Proc. Natl. Acad. Sci. USA Vol. 92, pp. 2553-2557, March 1995 Physiology

Pancreatic digestive 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 , 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 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 . 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). Enzymes proximately doubled. CCK and MCh differed subtly but were also separated by two-dimensional 'isoelectric focusing significantly from each other in their effects on the overall and SDS electrophoresis by the method of Scheele (3). As secreted percentages of amylase, chymotrypsinogen, and tryp- expected in the two-dimensional system, lipase I was a unique sinogen. band, amylase separated into a and y bands, and chymo- Stimulation with CCK and MCh caused major changes in the trypsinogen and trypsinogen showed acidic and basic forms. period-to-period variability of the enzyme percentages (Table Testing of Systematic Errors. To test the inherent variability 1), as confirmed by an F test that estimated the significance of and error in the measurements of the enzyme bands in the gel differences in the standard deviation of the enzyme outputs. system, control gels were prepared from each sample group from After CCK stimulation, the variability of three of the four each individual animal by mixing an equalvolume ofthe secretion enzymes was less than under basal conditions. Significant from all 36 collection periods and redistributing them into 36 changes in overall variation were also evident for lipase and identical aliquots for separation and densitometry, thereby caus- trypsinogen when CCK and MCh treatments were compared. ing loss of any true variation in enzyme proportions. The residual Despite equal protein outputs, the variability after CCK systematic variability was then determined by using controls treatment'was less than that after MCh in every case. The under identical experimental conditions. variation in enzyme percentages was not due to systematic Statistical Analysis. Statistical analyses were done by using errors in their determination; under all experimental condi- SAS programs (SAS Institute, Cary, NC). Means and F testing tions, the overall variability in enzyme percentage was at least were done by analysis-of-variance programs and were applied 2-fold greater than that due to systematic error (P < 0.005; to both inter- and intraexperimental comparisons. F and P data not shown). values were considered not significant if >0.05. Enzyme out- The mean ratios of the percentages of the four hydrolases puts and ratios were also independently compared by corre- differed greatly (Table 1). The ratio of lipase to amylase lation and regression analysis and tested for homogeneity of dropped to -33% of the basal value after MCh and to 50% slopes by the least-squares method. Cyclic variations in enzyme after CCK. The ratios of lipase to chymotrypsinogen and

Origin - kDa

LIP I - 66 a-AMY - LIP II - 45 CPASE B+A - _36 FIG. 1. Protein output (mean ± CC- SEM) (periods are 5 min long ex- 0 cept periods 1-3, which are 20 min 29 CHTG - 29 long) during continuous infusion of TRPG - 24 CCK (A), MCh (0), or Ringer's lactate (O), beginning at arrow, as described in the text; n = 4 for each : -20.1 series. (Inset) SDS/PAGE gel of secretion from a single 5-min col- lection period (MCh) and molecu- lar mass standards (MWT). Abbre- _1414.22 viations are as in Table 1, except 10 20 30 40 that CPASE is carboxypeptidase Collection period Front - and TRPG is trypsinogen. Downloaded by guest on October 1, 2021 Physiology: Adelson et aL Proc. NatL Acad Sci USA 92 (1995) 2555

Table 1. Enzyme percentages and enzyme ratios in basal and Basal A stimulated pancreas m-O.36±0.03 300 - r2=0.59 Value 20 /S' CCK** o m-0.2±0.02 Parameter Basal MCh CCK ^ 200 q/ / A-0 ~~~~~86 Enzyme U, 20 40 60S* * percentage 100 8. -X - m=0.19±0.02 Lip I, % 8.25 ± 0.53 5.19 ± 0.32*t 5.84 ± 0.19*t§ * w_ @0wo 6 Amyl, % 6.43 ± 0.25 10.59 ± 0.30* 8.20 ± 0.24*t# r2-0.37 ChTg, % 5.41 ± 0.24 10.42 ± 0.34*t 12.72 ± 0.24*t Tg, % 9.52 ± 0.45 17.26 ± 0.45* 19.15 ± 0.30*tt§ 100 300 500 700 Amylase, ,ug Enzyme CT, ratio Lip/Amyl 1.43 ± 0.09 0.54 ± 0.04*t 0.77 ± 0.03*14 700 .Baal m=0.47±O.O1 B Lip/ChTg 2.04 ± 0.19 0.61 ± 0.06*t 0.52 ± 0.04*t§ *-40 r2-0.98 ± ± ± * m=0.78±O.03 Lip/Tg 1.11 0.08 0.35 0.04*t 0.33 0.04*t§ t 500 Amyl/ChTg 1.46 ± 0.12 1.14 ± 0.05*t 0.69 ± 0.03*tt§ --r2o.s7r Amyl/Tg 0.77 ± 0.05 0.65 ± 0.02*t 0.44 ± 0.02*tt§ 20 04060 t ChTg/Tg 0.60 ± 0.02 0.63 ± 0.02 0.66 ± 0.01*t§ i30 . 3 * ~~~~C:CK** Lip I, lipase I; Amyl, amylase; ChTg, chymotrypsinogen; Tg, ;l- m-O.46±0.01 trypsinogen. Enzyme percentages and ratios are given as the mean ± 100 ,-. . . . i . r2-.94 SEM of all 36 stimulated periods for four animals. *, P < 0.05 vs. basal; 4:, P < 0.05 vs. MCh; t, F < 0.05 vs. basal; §, F < 0.05 vs. MCh. 100 300 500 700 900 trypsinogen dropped 3- to 4-fold after treatment with either Trypsinogen, ,ug secretagogue. The ratio of amylase to each protease was dimin- FIG. 2. Examples of pairwise regression and correlation analyses of ished as well. In contrast, the chymotrypsinogen/trypsinogen enzyme outputs for all 36 periods for all animals (for each series, n = ratio changed little after secretagogue stimulation. 4) during constant CCK or MCh infusion. Slopes (m) and correlation The overall variation in the enzyme ratios was also highly coefficients (r2) were derived and tested for heterogeneity. (Insets) sensitive to the type of stimulus. In 9 of the 10 enzyme Infusion of Ringer's lactate alone plotted on a proportionally equal scale to allow comparison of slopes. (A) Lipase vs. amylase. (B) Amy- pairs-except the chymotrypsinogen/trypsinogen ratio-the lase vs. trypsinogen. *, P < 0.05 vs. basal; **, P < 0.05 vs. MCh for SEM for the ratio was significantly lower during continuous heterogeneity of slopes. MCh stimulation than under basal conditions and differed in every case after CCK. CCK also differed in variation from Rapid cyclic variations in enzyme proportions and ratios MCh in each instance except for the lipase/amylase pair. Thus, were examined. The period-to-period changes in the secreted the period-to-period variations in ratio were decreased by enzyme percentage of the four enzyme species under different stimulation with either secretagogue, even though protein experimental conditions, each in a typical animal, are depicted output was highly stimulated, which would usually be expected in Fig. 3; the ratios of the enzyme percentages for the same to lead to increased variation; despite equal protein outputs, animals are presented in Fig. 4. Under basal or MCh- CCK stimulation showed a consistently lower ratio variability stimulated conditions, the period-to-period changes in the than MCh (P < 0.05, data not shown). enzyme percentage of lipase I were cyclic and of large mag- Examples of regression and correlation analyses of the nitude. Cyclic bursts of lipase I enrichment (average duration, period-by-period output data for two of the six enzyme pairs =12 min) occurred wherein the lipase percentage reached are shown in Fig. 2. Other data and slopes were similar and are 8-10 times the minimal level observed during the same not presented here due to space limitations. All regression experiment. Amylase, chymotrypsinogen, and trypsinogen be- slopes were significant at P < 0.0001. The slopes of lipase vs. haved similarly. The cyclicity in the experimental samples was amylase, chymotrypsinogen, or trypsinogen did not differ directly confirmed by Fourier analysis, which showed a signif- significantly when either of the secretagogues was compared icant rhythmic peak of the individual enzyme percentages and with basal secretion due to large period-to-period variations in their ratios between 10 and 15 min. In contrast, constant lipase output. In contrast, the slopes of lipase vs. all other stimulation of the gland with CCK resulted in a change in the enzymes differed in every case when CCK infusion was secretory pattern whereby the magnitude of the cyclic varia- compared to MCh (P < 0.0001 in each case). Regression tions was greatly reduced, but with apparent preservation of analysis of amylase vs. chymotrypsinogen showed significant the cyclicity. Additional experiments (not shown) showed heterogeneity for CCK vs. MCh; for amylase vs. trypsinogen, exactly the same features. The enzyme ratios (Fig. 4) also all three slopes were different, as were slopes for the chymo- showed a regular cyclic periodicity, wherein the ratios for trypsinogen/trypsinogen pair, where the enzyme outputs were lipase to amylase and the proteases varied by >30-fold. The so highly correlated to each other that even a minor difference chymotrypsinogen/trypsinogen ratio was relatively stable un- in slope was significant [for CCK, m = 0.71 + 0.01 and r2 = der all conditions, demonstrating that parallel secretion of two 0.99; for MCh, m = 0.78 ± 0.03 and r2 = 0.88]. The CCK- enzymes could be observed while nonparallel secretion was stimulated slope was >2.5 times the MCh-stimulated slope for observed with other pairs. lipase vs. amylase (Fig. 2A). For amylase vs. chymotrypsinogen Since the period-to-period variability could, theoretically, be and trypsinogen (Fig. 2B), the CCK slopes were =60% of the due to true cyclicity in these parameters or to artifactual MCh slopes, indicating an enrichment of proteases during fluctuations due to variability inherent in the measurement of CCK stimulation. The chymotrypsinogen/trypsinogen pair the densities of protein bands in the stained gels, we explicitly was highly correlated under all conditions. Even in the low controlled for such potential artifacts. The mean enzyme output range, as defined by the range of the basal samples, 8 percentages of the blended controls were found to be virtually of 10 slopes differed significantly between CCK and MCh; identical to the means of the experimental samples, as ex- other differences in slope were evident when basal and stim- pected, but the controls showed very little variability from ulated conditions were compared. sample to sample (Figs. 3 and 4). Further, the standard Downloaded by guest on October 1, 2021 2556 Physiology: Adelson et al. Proc. NatL Acad. Sci USA 92 (1995)

Lipase Amylase ChTg Tg

U, C)

U a) Ct C.) > CZ C) U C)

Li-

30 10 Collection period FIG. 3. Examples of enzyme percentages on a period-by-period basis in three animals. Constant secretagogue infusions and data normalization were carried out. Cyclicity is readily apparent for all enzymes in the series under both basal and MCh-stimulated conditions and was confirmed by Fourier analysis. The magnitude of the cyclicity in enzyme percentages can be seen to have greatly diminished after CCK stimulation. Each series consists of experimental points (A) accompanied by an explicit control for systematic errors (0). The dotted line indicates missing data points. ChTg, chymotrypsinogen; Tg, trypsinogen. deviation of the controls from the enzyme percentage of the of the output of a single enzyme to overall protein secretion, mean, as determined by the F test, was significantly lower for due to the effects of time or a stimulus; this would be all basal and MCh-stimulated enzymes and for three out of concordant with Pavlov's original observations that the feeding four of the CCK-stimulated samples compared to experimen- of specific digestive substrates rapidly altered the proportions of tal variability. Thus, the rapid fluctuations in enzyme percent- ages and ratios observed were not the result of systematic the secreted "digestive ferments" (18). After Pavlov, the question errors but, instead, were due to real changes in the proportions of the existence of nonparallel secretion shifted back and forth of the digestive enzymes. repeatedly between affirmation and denial. Recently, nonparallel secretion has been viewed as conflicting with the exocytosis DISCUSSION mechanism of enzyme secretion wherein prestored digestive Nonparallel pancreatic secretion can be defined as a change in enzymes are secreted in parallel by a single secretory pathway the ratio of the outputs of a pair or the ratio from the homogeneous pancreas (4). In fact, nonparallel secre- Lip/Amy Lip/ChTg Lip/Tg Amy/ChTg Amy/Tg ChTg/Tg i00-a .-t 200- CZ 100 m

U) C., 200

> 100 "A .t -, 2200 At;, V

100 P%P- uV

10 30 10 30 10 30 10 30 10 30 10 30 Collection period FIG. 4. Ratios of the enzyme percentages for each enzyme pair shown in Fig. 3 are represented. The cyclicity is apparent. The relative constancy of the ratio of chymotrypsinogen (ChTg) to trypsinogen (Tg) is apparent in comparison to all other ratios. The period-by-period variation in ratio of the experimental points (A) under CCK stimulation is seen to nearly diminish to nearly equal the minimal variability observed with the controls (-). Lip, lipase; Amy, amylase. Downloaded by guest on October 1, 2021 Physiology: Adelson et al. Proc. Natl. Acad Sci. USA 92 (1995) 2557 tion and the exocytosis mechanism need not be viewed as direct function of the stimulus and not of the level of protein incompatible (8), since the digestive enzymes have been shown to output. This observation therefore supported the concept that be secreted from heterogeneous prepackaged sources within the CCK elicited a shift in the pools contributing to secretion from gland (9, 19). Despite this uniting of the exocytosis mechanism a more heterogeneous group of pools observed under both with nonparallel secretion, the literature continues to indicate basal and MCh-stimulated conditions to more unique or that nonparallel secretion of digestive enzymes by the pancreas is homogeneous pools after CCK stimulation (19). Distinct dif- neither settled nor accepted (19-23). ferences between CCK- and acetylcholine-induced intracellu- Two of the strongest objections raised to nonparallel secre- lar Ca2+ signals in acinar cells have been described, with tion have concerned putative errors in the measurement of differences in both the type and localization of the intracellular enzymatic activity due to potential problems with zymogen Ca2+ signal (26, 27). activation or the presence of extraneous protein in the assay Traditional reasoning has long accepted that pancreatic (4-7). It has been proposed that the separation of secreted enzyme secretion is regulated in the short term with respect to enzymes followed by direct measurement of the relative mass the overall rate of enzyme output by both CCK and cholinergic of the individual enzymes be employed, thereby avoiding stimulation. The early work of Pavlov (18) and the basic enzyme activity measurements (3). By using such a mass assay observations of nonparallel secretion by Rothman and col- of enzyme secretion, we report here large changes in enzyme leagues (2, 28) are both validated and greatly extended by these percentages and ratios of basal and MCh- and CCK-stimulated studies that show that in the short term, pancreatic secretion conditions (Table 1 and Figs. 2-4), as determined by para- is also closely and rapidly regulated with respect to enzyme metric comparisons and by pairwise regression analysis of the composition. data. Under all conditions, the outputs of the enzymes were We thank Dr. P. E. Miller for participation in the initial experiments highly correlated with each other at a high level of significance, and Dr. Joseph Vasselli (Miles) for the acarbose. This work was indicating lack of independence of enzymes during secretion as supported by the Canadian Cystic Fibrosis Foundation, The Montreal expected of exocytosis, but not of an enzyme secretory mech- Children's Hospital/McGill University Research Institute, the Med- anism capable of releasing each individual species (see ref. 2). ical Research Council of Canada, and the Department of Pediatrics, This confirms the observations of cyclic nonparallel exocytotic Brown University. secretion, as determined by enzyme assays in the rabbit (8) and 1. Rothman, S., Liebow, C. & Grendell, J. (1991) Biochim. Biophys. the conscious rat (24, 25). The degree of correlation between Acta 1071, 159-173. enzymes during secretion was dependent upon both the en- 2. Rothman, S. S. (1985) in Protein Secretion: A Critical Analysis of zyme species and the stimulus. For example, lipase I was the Vesicle Model (Wiley, New York). loosely paired with other enzymes, whereas chymotrypsinogen 3. Scheele, G. A. (1975) J. Biol. Chem. 250, 5375-5385. and trypsinogen were paired very tightly; the linkage between 4. Scheele, G.A. & Palade, G. E. (1975) J. Biol. Chem. 250, pairs changed with CCK and MCh stimulation. 2660-2670. Thus, under conditions of constant stimulation with differ- 5. Gilliland, E. L. & Glazer, G. (1980) J. Physiol. (London) 303, ent or basal 33-41. secretagogues conditions, pancreatic exocrine 6. Glazer, G. & Steer, M. L. (1977) Anal. Biochem. 77, 130-140. secretion differed with respect to the percent of each enzyme 7. Tartakoff, A. M., Jamieson, J. D. & Palade, G. E. (1975) J. Biol. secreted as a proportion of the total enzyme species, with Chem. 250, 2671-2677. respect to each other, and with respect to the overall variation 8. Adelson, J. W. & Miller, P. E. (1985) Science 228, 993-996. from the mean in both enzyme percentage and ratio. The fact 9. Miller, P. E. & Adelson, J. W. (1987) Am. J. Physiol. 252, that nonparallel secretion occurred among and between basal G768-G775. conditions and the two classical types of stimulant, hormonal 10. Mroz, E. A. & Lechene, C. (1986) Science 232, 871-873. (CCK) and neurotransmitter (cholinergic stimulation), indi- 11. Bradford, M. (1976) Anal. Biochem. 72, 248-254. cates that nonparallel secretion ofdigestive enzymes is likely the 12. Laemmli, U. K. (1970) Nature (London) 227, 680-685. not 13. Davis, B. J. (1964) Ann. N.Y Acad. Sci. 121, 402-427. rule, the exception. 14. Gabriel, 0. (1971) in Methods in Enzymology, ed. Jacoby, W. B. With respect to the individual animal data, we found that (Academic, New York), Vol. 22, pp. 578-604. exocrine pancreatic secretion of digestive enzymes is a far 15. Imamura, S., Hirayama, T., Arai, T., Takao, K. & Misaki, H. more regulated and complex process than heretofore under- (1989) Clin. Chem. 35, 1126. stood. Thus, these and previous (8, 9, 19) data show that 16. Burrill, P. H., Brannon, P. M. & Kretchmer, N. (1981) Anal. multiple discrete secretagogue-specific intrapancreatic sources Biochem. 117, 402-405. contribute, in a cyclic neurosecretory-like fashion, to large 17. Caraway, W. T. (1959) Am. J. Clin. Pathol. 32, 97-99. changes in the proportions of the secreted enzyme mixture. 18. Pavlov, I. P. (1902) The Work of the Digestive Glands (Griffin, The relatively rapid periodic changes observed here contrib- London). 19. Adelson, J. W. & Miller, P. E. (1989) Am. J. Physiol. 256, uted to the major overall changes in mean enzyme percentages G817-G825. and ratios (Figs. 3 and 4) observed over the entire infusion 20. Erlanson-Ablertsson, C., Larsson, A. & Duan, R. (1987) Pancreas period (Table 1). In all cases in the individual animal, the 2, 531-535. enzyme proportions, determined by the percentage of a single 21. Keim, V. & Rohr, G. (1987) Pancreas 2, 562-567. enzyme specie secreted in a given period (Fig. 3) or by the ratio 22. Liebow, C. (1988) Pancreas 3, 343-351. of one enzyme to another (Fig. 4), were found to vary 23. Owyang, C. & Williams, J. (1991) in TextbookofGastroenterology, dramatically on a cyclic period-by-period basis, under constant ed. Yamada, T. (Lippincott, Philadelphia), Vol. 1, pp. 294-314. basal or MCh stimulation. Enzyme percentages varied by 24. Maouyo, D., Sarfati, P., Guan, D., Morisset, J. & Adelson, J. W. >8-fold and ratios varied by >30-fold among collection peri- (1993) Am. J. Physiol. 264, G792-G800. of 25. Maouyo, D., Guan, D., Rivard, N., Adelson, J. W. & Morisset, J. ods in a single animal. Importantly, the magnitude periodic (1995) Am. J. Physiol. 31, G251-G259. variability decreased almost to the level of the systematic 26. Petersen, O., Petersen, C. & Kasai, H. (1994) Annu. Rev. Physiol. controls when CCK-stimulated secretion was compared to 56, 297-319. either MCh-stimulated or basal conditions. Since protein 27. Thorn, P., Lawrie, A., Smith, P., Gallacher, D. & Petersen, 0. outputs were nearly identical under MCh and CCK stimulation (1993) Cell 74, 661-668. (Fig. 1), the different patterns of cyclic secretion were thus a 28. Rothman, S. S. (1977) Annu. Rev. Physiol. 39, 373-389. Downloaded by guest on October 1, 2021