Pancreas Acinar Cell Regeneration

Pancreas Acinar Cell Regeneration

VIII. Relationship of Acid Phosphatase and[-Glucuronidase to Intracellular Organellesand Ethionine Lesions

Akio Horie, MD, Tatsuro Takino, MD, Lawrence Herman, PhDand Patrick J. Fitzgerald, MD

IN RECENT YEARS, the role of the lysosomal enzymes indegenerative, necrotic and inflammatory processes has been exten-sively investigated.1- Most reports concern the liver 15-7 or kidney,6 8but joints,9 pancreas,1112 parotid gland 4 and other tissues 3 have beenstudied. In this laboratory, the effects of ethionine on pancreas acinarcells of the rat have been used as a model for the study of acinar celldegeneration, necrosis, repair and regeneration; 12-15 and the enzymaticactivities of acid phosphatase and j-glucuronidase, two lysosomalenzymes, have been measured biochemically in homogenates of thepancreas tissue throughout the ethionine effect.14

In this report, electron microscopic cytochemical technics were used tostudy the localization of enzyme reaction prodvucts of acid phosphataseand 3-glucuronidase in intracellular organelles of the normal gland andin lesions of the pancreas after ethionine was injected.16.17

Materials and Methods

General

Mtale albino Wistar rats, (Carworth Farms) each weighing 160 + 20 g weredivided into four groups.

Group 1 (PFE-PF). Animals received up to ten daily intraperitoneal (IP) injec-tions of DL-ethionine, 0.7 mg/g body weight (TBW), while on a protein-free diet; thereafter, they received no more ethionine but were maintained on aprotein-free diet for varying periods up to day 36.Group 2 (PFE-SD). Animals received up to 10 daily injections of ethionine

while on a protein-free diet; thereafter they received no more ethionine but weregiven a stock diet at day 11 and sacrificed after varying periods up to day 36.

From the Department of Pathology, State University of New York, Downstate MedicalCenter, Brooklyn, New York.

Supported by research grants CA-06801 and 2RO1-AM05556 from the US PublicHealth Service.

Accepted for publication January 15, 1971.Address for reprint requests: Dr. Lawrence Herman, Department of Pathology, State

University of New York, Downstate Medical Center, 450 Clarkson Avenue, Brookl-n, NewYork 11203.

299

300 HORIE ET AL American Journaiof Pathology

Group 3 (PF). Animals were maintained on a protein-free diet without ethio-nine for 36 days.

Group 4 (SD). Animals were maintained on a stock diet for 36 days.Animals from all four groups were sacrificed at 2, 6 and 24 hours after ethionine

was first injected (called day 1 of the experiment) and at days 2, 5, 8, 10, 12, 15, 18,28 and 36. Tissues from two similar experiments were used.

Cytochemistry

After the rats were sacrificed by guillotining. the pancreas was removed and rap-idly cut into approximately 1-mm cubes and fixed in cacodylate-buffered (pH 7.4)3% glutaraldehyde for 1 hour.18 The tissue blocks were then rinsed 3 times, 10minutes each rinse, with 7.5% sucrose and 0.1 M cacodylate buffer, pH 7.4. Forcytochemical procedures, the tissues were cut with a tissue sectioner (Smith-Far-quahar, Ivan Sorvall, Norwalk, Conn) into 50-v slices.

Acid Phosphatase. The tissue slices were divided into three groups and eachwas incubated 30 minutes at 37 C in one of the following media: (A) 0.1% (20 mg)cytidine monophosphate (Sigma), 5.6 ml of distilled water, 8 ml of 0.05 M acetatebuffer (pH 5.0), 2.4 ml of 1% lead nitrate, 4 ml of 0.025 M cobalt chloride and 1 gof sucrose,1'J (B) same as media A except that the substrate, cytidine monophos-phate, was omitted; (C) same as media A except that an inhibitor, 2 ml of 0.2 Msodium fluoride, was added. The 1% lead nitrate was prepared immediately prior touse. Media B and C served as controls.To evaluate further the possibility of artifacts such as nonspecific precipitation of

lead or diffusion of reaction products, specimens with the most enzymatic activity(day 12 PFE-PF) were examined after boiling for minutes, or, at either shorterincubation times (5, 10, 15 and 20 minutes), or at lowered concentrations of sub-strate-eg, 0.05% cytidine monophosphate.

After the tissue slices were incubated, they were washed three times with 7.5%sucrose-cacodylate buffer solution adjusted to pH 7.4. Some of the tissues werestained with 0.5% aqueous uranyl acetate solution for 1 hour and again washedthree times with the sucrose-cacodylate buffer solution. Tissues were postosmicatedwith 1% Veronal acetate-buffered osmium for 20 minutes.

(-Glucuronidase. Preincubation and postincubation procedures for the demon-stration of 3-glucuronidase were the same as those for acid phosphatase except thatnaphthol AS-BI glucuLronide was used as a substrate.2' As in the procedures foracid phosphatase, three different media were used: media A contained the substrate,naphthol AS-BI glucuronide as well as other required reagents; media B contained allthe reagents of media A except the substrate; media C contained the substrate, and,in addition, an inhibitor, glucosaccharo-1,4-lactone. Groups B and C served ascontrols. In addition to postosmicated specimens, nonosmicated tissues were alsoexamined to determine the degree of electron scatter of the coupled azo dye.

Electron Microscopy

The tissues were dehydrated through a sequence of graded alcohols and embeddedin Epon. Ultrathin sections were cut with an LKB microtome; unstained or uranylacetate-stained sections21 were examined in an RCA-EMU 3F electron microscope.Electron micrographs were taken at original magnifications of 1500-10,000 diam-eters and subsequently enlarged.

Correlative Light and Electron Microscopic Technics (5-Glucuronidase)Although the red reaction product used to demonstrate this enzyme could be

seen by light microscopy in lesions of tissue (in plastic 1-b-thick section), the

Vol. 63, No. 2 CYTOCHEMICAL STUDIES OF LYSOSOMAL ENZYMES 301May 1971

electron microscopic thin section lacked sufficient electron scatter to reveal thereaction product. Therefore, the thick-thin correlative technic was used to relatethe reaction product, visible in the thick section of light microscopy, with itscorresponding ultastructural continuum, the adjacent thin section of the electronmicrograph.

Observations

Acid PhlsphataseControls

All pancreatic tissue (SD, PF, PFE-PF and PFE-SD groups) incu-bated in media lacking the substrate, or containing substrate and in-hibitor, failed to exhibit reaction product within acinar cells. Some-times a fine precipitate was observed on the surface of tissue blocks orwithin intracellular spaces.

Normal Acinar Cell (SD Group)

Most normal pancreas acinar cells contained minimal amounts of reac-tion product, usually restricted to prozymogen vacuoles adjacent to theGolgi saccules, or to Golgi saccules and related vesicles (Fig 1). Occa-sionaly, reaction product completely covered a round, membrane-boundbody that was the size and shape of a zymogen granule. A rare acinarcel in the "normal" animal contained a condensed zone of ergastoplasmicdamage, which had lead phosphate deposited within and along itsouter surface (Fig 2).

Occasionally, macrophages contained reaction product within mem-brane-bound dense bodies.

Nuclear deposition of lead phosphate was noted, although, rarely, inthe nucleus of normal acinar cells that exhibited focal cytoplasmicvacuolar change.

Acinar Cell of Protein-Free (PF) Group

Beginning at day 5 and thereafter, the cytoplasm of acinar cells fre-quently contained membrane-bound cytoplasmic vacuoles, concentriclamellar arrangements of agranular membranes and large lipid drop-lets.2 Reaction product often appeared at the periphery of, or between,the whorls of smooth membranes (Fig 3).

Occasionally a whorl of ergastoplasm 17 contained deposits of leadphosphate along its periphery as well as deposits, extracisternally, be-tween lamellae of endoplasmic reticulum (Fig 4). Although, occasionally,deposits of lead phosphate could be seen along the outer edges of lipiddroplets, within immature secretory granules or near the Golgi zone,

302 HORIE ET AL American Journalof Pathology

such localization was rare in early stages (day 5). At later days (days12-15), the reaction product was greatly increased within lesions. Thelesions and reaction product were still present at day 36.

Acinar Cell of PFE-PF Group

At 2 hours after the first injection of ethionine, reaction product wasnot observed in most lesions although, infrequently, a few of the smallcytoplasmic lesions contained minimal deposits of lead phosphate (Fig5). With the increase of multiple daily injections of ethionine, the cyto-plasmic lesions increased in size and number. At day 5, the presencewithin lesions of reaction product from acid phosphatase varied. Withina cell, one lesion might contain considerable deposit of lead phosphatewhile other lesions contained little or none (Fig 6). Some ergastoplasmiclesions were not fully circumscribed and consisted of fused plaques ofosmiophilic membranes, many of which exhibited an intense focal cyto-chemical reaction (Fig 7 and 9). Some capillary endothelial cells con-tained vacuoles and fragmented portions of irregularly arrangedagranular membranes with deposits of lead phosphate in the adjacentinterstitium.At later stages of ethionine damage (days 8-10), ergastoplasmic lesions

involved larger portions of the acinar cell and occasionally occupied theentire cell cytoplasm, with more extensive deposition of reaction productthan in earlier stages.At stages of maximum degeneration (days 10-12), macrophages were

conspicuous and most contained numerous round granules, fragmentedmembranes and filamentous structures heavily coated with lead phos-phate (Fig 8). At day 12, the acinar cell ergastoplasm adjacent tolesions was seen to contain reaction product. Such nonlesion depositionwas restricted to the cytoplasmic matrix (between membranes of theER) and did not appear within ER cisternae. Such localization of enzymeactivity was noted regardless of reduction in incubation time or reduc-tion of substrate concentration. Plaque lesions often had heavy focalprecipitate (Fig 9).At day 12, centroductular cells damaged by ethionine possessed cyto-

plasmic vacuoles containing an electron-lucent homogeneous material,agranular membranes and dense bodies associated with deposits of leadphosphate, which were also present in the extracisternal cytoplasmicmatrix, particularly adjacent to lesions.

Lesions in the PFE-PF group disappeared slowly so that only at day28 did lesions and reaction product become rare.


Acinar Cell of PFE-SD Group

In the PFE-SD and PFE-PF groups, localization of reaction productfor the first 10 days was similar since during this time the regimens wereidentical. The feeding of a stock diet at day 11 in the PFE-SD groupdid not lead to a significant difference between the two groups untilafter day 12 when the lesions of the PFE-SD animals regressed morerapidly and the associated reaction product was less intense than in thePFE-PF group. By day 18, lesions and reaction product in the PFE-SDgroup were rare.

Islet Cells

Islet cells remained relatively intact after ethionine was injected. Oc-casionally, cells at day 15 in the PFE-PF animals contained small densebodies and whorls of agranular membranes with deposits of lead phos-phate. Golgi sacs of islet cells and, rarely, related secretion granulesalso exhibited reaction product.

I0-GucuronidaseUght Microscopy

Normal acinar cells failed to exhibit deposition of the red reaction pro-duct. Control groups incubated in media lacking substrate or in mediacontaining substrate and inhibitor failed to demonstrate the presence ofreaction product as did tissues boiled for 20 minutes prior to incubation.Thick plastic sections (1 pt) of nonosmicated specimens incubated

with substrate revealed staining of macrophages with red hexazoniumpararosaniline and severely damaged acinar cells of the PFE-PF, PFE-SD and PF groups.

Electron Microscopy

Specimens incubated in substrate and postosmicated contained rounddense granules in degenerating acinar cells (Fig 10) and within cyto-plasmic debris of macrophages. However, without postosmication, simi-lar granules or cytoplasmic lesions failed to exhibit sufficient density toindicate the presence of reaction product, presumably because of lack ofsufficient electron scattering. Control groups postosmicated, with or with-out boiling of tissue, exhibited comparable electron densities, suggestingthat the density was associated with the osmication and was not a resultof enzyme activity alone. Localization of reaction product using thick-thin correlative microscopy of nonosmicated tissue (Fig 11 and 12) re-vealed that localization of 0-glucuronidase was seen to follow the samepattern as that of acid phosphatase-ie, reaction product became in-

---


creasingly evident within acinar cell lesions during the progress ofdegeneration, reaching a peak at days 10-12 in the PFE-PF, PFE-SDand PF groups and receding thereafter.

Lesions and reaction product disappeared quicker in the PFE-SDgroup than in the PFE-PF animals. No reaction product was noted in thenormal acinar cell.

Discussion

The presence of the reaction product of acid phosphatase in prozymo-gen vacuoles, which are probably the condensing vacuoles of Jamiesonand Palade,22 and in the Golgi apparatus of the normal pancreas acinarcell suggests that in these areas the active site of the enzyme is exposedand elsewhere it is blocked. The absence of reaction product might alsoindicate diffusion of enzyme, lack of sensitivity of the technic, loss ofenzymatic activity in tissue processing or some combination of these, orother factors.

Enzyme and Lysosome

The fact that the early changes caused by ethionine reported byus 16.17 were ergastoplasmic, the relatively high concentrations ofacid phosphatase and 3-glucuronidase activities in our pancreas homog-enates 14 and the present demonstration of reaction products in theergastoplasmic ethionine-induced lesions are consistent with the as-sumed hydrolytic action of these enzymes in degenerative lesions.1"l5-23Many terms have been used to designate the lysosomal lesion,1' 3'5'7'2324all of which have in common the features of a normal cellular struc-ture,1 2325 or degenerated organelle fragments,57 an enveloping mem-brane and the presence of acid phosphatase reaction product.1'3-7 Theoriginal lysosomal hypothesis as a general widespread phenomenonhas been questioned by some authors 2.7,11,12,26.27 and supported byothers.1'5,23'25

Wachstein et al 28 found, by light and electron microscopic histochemi-cal technics, a high activity of acid phosphatase in the ethionine-inducedlesions of the acinar cells and in the macrophages of the rat pancreas.Fedou et al 29 reported that acinar cells of the dog pancreas, 48 hoursafter ethionine (or orotic acid) was administered, contained reactionproduct of acid phosphatase within "crystals" of the focal cytoplasmicdegenerative lesions (the crystals resemble the plaques of ethionine "I oractinomycin D 30 ) .Our ethionine lesions had many of the general features of lysosomes.

The earliest ethionine lesions, however, usually did not exhibit reaction


product, although a rare one did (Fig 5). Many stages of lesion werepresent with varying amounts of reaction product, although, in general,the more pronounced and extensive the lesion, the more reaction prod-uct found in it. Limitations of the technic may have prevented demon-stration of very low levels of enzyme activity. In the normal rat pancreasacinar cell, there is usually no consistent, discrete intracellular organellesimilar to any of the lysosomes described in the literature or resemblingthe ethionine lesion, although rarely, such a lesion is seen in the normalanimal (Fig 2). Reaction product, when present in normal acinar cells,is usually found in the prozymogen vacuole and Golgi apparatus and notin the ergastoplasm-the primary site of the ethionine lesion. We foundno significant increase in activity of either enzyme in the pancreas homo-genate during an early period of the PFE regimen.14 It would appear,therefore, that the enzymatic activity found in ethionine lesions occurssecondarily to the lesion and that the lesion did not represent an activa-tion of a preexisting lysosome. Others have reached similar conclu-sions.10"°1

Site of Synthesis of Acid Phosphatase

Acid phosphatase reaction product was noted often at an unusual siteat day 12 in the PFE-SD animals (and rarely in PF animals (Fig 4)).Ergastoplasm adjacent to prominent cytoplasmic lesions, or to ergasto-plasmic whorls,17 manifested the reaction product in the cytoplasmicmatrix external to the ergastoplasmic cisternae (Fig 9). Such foci oflead phosphate may have represented diffusion from an adjacent site ofhigh enzymatic activity, possibly from damaged ergastoplasm. The ab-sence of localization in boiled tissue, in tissue to which an enzyme in-hibitor was added or in lesions at other days makes the possibility ofnonspecific deposition less likely. It is possible that the presence of acidphosphatase reaction product in the cytoplasm between cisternae of theergastoplasm may have represented the site of synthesis of this enzyme,which became apparent only at day 12 when there was a marked in-crease of protein synthesis. Conceivably, a relative block in the transferof enzyme from an extracisternal cytoplasmic matrix site of synthesis tothe intracisternal space could have been operative at this time when thepeak of acid phosphatase activity in the pancreas homogenates of thePFE-SD animals occurred.'l

Brandes' study 31 suggests an extracisternal site of synthesis for acidphosphatase. Other studies have shown acid phosphatase reaction prod-uct to be localized in the ergastoplasmic cisternae.32'4 Arstila andTrump have reported acid phosphatase reaction product to be localized


in the cisternae of the ergastoplasm after prolonged incubation of thesubstrate, or after reduction of ATP in liver parenchymal cells.7 Suchdemonstrations do not necessarily indicate the site of synthesis but onlythe area of highest enzymatic activity. Either an intracisternal or extra-cisternal site of synthesis would be compatible with adsorption of enzymeby cytoplasmic lesions.

,-Glucuronidase

Since the localization of reaction product of 3-glucuronidase in thelesions was identical to that of acid phosphatase, it is possible that thetechnic indicates the presence of the enzyme. The finding that the coloredreaction product of 0-glucuronidase seen by optical microscopy was cor-related with a dense, homogeneous deposit seen electron microscopi-cally in lesions strengthens this assumption.Whereas with acid phosphatase all three parameters-lesions, reaction

product and homogenate activity-reached a peak at day 12, the bio-chemical analyses of homogenates for P-glucuronidase peaked at day10 and the number of lesions and amount of reaction product weremaximal at day 12. The biochemical values of homogenates were farhigher for 3-glucuronidase than for acid phosphatase 14 although muchmore reaction product was discernible after the acid phosphatasetechnic than was noted after the 3-glucuronidase method at comparabletimes; this is possibly a reflection of the relative sensitivities of the cyto-chemical technics.

Dietary Protein and Enzymatic Activity

Desnuelle and colleagues have shown that a diet very high in proteincaused increased activities of chymotrypsin, trypsin and amylase in thenormal rat pancreas and pancreatic juice.3'36 The stock diet (SD) portionof our PFE-SD regimen, a diet moderately high in protein (25%), wasassociated with a more rapid rise in the activity of the depressed amylase,lipase and chymotrypsin than that occurring when a protein-free diet fol-lowed the PFE regimen (PFE-PF ).14 The elevated activities of acidphosphatase and f-glucuronidase in the pancreas homogenates 14 andthe increase in enzyme reaction products of these enzymes reported inthis paper returned to normal levels in the PFE-SD animals muchquicker than they did in the PFE-PF animals. A diet higher in protein,therefore, not only restored the decreased activities of the exportenzymes quicker but it also lowered the elevated lysosomal enzymeactivities more rapidly to normal. These different responses of the twogroups of enzymes indicate the existence of separate control mechanisms.


A significant part of the enzyme activities of the pancreas homogenatewas derived from infiltrating macrophages, as indicated by the cyto-chemical technics, so that the presence of such inflammatory cells mustbe considered in evaluating homogenate findings.

SummaryCytochemical localization of acid phosphatase and f-glucuronidase

reaction products has been studied in the pancreas of ( 1) normal ratson a stock diet (SD), (2) animals maintained on an ethionine protein-free diet and then refed a stock diet (PFE-SD), (3) animals on theethionine protein-free diet and refed a protein-free diet (PFE-PF) and(4) rats kept on a protein-free diet (PF).In most normal pancreas acinar cells, small amounts of the acid phos-

phatase reaction product were found over prozymogen vacuoles andGolgi saccules. Only rarely was a 'lysosome" present in the normalacinar cell and, in these, enzyme reaction product was demonstrated.The earliest ethionine lesions in the PFE groups often had no reaction

product of acid phosphatase in the lesion, but a few lesions at 2 hoursafter ethionine was injected contained the precipitate. With the progres-sion of the degenerative process, more reaction product was present inlesions of the acinar cell, although some macrophages and duct cellscontained lesions and enzyme reaction product. The peak in the numberof ethionine lesions and amount of enzyme reaction product was at day12. Lesions and reaction product decreased as repair and regenerationprogressed.

Animals on the PFE-SD regimen resolved the ethionine lesions quickerand retained less enzyme reaction product than did the PFE-PF animals.The number of lesions with acid phosphatase reaction product and the

intensity of the enzyme reaction product generally paralleled the changesin the previously determined enzymatic activity of the pancreas homo-genate. With f-glucuronidase, the correlation was much poorer.

Since the reaction product of acid phosphatase was localized in theprozymogen vacuoles and Golgi apparatus in the normal acinar cell andin the ethionine ergastoplasmic lesion in the PFE animals, there was nocorrelation of lysosomal enzyme reaction product with any postulatedpreexisting lysosomal organelle.

References1. Novikoff AB: Lysosomes in the physiology and pathology of cells: contribu-

tions of staining methods, Ciba Foundation Svmposium on Lysosomes Editedby AVS de Reuck, NIP Cameron. Boston, Little, Brown & Co, 1963, pp 36-77

2. Van Lancker JL: Pathology symposium on lysosomes, Fed Proc 23:1009-1052, 1964


3. Dingle JT, Fell HB: Lysosomes in Biology and Pathology. Vol 1, 2. Amster-dam, North-Holland Publishing Co, 1969

4. Novikoff AB: Cytochemical staining methods for enzyme activities: theirapplication to the rat parotid gland. Jewish Mem Hosp Bull 6:70-93, 1962

5. Novikoff AB, Essner E: Cytolysomes and mitochondrial degeneration. J CellBiol 15:140-146, 1962

6. Straus W: Cytochemical observations on the relationship between lysosomesand phagosomes in kidney and liver by combined staining for acid phosphataseand intravenously injected horseradish peroxidase. J Cell Biol 20:497-507,1964

7. Arstila AU, Trump BF: Studies on cellular autophagocytosis. The formationof autophagic vacuoles in the liver after glucagon administration. Amer J Path53:687-733, 1968

8. Trump BF, Bulger RE: Studies of cellular injury in isolated flounder tubules.I. Correlation between morphology and function of control tubules and ob-servations of autophagocytosis and mechanical cell damage. Lab Invest 16:453-482, 1967

9. Thomas DPP: Lysosomal enzymes in experimental and rheumatoid arthritis.3Vol 2, Chap 5, pp 87-110

10. Holtzer RL, Van Lancker JL: Early changes in pancreas autolysis. Amer JPath 40:331-336, 1962

11. Hruban Z, Swift H, Wissler RW: Analog-induced inclusions in pancreaticacinar cells. J Ultrastruct Res 7:273-285, 1962

12. Fitzgerald PJ: The problem of the precursor cell of regenerating pancreaticacinar epithelium. Lab Invest 9:67-85, 1960

13. Fitzgerald PJ, Herman L, Carol B, Roque A, Marsh WH, Rosenstock L,Richards C, Perl D: Pancreatic acinar cell regeneration. I. Cytologic, cyto-chemical and pancreatic weight changes. Amer J Path 52:983-1011, 1968

14. Marsh WH, Goldsmith S, Crocco J, Fitzgerald PJ: Pancreatic acinar cell re-generation. II. Enzymatic, nucleic acid, and protein changes. Amer J Path52:1013-1037, 1968

15. Fitzgerald PJ, Vinijchaikul K, Carol B, Rosenstock L: Pancreatic acinar cellregeneration. III. DNA synthesis of pancreas nuclei as indicated by thymidine-H3 autoradiography. Amer J Path 52:1039-1065, 1968

16. Herman L, Fitzgerald PJ: The degenerative changes in pancreatic acinarcells caused by DL-ethionine. J Cell Biol 12:277-296, 1962

17. Herman L, Fitzgerald PJ: Restitution of pancreatic acinar cells followingethionine. J Cell Biol 12:297-312, 1962

18. Sabatini DD, Bensch K, Barrnett RJ: Cytochemistry and electron micros-copy: the preservation of cellular ultrastructure and enzymatic activity byaldehyde fixation. J Cell Biol 17:19-58, 1963

19. Friend D: Personal communication, 196720. Hayashi M, Shirahama T, Cohen AS: Combined cytochemical and electron

microscopic demonstration of P-glucuronidase activity in rat liver with the useof a simultaneous coupling azo dye technique. J Cell Biol 36:289-297, 1968

21. Stempak JG, Ward RT: An improved staining method for electron micros-copy. J Cell Biol 22:697-701, 1964

22. Jamieson JD, Palade GE: Intracellular transport of secretory proteins in the


pancreatic exocrine cell. II. Transport to condensing vacuoles and zymogengranules. J Cell Biol 34:597-615, 1967

23. deDuve C: The lvsosome. Sci Amer 208:64-72, 196324. Ashford TP, Porter KR: Cvtoplasmic components in hepatic cell Ivsosomes.

J Cell Biol 12:198-202, 196225. Cohn ZA, Fedorko ME: The formation and fate of lvsosomes.3 V7ol 1, Chap

5, pp 43-6396. Slater TF, Greenbaum AL: Changes in lvsosomal enzymes in acute experi-

mental liver injury. Biochem J 96:484-491, 196527. Levvy GA, Conchie J: The subcellular localization of the "lvsosomal" enzyme

and its biological significance. Progress Biophy-s Molec Bio 14:107-129, 196428. Wachstein M, Femandez C, Ortiz J: Enzvmatic light and electron micros-

copy of ethionine induced pancreatic degeneration. J Histochem Cytochem13:21-22, 1965

29. Fedou R, Frexino J, Ribet A: The sites of acid phosphatase activity in theacinar cells of dog's pancreas, following the administration of ethionine andorotic acid. Proc Roval Microsc Soc 3:80, 1968

30. Yamaguchi K, Kobavashi Y, Sato T, Herman L, Marsh WH, Rosenstock L,Fitzgerald PJ: Pancreas acinar cell regeneration. IX. Effect of actinomycinD on microstructure. Amer J Path (in press)

31. Brandes D: Observation on the apparent mode of formation of "pure" E!-so-somes. J Ultrastruct Res 12:63-80, 1965

32. Goldfischer S, Carasso N, Favard P: The demonstration of acid phosphataseactivitv bv electron microscopy in the ergastoplasm of the ciliate Campanellaumbellaria L. J MIicroscopie 2:621-628, 1963

33. Carasso N, Favard P, Goldfischer S: Localisation, 'L r&chelle des ultrastruc-tures, d'activites de phosphatases en rapport avec les processus digestifs chezun cilie (Campanella umbellaria). J Microscopie 3:297-322, 1964

34. Lane NJ, Novikoff AB: Effects of arginine deprivation, ultraviolet radiationand X-radiation on cultured KB cells: a cvtochemical and ultrastucturalstudv. J Cell Biol 27:603-620, 1965

35. Reboud JP, Mfarchis-Mouren G, Pasero L, Cozzone A. Desnuelle P: Adapta-tion de la vitesse de biosvnthese de l'amvlase pancreatique et du chymo-trypsinogene 'a des regimes riches en amidon ou en proteines. Biochim BiophvsActa 117:351-367, 1966

36. Reboud JP, Ben Abdeljlil A, Desnuelle P: Variations de la teneur en enzymesdu pancreas de rat en fonction de la composition des retgimes [Variations in theenzyme content of the rat pancreas as a function of the composition of thediet]. Biochim Biophys Acta 58:326-337, 1962

Dr. Horie's present address is the Department of Pathology, Kvushu Universiht Schoolof NMedicine, Fukuoka, Japan.

Dr. Takino's present address is the Department of Medicine, Kvoto Prefectural Univ er-sitv, School of Medicine, Kyoto, Japan.

All figures are electron micrographs of cytochemical preparations of rat pancreas.Figures 1-9 are acid phosphatase preparations; Figures 1O-12 are I-glucuronidasepreparations. See text for details.

Fig 1-Normal pancreas, stock diet (SD) group. Reaction product appears as denseprecipitate restricted to small foci within prozymogen vacuoles and cisternae of theGolgi apparatus (upper right) (x 32,000).

Fig 2-Normal pancreas (SD). Rare cytoplasmic lesion in normal animal containingwhorl of ergastoplasm with reaction product in it and along perimeter of lesion(x 32,000).

2

3 II

Fig 3-Protein-free diet (PF) group, day 15. Large cytoplasmic lesion containing manysmaller components of vacuoles, amorphous debris and tightly coiled whorls of smoothmembranes, and osmiophilic strands. Reaction product scattered mostly between whorls(X 25,000).

Fig 4-PF group, day 15. Whorl of ergastoplasm (ER) distinct from surrounding,more normal ER. Small deposits of lead phosphate are localized in cytoplasmic matrixbetween adjacent membranes of ER whorl, extracisternally; larger deposits are presentat periphery (x 24,000).

Fig 5-Protein-free ethionine (PFE) group 2 hours after ethionine was injected. Re-action product is concentrated in small cytoplasmic lesion. Most lesions at this timehad no reaction product (x 56,000).

5

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6-PFE 2. K6 haveFig PFE group, day 2. Different lesions have different amounts of reaction product.Lesion 1 contains heaviest deposit; lesion 2 contains moderate amount; lesion 3 containsminimal precipitate and lesion 4 is free of reaction product (X 25,000).

Fig 7-PFE group, day 10. Intense lead phosphate deposits are associated withplaques (x 28,000).

Fig 8-PFE-PF group, day 12. Macrophage with intense reaction product betweencytoplasmic vacuoles (x 9500).

7

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Fig 11-Protein-free ethionine, stock diet group (PFE-SD), day 15. a-Glucuronidase prep-aration, postosmicated and stained with uranyl acetate. Portion of macrophage containingdebris with varying degrees of staining. (X 23,000). Fig 12-PFE-PF regimen, day 12.Correlative thick (A) and thin (B) sections of lesion within macrophage. A illustratesp8-glucuronidase reaction product seen by light microscopy (dense area at arrows). B, theadjacent thin section, demonstrates corresponding zones with increased electron scatter-ing (A, x 2100; B, x 15,000).

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HORIE ET ALCYTOCHEMICAL STUDIES OF LYSOSOMAL ENZYMES

American Journalof Pathology

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Pancreas Acinar Cell Regeneration