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Pancreatic Digestive Enzyme Secretion in the Rabbit: Rapid Cyclic Variations in Enzyme Composition JOEL W

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Pancreatic Digestive Enzyme Secretion in the Rabbit: Rapid Cyclic Variations in Enzyme Composition JOEL W Proc. Natl. Acad. Sci. USA Vol. 92, pp. 2553-2557, March 1995 Physiology Pancreatic digestive enzyme secretion in the rabbit: Rapid cyclic variations in enzyme composition JOEL W. ADELSON*t, RINO CLARIZIOt, AND JESSICA A. COUTU* *Departments of Pediatrics and Physiology, Rhode Island Hospital, Brown University School of Medicine, Providence, RI 02903; and tMontreal Children's Hospital Research Institute, Montreal, PQ, Canada, H3H 1P3 Communicated by Viktor Mutt, Karolinska Institute, Stockholm, Sweden, December 6, 1994 (received for review June 14, 1994) ABSTRACT The role and mechanism of nonparallel pan- Others (3-7), citing the requirement ofthe exocytosis mechanism creatic secretion of digestive enzymes, in which enzyme pro- for parallel discharge of granule contents, have disputed the very portions change in rapidly regulated fashion, remain contro- existence of nonparallel secretion, claiming it to be due to versial. Secretion was collected from male 2.2-kg New Zealand experimental artifact. Both schools have assumed that the exo- rabbits in 5-min intervals for 3 h under basal conditions or crine pancreas is a homogeneous organ in which the mixed constant stimulation with cholecystokinin (CCK; 0.1 ,ig per enzymes are distributed in secretion granules throughout the kg per h i.v.) or methacholine chloride (MCh; 40 ,ug per kg per gland. Recently, a way has been found that may resolve the h i.v.). Both CCK and MCh produced an 8-fold stimulation of apparent paradox: this laboratory has shown (8, 9) that digestive protein output. Enzymes were separated by SDS/PAGE and enzymes can be secreted in a regulated but nonparallel fashion quantitated by densitometry of Coomassie blue-stained gels. consistent with exocytosis from heterogeneous sources in the Under both basal conditions and constant MCh infusion, pancreas, and differential secretion from these sources would rapid neurosecretory-like 12-min cyclic changes occurred in accommodate both exocytosis and nonparallel secretion. Others the proportions of amylase, lipase I, chymotrypsinogen, and (10) have shown that individual zymogen granules may contain trypsinogen. During constant infusion their percentages differing proportions of enzymes. We show here that nonparallel changed as much as 10-fold, and their ratios cycled by as much secretion is a primary characteristic of pancreatic function and, as 30-fold. The mean percentage for the entire infusion period surprisingly, that under constant conditions, digestive enzymes for lipase I declined >25% with CCK or MCh, for amylase it are secreted in a cyclic neurosecretory-like fashion. rose -30%Yo, and for chymotrypsinogen and trypsinogen it doubled (for all, P < 0.05). CCK and MCh elicited subtly but MATERIALS AND METHODS significantly different mean enzyme percentages and enzyme Collection of Pancreatic Juice. Male albino New Zealand ratios (P < 0.05) for amylase, chymotrypsinogen, and rabbits (2.2 kg), fed chow (Purina), were fasted overnight but trypsinogen; these differences were also confirmed by regres- allowed water before cannulation of the pancreas as described sion and correlation analyses. The changes in enzyme per- (9). The rabbits were anesthetized by i.p. administration of centages and ratios were explicitly consistent with secreta- xylazine (12.5 mg/kg) and inhaled methoxyflurane and 02- gogue-caused shifts in the intrapancreatic enzyme secretory Ringer's lactate solution and secretagogues dissolved in Ring- sources. Nonparallel secretion of digestive enzymes occurs er's lactate were infused at 15 ml/h via the femoral vein. A 1-h routinely, even during constant stimulation, and is due to constant infusion of Ringer's lactate was given followed by cyclic neurosecretory-like secretion from heterogeneous in- stimulation with cholecystokinin 8 (CCK-8; 0.10 jig per kg per trapancreatic sources. h, n = 4; Pharmacia), methacholine chloride (MCh; 40 ,tg per kg per h in Ringer's lactate, n = 4; Sigma), or Ringer's lactate The pancreas is a major organ of digestion, producing gram alone (n = 4, "basal" animals). Samples were collected in quantities of digestive enzymes daily in the human. The 5-min periods for 3 h and stored at -20°C. enzyme mixture is composed of -24 species that hydrolyze a SDS/PAGE Separation and Densitometry. Protein was diverse mixture of polymeric substrates. The basic question of measured with bovine serum albumin (fraction V, Sigma) as whether the relative proportions of the specific enzyme species standard (11). Enzymes were separated on discontinuous in the mixture can be rapidly adjusted to reflect the immediate SDS/15% polyacrylamide gels (18 x 13 cm) (12), with a 5% requirements for intraluminal intestinal hydrolysis dates back stacking gel (3 cm). Pancreatic secretion was diluted 1:1 with to the work of Pavlov who demonstrated "nonparallel" 0.125 M Tris-HCl, pH 6.8/4% (wt/vol) SDS/20% (vol/vol) changes in the enzyme mixture after administration of differ- glycerol/10% (vol/vol) 2-mercaptoethanol and boiled for 5 ent digestive substrates. Nearly a century later, the subject is min. Protein at 50-100 ,gg was loaded per lane; calibration was still disputed (1). with 14- to 70-kDa markers (MW-SDS-70L, Sigma). Gels were Recently, the debate has been reformulated in terms of cell electrophoresed at 10°C (constant current, 30 mamp per gel biological secretory mechanisms, especially the vectorial se- per 1.5 mm of thickness) against running buffer (0.025 M cretion of cellular products by exocytosis. On the one hand, a Tris HCl, pH 8.3/0.192 M glycine/0.1% SDS) and stained with substantial number of observations have been made, usually in Coomassie brilliant blue R-250 (Bio-Rad) as in the Hoefer the intact pancreas, of rapid nonparallel secretion of enzymes manual. Proteins were quantified by scanning (Hoefer model after administration of digestive substrates, end products, GS-300 densitometer with the GS-360 program). Protein bands gastrointestinal hormones, and hormone-like factors (2). These in each lane were quantified as the percentage of the total observations have led to a dispute concerning the basic concept protein in the lane and denoted the "enzyme percentage." The that the prepackaged digestive enzymes are secreted en masse by assay showed linearity of staining density for total protein and exocytosis of the mixed contents of the secretory (zymogen) each individual band in the concentration range used here; granules, since this would necessarily result in parallel secretion. addition of single enzymes to the mixture gave the expected incremental increase in signal for the appropriate band. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviations: CCK, cholecystokinin; MCh, methacholine chloride. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 2553 Downloaded by guest on October 1, 2021 2554 Physiology: Adelson et aL Proc. NatL Acad Sci USA 92 (1995) Enzyme Assays and Identification. Several steps were taken outputs and ratios were confirmed by Fourier analysis with the to assure the identification of the enzyme bands. Native pancre- IGOR program (WaveMetrics, Lake Oswego, OR). atic juice, purified rabbit amylase, porcine amylase and lipase To place variations in enzyme percentages and ratios on a (Sigma), and molecular mass standards (MW-ND-500, Sigma) reasonable basis for interanimal comparisons, data were nor- were diluted with 0.1 vol of 0.1% bromophenol blue in 50% malized for each experiment by calculating a mean percentage (wt/vol) sucrose and electrophoresed in a nondenaturing gel sys- for each enzyme species and a mean ratio of the percentages tem with a 7% separation and a 3.125% stacking gel as described for each enzyme pair; these means were denoted as 100%. (13). Adjacent duplicate samples were electrophoresed to allow Data were expressed as fractions or multiples of the mean by staining of one lane and elution of material from the other for the formula: percent of normalized mean of enzyme percent- determination of enzyme activity (14). The unstained lane was age or ratio = [(x - x)/x + 1](100), where x is the enzyme sliced into 1-cm sections, which were homogenized on ice with a percentage or ratio observed and X is the experimental mean. Kontes minitube homogenizer, and material was eluted at 4°C Thus, if the enzyme percentage or ratio in a single period was overnight in 1 ml of 0.1 M Tris-HCI, pH 8.0/0.1 M KCl/0.02 M found to be half the mean, the value would be 50%. CaCl2 and then centrifuged (3000 x g, 4°C per 10 min). Lipase activity was determined by hydrolysis of diglycerides RESULTS (15). Rabbit pancreatic juice showed two lipase activ'ity peaks The gel system resolved the secreted proteins into -13 bands on the nondenaturing gels that migrated to the bands shown as from 15 to 70 kDa (Fig. 1). Lipase I and II, amylase, chymo- lipase I and lipase II (Fig. 1) and comigrated with porcine trypsinogen, and trypsinogen were identified as described lipase on SDS/PAGE. Amylase was purified by binding to an above. Stimulation with CCK or MCh at the doses used gave acarbose (Bay g5421, Miles)-conjugated AH-Sepharose 4B equal '8-fold increases in protein output, which diminished affinity column (16). Activity was determined by the Caraway over time, as did the basal output. method (17). The amylase band (Fig. 1) comigrated with The mean percentage of each enzyme was significantly porcine amylase and purified rabbit amylase in a single band. altered from the basal condition after treatment with either Trypsinogen, chymotrypsinogen, and the carboxypeptidases CCK or MCh (Table 1); the percentage of lipase fell by >25%, migrated to their expected molecular weight positions; the percentage of amylase rose by as much or more, and the trypsinogen and chymotrypsinogen comigrated with their percentages of chymotrypsinogen and trypsinogen were ap- counterparts from porcine sources (Worthington).
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