Independent secretion of different digestive enzymes by the pancreas

Transcription

1 HCl AMERICAN JOURNAL OF PHYSIOLOGY Vol. 231, No. 6, Decembe Pinted in U.S.A. Independent secetion of diffeent digestive enzymes by the panceas S. S. ROTHMAN Depatment of Physiology, Univesity of Califonia, San Fancisco, Califonia ROTHMAN, S. S. Independent secetion of diffeent digestive enzymes by the panceas. Am. J. Physiol. 231(6): l&u Panceatic secetion was collected fom the cannulated duct of anesthetized abbits duing the pefusion of the uppe duodenum with a balanced salt solution with o without 5 mm glucose. The secetion of amylase, chymotypsinogen, typsinogen, and total potein was measued. While glucose did not incease oveall digestive enzyme secetion, it did change the popotions of the enzymes in secetion. In addition, the following was obseved: 1) non-o,0 intecepts when the output of one enzyme was plotted against that of anothe (an enzyme-pai plot), 2) changes in the vaiance of the slope and intecept of enzyme-pai plots, and 3) an incease in the vaiance aound the slope of an enzyme-pai plot, concuent with a decease in the vaiance aound the slope of a plot fo anothe enzyme pai that contained a common membe. These obsevations suggest that diffeent digestive enzymes can be seceted independently of each othe. potein tanspot; nonpaallel secetion; paallel secetion; exocytosis IT IS A MATTER OF COMMON AGREEMENT that individual diffeences in the amount of digestive enzyme seceted by the panceas of animals pesumably in the same physiological state cannot be ascibed simply to measuement eo, but esult in pat fom the vaiable pesence of secetoy stimuli o inhibitoy influences that the investigato has been unable to contol. On the othe hand, unexplained diffeences seen fom animal to animal in the popotions of the vaious digestive enzymes in secetion ae often attibuted entiely to a lack of pecision in enzyme assays and not, to specific biological causes (most ecently, 27, 33, 34). Recent obsevations fom the autho s laboatoy (1, 2, 16-19, 25), and elsewhee (4, 5, 6, 26, 29, 32, 36), demonstate that some of these diffeences, like the diffeences in the oveall amount of enzyme seceted, ae indeed of biological oigin. We have suggested that this vaiability in the popotions of diffeent enzymes in secetion may eflect specific digestive o hydolytic needs in the intestine and povide fo the egulation of digestion at the molecula level, namely, the level of the specific polyme o substate bonds pesent in the intestinal contents (19). In the pesent wok, when glucose, an end poduct of digestion whose pesence in the intestine does not augment, oveall enzyme secetion (35)) was added to a duodenal pefusion medium, the popotions of cetain enzymes in the panceatic secetion changed, We pevi ously epoted simila effects with lysine (19). In addition to changes in the popotion of the enzymes in panceatic juice, glucose pefusion alteed the degee to which the secetion of diffeent enzymes covaied. The obsevations pesented below suggest, that the paticula mixtue of enzyme in secetion, at least in nonaugmented secetion, is a consequence of the independent tanspot of each enzyme, athe than a esult of thei linked tanspot. METHODS l AnimaZ pepaation. The uppe duodenum (between the pylous and the entance of the panceatic duct into the duodenum) of anesthetized (Dial-uethan, CIBA Phamaceutical Co., 0.7 ml/kg body wt SC) male New Zealand abbits was pefused with a bicabonatebuffeed Kebs-Henseleit medium (ph 7-4) (8) with o without the addition of 5 mm glucose. The flow of the pefusion fluid though the intestine was poduced by using gas pessue (95% 0, + 5% CO,) as the diving foce. The technique has been descibed elsewhee (19). Panceatic secetion was collected fom the cannulated duct (polyethylene tubing, PE-10) in taed vessels (19). Assays. Typsinogen, chymotypsinogen, amylase, and potein wee estimated in panceatic juice. Chymotypsinogen and typsinogen wee activated by incubation in 40 mg puified enteopeptidase (EC ) (gade B, Calbiochem) pe 100 ml 0.1 M sodium phosphate buffe (ph 7.4) fo 30 min at 37 C. The pesence of typsin and chymotypsin in activated samples was detemined fom the esteolytic activity of each enzyme usingp-toluenesulfonyl+aginine methyl este as the substate fo typsin (EC 3.4X.4) (28) andn-acetyl- L-tyosine ethyl este*h,o as the substate fo chymotypsin (EC l) (31). Both eactions wee measued as initial ates, Amylase activity was estimated by the solubilization of a dye (Remazol Billiant Blue R) fom an amylose substate to which it was covalently linked (14). The details of these enzyme assays fo panceatic secetion have been descibed elsewhee (2, 3, 15, 18, 20). The potein content of samples was estimated using the Folin phenol eagent (10). RESULTS Goss esponse to duodenal pefusion of glucose. The addition of 5 mm glucose to the pefusion medium fo 60 min did not manifestly alte the ate of digestive en-

2 1848 S. S. ROTHMAN zyme secetion by the panceas; that is, enzyme secetion in geneal deceased gadually ove time in the pesence of glucose at much the same ate as it did in its absence (Fig. 1). Thus, the aveage secetoy outputs of typsinogen, chymotypsinogen, amylase, and total potein wee unalteed by glucose (Fig. 1). Changes in popotions of enzymes seceted in pesence of glucose. As mentioned above, a potion of the vaiability obseved in the amount of enzyme seceted by animals, ostensibly in the same physiological state, is elated to diffeences in the geneal secetoy activity of diffeent glands. The aveaging of enzyme outputs incopoates this souce of vaiance into the calculated standad deviation (Fig. 1) and makes it difficult to discen changes in the popotions of the enzymes seceted because of the esultant lage and often ovelapping vaiance (Fig. 1). This souce of expeimental vaiance can be isolated fom the data by plotting the individual sample points fo one enzyme measue against anothe, and detemining the elationship between the two vaiables (Figs. 2 and 3). When this is done, the amount of one enzyme seceted elative to anothe is defined by the slope of the line (b), as well as by the intecept (a) if the line does not pass though the oigin and is independent of the geneal secetoy activity of the gland. When the pesent data wee teated in this way and the elationship between the two vaiables was estimated by linea egession, substantial changes in the popotions of the enzymes in secetion wee disclosed as a esult of the glucose pefusion (Table l), diffeences that wee not appaent when aveages wee compaed (Fig. 1). Thee was a geate than theefold incease in typsinogen secetion elative to chymotypsinogen, an appoximately twofold incease in amylase 3- GLUCOSE Time Iminuted Time (mlnuteal FIG. 1. Secetion of amylase, typsinogen, chymotypsinogen, and total potein by panceas as a function of time. Duodenum was pefused fo all six 20-min peiods shown, both in the absence of glucose (0) (n, 9, values ae means t SE) and in its pesence (0) (n, 6). When 5 mm glucose was added to pefusion medium (open cicles), it was added at 60 3) and emoved afte 5th collection peiod (100 min). Fo the + glucose seies (0) only mean values ae-pesented. None of points fo diffeent times o measues was significantly diffeent fom time-paied contols, 0, when the mean is the same fo both - and + glucose. 22 I / / x 0~ 1 I I I I 1 I J 0 I Chymotypsinogen output /20 min. Kg b.w., pmoles ATEe splitlmin FIG. 2. Plot of secetion of enzyme pai chymotypsinogen-typsinogen befoe glucose was added to pefusion medium (x) (peiods 1 and 2 in Fig. 1) and afte (0) (peiods 5 and 6). Solid lines ae calculated linea egessions fo both goups. Cuved (dashed) line is dawn to show that the - glucose data fit a nonlinea function about equally as well as they fit a linea function (see RESULTS). secetion elative to chymotypsinogen, and a smalle, but significant, incease in typsinogen secetion elative to amylase (Table 1). Unde contol conditions (no glucose in the pefusion medium), thee was a positive intecept fo typsinogen secetion elative to both amylase and chymotypsinogen (Table 1). This could mean eithe that typsinogen secetion occus in the complete absence of secetion of the othe two enzymes (i.e., typsinogen secetion can be completely independent of thei secetion), o that the elationship between the espective outputs was actually nonlinea and the tue intecept was still at the oigin even though fitting the data to a linea egession eoneously indicated a non-o,0 intecept. Thee is evidence fo both. The contol values (x) in Fig. 2 (chymotypsinogen vs. typsinogen) can be fit about as well to a linea o nonlinea function (dashed line) (linea leastsquaes, = 0.89; nonlinea least-squaes, = 0.84), and although we cannot distinguish between them statistically with the pesent data (N.B., a linea function is assumed in Table l), in anothe study a statistically distinguishable nonlinea elationship has been obseved between the secetion of these two enzymes by abbit panceas (19). Thus, an obseved change in the popotions of typsinogen and chymotypsinogen in secetion might be the esult of the displacement of data points towad the oigin of a cuvilinea elationship, as well as the esult of a change in the slope of a function, o both may occu. On the othe hand, the + glucose data in Fig. 3 (amylase vs. chymotypsinogen) ae

3 Kg SECRETION OF DIGESTIVE ENZYME 1849 c 8 I 2 3 Amylase output / 20 min l b.w., units FIG. 3. Plot of secetion of the enzyme pai amylase-chymotypsinogen befoe glucose was added to pefusion medium ( X) (peiods 1 and2 in Fig. 1) and afte (0) (peiods 5 and 6). Lines ae calculated linea egessions. The + glucose intecept is significantly diffeent fom oigin (see Table 1). TABLE 1. Effect of duodenal pefusion of 5 mawglucose on popotions of thee enzymes in secetion as detemined by linea egession of enzyme pais Typsinogenlchymotypsinogen C hymotypsinogenl amylase Typsinogenlamylase - Glucase Slope, b Intecept, a + Glu- CO8e A%b - Glu- +Glucase case % *0.3* k 1.2-b k 1.32 e % ko O.l$ % tl.o $ kl.o$ Numbe is 12 fo each function. Goodness of fit, P < 0.01 fo all functions;, coelation coefficient; -glucose is data fom peiods I (20 min) and 2 (40 min) in Fig. 1; +glucose is data fom peiods 5 and 6 in Fig. 1; no changes in b o a wee seen in time-paied contols. * Standad eo of the estimate. t Diffeence (-vs. -t- glucose) in all slopes significant at P < $ Diffeent fom zeo intecept, significant at P < clealy linea ove the obseved ange and have a positive intecept (Table 1). In any event, both a positive intecept and a nonlinea function indicate that the popotions of the paticula enzymes in secetion ae not fixed o invaiant. Futhemoe, the addition of glucose to the pefusion medium alteed the appaent intecept. A positive typsinogen intecept was no longe calculated elative to chymotypsinogen (Fig. 2 and Table I), but still was elative to amylase (Table I). Also, the slight positive chymotypsinogen intecept (elative to amylase) that was statistically indistinguishable fom the oigin in the contols (Table 1) now appeaed as a tue positive intecept by statistical citeia, because the standad deviation of the intecept was sufficiently educed by teatment (Table 1). Souces of vaiance. If the geneal level of secetoy activity wee the sole souce of vaiance in the measuement of enzyme secetion, then plotting the output of one enzyme against anothe should take that vaiance into account and poduce a calculated egession line that fits the data points pefectly, i.e., the coelation coefficient should be 1. Although the data fit a egession line fo the thee enzyme pais within 1% eo fo all of the conditions studied (Table l), a substantial vaiance aound the calculated functions was still obseved (Figs. 2 and 3). Theefoe, a potion of the vaiance is attibutable to souces othe than the geneal level of secetoy activity, that is, it is eithe of biological oigin o due to measuement eo (pedominantly assay eo), o both. We can estimate the vaiance due to measuement eo if we assume that the vaiance of the best-fit egession line fo a given enzyme pai eflects measuement eo alone, that is, no natual o biological vaiance exists in this case. This estimate gives a maximum value fo this eo. Since the estimate of eo is deived fom a plot of two enzyme activities, it is the sum of both assay eos. The squae of the coelation coefficient can be used as an index of this eo (30). If 2 = 1, then thee is no eo. If 2 = 0.9, then we estimate the eo to be lo%, etc. When calculated in this way the eo fo typsinogen (ET$) plus chymotypsinogen (EC& was 21%. The eo fo typsinogen plus amylase (Eamylase) was 17% and the eo fo chymotypsinogen plus amylase was 8%. Since thee ae thee equations (the sum of the individual eos equals the eo of the pai fo the thee pais) and thee unknowns (the individual assay eos: ETg, E ChTg? E atmylase ) 9 each individual maximum eo can be calculated algebaically: Eamylase = 2%, EChTg = 6%, ET, = 15%. When the thee enzymes wee egessed togethe by multiple linea egession, the best fit gave an eo of 24% ( = 0.87), which is in good ageement with the combined additive eo of 23% fo the enzymes calcu- lated individually above. In othe expeiments, using I a simila chymotypsinogen assay (2), the maximum combined andom assay eo fo chymotypsinogen plus A lipase was appoximately 8%, as compaed to &htg = 6% fo the pesent data. Any othe assay eo would have to be systematic o nonvaiable and would alte an obseved slope in a constant manne and, thus, not affect compaisons between slopes. If thee wee no additional vaiance beyond that due to the geneal level of secetoy activity and measuement eo, then the vaiance obseved aound the calculated line should have been oughly equivalent fom time to time and unde diffeent expeimental conditions, at least within the limits of sampling vaiability. This was not the case and a consideable, stimulusinduced vaiation in the standad deviation aound the slope was obseved (Fig. 4). Such diffeences can only be attibuted to eal diffeences in the popotions of the diffeent enzymes seceted (see efs, 2 and 19 fo othe examples of the same situation). Put anothe way, the degee of elatedness o coelation between the secetion of diffeent enzymes vaies, as does the slope, as a esult of expeimental conditions (Figs. 2 and 3). If we compae the vaiances aound the calculated slopes, we

4 1850 A. TgKhTg Glucose f B. ChTg /Amylase Glucose FIG. 4. Vaiance aound slope of 2 enzyme-pai plots chymotypsinogen-typsinogen (Tg/ChTg) and amylase-chymotypsinogen (ChTglamylase) befoe (clea bas) and afte (shaded bas) addition of glucose to pefusion medium. Teatment alteed vaiance aound calculated slope in both cases, inceasing it tansiently fo Tg/ChTg (A) (F test, P < 0.05) and deceasing it fo ChTglamylase (B) (P < 0.05). can distinguish two types of appaently stimulus-induced changes in the coelation of the secetion of enzyme pais. The fist was an initial incease in the vaiance, followed by a decease towad pestimulus contol levels (Fig. 4, A) (fo typsinogen vs. chymotypsinogen, F = 6.3, P < 0.05). (This was also seen fo typsinogen vs. amylase, whee F appoached significance at the 5% level). The second was a maintained decease in the vaiance (chymotypsinogen vs. amylase, F = 4.0,P < 0.05) (Fig. 4, B). DISCUSSION A diffeential o nonpaallel secetion of digestive enzyme was elicited by a stimulus (the intestinal pefusion of glucose) that did not incease the oveall secetion of digestive enzyme above basal levels. Since, in the pesent expeiments, the acina cells wee filled with secetoy potein and enzyme-containing zymogen ganules as the esult of an ovenight fast, the amount of potein seceted in esponse to glucose was only a small faction of the available digestive enzyme in the cell (20, 24). Thus, the diffeential secetion was in all likelihood the esult of selective enzyme tanspot out of the cell and not the esult of a tunove in the enzyme content of the tissue, as would be equied if secetion occued by a andom o nonselective tanspot mechanism, which has been poposed fo some yeas now (13). Even in the absence of a substantial tunove in tissue enzyme content, new potein might nevetheless account fo the esponse if it could bypass the majo stoage pools (pesumably the zymogen ganules in patitula) on its way out of the cell. Indeed, such a bypass system has been demonstated in this tissue (22, 24). If S, S. ROTHMAN this occued, then the effect would still be due to the diffeential tanspot of potein as well as its diffeential synthesis, since the secetion of new enzyme would have been favoed ove stoed enzyme. Whicheve intacellula enzyme is seceted (new o old, o fom one stoage pool o anothe) and by whateve mechanism (exocytosis o membane tanspot), the pesent obsevations indicate that the ate of secetion of one digestive enzyme can be independent of anothe. That is, the selective secetion appeas to involve, at least in pat, the independent tanspot of diffeent enzymes. This is suggested by, among othe findings, the pesence of non-o,0 o positive intecepts when the output of an enzyme is plotted against that of anothe (an enzyme-pai plot) (Table 1). One enzyme may be seceted in the absence of othes. Futhemoe, glucose pefusion inceased the vaiance aound the slope of one enzyme-pai plot, while simultaneously deceasing the vaiance aound the slope of a plot of anothe enzyme pai that contained a common membe (Fig. 4). This indicates that the secetion of at least two of the thee enzymes was independently detemined. The independent secetion of diffeent enzymes by the abbit panceas is also suggested by ou ecent epots (1, 2) that chymodenin, a peptide puified fom hog duodenum, substantially augments the secetion of chymotypsinogen, wheeas it only modestly, if at all, inceases the secetion of othe digestive enzymes in this species. These obsevations ae consistent with the equilibium o membane tanspot hypothesis fo potein secetion, which poposes that enzyme is seceted as the esult of its vaiable and diffeential elease fom stoage pools into the cytoplasm and its subsequent tanspot out of the cell acoss the luminal plasma membane by independent equilibium pocesses fo each enzyme (2). The pesent obsevations ae also consistent with an exocytosis hypothesis if zymogen ganules contain single enzyme species (and, in addition, if the exocytosis of these ganules wee independently egulated), This does not seem likely on the basis of cuent knowledge, all of which suppots the view that the enzyme contents of the zymogen ganule ae mixed (7, 12, 23, 37), although this view cannot be igoously excluded. It is also conceivable that cetain enzymes ae excluded fom specific zymogen ganules in all cells o in cetain goups of cells in such a way as to pemit the independent secetion of enzyme by the pefeential exocytosis of ganules that ae of mixed content but lack a specific enzyme o goup of enzymes. Futhemoe, a ecent epot (11) shows diffeences in the enzyme content of acina cells fom diffeent pats of the panceas, and theeby suggests that egional diffeences could be anothe way in which nonpaallel tanspot could be accounted fo by exocytosis. Howeve, such diffeences by themselves could only account fo the pesent obsevations if specific enzymes wee wholly excluded fom distinct egions of the gland. Thee is no evidence that this is the case, In fact, while the ecent study cited above demonstated that the popotions of cetain enzymes wee diffeent in acina cells in close poximity to the islets of Langehans, lage diffeences wee not

5 SECRETION OF DIGESTIVE ENZYME obseved, and neithe wee specific enzymes found to be wholly lacking fom one aea o the othe. It should also be appeciated that peinsul.a cells epesent only a minute faction of the mass of secetoy cells (of the ode of the islets themselves) and thee is no indication that othe egional diffeences exist which could account fo the vaiety of selective I effects that have been obseved. Finally, in the absence of convincing evidence that exocytosis occus in this system at all, no less than it occus in a fom consistent with the constaints that obsevations such as these would place on such a pocess, we should apply the law of pasimony in constuct ing hypotheses fo potein secetion, That is, if diffeent enzyme species can be seceted at ates that ae independent of each othe, as the pesent obsevations suggest, then a diffusional pocess should be the fist consideed, because diffusional pocesses account fo this behavio the most simply. These expeiments wee caied out in 1969 at Havad Medical School with the excellent technical assistance of Ms. Magaet Coppe. The wok was suppoted by National Institutes of Health Gants AM10455 and AM Received fo publication 12 Mach REFERENCES 1, ADELSON, J. W., AND S. S. ROTHMAN. Selective panceatic enzyme secetion due to a new peptide called chymodenin. Science 183: , ADELSON, J. W., AND S. S. ROTHMAN. Chymodenin, a duodenal peptide: specific stimulation of chymotypsinogen secetion. Am. J. Physiol. 229: , 1975, 3. BURWEN, S J. AND S. S. ROTHMAN. Zymogen ganules: osmotic popeties, inteactions with ions, and some stuctual implications. Am. J. Physiol. 222: , DICK, J., AND J. P. FELBER. Specific homonal egulation, by food, of the panceas enzymatic (amylase and typsin) secetions. Homone Metab. Res. 7: , FELBER, J. P., A. ZERMATTEN, AND J. DICK. Modulation, by food, of homonal system egulating at panceatic secetion. Lancet II: , GOLDBERG, D. M., J. K. SALE, AND K. G. WORMSLEY, Ratio of chymotypsin to typsin in human duodenal aspiate. Digestion 8: , KRAEHENBUHL, J, P., AND J. D. JAMIESON. Solid-phase conjugation of feitin to Fab-fagments of immunoglobulin G fo use in antigen localization on thin sections, Poc. Nutl. Acad. Sci. US 69: , KREBS, H, A., AND K. HENSELEIT. Untesuchungen iibe die Hanstoffbildung im Tiekope. 2. Physiol. Chem. 210: 33-66, LIEBOW, C., AND S. S. ROTHMAN. Membane tanspot of poteins. Natue New Biol. 240: , LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RAN- DALL. Potein measuement with the folin phenol eagent. J. Bio2. Chem. 193: , MALAISSE-LAGAE, F,, M. RAVAZZOLA, P. ROBBERECHT, A. VAN- DERMEERS, W. J. MALAISSE, AND L. ORCI. Exocine panceas: Evidence fo topogaphic patition of secetoy function, Science 190: , MARSHALL, J. M., JR. Distibutions of chymotypsinogen, pocaboxypeptidase, desoxyibonuclease, and ibonuclease in bovine panceas. &@Z. CeZZ. Res. 6: , PALADE, G. E. Intacellula aspects of the pocess of potein synthesis. Science 189: , RINDERKNECHT, H., P. WILDING, AND B. J. HAVERBACK. A new method fo the detemination of cu-amylase. Expeientia 23: 805, ROTHMAN, S. S. Typsin and chymotypsin secetion fom abbit panceas in vito. Am. J. Physiol. 211: , ROTHMAN, S. S. Non-paallel tanspot of enzyme potein by the panceas. Natue 213: , ROTHMAN, S. S. Tanspot of potein by panceatic acina cells: andom o selective? In: The Exocine GZunds, edited by S. Y. Botelho, F. P. Books, and W. B. Shelly. Philadelphia: Univ. of Pennsylvania Pess, 1969, p ROTHMAN, S S. Subcellula distibution of typsinogen and chymotypsinogen in abbit panceas. Am. J. Physiol. 218: , ROTHMAN, S. S. Molecula egulation of digestion: shot tem and bond specific. Am. J, PhysioZ. 226: 77-83, ROTHMAN, S. S. Enzyme secetion in the absence of zymogen ganules. Am. J. Physiol. 228: X , ROTHMAN, S. S. Potein tanspot by the panceas. Science 190: , ROTHMAN, S. S. Secetion of new digestive enzyme by panceas with minimal tansit time. Am. J. Physiol. 230: , ROTHMAN, S. S., S. BURWEN, AND C. LIEBOW. The zymogen ganule: intaganula oganization and its functional significance. In: Cytophamucology of Secetion, edited by B. Ceccaelli, J. Meldolesi, and F. Clementi. New Yok: Raven, 1974, vol. 2, p ROTHMAN, S, S., AND ISENMAN, L. D. The secetion of digestive enzyme deived fom two paallel intacellula pools. Am. J. Physiol. 226: , * ROTHMAN, S. S,, AND H. WELLS. Selective effects of dietay egg white typsin inhibito on panceatic enzyme secetion. Am. J. PhysioZ. 46: , SARLES, H., C. FIGARELLA, G. PREZALIN, AND C. SOUVILLE. Compotement diff&ent de la lipase, de l amylase et des enzymes pot&olytiques gpes diffbents modes d excitation du panceas humain. BuZZ. Sot. Chim. BioZ. 48: , SCEIEELE, G. A., AND G. E. PALADE. Studies on the guinea pig panceas. Paallel dischage of panceatic exocine enzyme activities. J. BioZ. Chem. 250: , SCHWERT, G. W., H. NEURATH, S. KAUFMAN, AND J. E. SNOKE. The specific estease activity of typsin, J. BioZ. Chem. 172: , SOBOL BROWN, R., M. B. BROWN, A. BDOLAH, AND E. KOCHVA. Accumulation of some secetoy enzymes in venom glands of Vipeu PaZuestinae. Am. J. Physiol. 229: , SNEDECOR, G. W., AND W. G. COCHRAN. StatisticaL Methods. Ames: Iowa State Univ. Pess, 1967, p SNOKE, J. E., AND H, NEURATH. The effect of tempeatue on the estease activity of chymotypsin. J. BioZ. Chem. 182: , 1950, 32, SULLIVAN, J. F., R. E. BURCH, AND D. G. MAGEE. Enzymatic activity and divalent cation content of panceatic juice. Am. J. Physiol. 226: , TARTAKOFF, A. M., L. 5. GREENE, J. D. JAMIESON, AND G. E. PALADE. Paallelism in the pocessing of panceatic poteins. In: Advances in Cytophamacology, edited by B. Ceccaelli, F. Clementi, and J. Meldolesi. New Yok: Raven, 1974, vol. 2, p TARTAKOFF, A. M., 5. D. JAMIESON, G. A. SCHEELE, AND G. E. PALADE. Studies on the panceas of the guinea pig. Paallel pocessing and dischage of exocine poteins. J. Biol. Chem. 250: , WANG, C. C., and M. I. GROSSMAN. Physiological detemination of elease of secetin and panceozymin fom intestine of dogs with tansplanted panceas. Am. J. Physiol. 164: , WORMSLEY, K. G,, AND D. M. GOLDBERG. The inteelationships of the panceatic enzymes, Gut 33: , YASUDA, K., AND A. H. COONS. Localization by immunofluoescence of amylase, typsinogen, and chymotypsinogen in the acina cells of the pig panceas. J. Histochem. Cytochem. 14: , 1966.