Gut: first published as 10.1136/gut.28.10.1208 on 1 October 1987. Downloaded from Gllt, 1987, 28, 1208- 1214 Alimentary tract and pancreas Demonstration of pepsinogen C in human pancreatic islets P B SZECSI, LI HALGREEN, SS POULSEN, C K AXELSSON, M DAMKJ/ER-NIELSEN, T KJ/ER, AND B FOLTMANN From The Department of Clinical Chemistry, Hvidovre Hospital; Institute of Pathology, Bispebjerg Hospital; Institute of Biochemical Genzetics, In.stituite of Medical Anatomy, Departnzent B and Department of Clinical Physiology, Glostrup Hospital, University of Copenhagen. Department K of Surgery, Vejle Hospital and Department L ofSurgery, Arhu.s Municipal Hospital, University ofArhus, Denmark. SUMMARY Pancreatic tissue from 16 post mortem kidney donors have been examined for the content of pepsinogens. A zymogen with electrophoretic mobility, isoelectric point and molecular weight equal to that of pepsinogen C of gastric origin was found in all specimens. A comparison between pepsinogen C extracted from pancreatic tissue and gastric mucosa demonstrated immunological identity. Quantitative measurements with a radioimmunoassay showed pepsinogen C concentrations in pancreatic tissue three to 80 times higher than those of blood serum. Immunohistochemical staining gave positive reaction for pepsinogen C only in the alpha cells of the pancreatic islets. http://gut.bmj.com/ Gastric proteases belong to the superfamily of superfamily of aspartic proteases localised outside aspartic proteases (EC 3.4.23). The predominant the gastrointestinal tract have traditionally been gastric proteases of adult mammals are designated designated cathepsin D or E.'" During a series of pepsin A (EC 3.4.23.1) and pepsin C (EC 3.4.23.3). investigations on the general distribution of aspartic For both proteases several isoenzymes and iso- proteases, we observed that several tissues contained zymogens may be observed. In native state the two zymogens and proteases with electrophoretic and types show no immunochemical cross reactions. immunochemical properties that were indistinguish- Within each type the isozymogens cannot be distin- able from gastric pepsinogen A and C.2 19 on September 29, 2021 by guest. Protected copyright. guished with polyclonal antisera.' This study deals with our findings of a proenzyme The proenzyme of pepsin A (pepsinogen A) is in the human endocrine pancreas that is indistin- synthesised in the chief and mucous neck cells of the guishable from gastric pepsinogen C. fundic mucosa, whereas the proenzyme of pepsin C (pepsinogen C) is found in the same cells as well as Methods in cells of cardiac, pyloric and Brunner's glands.4 Furthermore, both pepsinogen A and C have been REAGENTS shown in serum,i in gastric gland heterotopia and Agar Noble (Difco, Detroit, USA); Agarose type II metaplasia anywhere in the gastrointestinal tract3 (Sigma, St Louis, USA); Agarose HSA (Litex, and in a Meckel's diverticulum.' Pepsinogen A has Glostrup, Denmark); Aprotinin (Bayer, Lever- been demonstrated in normal urine' "''" and pepsi- kusen, FRG); Acrylamide (BioRad, Richmond, nogen C in seminal fluid,'"' the seminal vesicles2 and USA); N,N'-metylene-bis-acrylamide (Sigma, St the prostate gland."' Louis, USA); Benzamidiniumchloride (Merck, Apart from renin (EC 3.4.23.16) members of the Darmstadt, FRG); Human albumin (Behringwerke, Marburg, FRG); "'5I-Nal (Hoechst, Frankfurt am Main, FRG); Lactoperoxdidase (Sigma, St Louis, Addrcss for (orrcsporlndcncc 13B Szcc.s.. D)pairticrit oC(hlical (Chemistry 339. USA); Phenylmethylsulfonylfluoride (Sigma, St 11v idovrc l lospit.t. UnIvcrsity otfCopenhagcn. Kctteg.aardsa llc 23. DK-265(0 1 idowrc Dct.mrkI) Louis, USA); Sephadex G-25 (Pharmacia, Uppsala, Reccivcd tor puhlication 'S Mi.rth 1987 Sweden); Triton X-100 (Serva, Heidelberg, FRG); 1208 Gut: first published as 10.1136/gut.28.10.1208 on 1 October 1987. Downloaded from Demtionistration ojpepsinogen C in hlumlan pancreatic islets 129() 3-3' diaminobenzidine tetrahydrochloride (Sigma, St E LECTROPHORESES Louis, USA); HAWP nitrocellulose (Millipore, Agar gel zone electrophoresis was carried out in l1(o Molsheim, France); Sodium dodecylsulfate (Merck, (w/v) agar gels in 0(05 M sodium phosphate pl I 6() Schuchardt, FRG); Donkey antirabbit antibody for two hours at a gradient of 15 V/cm at constant coated cellulose (Wellcome, Dartford, UK). voltage as described previously."' Crossed tiindeiii immunoelectrophoresis was carried out in 1 %O (w/v) SAMP1 ES OF TISSUES agarose gels." Pancreatic tissue was obtained from post mortem Sodium dodecylsulphate (SDS) polyacrylamide kidney donors. The donors were victims of accidents gel electrophoresis was carried out in vertical slab and without systemic disease. The time of ischaemia gels (T=15%, C=0-5%).- The proteases were de- was kept as short as possible (15-120 min). The tected by indirect peroxidase staining after trans- pancreas were dissected free from fat and connective ferring of proteins to nitrocellulose." tissue, washed with ice cold 0-05 M sodium phos- Isoelectric focusing was performed in I mmin thick phate p1i 6-0 and cut into pieces of 0-5 cm.' polyacrylamide gels (T=7.5%0, C=3(o ) coiitdiiniflg Extraction of 1 g wet weight tissue took place in a 500 (v/v) Pharmalyte (pH 2-5-5) for 3 h at 1500 V Potter-Elvehjem homogeniser with 3 ml of 0-05 M (constant voltage) after one hour of prefocuising. sodium phosphate pH 6-0 made 5 mM with phenyl- methylsulfonylflouride, 20 mM with benzamidine DETECTION OF ACTIVITY AFTER and 40 FtM with aprotinin. Triton X-100 was then El ECTROPHORESIS added to the homogenate at a final concentration of After electrophoresis the protease containing zonles 2% (v/v) and sonication took place for three times 1Os were visualised as caseograms - that is, overlaying a in an ice bath. Cell debris were removed by centri- skim milk containing gel on top of the electrophoresis fugation at 20000 g at 5°C for 30 min. The super- gel and observation of clotted casein. natant was stored at -20°C until assayed. Extraction of samples of liver and spleen tissues for QUANTIFICATION OF PtEPSINOGFNS control experiments took place as described for Pepsinogen C was measured by a double antihody pancreatic tissues. Fixation for immunohistochemis- solid phase radioimmunoassay (RIA) with stalndards try took place in Bouins fixative for six hours at 4°C. purified from human gastric mucosa.-` The tissues were kept in 70% (v/v) alcohol until final Pepsinogen C was labelled with ''2I by a lacto- http://gut.bmj.com/ embedding in paraffin. peroxidase method.4 In brief, 1-63 ug pepsinogen C dissolved in 115 Rl 0-5 M sodium phosphate pH1 7-5 ENZYMES AND HORMONES was mixed with 18X5 MBq "'lI in 100 RI water, 5 [tg Pepsinogen A was prepared from human gastric lactoperoxidase in 10 1t 0.5 M sodium phosphate p1i mucosal or human urine"'; pepsinogen C was pre- 7-4 and 10 RI 352 RM solution of hydrogen peroxide. pared from human gastric mucosa"; human renin lodination was terminated after 30 min by addition of was a gift from Dr I Rubin, Copenhagen, Denmark; I ml water. '2I-pepsinogen C was obtained by insulin and glucagon was from NOVO, Bagsvxrd, gelfiltration on Sephadex G-25 eluted with ()0I M on September 29, 2021 by guest. Protected copyright. Denmark; somatostatin was from Cambridge Re- sodium phosphate pH 7-4. Hluman albumin (200 itl search Biochemicals, Harston, UK. 2% (v/v)) was added to 6 ml of tracer and aliquots of 500 tl were stored frozen. ANTISERA Immediately before use the tracer was diluted with Antisera against pepsinogens were raised in rabbits'' the RIA buffer: 0-04 M sodium phosphate pl1 7-4 at Statens Seruminstitut, Copenhagen, Denmark by with 0-15 M sodium chloride, 0-01 M EDTA, 5 g/l the courtesy of Dr N H Axelsen. The immuno- human albumin and 5 mg/l phenylmercuric acetate. globulin fractions of the antisera were purified by salt With freshly prepared tracer the dilution factor was precipitation and ion exchange chromatography.' determined allowing a radioactive concentration of The following antibodies were obtained from 1.1 MBq/l of diluted tracer solution. The radio- Dakopatts, Copenhagen, Denmark: horseradish immunoassay procedure was as follows: 50 Itl pepsi- peroxidase conjugated porcine antirabbit IgG nogen C standard (2-6-191 [g/l) or test substance (P 217), porcine antirabbit IgG (Z 196), peroxidase were mixed with 200()l "'"I-pepsinogen C solution, antiperoxidase complex (Z 113). Rabbit antiinsulin 200 itl rabbit antipepsinogen C (1:30000) and 300 [dl and rabbit antiglucagon was a gift from Dr J J Hoist, RIA-buffer. The mixture was incubated for 48 h at Copenhagen, Denmark. Rabbit antisomatostatin room temperature. Counting of bound pepsinogen C was from Cambridge Research Biochemicals, was performed after precipitating with 50 Fil of at Harston, UK. Rabbit antirenin was a gift from Dr K suspension of donkey antirabbit aintibody coated Poulsen, Copenhagen, Denmark. cellulose. Detection limit was 2-6 Ftg/l. The intra- Gut: first published as 10.1136/gut.28.10.1208 on 1 October 1987. Downloaded from 121() Szecsi, Halgreen, Poulsen, Axelsson, Damkjwr-Nielsen, Kjwr, and Foltmann Pg Pn A3 - A3- C- A4. A5 C1l C2 - C3 - ,..,u,,,.,.;/!i!;-_ &is«,;;'. : g C ii ':. ..:..'::.;. .1-' 4..~~~..:.;.t ... .o Fig. 2 Tandet-em crossed immunoelectrophoresis of' panc reatic extract C. The saimples appi ainidpepsinogeni followinig http://gut.bmj.com/ were applied: P well (extended), 25 Fl of'humatn panicreatic extrtrct; G well, 5 [tl chromatographically puirified gastric pepsinogen C (09 gll). First dintiensioni electrop)horesis (anode to the right) was runl (it 10 V/cmfor 60 mimi at 12°C in "' agarose gel (10%) with TrislBarbital bufferpH 8-6. Se-otnd Fig. I (aseogramn (ijier aigair gel electrophoresis in 0-05 M dimenisioni electrophoresis (aniode (it the top) was run at .sodlii,n Phosl)lphtae, pH 60(andtl 15 V/cm ftor two houirs.