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Biochimica et Biophysica Acta, 584 (1979) 33--42 © Elsevier/North-Holland Biomedical Press

BBA 28871 R A T PANCREAS A D E N Y L A T E CYCLASE VI. R O L E O F THE ENZYME IN SECRETIN STIMULATED ENZYME SECRETION

J.J.H.H.M. DE PONT, DINY LUYBEN and S.L. BONTING Department of Biochernistry, University of Nijmegen, Nijrnegen (The Netherlands) (Received August 21st, 1978)

Key words: Cyclic AMP; Secretin; Adenylate cyclase; (Rat pancreas)

Summary 1. The responsiveness of adenylate cyclase and enzyme secretion for secretin and the C-terminal octapeptide of pancreozymin has been investigated in particulate fractions of the pancreas of five different species. 2. The adenylate cyclase is sensitive to the C-terminal octapeptide of pancreozymin in all species investigated. 3. The enzyme is much more sensitive to secretin in rat and cat than in mouse and rabbit, whereas with guinea pig intermediate values are obtained. 4. The enzyme secretion is stimulated by secretin in pancreatic fragments of rat and cat, b u t n o t in those of mouse and rabbit. 5. These results suggest that in species where secretin stimulated enzyme secretion, it does so by stimuiating the adenylate cyclase system.

Introduction In rat pancreas there is an adenylate cyclase system, which is sensitive towards the hormones secretin and pancreozymin in a non-additive way [1--5]. Most of the activity is located in the acinar cells [6]. The different physiological effects of the two hormones on secretory processes makes the assessment of the function of the adenylate cyclase system in the pancreas rather difficult. A role for this enzyme in pancreozymin-stimulated enzyme secretion has been postulated in the past [7--9]. There are some findings that make a primary role in this process unlikely. Cholera toxin, which stimulates the pancreatic adenylate cyclase [10], does n o t evoke enzyme secretion [10,11]. An increase

34 in cyclic AMP content in acinar cells upon application of pancreozymin or its octapeptide can only be found when phosphodiesterase inhibitors are present [6,12], but even then the increase is much smaller than that obtained with secretin. The addition of cyclic AMP, derivatives of this c o m p o u n d or cyclicAMP phosphodiesterase inhibitors to various pancreatic preparations leads in many cases to a stimulation of enzyme secretion, but the results are variable and the level of secretion is always lower than obtained with pancreozymin or acetylcholine (for a review, see Ref. 13). In addition, the concentrations of pancreozymin required for stimulation of adenylate cyclase activity are much higher than those necessary for enzyme secretion. Finally, there is the fact that the adenylate cyclase system can be stimulated by secretin, although it has only a limited effect on the enzyme secretion• An involvement of the adenylate cyclase system in the pancreozymin-stimulated fluid secretion process is also unlikely. This type of fluid secretion differs from the secretin-stimulated fluid secretion in the sense that the former appears to originate from the acinar cells, is independent of the presence of HCO~ and dependent on the presence of extracellular Ca 2÷, and yields a secretion which has a low bicarbonate content [14--16]. The secretin-stimulated fluid secretion originates from the ductular/centroacinar cells, depends on the presence of HCO~, does n o t need extracellular Ca 2* and results in a bicarbonate-rich secretion [ 14,15]. It is thus likely that an adenylate cyclase system, which is sensitive to both hormones and is located in the acinar cells, is involved in either one of these fluid secretion processes. A third possibility is that the enzyme system would play a role in the secretin-stimulated enzyme secretion process, which requires relatively high concentrations of secretin [10,12,16]. The same system could also function in the enzyme secretion evoked by very high concentrations of pancreozymin, while for the stimulation by pancreozymin in low concentration an increase in cytoplasmic calcium level would primarily be involved (for a review, see Ref. 13). We have investigated this possibility by a comparative study of five different species, in which we have determined the effects of secretin on both adenylate cyclase activity and enzyme secretion. The results presented here show that secretin only stimulates enzyme secretion in the species, where it also stimulates the adenylate cyclase system. This supports the hypothesis that the adenylate cyclase system is involved in the secretin-stimulated enzyme secretion process• Materials and Methods

Animal preparations. The animals used in this study, Wistar rats, albino guinea pigs, Swiss Cpb : SE mice, New Zealand white rabbits and cats, are of either sex and are fed ad libitum. The animals are killed by decapitation, with the exception of the cats, where cardiac puncture is applied. The pancreas is immediately removed and freed from adhering fat. It is then used for adenylate cyclase assays or enzyme secretion measurements. Adenylate cyclase assay. The pancreas is minced with scissors and homogenized in a loosely fitting glass Potter-Elvehjem homogenizer in 4 vols. of a solution, containing 10 mM Tris-HC1/2.5 mM MGC12/2.5 mM Na2EDTA and 0.2 mg/

35 ml soybean trypsin inhibitor, adjusted to pH 7.4. The homogenate is filtered through four layers of medical gauze and centrifuged at 8000 × g for 5 min at 0°C. The pellet is washed twice with 5 vols. and then suspended in 3 vols. of the same solution. The resulting suspension is immediately used for the adenylate cyclase assay. Protein is determined in an aliquot of the suspension according to Lowry et al. [20], using bovine serum albumin as a standard. The average protein content of the suspension is 6--15 mg/ml. For each experiment one pancreas is used, except for the mouse, where five are pooled. The adenylate cyclase assay is carried out according to Rutten et al. [1], with the exception that when hormones are present 1 mg/ml phosphatidylserine is added to the incubation medium in order to protect the hormone receptors [3]. In this m e t h o d [1] cyclic AMP formed is separated from unconverted ATP by thin-layer chromatography on Chromar-Sheet 500 (Malinckrodt Chemical Works, St. Louis, MO, U.S.A.). In later experiments, when ChromarSheet was not available anymore, the separation of cyclic AMP from ATP is carried out according to Salomon et al. [ 21], using two successive column chromatographic procedures on Dowex 50 W-X4 cation-exchange resin and neutral alumina. The recovery of the chromatographic procedure is determined by adding approx. 3000 cpm cyclic [8-3H]AMP to each tube before centrifugation. Enzyme secretion measurement. The enzyme secretion is measured by means of a pulse chase method. For each experiment approx, nine fragments of 100-200 mg wet weight are cut from pancreas from one cat, one or two rabbits, two guinea pigs, two or three rats, or five or six mice. The fragments are incubated together in 2 ml of a medium containing (in mM): Na ÷, 144; K ÷, 4.9; Ca 2+, 2.5; Mg2+, 1.2; HCO~, 25; HPO~, 1.2; CI-, 131 and glucose, 5.5, and an amino acid mixture according to Eagle [22] with L-leucine omitted. The pH is adjusted to 7.4 and the medium is continuously gassed with O2/CO2 (95 : 5, v/v} and is maintained at 37°C. The pulse period is initiated by addition of 100/~Ci L-[3H]leucine. After 15 min of incubation the fragments are washed three times with the same incubation m e d i u m but with 0.4 mM L-leucine replacing L[3H]leucine. The fragments are then incubation for 3 h in this medium in order to allow incorporation of the newly formed proteins into mature zymogen granules. Thereafter each fragment is incubated separately in 2.5 ml of the non-radioactive incubation medium. After 0, 20, 40, 60 and 80 min incubation two samples of 200 gl are taken from the incubation medium and replaced by 400 pl fresh medium. After 40 min incubation either 1 • 10 -s M carbachol (carbamylcholine} or 3 • 10 -7 M secretin is added to the fragments. After the experiment 50 pl of an 8 mg/ml solution of bovine serum albumin is added to each of the medium samples to facilitate subsequent protein precipitation. Each fragment is then homogenized in 500 pl water and two 20-pl samples are taken. 2 ml 10% (w/v) trichloroacetic acid is added to each sample, whereupon the samples are stored overnight in the refrigerator. Next morning the samples are filtered by suction over a Selectron AE 95 filter (1.2 pm pore size, Schleicher and Schiill, Dassel, F.R.G.). After washing the filters three times with 3 ml icecold trichloroacetic acid, they are placed in a liquid scintillation vessel to which 1 ml 0.5 M NaOH is added. After 2 h 10 ml scintillation fluid (Pico-Fluor TM,

36 Packard) is added, and the samples are counted in a liquid scintillation analyser. By adding the radioactivity of the samples (after correction for sampling and readdition of fresh medium) to that of the final homogenate the radioactivity present in each slice at different times can be calculated. This yields the amount of protein secreted in each 20 min period, expressed as percent of the initial amount present in the slice at t = 0. Materials. Bovine serum albumin (Cohn fraction V) and soybean trypsin inhibitor are from Sigma Chemical Co., St. Louis, MO, U.S.A. Synthetic secretin is a kind gift of Dr. H. Beyerman (Department of Organic Chemistry, Technical University, Delft, The Netherlands), while the C-terminal octapeptide is generously donated by Dr. M. Ondetti (The Squibb Institute for Medical Research, Princeton, NJ, U.S.A. [~-32p]ATP (5--10 Ci/mmol), cyclic [8-3H] AMP (20--30 Ci/mmol) and L-[4,5-3H]leucine (1 Ci/mmol) are obtained from The Radiochemical Centre, Amersham, U.K. Carbachol, the carbamyl analogue of acetylcholine, is purchased from Brocades-ACF Holland. Phosphatidylserine is prepared from bovine white matter according to Sanders [23]. All other chemicals are of reagent grade. Results

Adenylate cyclase activities Fig. la--e shows the dose-response curves for the effect of secretin and pancreozymin-C-octapeptide on the adenylate cyclase activation in the 5 min 8000 × g pellets of the pancreas of five different species. The absolute value of the specific fluoride-stimulated adenylate cyclase activity differs from species to species (upper line, Table I). The shape of the dose-response curve for pancreozymin-C-octapeptide is approximately the same in all species. The curves reach half of the maximal obtainable activity at a concentration in the order of 1 • 10 -7 M (somewhat higher in the cat), whereas at higher concentrations there tends to be a leveling off of the response. The maximal activity obtained with pancreozymin-C-octapeptide in percent of the fluoride-stimulated activity varies from 45% (cat) to 110% (guinea pig). The ability of synthetic secretin to stimulate the adenylate cyclase activity differs markedly from species to species. In rat and cat pancreas secretin is a somewhat better stimulus than pancreozymin-C-octapeptide, in guinea pig it stimulates but less efficiently than pancreozymin-C-octapeptide, whereas in mouse and rabbit pancreas it stimulates very weakly. In Table I the activities obtained without stimulus and with 1 • 10 -6 M secretin and pancreozymin-C-octapeptide are expressed as percent of the fluoridestimulated activity. This table shows again the discrepancy between the effects of pancreozymin-C-octapeptide and secretin. In addition it shows that the secretin-stimulated activities are significantly (P < 0.05) higher than the basal activities in all species, even in mouse and rabbit. Enzyme secretion Enzyme secretion from fragments of the different species is measured after 15 min pulse labeling with L-[3H]leucine, followed by a 3 h chase period in non-radioactive medium. The secretion of 3H-labeled protein is measured in

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33 Biochimica et Biophysica Acta, 584 (1979) 33--42 © Elsevier/North-Holland Biomedical Press BBA 28871 R A T PANCREAS A D E N Y L A T E CYCLASE VI...
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