(Received 6 August 1979)

Transcription

1 J. Phyoiol. (1980), 303, pp With 2 text-figurew Printed in Great Britain PARALLEL SECRETION OF ENZYMES BY THE RABBIT PANCREAS BY E. L. GILLILAND* AND G. GLAZER From the Academic Surgical Unit, St. Mary's Hospital Medical School, London W.2 (Received 6 August 1979) SUMMARY 1. The purpose of this study was to determine whether or not the secretion of pancreatic enzymes by the rabbit remained proportional (parallel) after acute stimulation. 2. Hourly samples of pancreatic juice were collected from anaesthetized rabbits, each sample being analysed for volume, protein, amylase, trypsinogen and chymotrypsinogen. 3. Four groups of animals were studied for 2 hr before and 2 hr after stimulation with either saline (controls), methacholine, sincalide (C-terminal active octa-peptide of CCK-PZ) or CCK-PZ. 4. Despite a rise in protein output of more than 100 % in the hour after stimulation in all experimental groups, there was no change in the specific activity (u./mg protein) of the three enzymes monitored, and the ratios of these enzymes to each other remained constant. 5. These results in the in vivo rabbit confirm our previous observation of parallel secretion in the in vitro rabbit pancreas; they are at variance with other studies in the rabbit (predominantly in vitro but also in vivo) which showed non-parallel secretion after CCK-PZ stimulation. 6. Our results tend to support the theory of mass transport and parallel secretion of pancreatic enzymes though no firm deductions on intracellular events can be made from juice analysis alone. INTRODUCTION There are two principal theories describing the process of enzyme secretion by the pancreas. One theory (cisternal packaging - exocytosis theory) proposes that enzymes are synthesized, stored and transported across the acinar cell through a series of intracellular, but extracytoplasmic, compartments; the mixture of enzymes within this system remains constant, and the proportion of different enzymes discharged into the duct remains the same (parallel) over the short term, despite stimulation of the gland (Palade, 1975; Scheele & Palade, 1975). The other theory suggests that in addition to this compartment enzymes may also be pooled and transferred within the cytoplasm of the acinar cell and that under certain conditions the discharge of enzymes from the pancreas may be non-proportional (Rothman, 1975; Rothman 1977). * Wellcome Research Fellow /80/ $ The Physiological Society 2 PHY 303

2 34 E. L. GILLILAND AND G. GLAZER The in vitro rabbit pancreas has been used to examine this problem with conilicting results. In the original work with this model, Rothman (1967) found nonparallel secretion of trypsinogen relative to chymotrypsinogen following stimulation with cholecystokinin-pancreozymin (CCK-PZ) but this finding could not be confirmed (Steer & Glazer, 1976). Rothman subsequently repeated his experiments (Rothman & Wilking, 1978) and again obtained non-parallel transport of trypsinogen relative to chymotrypsinogen with the whole molecule of CCK-PZ, but parallel transport of these enzymes with sincalide (the synthetic COOH-terminal active octapeptide of CCK-PZ) and methacholine stimulation; this led him to the hypothesis that the whole molecule of CCK-PZ acted as a stimulator and inhibitor of the secretion of these enzymes by differential binding at various receptor sites. Further repetition of these experiments using similar doses of CCK-PZ (Steer & Manabe, 1979) has again demonstrated parallel discharge of enzymes and so the situation remains confused. As the rabbit pancreas may react differently in an organ bath and in vivo, we have re-examined this problem, using anaesthetized rabbits, and have demonstrated parallel secretion of trypsinogen, chymotrypsinogen and amylase both in control animals and in animals stimulated with methacholine, CCK-PZ and sincalide. METHODS New Zealand white rabbits (2-4 kg) were fasted for 24 h except for water, and then anaesthetized with i.p. sodium pentobarbitone. A midline laparotomy was performed and the pancreatic duct was cannulated extraduodenally with fine polyethylene tubing (length 25 cm; o.d mm, i.d. 0 5 mm). The animals were maintained in an isothermic environment and breathing spontaneously. The polyethylene tubing was led out of the abdomen and after a twohour period of equilibration, hourly samples of pancreatic juice were collected in tared capped vessels kept on ice. A needle was inserted into an ear vein to allow the intravenous injection of various stimulants. Four groups of experiments were performed. Group A - controls (n = 6); pancreatic juice samples were collected for four one-hour periods; at the beginning of the third hour of juice collection isotonic saline (3 ml./kg body wt.) was infused intravenously over a ten-minute period. In the other groups of experiments, a pancreatic stimulant dissolved in an equivalent volume of saline was infused over the same period of time. Thus in all groups of experiments there were two one-hour periods of pre-stimulatory juice collection, and two one-hour periods of poststimulatory juice collection. The number of experiments performed, and the type and dose of stimulants used were as follows: group B - methacholine (n = 6); 6 #sg/kg body wt.; group C - sincalide (the synthetic C-terminal octapeptide of CCK-PZ) (n = 8), 0 06,ug/kg body wt.; group D - CCK-PZ (n = 8), 2 Ivy dog units/kg body wt. The doses used were found to cause an increase of at least 100% in protein output. (Sincalide (SQ 1984) was obtained from E. R. Squibb Ltd. Purified CCK-PZ was obtained from the GIH Research Institute, Chemistry Department, Karolinska Institutet, Stockholm, Sweden. Methacholine chloride (acetyl-bmethyl choline chloride) was obtained from Sigma Chemical Company.) A88ay method Protein concentration was determined by the method of Lowry (Lowry, Rosebrough, Farr & Randall, 1951) using bovine serum albumin (BSA) as a standard, and a-amylase was assayed by the method of Bernfeld, (Bernfeld, 1955). One unit of amylase activity is defined as that which liberated 1 mg equivalent of maltose from starch in 3 min at 30 IC.

3 PARALLEL ENZYME SECRETION IN RABBIT PANCREAS 35 Activation of zymogens The conditions for optimal activation of trypsinogen and chymotrypsinogen in rabbit pancreatic juice have been reported (Glazer & Steer, 1977). In brief, the activation of trypsinogen and chymotrypsinogen in the current experiments was accomplished as follows. The pancreatic juice was initially diluted to a final protein concentration of 50,sg/ml. in 50 m-mole Tris-HCl buffer, ph 8-1, containing CaCl2 50 m-mole and BSA, 100,ug/ml. For trypsinogen activation, one portion of the diluted pancreatic juice was added to an equal volume of an 0 05 #sg/ml. aqueous solution of purified porcine enterokinase (Maroux, Baratti & Densuelle, 1971). This mixture was allowed to incubate for 2 hr at 30 0C before aliquots were removed for trypsin assay. For chymotrypsinogen activation, another portion of the pancreatic juice was used, which was diluted to a final protein concentration of 25,tg/ml. in 50 m-mole Tris-HCl buffer, ph 8-1, containing CaCl2 50 m-mole and BSA 100 jug/ml. To each 0-2 ml. of this diluted juice, 1 /1d. of a 1 mg/ml. solution of crystalline bovine trypsin (Sigma Chemical Co TAME u./mg; TAME = p- toluene sulphonyl-l-arginine methyl ester) in 1 m-mole HCl were added. The resulting solution was incubated for 2 hr at 4 'C and then aliquots were removed for chymotrypsin assay. The concentration of juice protein used in the chymotrypsinogen activation was different to that in our previous reports in which the final juice protein concentration was double that used in the present group of experiments. The activation procedure was otherwise the same, the the reason for this change was a modification in the assay of chymotrypsin activity (see below). As8ay of trypsin and chymotryp8in activity These assays were based on the method of Hummel (Hummel, 1959) using a Pye Unicam SP 1800 spectrophotometer, thermostated at C and recording the initial (linear) phase of substrate hydrolysis. For trypsin determination 0-1 ml. of the diluted and activated juice was added to 0-9 ml. of a mixture containing 0-2 M-Tris-HCl, ph 8-1, 0 01 M-CaCl2 and TAME, mg/ml. and substrate hydrolysis measured at 247 nm. Chymotrypsin activity was measured by adding 0.1 ml. of the diluted and activated juice to 0-9 ml. of a mixture of 0-08 M-Tris- HCl, ph 7-8, 0 05 M-CaCl2, 1% methanol, 0-1% Triton-X-100 and BTEE mg/ml. (Nbenzoyl-L-tyrosine ethyl ester); the addition of Triton-X-100 results in greater sensitivity of the assay (Rao & Lombardi, 1975). Substrate hydrolysis was recorded at 256 nm. Standard formulae were used to convert the change in optical density/min into trypsin and chymotrypsin units. Results were expressed as u./hr (output), and as u./mg juice protein (specific enzyme activity). The increased sensitivity of the chymotrypsin assay results in different units when compared to our previous work, and consequently the numerical value of enzyme ratios are different to those previously reported (Steer & Glazer, 1976). AnalySi8 of data All assay results reported in this publication were performed in duplicate and the mean result used for the final analysis. Preliminary studies on juice protein and enzyme assays confirmed the accuracy of these measurements to within + 5 % when performed in triplicate. For each experiment, collections were made both before and after the injection of a stimulating agent, and thus each animal served as its own control. Within each experimental group, values for juice volume, protein concentration, protein output and each individual enzyme output were compared, using a paired t test; the second hour of basal juice collection (hour 2) was compared with the first hour of basal collection (hour 1) and with both the hours after stimulation (hours 3 and 4). Protein output and individual enzyme output were compared between the groups in identical time periods using an unpaired t test. The ratio of enzyme outputs and the specific activity of each enzyme (expressed in u./mg juice protein) were separately analysed during the four collection hours within each group by a two-way analysis of variance (Hartley, 1960). Changes were considered as statistically significant with a P value of less than

4 36 E. L. GILLILAND AND G. GLAZER RESULTS The values for juice volume, protein concentration and protein output are illustrated in Fig. 1, and the individual enzyme outputs in Fig. 2. Controls (saline) Volume did not change significantly, but protein concentration fell in hours 3 and 4 (P < 0-05). Protein output fell in hour 3 (P < 0 05) as did the output of each enzyme (P < 0-05). In hour 4 there was no significant differences in protein or enzyme output compared with hour 2. Saline (6) Sincalide (8) Methacholine (6) CCK-PZ (8) 1000 I E 400 *5200 _ so ' t50 cm! ~- AL"J Collection period Fig. 1. Volume, protein concentration and protein output in hourly samples of pancreatic juice. Results expressed as mean + s.e. Methacholine Volume increased after methacholine stimulation, this being significant in hour 4 (P < 0 05). Protein concentration, protein output and individual enzyme outputs rose significantly in the hour after stimulation (P < 0 001). All values returned to basal levels in the second hour after stimulation (hour 4). Sincalide Volume, protein concentration, protein output and individual enzyme outputs all rose significantly in hour 3 (P < 0.001). In the second hour after stimulation (hour 4) values returned to basal levels.

5 PARALLEL ENZYME SECRETION IN RABBIT PANCREAS 37 CCK-PZ A similar pattern of results were obtained with the whole peptide molecule of CCK-PZ as were obtained with sincalide. Volume, protein concentration, protein output and individual enzyme outputs all rose significantly in hour 3 (P < 0001) and fell to basal values in hour 4. Saline (6) Sincalide (8) Methacholine (6) CCK-PZ (8) 1500 I I 1 1, hj + I 500 u 6o b h11 x 8 h l h8 t 2l~ Ant '2`3 4 1'2 34 Collection period Fig. 2., chymotrypsinogen and amylase outputs in hourly samples of pancreatic juice. Results expressed as mean + s.e. Comparison between groups There were no significant differences of protein or enzyme outputs between any of the groups in the basal periods of collection (hours 1 and 2), or in the second hour after stimulation (hour 4). In the first hour after stimulation (hour 3) the output of protein and enzymes was significantly increased over controls in all three groups (P < 0-001). The output of protein and enzymes in hour 3 was significantly higher in the CCK-PZ group when compared with the methacholine (P < 0 05) and sincalide (P < 0-01) groups. There were no significant differences between the sincalide and methacholine groups in hour 3.

6 38 E. L. GILLILAND AND G. GLAZER TABLE 1. Specific activity of trypsinogen, chymotrypsinogen and amylase expressed as mean + Stimulants were infused i.v. for 10 mi at the beginning of hour 3 collection period. Hour Saline *07 (controls) (n = 6) (TAME u./mg protein) *46 + 1* (BTEE u./mg protein) Amylase * (u./mg protein) + 21* Sincalide (n = 8) P72 (TAME u./mg protein) s.e (BTEE u./mg protein) Amylase (u./mg protein) *6 +17*6 +18*2 + 22*2 Methacholine (n = 6) (TAME u./mg protein) (BTEE u./mg protein) s.e Amylase (u./mg protein) CCK-PZ (n = 8) * (TAME u./mg protein) * (BTEE u./mg protein) Amylase (u./mg protein) TABLE 2. Ratios of enzyme outputs in rabbit pancreatic juice expressed as mean ±.E. The mean results represent the means of each individual enzyme ratio for the particular collection period. Saline (controls) (n = 6) Sincalide (n = 8) Methacholine (n = 6) CCK-PZ (n = 8) / / Amylase / Hour 8.E. 8.E *07 3* * *32 +0*34 6*59 6*95 6* *86 +0*92 +0*59 25*6 26* * *85 3*64 3*86 +0* *18 +0' W20 +0*56 +0* *7 + 2* *7 + 2*10 4*20 3*90 3*84 4*12 +0* *20 +0*50 6*25 6*99 6* *44 +0* *42 25*5 26*3 25B * *6 +1* *48 3* *20 + 0*23 + 0*20 + 0* * * * *7 21* *1 +1*7 +1*5 +1*2 +1-6

7 PARALLEL ENZYME SECRETION IN RABBIT PANCREAS 39 Specific activity of enzymes and enzyme ratios The mean specific activity of amylase, trypsinogen and chymotrypsinogen in units/mg juice protein for each collection period in each group are given in Table 1. The ratios of trypsinogen to chymotrypsinogen, a-amylase to trypsinogen and a-amylase to chymotrypsinogen for each collection period are given in Table 2; these are not the ratios of the mean enzyme outputs, but are the mean results of each individual ratio calculated for each animal in each period of collection. A two-way analysis of variance was used to analyse the results of individual enzyme specific activities and enzyme ratios. The sum of squares for individual effects were isolated and their mean square variation was computed. From these values, the F statistic for the individual effects was calculated. The effects of time (and hence intervention by a stimulus) and replication (variation between animals) were thus determined. In all cases, both for enzyme specific activity and for enzyme ratios, there were significant animal variations (P < 0 01). The specific activity of all enzymes remained constant with time within groups, except for chymotrypsinogen, which fell in the second hour after CCK-PZ stimulation, hour 4 (P < 0.05). The only significant variation in enzyme ratios occurred in the first hour of basal collection (hour 1) in the sincalide group (P < 0.05); the variations in enzyme ratios in hours 2, 3 and 4 in the sincalide group were not significantly different from each other. DISCUSSION The results of these experiments in the anaesthetized rabbit show that the secretion of the pancreatic enzymes, amylase, trypsinogen and chymotrypsinogen, remains proportional (parallel) in control animals given saline, and in animals stimulated with either methacholine, sincalide (the C-terminal active octapeptide of CCK-PZ) or the purified whole molecule of CCK-PZ. The specific activities of these enzymes (u./mg juice protein) remained constant in all periods of collection, except for chymotrypsinogen, which was slightly reduced in the second hour after CCK-PZ stimulation (P < 0.05). The ratio of enzymes in this collection period did not however, change significantly as the fall in chymotrypsinogen activity was paralleled by falls in trypsinogen and amylase, though these falls in enzyme activity were not statistically significant. This slight fall in chymotrypsinogen activity occurred after the major protein response to CCK-PZ had subsided. The only period of disproportional secretion of enzymes occurred during the initial unstimulated period (hour 1) in the animals subsequently given sincalide. The ratio of trypsinogen to chymotrypsinogen was different to that in subsequent collection periods in this group (P < 0.05); however, the specific activities of the enzymes were not statistically different and the ratios of amylase to trypsinogen and amylase to chymotrypsinogen did not alter. As this small variation in trypsinogen and chymotrypsinogen secretion was not related to any stimulus of secretion and did not occur in the other groups of animals during the same pre-stimulatory period, it is unlikely to be of any biological significance.

8 40 E. L. GILLILAND AND G. GLAZER These results are thus at variance with those reported by Rothman (1967) in the in-vitro rabbit pancreas, but confirm our findings in the same in-vitro model of parallel secretion of amylase, trypsinogen and chymotrypsinogen following stimulation by either methacholine, sincalide or CCK-PZ (Steer & Glazer, 1976). In the original work with this organ bath preparation Rothman (1967) had demonstrated non-proportional secretion of trypsinogen relative to chymotrypsinogen after CCK- PZ stimulation, but not after methacholine; in addition, a similar response was reported from three anaesthetized rabbits, Using the same in vitro model (Steer & Glazer, 1976), but different enzyme techniques which gave linear responses with sample size (Glazer & Steer, 1977), we were unable to find any evidence of nonparallel discharge of enzymes. This work led to further studies by Rothman (Rothman & Wilking, 1978) which confirmed the validity of our assay techniques, but which again showed that the whole peptide molecule of CCK-PZ (in both its impure and relatively pure forms) resulted in an average fifteenfold increase of trypsinogen, whilst chymotrypsinogen was only increased fourfold. As this effect was not observed after stimulation with methacholine or sincalide the authors proposed that the whole peptide molecule of CCK-PZ (as opposed to its active octapeptide radicle) was acting as a partial inhibitor of chymotrypsinogen through a second receptor site. The difference between these results and those reported by us (Steer & Glazer, 1976) was attributed by Rothman and his colleague (Rothman & Wilking, 1978) to the fact that the dose of CCK-PZ used in our experiments did not result in a very large protein response. This work was therefore repeated using identical techniques and doses of CCK-PZ (pure 39 amino acid polypeptide provided by Professor Mutt of the Karolinska Institutet) sufficient to cause a three-to-fourfold increase in protein output (Steer & Manabe, 1979); once again the results demonstrated only proportional secretion of enzymes. It is difficult to reconcile these results with those of Rothman and Wilking. There is no evidence from the in vivo experiments reported in this communication of an inhibitory effect of CCK-PZ on chymotrypsinogen secretion, nor of any nonparallel secretion of enzymes after cholinergic or hormonal stimulation. This finding does not necessarily exclude Rothman's previous theory (Rothman, 1975) of transport of digestive enzymes both in the cytoplasm and cisternal space within the acinar cell. However, as the enzymes are discharged in parallel it follows that the mixture of enzymes within these two compartments must be identical or that the discharge of enzymes from both spaces is affected in a parallel fashion by secretary stimuli. The debate over parallel Vs non-parallel discharge of pancreatic enzymes continues and is one of fundamental biological importance. Some workers have attempted to reconcile the opposing views by showing the exocrine pancreas to be functionally heterogenous (Malaisse-Lagae, Ravazzola, Robberecht, Vandermeers, Malaisse & Orci, 1975), whilst others (Daghorn & Estival, 1979) believe from experiments on rat pancreatic lobules and the rat pancreas in vivo and in vitro, that methodological differences, giving rise to different basal levels of protein secretion, can explain certain discrepancies in the literature: such arguments, however, cannot reconcile different results obtained in identical experiments. In an extensive review of the

9 PARALLEL ENZYME SECRETION IN RABBIT PANCREAS 41 work previously published on this subject, Case (1978) concluded, with some reservation, that the evidence in favour of non-parallel discharge of pancreatic enzymes was scant. The results of our present experiments in the rabbit, showing only parallel discharge of enzymes do not contradict his conclusion. However, there remains uncertainty which will only be resolved by further experimentation. The authors wish to thank Professor H. A. F. Dudley for helpful advice in this work. Mr E. L. Gilliland was supported by a Wellcome Research Fellowship and we gratefully acknowledge the support given by the Wellcome Trust. REFERENCES BERNFELD, P. (1955). Amylase a and fi. Meth. Enzym. 1, CAsE, R. M. (1978). Synthesis, intracellular transport and discharge of exportable proteins in the pancreatic acinar cells and other cells. Biol. Rev. 53, DAGHORN, J. C. & EsTIvAL, A. (1979). Non-parallel enzyme secretion from rat pancreas in vitro studies. J. Phy8iol. 290, GLAZER, G. & STEER, M. L. (1977). Requirements for activation of trypsinogen and chymotrypsinogen in rabbit pancreatic juice. Analyt. Biochem. 77, HARTLEY, H. 0. (1960). Analysis of variance. In Mathematical Method8 for Digital Computers, ed. RAUSTON, A. & Wnyr, H., New York: Wiley. pp HUMMEL, B. C. W. (1959). A modified spectrophotometric determination of chymotrypsin, trypsin and thrombin. Can. J. Biochem. Physiol. 37, LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with the folin phenol reagent. J. biol. Chem. 193, MALAissE-LAGAE, F., RAVAZZOLA, M., ROBBERECHT, P., VANDERMEERs, A., MALAISSE, W. J. & ORcI, L. (1975). Exocrine pancreas. Evidence of topographic partition of secretary function. Science, N.Y., 190, MAROUX, S., BARATrI, J. & DESNUELLE, P. (1971). Purification and specificity of porcine enterokinase. J. biol. Chem. 246, PALADE, G. E. (1975). Intracellular aspects of the process of protein synthesis. Science, N. Y. 189, RAO, K. N. & Lom-BARDI, B. (1975). Substrate solubilization for the Hummel a chymotrypsin assay. Analyt. Biochem. 65, ROTHMAN, S. S. (1967). 'Non-parallel transport' of enzyme protein by the pancreas. Nature, Lond. 213, ROTHMAN, S. S. (1975). Protein transport by the pancreas. Science, N.Y. 190, ROTHMAN, S. S. (1977). The digestive enzymes of the pancreas; a mixture of inconstant proportions. A. Rev. Physiol. 39, ROTHMAN, S. S. & WILKING, H. (1978). Differential rates of digestive enzyme transport in the presence of cholecystokinin-pancreozymin. J. biol. Chem. 253, SCHEELE, G. A. & PALADE, G. E. (1975). Studies on the guinea pig pancreas. Parallel discharge of exocrine enzyme activities. J. biol. Chem. 250, STEER, M. L. & GiTaZx, G. (1976). Parallel secretion of digestive enzymes by the in vitro rabbit pancreas. Am. J. Physiol. 231, STEER, M. L. & MANABE, T. (1979). Cholecystokinin-pancreozymin induces parallel discharge of digestive enzymes from the in vitro rabbit pancreas. J. biol. Chem. 254,