stimula- Received September 23, Accepted December 20, Address requests for reprints to: Manjit Singh, M.D., VA Hospital509/151, Augusta, Georgia This work was supported by the Veterans Administration (MRIS Nos and 9274) and Grant AM from the National Institutes of Health tion of zymogen granule discharge by carbamylcholine in vitro primarily affects the discharge of secretory proteins and does not, directly or indirectly, influence their rate of synthesis or intracellular transport. 4 This study was undertaken to examine the effects of acute and chronic administration of cholecystokinin-pancreozymin (CCK-PZ), pentagastrin, and secretin on pancreatic macromolecular synthesis, transport, and secretion. These studies demonstrate that CCK-PZ and pentagastrin, used in vitro in pancreatic slices prepared from fed animals, do not alter the intracellular transport of proteins, although they enhance secretion. Chronic stimulation of the pancreas with CCK-PZ, which has been shown to be associated with increased protein, RNA, DNA content, and rate of DNA synthesis,

2 June 1975 PANCREATIC MACROMOLECULAR TRANSPORT 1537 does not alter the rates of intracellular transport. 5 Likewise, secretin was found to have no effect on the transport and secretion of pancreatic macromolecules. Materials and Methods Male Sprague-Dawley rats (280 to 300 g body weight) obtained from Holtzman Company, Madison, Wisconsin, were used. The rats were maintained in an air-conditioned environment. They were fed Purina rat chow ad libitum and had free access to water. Rats used for studies with secretin and controls were fasted for 24 hr before L-[U- 14 C ]Phenylalanine (466 me per mmole) was purchased from the New England Nuclear Corporation, Boston, Massachusetts. Composition and preparation of tissue culture media have been described. CCK-PZ was obtained from the Gastrointestinal Hormone Research Unit, Karolinska Institute, Stockholm, Sweden. Pentagastrin was a gift from Ayerst Chemicals, New York, New York. Synthetic secretin was purchased from Schwarz/Mann, Orangeburg, New York, Preparation of tissue. For each experiment, 8 rats were killed, and the pancreas was removed and placed in freshly oxygenated, ice-cold, Krebs-Ringer phosphate buffer, ph 7.4. The "head" portion of the pancreas was cut into four 125-mg slices each measuring 10 by 10 by 1 mm. One slice from each pancreas was placed in each of four Erlenmeyer flasks (25 ml). Each flask contained approximately 500 mg of pancreatic tissue suspended in 5 ml of tissue culture medium. Incubation of tissue. Two different incubation periods were employed: the first, a pulselabeling incubation, and the second, a chase incubation. For pulse-labeling incubations, 10 IJ.C (22!J.M) of L-[ 14 C]phenylalanine was added to 5 ml of the medium in control and experimental flasks, and the flasks were incubated in a shaking water bath (60 oscillations per min) at 37 C under a constant flow of 100% oxygen. At termination of a 5-min pulse-labeling period, the media were decanted, the tissue was washed with warm, unlabeled, aerated "chase" medium, and transferred to similar 25-ml Erlenmeyer flasks containing 5 ml of unlabeled "chase" medium. The selected peptide hormone under study was added in appropriate concentrations. The "chase" medium contained 780 times excess unlabeled phenylalanine. The "chase" incubation was performed in shaking water bath (60 oscillations per min.) at 37 C under constant flow of 100% oxygen. Periods of incubation were 15, 30, 60, and 120 min. Preparation of tissue homogenates, subcellular fractionation, preparation and washing of pellets, and supernatant proteins have been described. 7 Proteins were estimated by biuret and Lowry methods. 9 Radioactivity was assayed in a Packard Tri-Carb liquid scintillation counter using a phosphor developed by Patterson and Green. 10 Results The data validating this in vitro incubation system for the study of synthesis, transport, and secretion of digestive enzymes have been published. 7 A reasonable degree of purity for subcellular prepared by these methods was established by biochemical methods, 7 marker enzyme assays, and electron microscopy. Table 1 shows effects of 0.1 U per ml of CCK-PZ on protein transport and secretion in rat pancreas. A comparison of control and CCK-PZ-treated slices revealed that this concentration of hormone was not associated with alteration of rate of transport of L- ( 14 C ]phenylalanine from microsomal to zymogen granule. There was a significant increase in specific activity of proteins in the medium in drugtreated compared with control slices at 60 and 120 min. Table 2 shows effects of 1.. U per ml of CCK-PZ on protein transport and secretion in rat pancreas. There was a decrease in specific activity of zymogen granule at 60 and 120 min and a significant increase in secretion of labeled proteins into media at these time points. Table 3 shows effects of 5 U per ml of CCK-PZ on protein transport and secretion in rat pancreas. There was a significant decrease in the specific activity of the zymogen granule at 60 min, and 120 min and an increase in the secretion of labeled proteins. Table 4 shows effects of 0.1!J.g per ml of pentagastrin on protein transport and secretion in rat pancreas. There was no alteration of rate of transport of L [14C]phenylalanine from microsomal to zymogen granule. There was a significant increase in specific activity of proteins in the medium from drug-treated slices over controls at 60 and 120 min.

3 1538 SINGH AND WEBSTER Vol. 68, No.6 TABLE 1. Effects of cholecystokinin-pancreozymin (CCK-PZ) on protein transport and secretion in the rat pancreas (0.1 U per ml) Homogenate ± ±156< 1717± ±122< 1638± ±146< 1881± ±154< Microsomes (ER ) 2338± ±248< 2219± ±223< 2130± ±353< 1998± ±199< Zymogen granules 595±84 602±87< 966± ±86< 1096± ±119< 1259± ±188< supernatant ±78 453±37< 777±83 532±37< 878± ±78< 803± ±40< Medium ±8 135±10< 145±3 149±12< 361±33 710±67" 998± ±186' Data are expressed as counts per min per mg of protein. Control values are the mean ± SE of four p < O.ol. e p < O.Ql. TABLE 2. Effects of cholecystokinin-pancreozymin (CCK-PZ) on protein transport and secretion in the rat pancreas (1 U per ml) Homogenate ± ±47< 2042± ±140< 2099± ±194< 2414± ±196< Microsomes (ER") 2984± ±254< 2176± ±155< 1825± ±338< 1664± ±355< Zymogen granules 895± ±115< 1373± ±151< 1851± ±124" 2561± ±87' supernatant.. 668± ± 72< 769±66 595±42< 1043± ±78< 1053± ±95< Medium ±15 114±26< 151±20 174±22< 320±17 842± ± ±143 a Data are expressed as counts per min per mg of protein. Control values are the mean ± SE of five d p < e P < I p < p < Table 5 shows effect of 2 f.lg per ml of pentagastrin on protein transport and secretion in rat pancreas. There was a decrease in the specific activity of zymogen granule at 60 and 120 min in pentagastrin-treated slices. This was associated with a significant increase in secretion of labeled proteins into media at these time points. Table 6 shows the effects of 10 f.lg per ml of pentagastrin on protein transport and secretion on rat pancreas. There was a decrease in the specific activity of zymogen granule fraction at 60 and 120 min, and a significant increase in secretion of labeled proteins into media at these time points in pentagastrin-treated slices. Table 7 shows the effect of chronic stimulation with CCK-PZ (20 U per kg intraperitoneally, twice a day for 5 days) on protein transport and secretion in rat pancreas. Parallel sets of slices were obtained from animals stimulated chronically with CCK-PZ and control animals injected with saline. These sets of slices were stimulated in vitro with 1 U per ml of CCK-PZ. No significant difference was found when specific radioactivity of the microsomes, zymogen granules, and proteins in the medium derived from chronically stimulated animals was compared with control animals at various time points. Tables 8, 9, and 10 show effects of 0.1, 1, and 10 U per ml of secretin respectively, on

4 JuJl(! 1975 PANCREATIC MACROMOLECULAR TRANSPORT 1539 TABLE 3. Effects of cholecystokinin-pancreozymin (CCK-PZ) on protein transport and secretion in the rat pancreas (5 U per ml) Homogenate ± ±281 c 2126± ±95C 1910± ±160C 1954± ±232c Microsomes (ER ) 3559± ±338c 2755± ±167C 2234± ±178C 1420± ±434C Zymogen granules 510± ±88C 929±78 890±57c 1722± ±67" 2277± ±66' supernatant ± ±105c 814±95 867±53c 845±63 814±40C 855± ±103C Medium ±15 114±11" 187±28 260±11c 283±7 753± ± ±108 Data are expressed as counts per min per mg of protein. Control values are the mean ± SE of five d p < e P < I p < p < TABLE 4. Effects of pentagastrin on protein transport and secretion in the rat pancreas (0.1 1-Lg per ml) Control Pentagastrin Control Pentagastrin Control Pentagastrin Control Pentagastrin Homogenate ± ±178c 1400± ±257c 1349± ±189c 1475± ±193c Microsomes (ER ) 2490± ±221" 2198± ±273C 1828± ±2llc 1759± ±180c Zymogen granules 552± ±128c 707±93 773±206c 948± ±140c 1274± ±259C supernatant 555±68 516±58C 625±73 635±92c 731± ±88C 949± ±38C Medium.... '.. 148±15 138±18C 134±18 163±19c 300±18 533±60" 915± ±107' a Data are expressed as counts per min per mg of protein. Control values are the mean ± SE of seven d p < e P < TABLE 5. Effects of pentagastrin on protein transport and secretion in the rat pancreas (2 J.Lg per ml) Control Pentagastrin Control Pentagastrin Control Pentagastrin Control Pentagastrin Homogenate ± ±176c 1714± ±174c 1773± ±251c 1706± ±252c Microsomes (ER ) 2615± ±63c 2376± ±44C 1702± ±179c 1403± ±65c Zymogen granules 568±68 548±91" 885±44 802±114c 1739±53 991±51" 2449± ±95' supernatant ±98 548±44c 773±74 635±57C 862±53 801±60C 1107±73 857±87c Medium ±23 155±25c 243± ±70C 614± ± ± ±49 a Data are expressed as counts per min per mg of protein. Control values are mean ± SE of four d p < e P < f p < p < 0.01.

5 1540 SINGH AND WEBSTER Vol. 68, No.6 TABLE 6. Effects of pentagastrin on protein transport and secretion in the rat pancreas (10 p.g per ml)" Control Pentagastrin Control Pentagastrin Control Pentagastrin Control Pentagastrin Homogenate ± ±182< 1840± ±60< 1808± ±137< 1851± ±90< Microsomes (ER ) 2693± ±184< 2251± ±148< 2130± ±160< 1697± ±187< Zymogen granules 396±34 490±67< 1056± ±64< 1541± ±128" 1725±80 851±87' supernatant ± ±60< 670±96 621±93< 656± ±87< 823± ±61< Medium ±9 149±44< 159±12 232±109< 347± ±108' 1125± ±35 a Data are expressed as counts per min per mg of protein. Control values are the mean ± SE of five 'None of these values were significant (P value determined by Student's t-test). d p < e P < I p < P < TABLE 7. Effects of chronic in vivo stimulation with cholecystokinin-pancreozymin (CCK-PZ) on protein transport and secretion in rat pancreas Fractions Homogenate 1650± ± ± ± ± ± ± ±142 Microsomes 2785± ± ± ± ± ± ± ±136 Zymogen granules 558±56 499±49 969± ± ± ± ± ±160 supernatant.. 570±57 436±26 606±78 575± ± ±41 838± ±102 Medium ±18 105±13 226±21 290± ±85 977± ± ±145 a Data are expressed as counts per min per mg of protein. Control values are means ± SE of five The slices marked as control were obtained from rats injected with normal saline intra peritoneally. The slices marked as CCK-PZ were obtained from rats stimulated in vivo with 20 units of CCK-PZ per kg twice a day for 5 days. Both sets of slices were stimulated in vitro with 1 U of CCK-PZ per ml of the medium. None of these values were significant (P value determined by Student's t-test). TABLE 8. Effects of secretin on protein transport and secretion in rat pancreas (0.1 U per ml) Control Secretin Control Secretin Control Secretin Control Secretin Homogenate ± ±19!)< 1005± ±208< 1146± ±179< 969±64 996±177< Microsomes (ER") 1842± ±116< 1699± ±224< 1429± ±173< 829±32 920±88< Zymogen granules 276±23 297±58< 398±59 421±62< 617±52 587±68< 850±77 899±89< supernatant 319±28 368±58< 411±36 475±59< 459±65 415±34< 536±98 521±82< Medium ±28 123±25' 147±6 133±13< 302±25 320±16< 864±87 847±88< a Data are expressed as counts per min per mg of protein. Control values are mean± SE of three 'None of these values were significant (P value determined by Student's t-test). protein transport and secretion in rat pancreas. Comparison of secretin-treated slices with controls revealed no difference in specific activity of the microsomal zymogen granule. There was no difference in the rate of secretion as shown by

6 June 1975 PANCREATIC MACROMOLECULAR TRANSPORT 1541 lack of significant difference in appearance of labeled proteins in the media supporting the control and secretin-treated slices. Discussion In this study, we have used the in vitro model reported previously 7 to examine effects of pentagastrin, secretin, and acute and chronic administration of CCK-PZ on pancreatic macromolecular transport and secretion. Slices obtained from fed animals, stimulated in vitro by the indicated dosages of CCK-PZ and pentagastrin showed significant increase in specific activity of labeled proteins in the media indicating enhanced secretion. There was, however, no increase in the rate of decline of specific radioactivity of microsomes in treated compared to control slices, demonstrating no stimulation of the process of transport. The zymogen granules showed significant decline in specific activity at 60 and 120 min with CCK-PZ (dose: 1 per ml and 5 U per ml) and pentagastrin (dose: 2,.,g per ml and 10,.,g per ml). This signifies that these hormones do not alter the rate of transport of proteins from microsomes to zymogen granules but stimulate the discharge of labeled proteins from the zymogen granules into the medium. Chronic stimulation of the pancreas with CCK-PZ has been shown to alter pancreatic weight, protein content, DNA content, and rates of DNA synthesis. The effects of chronic stimulation with hormones on transport and secretion in the pancreas have not been described. The present data point out that the rate of intracellular transport and secretion of proteins is not affected by chronic stimulation of the pancreas with pharmacological doses of CCK-PZ. Although there is a unanimity of opinion regarding the effect of secretin on water and electrolyte secretion from the pancreas, there is a divergence of opinion regarding its effect on protein secretion. TABLE 9. Effects of secretin on protein transport and secretion in rat pancreas (1 U per ml) Control Secretin Control Secretin Control Secretin Control Secretin Homogenate ± ±131< 952± ±28< 977± ±103C 1077± ±96" Microsomes (ER"). 1711± ±130c 1503± ±50< 1384± ±151< 905±97 952±170< Zymogen granules. 306±36 359±43c 432±53 452±68c 693± ±81C 1054± ±130c supernatant ±72 295±52c 357±41 343±4F 594±85 539± 72< 697±68 726±61< Medium ±25 116±16" 124±11 108±11c 267±12 288±18< 909±33 925±26" a Data are expressed as counts per min per mg of protein. Control values are mean± SE of three TABLE 10. Effects of secretin on protein transport and secretion in rat pancreas (10 U per ml)" Control Secretin Control Secretin Control Secretin Control Secretin Homogenate ±95 988±79< 969±26 978±34C 937±36 997±74C 964± ±51< Microsomes (ER") 1168± ±54< 1239± ±77c 945± ±127< 890±20 867±85C Zymogen granules 310±27 272±87< 471± ±143< 665± ±159< 764±30 781±52< supernatant ±44 307±43< 430± ±97< 496±51 508±67C 516±34 554±80c Medium ±7 97±12< 112±27 107±2F 401±10 420±34c 898±37 938±63c a Data are expressed as counts per min per mg of protein. Control values are mean ± SE of three

7 1542 SINGH AND WEBSTER Vol. 68, No. 6 Injection of secretin in cat, dog, and man results in a moderate secretion of enzymes which has been interpreted as being due to "wash-out phenomenon," i.e., a flushing out of the enzymes already present in the ducts ' When dose and response relationship to pure secretin was studied in chronic gastric and pancreatic fistulae dogs, it was found that all doses of secretin were associated with a "wash-out" phenomenon during the first 15 min infusion period. However, in all time periods, the output of proteins was significantly above the basal level, thereby indicating that secretin caused a weak stimulation of enzyme secretion from the dog pancreas. 15 These studies have been done in large animal models by cannulation of the pancreatic duct, by construction of pancreatic fistulae, or by aspiration of duodenal secretions. Such large animal models are of limited value in studies designed to provide information concerning processes at cellular level, coincident with protein transport or secretion. In this study, which was specifically designed to provide such information, secretin, in doses of 0.1, 1, and 10 U per ml, was found to have no effect on the intracellular rate of transport and secretion of nascent proteins labeled with L [14C]phenylalanine. This confirms that secretin has no effect on the intracellular transport and secretion of pancreatic macromolecules in the pancreatic acinar cells. REFERENCES 1. Jamieson JD, Palade GE: Intracellular transport of secretory proteins in the pancreatic exocrine cell. I. Role of peripheral elements of the Golgi complex. J Cell Bioi 34: , Jamieson JD, Palade GE: Intracellular transport of secretory proteins in the pancreatic exocrine cell. II. Transport to condensing vacuoles and zymogen granules. J Cell Bioi 34: , Palade GE, Siekevitz P, Caro LG: Structure, chemistry and function of the pancreatic exocrine cell. In Ciba Foundation Symposium on the Exocrine Pancreas. London, J and A Churchill Ltd, 1962, Jamieson JD, Palade GE: Synthesis, intracellular transport and discharge of secretory proteins in stimulated pancreatic exocrine cells, J Cell Bioi 50: , Mainz DL, Black 0, Webster PD: Hormonal control of pancreatic growth. J Clin Invest 52: , Morisset JA, Webster PD: Effect of fasting and feeding on protein synthesis by the rat pancreas. J. Clin Invest 51:1-8, Singh M, Black 0, Webster PD: Effects of selected drugs on pancreatic macromolecular transport. Gastroenterology 64: , Gornall AG, Bardawill CF, David M: Determina tion of serum proteins by means of biuret reaction. J Bioi Chern 177: , Lowry OH, Rosenbrough NJ, Farr AL, et a!: Protein measurement with the folin phenol reagent. J Bioi Chern 193: , Patterson MS, Green RC: Measurement of low energy beta-emitters in aqueous solution by liquid scintillation counting of emulsions. Anal Chern 37: , Wang CC, Grossman MI, Ivy AC: Effect of secretin and pancreozymin or amylase and alkaline phosphatase secretion by the pancreas in dogs. Am J Physiol 154: , Henerickson FW: The effect of snythetic secretin on the external pancreatic secretion in dogs. Acta Physiol Scand 72: , Wormsley KG: The action of secretin on the secretion of enzymes by the human pancreas. Scand J Gastroenterol 3: , Case RM, Harper AA, Scratcherd T: On the mode of action of secretin and pancreozymin. In Proceedings of the Third Symposium, European Pancreatic Club. Praha, Czech Med Press, 1970, Stening GF, Grossman MI: Gastrin related pep tides as stimulants of pancreatic and gastric secretion. Am J Physiol 217: , , 1969