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Lipolytic sites in crayfish hepatopancreas and correlation with fine structure
Comp. Biochem. Physiol., 1971, Vol. 39B, pp. 227 to 236, Pergamon Press. Printed in Great Bntain
LIPOLYTIC SITES IN CRAYFISH HEPATOPANCREAS AND CORRELATION WITH FINE STRUCTURE* ROBERT
F. LO1221
and DARRYL
R. PETERSON
Department of Physiology, University of Illinois at the Medical Center, Chicago, Illinois 60680 (Received
29 September 1970)
Abstract-l. Tween 60-esterase activity was localized in crayfish hepatopancreas, primarily in tubule lumina, F-cell Fe-granules, B-cell vacuoles and R-cell striated borders. 2. All sites exhibited maximal activity at pH 7.1-7.3 and 8.1-8.4. 3. Na-taurocholate inhibited activity at all sites. 4. NaF slightly activated striated border activity; quinine had no effect. 5. NaF had no effect on luminal and B-cell activities; quinine inhibited both. 6. Extent of the surface enteric coat (fuzz layer) on various cell types correlated with intensity of enzyme reaction. 7. The results indicate the presence of at least two hepatopancreatic Tween 60-esterases and that B-cells secrete one of these into the lumen. INTRODUCTION THE INTERDEPENDENTquestions
of cell lineage in the hepatopancreas tubule and which cell is the source of digestive enzymes have been the subjects of much research since Huxley first recommended this gland as a secretory model in 1880. Jacobs (1928) c1assified the four epithelial cell types in the tubule: E-cells (embryonic) arise mitotically in the blind, distal tip ; R-cells (resorptive) contain much lipid and glycogen and are the most numerous of the four; F-cells (fibrillar) are highly basophilic; and B-cells (blister-like) are immense due to a single, large vacuole which squeezes all cytoplasm to the periphery. By their presence, the latter demarcate a long, middle region of the tubule. Hirsch & Jacobs (1928, 1930) correlated oscillating cell counts with enzyme activities at intervals after feeding and concluded that B-cells secrete the digestive enzymes of the hepatopancreas. They also concluded that cell differentiation followed the scheme: E + F -+ B + holocrine secretion
* Supported in part by Institutional Grants from USPHS and ACS. Preliminary results of this study were presented at the 1969 Summer Meeting of the American Society of Zoologists, University of Vermont, Burlington. 227
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More recent studies, however, disagree that the B-cell is secretory. The basophilia of the F-cell is a characteristic of most enzyme-secreting cells, and F-cell RNA content has been correlated with hepatopancreatic amylase activity (Fingerman et al., 1967). It has been suggested by Travis (1957) on the basis of light microscopy and by Bunt (1966) using electron microscopy that B-cells are actually degenerating R-cells and that F-cells synthesize and secrete the enzymes. Intestinal absorption cells observed in newborn rats (Wissig & Graney, 1968) closely resemble hepatopancreatic B-cells. In addition, several newer theories of cell lineage have been proposed which argue against the F-cell-B-cell sequence (Van Weel, 1955; Ogura, 1959; Davis & Burnett, 1964); however, all of these do assign an enzymesecreting role to the B-cell. In contrast to the above studies, Loizzi (1968) using electron microscopy, concluded that B-cells were formed from F-cells by continuous engorgement of a supranuclear vacuole in the latter, via apical pinocytosis of luminal contents. Moreover, the fine structural characteristics of R-cells differed considerably from those of F- and B-cells. This evidence tends to confirm the conclusions of Hirsch and Jacobs with regard to cell lineage but it leaves the identity of the secretory cell unanswered. The most direct approach to identifying the source of digestive enzymes, in the case of the hepatopancreas, would seem to be histochemical localization. No zymogen granules are present to give clues, and a lack of precise biochemical knowledge concerning hepatopancreatic enzymes has up to now prevented use of radioactive precursors. Crayfish trypsin has been successfully isolated (De Villez, 1965; Zwilling et al., 1969), but its amino acid composition is yet unknown. With respect to lipolytic enzymes, four non-specific esterases have been identified in hepatopancreatic extracts of Astacus (Kleine, 1967), and one of these has the same electrophoretic properties as gastric juice lipase. The pH optimum for this enzyme is acidic in gastric juice extracts but alkaline (pH 8-9) in the hepatopancreas extracts. The Gomori "Tween" method (Gomori, 1949) has been used to localize lipolytic enzyme activity with Tween 60 (a monostearate ester of sorbitol) as the substrate. Although Tween 60-esterase localization does not readily distinguish between true lipases and short-chain fatty acyl esterases (Mark, 1950; Bufio & Marifio, 1952), it does demonstrate those enzymes which are most likely to take part in the digestion of lipids. Activators and inhibitors such as NaF, bile salts and quinine can then be used in conjunction with Tween 60 to further differentiate enzymes (Gomori, 1952). In the present study, Tween 60-esterase activity in the hepatopancreas was characterized with respect to cellular and extraceUular sites, pH optima, and response to the above activators and inhibitors. Regions exhibiting lipolytic activity were then examined with electron microscopy. From the results of these studies, it is concluded that the source of luminal hepatopancreatic lipase is the B-cell, and that other esterases are located on the R-cell striated borders.
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MATERIALS AND METHODS Specimens of Procambarus clarkii were obtained in Spring from commercial suppliers and maintained in the laboratory for at least three weeks prior to the experiments. Control animals were fed oatmeal and liver ad lib. ; fasted animals were deprived of food 1 week prior to the experiments ; fed animals were given a stimulus meal of liver following 1 week of food deprivation at various times preceding the experiment. Hepatopancreases were obtained and prepared as follows: The crayfish was immersed in an ice-water mixture until its responses to stimulation were either absent or very sluggish. T h e carapace was removed, the alimentary tract was severed anteriorly at the esophagus and posteriorly through the hindgut, and the entire gut with both sides of the hepatopancreas attached was transferred to chilled van Harreveld's solution (van Harreveld, 1936). Each hepatopancreas side was quickly divided into three parts, rinsed in fresh solution, drained, and dropped into vials of ice-cold fixatives. These included acetone for the lipase technique, and 3 : 1 ethanolacetic acid and buffered 10% formalin for routine histology.
Light microscopy Lipolytic activity was localized using a modification of the Gomori method (1949) with Tween 60 as the substrate and Fast Green as the counterstain. T h e final incubation media, containing Tween 60 (polyoxyethylene sorbitan monostearate, Sigma), CaCI, and T r i s maleate buffer, was filtered through a Millipore filter with 0'45/~ pores to remove any precipitated calcium stearate. Pilot studies indicated that incubation for 24 hr at 37°C yielded optimum results. At room temperature, 48 hr were required to produce the same intensity, but it resulted in spurious deposits; therefore, the shorter incubation procedure was used throughout. Controls included sections incubated in substrate-free media and sections immersed in boiling water five minutes prior to incubation. False positives were checked using the Berlin Blue reaction for iron, and citric acid treatment for calcium (Humason, 1962). The effects of p H were studied by incubating serial sections in 14 different media over the p H range 6.2-8"4. Further characterization of the enzymes was carried out by incubating sections in NaF, Na-taurocholate or quinine hydrobromide at 10 -3 M (Gomori, 1952). Enzyme reaction sites were compared with other histochemical and morphological data from the Azure A triple stain for D N A , polysaccharides and protein (Himes & Moriber, 1956) and the mercuric bromphenol blue technique for protein (Mazia et al., 1953).
Electron microscopy All preliminary dissections were carried out in 2"5% glutaraldehyde buffered to p H 7"8 with phosphate to the point of freeing individual hepatopancreas tubules from collecting ducts under a dissecting microscope. Tubules showing no evidence of damage were transferred to fresh fixative and kept at 0--4°C overnight, post-fixed for 1½ hr in 1% osmium tetroxide at p H 7"8, dehydrated in an alcohol series and propylene dioxide, embedded in Epon 812 (Luft, 1961) and sectioned through various regions along the tubule. Thin sections were mounted on parlodion-coated copper grids, stained with uranyl acetate followed by lead citrate (Reynolds, 1963) and examined on an RCA E M U - 3 H instrument at 50 kV.
RESULTS
General distribution of Tween 60-esterase activity G o l d e n b r o w n d e p o s i t s o f P b S i n d i c a t i n g l i p o l y t i c sites w e r e o b s e r v e d m a i n l y in t h r e e r e g i o n s o f h e p a t o p a n c r e a s t u b u l e s (Figs. 1 - 4 ) : in t h e t u b u l e l u m e n ; o n t h e l u m i n a l surface o f s t r i a t e d b o r d e r s ; a n d in t h e B-cell vacuole. T h e s t r i a t e d b o r d e r
ROBERT F. LoIZZl AND DARRYL R. PETERSON
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sites were restricted almost exclusively to R-cells. A distinct gap in the PbS deposit was often observed at the luminal surface of the F-cell (Fig. 4) and a similar though less obvious discontinuity was present at the apical surface of the B-cell. Positive reactions were observed occasionally in the lumen of collecting ducts, the lumen of large blood vessels and in the hemolymph space surrounding tubules. No reaction was found in the lumen of capillaries which run parallel to the tubules. Very discrete deposits were sometimes seen in the cytoplasm of R-cells. Large granules in the F-cells showed a very dense, positive reaction (Figs. 1, 2, 4); however, in control sections, the brownish black color was only reduced, not eliminated. The Berlin Blue technique showed that these were Fe-granules (Ogura, 1959); it was concluded, therefore, that the background deposit was probably FeS or F%S a and, superimposed on it, was a positive enzyme reaction. Among the fourteen animals used in this study there was considerable variation in the extent of lipolytic activity throughout the gland. While in some animals cross sections through the hepatopancreas revealed that nearly all the tubules exhibited lipolytic activity, in other animals only a few tubules showed a reaction. There was also variation with respect to how many of the above-mentioned sites showed activity in the same gland; that is, some glands exhibited only striated border activity, others only luminal and B-cell vacuole activity, and others all three.
Effects of pH Serial sections of hepatopancreases from two animals were incubated in Tween 60 media at 14 different pH's ranging from 6.2 to 8-4. The relative lipolytic activities at five sites (tubule lumen, B-cell vacuole, Fe-granule, striated border and hemolymph) were all maximal in two pH regions: pH 7.1-7.3 and pH 8-1-8.4 (Table 1). The Fe-granule values were highest due to the fact that activity is superimposed on the natural density of the granule.
Effects of 10-3M sodium tauroeholate When sections were incubated in media containing sodium taurocholate, activity at all major sites was markedly inhibited. This was best demonstrated by examining sections at various intervals during incubation from 1½ to 16 hr. Sections incubated in normal media showed PbS deposits as early as 1½ hr, and the reaction had increased considerably by 5½ hr. Sections incubated in the media with bile salt exhibited little or no activity prior to 16 hr. FIG. 7. Low-power electron micrograph of hepatopancreatic B-cell. Cytoplasm (arrows) is almost totally obliterated due to engorgement of vacuole (vac). Interior of vacuole is electron-transparent except for a few small particles, vesicles, myelin forms and needle-like structures. A vesicle-loaded apical complex (ac) separates the vacuolar contents from the tubule lumen. FxG. 8. Low-power electron micrograph of hepatopancreatic F-cell with developing Fe-granule (Fe). The cytoplasm of this cell is filled with rough endoplasmic reticulum and several Golgi bodies (arrows), indicating its capacity for synthesizing exportable protein. R, adjacent R-cells.
Ftt;. 5. Electron m i c r o g r a p h of R-cell microvillar surfaces. Surface enteric c~mt (fuzz layer) is thickest ~m these cells, l,, lumen. Fro. 6. l : l e c m m m i c r o g r a p h of adjacent F-cell (F) a n d R-cell (R). A l t h o u g h tile microvilli of these cells are similar, the surface enteric coat a b r u p t l y decreases in thickness (arrow) over the F-cell. C o m p a r e with ~ap in e n z y m e activity s h o w n in Fig. 4.
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TABLEI~EFFECTS OF pH ON RELATIVELIPOLYTICACTIVITYIN FIVESITESOF HEPATOPANCREAS FROM Procambarusclarkii Site
6.23 6"45 6.55 6"70 6"99 7.13 7.23 7.40 7.51 7.66 7.87 8"13 8"28 8.37
Striated border B-cell vacuole Lumen Fe-granule Hemolymph
1 1 1 3 1
1 1 1 2 1
1 1 1 2 1
1 1 1 3 1
2 1 2 3 2
2 2 2 3 2
3 3 3 4 3
1 1 1 2 1
2 1 2 3 1
2 1 2 3 2
3 2 2 4 2
4 3 3 5 2
4 4 3 5 3
4 3 3 4 3
Sections (6/~) of hepatopancreas were incubated 24 hr in Tween 60 media (Gomori, 1950) at each of the above pH's. Relative activity was measured by estimating microscopically the amount of brown PbS deposit at each site and assigning a score of 0-5. Each value given is the average of three sections x two animals, rounded to the nearest whole number. All sites showed a first activity peak at approximately pH 7"1-7"3 and a second at pH 8.1-8"4.
Effects of
IO-SM
sodium fluoride
T h e presence of sodium fluoride in the incubation media slightly increased lipolytic activity on striated borders. It had no effect on activity in the tubule lumen or in B-cell vacuoles.
Effect of
IO-aM
quinine hydrobromide
Striated border activity was not affected by the presence of quinine in the incubation media. However, activity in the lumen and in B-cell vacuoles was markedly inhibited as demonstrated by examining sections at intervals during incubation.
Electron microscopy All cells lining the lumen of the hepatopancreas tubule possess a striated or microvillar border including B-cells (Figs. 5-7). Extending from the tips of the microvilli is a surface enteric coat or fuzz layer whose thickness varies with cell type. T h e coat is thickest on R-cells (Fig. 5), decreases abruptly over F-cells (Fig. 6), and is virtually absent on B-cells. T h e B-cell vacuole (Fig. 7) appears to have a watery interior in which a variety of small vesicles, granules and needle-like forms are suspended. Separating the vacuole from the lumen is an apical complex filled with pinocytic vesicles and mitochondria. T h e Fe-granule of the F-ceU (Fig. 8) also contains dense granules and vesicles similar to the B-cell vacuole. T h e quantity of these inclusions as well as the size of the Fe-granule are determined by the development of the F-cell. T h e cytoplasm of the F-cell is rich in rough endoplasmic reticulum, indicating high protein synthetic activity, and several Golgi bodies are present in a single cell.
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ROBERTF. LoIzzI ANDDARRYLR. PETERSON
DISCUSSION Maximum Tween 60-esterase activity in these experiments was observed in two pH ranges: 7.1-7.3 and 8-1-8.4. The first peak coincides with the pH recommended for the Tween method (Gomori, 1949). The values are also in general agreement with the pH optima for lipolytic activity in crude gastric juice and hepatopancreas extracts, that of the former usually being more acidic than the latter (Vonk, 1960). With regard to enzymes of greater purity, four hepatopancreas esterase fractions, with pH optima between 8 and 9, have been separated electrophoretically from Astacus (Kleine, 1967), and one of these fractions has the same electrophoretic mobility as the single gastric esterase; however, maximal activity of the latter occurs at several pH's (viz. 6.0, 7.5, 8.0 and 8.5) depending on the substrate used. With tributyrin, two peaks (6.0 and 8.0-8.5) were observed. In view of the non-specificity of the Tween 60 technique with respect to lipases and esterases, it is likely that each major activity site observed in this study (striated border, lumen, Fe-granule, and B-cell vacuole) represents more than one lipolytic enzyme. The observance of lipolytic activity on the striated border is consistent with the results of many other studies (reviewed by Greenberger, 1969) indicating the presence of digestive enzymes associated with intestinal brush borders. It is thought that the microvillar network serves as an important site for the hydrolysis of numerous nutritive substrates within the intestine as well as a site for absorption into the ceil. Ugolev (1965) has suggested that the extracellular zone immediately luminal to the plasma membrane of the brush border is an important region for a terminal digestive process which he has termed "membrane or contact digestion". The latter is claimed to be more effective than "luminal or cavital digestion", since it results in a high concentration of hydrolyzed end products at the cell membrane, and thus, favors transport into the cell. The electron microscopic results of this study reveal the presence of an enteric surface coat or fuzz layer along the striated borders of R-cells within the crayfish hepatopancreas. This feature is present to a lesser extent on F-cells and virtually absent on B-cells. The surface enteric coat was first described by Ito (1965) on cat intestinal absorption cells and consists of a filamentous network originating mainly at the tips of the microvilli. Such an arrangement fits in well with Ugolev's concept of contact digestion. Results of the present study suggest that the enzymes responsible for high lipolytic activity on the R-cell striated borders are concentrated in the thick surface enteric coats of those cells and function in the final hydrolysis and absorption of lipid nutrients. Conversely, the reduced fuzz layer on F- and B-cells correlates with a lack of enzyme activity and suggests that these two cells are not major participants in contact digestion. Based on their responses to activators and inhibitors, hepatopancreatic lipolytic enzymes appear to fall into two categories. On the one hand, striated border activity was inhibited by bile salt, not affected by quinine, and slightly activated by fluoride. Conversely, both luminal and B-cell vacuole activities were inhibited by bile salt, inhibited by quinine, and not affected by fluoride. The conclusions,
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therefore, are (1) that the same enzyme or class of enzymes is represented by Tween 60-esterase activities in the lumen and B-cell vacuole, and (2) that the activity on the striated border represents an enzyme or class of enzymes different from that in the first two sites. Further identification of the two enzyme classes is limited by the data given. According to Gomori's classification (1952), most gastro-intestinal esterases have characteristics lying in between those of "true lipases" such as pancreatic lipase (favor fatty acids > C-12; inhibited by quinine; slightly inhibited by fluoride; and activated by bile salts), and those of "true esterases" such as liver esterase (favor fatty acids < C- 12; no effect by quinine; inhibited by fluoride; and inhibited by bile salt). Other differences in specificity were also described such as straight vs. iso-chains, aliphatic vs. aromatic fatty acids and glyceride vs. monohydric alcohol moieties. Although this classification does not allow as precise a differentiation between specific enzymes as does the classification based on using a series of related substrates (Pearse, 1960; George & Ambadkar, 1963), it does permit the grouping of different enzymes with similar physiological roles. Hence, in the present study, since all sites hydrolyzed Tween 60 (which contains an 18-carbon fatty acid), they would all be expected to play an important role in the hydrolysis of ingested fats. In addition, their responses to bile salt, quinine and fluoride suggest that the two enzyme classes observed in the hepatopancreas, like most gastrointestinal lipolytic enzymes, are intermediate between true lipases and true esterases. From the presence of lipolytic activity in the B-cell vacuole and the conclusion discussed above, that B-cell enzyme and luminal enzyme are probably one and the same, three possible mechanisms are suggested: First, B-cells themselves may synthesize the enzyme, sequester it in the B-cell vacuole, and later secrete it apically. However, based on the fine structural appearance of the B-cell, its rough endoplasmic reticulum is in a state of degeneration as a result of compaction by the enlarging B-cell vacuole and is probably no longer capable of synthesizing protein. A second possibility is that the enzyme is synthesized and stored in a precursor of the B-cell and secreted only after differentiation to the B-cell stage. In this regard, the F-cell has been suggested as a precursor of the B-cell (Hirsch & Jacobs, 1928, 1930). Its cytoplasm is rich in rough endoplasmic reticulum and its Fegranule has fine structural similarities to the B-cell vacuole (Loizzi, 1968). Finally, hepatopancreatic amylase content has been correlated with F-cell RNA (Fingerman et al., 1967). These characteristics suggest that F-cells synthesize digestive enzymes and sequester them in the supranuclear vacuole containing the Fegranule. The present study, for the first time, shows the presence of digestive enzyme activity in the Fe-granule, thus strongly supporting such a mechanism. Both of the above possibilities (synthesis by either B-cell or F-cell) define the B-cell as secretory in function. It is also conceivable that the enzyme is synthesized and also secreted into the lumen by the F-cell; later, it may be absorbed into the B-cell vacuole, along with partially digested nutrients, thus accounting for the presence of lipolytic activity in the B-cell vacuole. This interpretation favors the
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B-ceU as being primarily absorptive in nature. If this were the case, however, one might, in at least some animals, expect the lumen to be filled with lipase activity and protein, with a much lesser amount in the B-cell vacuole. In most cases, however, the reverse was true. Results of the present study alone, however, do not prove which of the above is the true mechanism because the experiments do not tell whether the enzyme has moved from B-cell to lumen or vice versa. Rather, the results provide a direct link between B-cells and digestive enzymes, a fact which was only assumed in previous studies where the secretory function of the B-cell was based on a correlation between B-cell number and enzyme activity in gland homogenate. Since this study shows that B-cells contain digestive enzymes, and that these are probably the same as the enzymes of the lumen, use of the correlative studies (Hirsch & Jacobs, 1928, 1930) to show enzyme movement from B-cell to lumen is now justified. Hence, it may be concluded that F-cells differentiate into B-cells, and that the latter are truly secretory. Finally, the presence of lipolytic activity in blood vessels and hemolymph is interesting in that it suggests the possibility of a circulating "clearing factor" in crustaceans such as the lipoprotein lipase found in mammalian serum (Engelberg, 1964) which functions in the removal of triglyceride from the bloodstream.
SUMMARY The Gomori Tween 60 technique was used to localize lipolytic enzyme activity in the hepatopancreas of Procambarus darkii. Regions showing activity were then examined with the electron microscope. Activity sites included the tubule lumen, B-cell vacuole, F-cell Fe-granule, R-cell striated border, and hemolymph bathing the tubules. All sites showed maximal activities in two pH regions: 7.1-7.3 and 8.1-8.4. Correlations between Tween 60-esterase results and fine structure indicates that the border activity is related morphologically to the surface enteric coat of the R-cell and functions in contact digestion. From their responses to quinine hydrobromide, sodium taruocholate and sodium fluoride, it was concluded that activity in the B-cell vacuole and in the lumen represent one class of esterases while that on the striated border constitutes a second class. The two classes occupy an intermediate position between "true lipases" and "true esterases" according to Gomori's classification; however, since both classes actively hydrolyzed Tween 60, an ester of stearic acid (C-18), all tubule sites localized in this study are probably involved in hepatopancreatic digestion of ingested fats. The presence of digestive enzyme activity in both the Fe-granule of the F-cell and in the B-cell vacuole supports the view that F-cells differentiate into B-cells. The similarities between luminal and B-cell enzyme responses to activators and inhibitors supports the view that B-cells secrete the digestive enzymes of the hepatopancreas.
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REFERENCES BuI~o W. & MARII~OR. G. (1952) Location of lipase activity in the chick embryo. Acta anat. 16, 85-92. BUNT A. H. (1966) An ultrastructural study of the hepatopancreas of Procambarus clarkii (Girard) (Decapoda, Astacidea). Crustaceana 15, 282-288. DAVIS L. E. & BURNETT A. L. (1964) A study of growth and cell differentiation in the hepatopancreas of the crayfish. Devel. Biol. 10, 122-153. DEVILLEZ E. M. (1965) Isolation of the proteolytic digestive enzymes from the gastric juice of the crayfish Orconectes virilis (Hagen). Comp. Biochem. Physiol. 14, 577-586. ENGELRERGH. (1964) Heparin and the removal of triglyceride from the blood stream. In Int. Symposium on Heparin, Los Angeles, 1963. Amer.ff. Cardiol. 14, 8-17. FINGERMANM., DOMINICZAKT., MIYAWAKIM., OGURO C. & YAMAMOTOY. (1967) Neuroendocrine control of the hepatopancreas in the crayfish Procambarus clarkii. Physiol. Zool. 40, 23-30. GEORGE J. C. & AMBADKARP. M. (1963) Histochemical demonstration of lipids and lipase activity in rat testis..7. Histochem. Cytochern. 11,420--425. GOMORI G. (1949) Histochemical localization of true lipase. Proc. Soc. exp. Biol. Med. 72, 697-700. GOMORI G. (1952) The histochemistry of esterases. In Int. Rev. Cytol. (Edited by BOURNE G. H. and DANIELLIJ. F.), Vol. 1, pp. 323-335. Academic Press, New York. GREENBERGERN. J. (1969) The intestinal brush border as a digestive and absorptive surface. Am..7. Med. Sci. 258, 144 149. HIMES M. & MORIBER L. (1956) A triple stain for desoxyribonucleic acid, polysaccharides and proteins. Stain Technol. 31, 67-70. Hlascrt G. C. & JACOBSW. (1928) Der Arbeitsrhythmus der Mitteldarmdriise yon Astacus leptodactylus--I. Methodik und Technik: Der Beweis der Periodizitiit. Ztschr. wiss. Biol. 8, 102-144. HIRSCH G. C. & JACOBSW. (1930) Der Arbeitsrhythrnus der Mitteldarmdriise yon Astacus leptodactylus--II. Wachstum als primiirer Faktor des Rhythmus eines polyphasischen organigen Sekretionssytems. Ztschr. wiss. Biol. 12, 524-558. HUMASON G. L. (1962) Animal Tissue Techniques, pp. 348-350. W. H. Freeman & Co., San Francisco. ITO S. (1965) The enteric surface coat on cat intestinal microvilli..7. Cell Biol. 27, 475--491. JACOBSW. (1928) Untersuchungen fiber die Cytologie der Sekretbildung in der MitteldarmdriJse yon Astacus leptodactylus. Ztschr. Zellforsch. mikro. Anat. 8, 1-62. KLEINE R. (1967) Vorkommen und Eigenschaften der Carbonsatireesterasen im Hepatopankreas und Magensaft des Flusskrebses Astaeus astaeus (L.) und Cambarus affinis (Say). Z. vergl. Physiol. 55, 333-353. LolzzI R. F. (1968) Fine structure of the crayfish hepatopancreas..7. Cell Biol. 39, 82a. LUFT J. H. (1961) Improvements in epoxy resin embedding materials..7. Biophys Biochem. Cytol. 9, 409--414. MARK D. D. (1950) Distribution of lipase in preneoplastic and neoplastic states induced in the rat liver by paradimethylaminoazobenzene. Archs Path. 49, 545-554. MAZlA D., BREWERP. A. & ALFERT M. (1953) The cytochemical staining and measurement of protein with mercuric bromphenol blue. Biol. Bull. 104, 57-67. OGURA K. (1959) Midgut gland cells accumulating iron or copper in the crayfish, Procambarus clarkii. Annotnes zool..Tap. 32, 133-142. PEARSE A. G. E. (1960) Histochemistry Theoretical and Applied, chap. XVI, pp. 456--490. Little, Brown & Co., Boston. REYNOLDS E. S. (1963) The use of lead citrate at high p H as an electron opaque stain in electron microscopy..7. Cell Biol. 17, 208-212. 9
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TRAVlS D. F, (1957) The molting cycle of the spiny lobster Panulirus argus latralle--IV. Post-ecdysial histochemical changes in the hepatopancreas and integumental tissues. Biol. Bull. 113, 451-479. U c o ~ v A. M. (1965) Membrane (contact) digestion. Physiol. Rev. 45, 555-595. VAN HAI~.WLD A. (1936) A physiological solution for fresh water crustaceans. Proc. Soc. exp. Biol. Med. 34, 428-432. VAN WFa~LP. B. (1955) Processes of secretion, restitution and resorption in gland of midgut of Atya spinipes newport. Physiol. Zool. 28, 40-54. VONK H. J. (1960) Digestion and metabolism. In Physiology of Crustacea--I : Metabolism and Growth (Edited by WATERMANT. H.), pp. 291-316. Academic Press, New York. WXSSlG S. L. & GRANEY D. O. (1968) Membrane modifications in the apical endocytic complex of ileal epithelial cells, ft. Cell Biol. 39, 564-579. ZWlLLING R., PFLEIDE~R G., SONN-EBOaNH., KRAFTV. & STUCKY I. (1969) The evolution of endopeptidases--V. Common and different traits of bovine and crayfish trypsin. Comp. Biochem. Physiol. 28, 1275-1287. Key Word lndex--Hepatopancreas; Procambarus clarkii; crayfish; lipase; esterase; Tween; histochemistry; fine structure; electron microscopy; cell differentiation; secretion; absorption; striated border; fuzz layer. While this article was in press, the following related reference was published: LoIzzI R. F. (1971) Interpretation of crayfish hepatopancreatic function based on fine structural analysis of epithelial cell lines and muscle network. Z. Zellforsch. 113, 420 n,~9.
LIPOLYTIC SITES IN CRAYFISH HEPATOPANCREAS AND CORRELATION WITH FINE STRUCTURE* ROBERT
F. LO1221
and DARRYL
R. PETERSON
Department of Physiology, University of Illinois at the Medical Center, Chicago, Illinois 60680 (Received
29 September 1970)
Abstract-l. Tween 60-esterase activity was localized in crayfish hepatopancreas, primarily in tubule lumina, F-cell Fe-granules, B-cell vacuoles and R-cell striated borders. 2. All sites exhibited maximal activity at pH 7.1-7.3 and 8.1-8.4. 3. Na-taurocholate inhibited activity at all sites. 4. NaF slightly activated striated border activity; quinine had no effect. 5. NaF had no effect on luminal and B-cell activities; quinine inhibited both. 6. Extent of the surface enteric coat (fuzz layer) on various cell types correlated with intensity of enzyme reaction. 7. The results indicate the presence of at least two hepatopancreatic Tween 60-esterases and that B-cells secrete one of these into the lumen. INTRODUCTION THE INTERDEPENDENTquestions
of cell lineage in the hepatopancreas tubule and which cell is the source of digestive enzymes have been the subjects of much research since Huxley first recommended this gland as a secretory model in 1880. Jacobs (1928) c1assified the four epithelial cell types in the tubule: E-cells (embryonic) arise mitotically in the blind, distal tip ; R-cells (resorptive) contain much lipid and glycogen and are the most numerous of the four; F-cells (fibrillar) are highly basophilic; and B-cells (blister-like) are immense due to a single, large vacuole which squeezes all cytoplasm to the periphery. By their presence, the latter demarcate a long, middle region of the tubule. Hirsch & Jacobs (1928, 1930) correlated oscillating cell counts with enzyme activities at intervals after feeding and concluded that B-cells secrete the digestive enzymes of the hepatopancreas. They also concluded that cell differentiation followed the scheme: E + F -+ B + holocrine secretion
* Supported in part by Institutional Grants from USPHS and ACS. Preliminary results of this study were presented at the 1969 Summer Meeting of the American Society of Zoologists, University of Vermont, Burlington. 227
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More recent studies, however, disagree that the B-cell is secretory. The basophilia of the F-cell is a characteristic of most enzyme-secreting cells, and F-cell RNA content has been correlated with hepatopancreatic amylase activity (Fingerman et al., 1967). It has been suggested by Travis (1957) on the basis of light microscopy and by Bunt (1966) using electron microscopy that B-cells are actually degenerating R-cells and that F-cells synthesize and secrete the enzymes. Intestinal absorption cells observed in newborn rats (Wissig & Graney, 1968) closely resemble hepatopancreatic B-cells. In addition, several newer theories of cell lineage have been proposed which argue against the F-cell-B-cell sequence (Van Weel, 1955; Ogura, 1959; Davis & Burnett, 1964); however, all of these do assign an enzymesecreting role to the B-cell. In contrast to the above studies, Loizzi (1968) using electron microscopy, concluded that B-cells were formed from F-cells by continuous engorgement of a supranuclear vacuole in the latter, via apical pinocytosis of luminal contents. Moreover, the fine structural characteristics of R-cells differed considerably from those of F- and B-cells. This evidence tends to confirm the conclusions of Hirsch and Jacobs with regard to cell lineage but it leaves the identity of the secretory cell unanswered. The most direct approach to identifying the source of digestive enzymes, in the case of the hepatopancreas, would seem to be histochemical localization. No zymogen granules are present to give clues, and a lack of precise biochemical knowledge concerning hepatopancreatic enzymes has up to now prevented use of radioactive precursors. Crayfish trypsin has been successfully isolated (De Villez, 1965; Zwilling et al., 1969), but its amino acid composition is yet unknown. With respect to lipolytic enzymes, four non-specific esterases have been identified in hepatopancreatic extracts of Astacus (Kleine, 1967), and one of these has the same electrophoretic properties as gastric juice lipase. The pH optimum for this enzyme is acidic in gastric juice extracts but alkaline (pH 8-9) in the hepatopancreas extracts. The Gomori "Tween" method (Gomori, 1949) has been used to localize lipolytic enzyme activity with Tween 60 (a monostearate ester of sorbitol) as the substrate. Although Tween 60-esterase localization does not readily distinguish between true lipases and short-chain fatty acyl esterases (Mark, 1950; Bufio & Marifio, 1952), it does demonstrate those enzymes which are most likely to take part in the digestion of lipids. Activators and inhibitors such as NaF, bile salts and quinine can then be used in conjunction with Tween 60 to further differentiate enzymes (Gomori, 1952). In the present study, Tween 60-esterase activity in the hepatopancreas was characterized with respect to cellular and extraceUular sites, pH optima, and response to the above activators and inhibitors. Regions exhibiting lipolytic activity were then examined with electron microscopy. From the results of these studies, it is concluded that the source of luminal hepatopancreatic lipase is the B-cell, and that other esterases are located on the R-cell striated borders.
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MATERIALS AND METHODS Specimens of Procambarus clarkii were obtained in Spring from commercial suppliers and maintained in the laboratory for at least three weeks prior to the experiments. Control animals were fed oatmeal and liver ad lib. ; fasted animals were deprived of food 1 week prior to the experiments ; fed animals were given a stimulus meal of liver following 1 week of food deprivation at various times preceding the experiment. Hepatopancreases were obtained and prepared as follows: The crayfish was immersed in an ice-water mixture until its responses to stimulation were either absent or very sluggish. T h e carapace was removed, the alimentary tract was severed anteriorly at the esophagus and posteriorly through the hindgut, and the entire gut with both sides of the hepatopancreas attached was transferred to chilled van Harreveld's solution (van Harreveld, 1936). Each hepatopancreas side was quickly divided into three parts, rinsed in fresh solution, drained, and dropped into vials of ice-cold fixatives. These included acetone for the lipase technique, and 3 : 1 ethanolacetic acid and buffered 10% formalin for routine histology.
Light microscopy Lipolytic activity was localized using a modification of the Gomori method (1949) with Tween 60 as the substrate and Fast Green as the counterstain. T h e final incubation media, containing Tween 60 (polyoxyethylene sorbitan monostearate, Sigma), CaCI, and T r i s maleate buffer, was filtered through a Millipore filter with 0'45/~ pores to remove any precipitated calcium stearate. Pilot studies indicated that incubation for 24 hr at 37°C yielded optimum results. At room temperature, 48 hr were required to produce the same intensity, but it resulted in spurious deposits; therefore, the shorter incubation procedure was used throughout. Controls included sections incubated in substrate-free media and sections immersed in boiling water five minutes prior to incubation. False positives were checked using the Berlin Blue reaction for iron, and citric acid treatment for calcium (Humason, 1962). The effects of p H were studied by incubating serial sections in 14 different media over the p H range 6.2-8"4. Further characterization of the enzymes was carried out by incubating sections in NaF, Na-taurocholate or quinine hydrobromide at 10 -3 M (Gomori, 1952). Enzyme reaction sites were compared with other histochemical and morphological data from the Azure A triple stain for D N A , polysaccharides and protein (Himes & Moriber, 1956) and the mercuric bromphenol blue technique for protein (Mazia et al., 1953).
Electron microscopy All preliminary dissections were carried out in 2"5% glutaraldehyde buffered to p H 7"8 with phosphate to the point of freeing individual hepatopancreas tubules from collecting ducts under a dissecting microscope. Tubules showing no evidence of damage were transferred to fresh fixative and kept at 0--4°C overnight, post-fixed for 1½ hr in 1% osmium tetroxide at p H 7"8, dehydrated in an alcohol series and propylene dioxide, embedded in Epon 812 (Luft, 1961) and sectioned through various regions along the tubule. Thin sections were mounted on parlodion-coated copper grids, stained with uranyl acetate followed by lead citrate (Reynolds, 1963) and examined on an RCA E M U - 3 H instrument at 50 kV.
RESULTS
General distribution of Tween 60-esterase activity G o l d e n b r o w n d e p o s i t s o f P b S i n d i c a t i n g l i p o l y t i c sites w e r e o b s e r v e d m a i n l y in t h r e e r e g i o n s o f h e p a t o p a n c r e a s t u b u l e s (Figs. 1 - 4 ) : in t h e t u b u l e l u m e n ; o n t h e l u m i n a l surface o f s t r i a t e d b o r d e r s ; a n d in t h e B-cell vacuole. T h e s t r i a t e d b o r d e r
ROBERT F. LoIZZl AND DARRYL R. PETERSON
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sites were restricted almost exclusively to R-cells. A distinct gap in the PbS deposit was often observed at the luminal surface of the F-cell (Fig. 4) and a similar though less obvious discontinuity was present at the apical surface of the B-cell. Positive reactions were observed occasionally in the lumen of collecting ducts, the lumen of large blood vessels and in the hemolymph space surrounding tubules. No reaction was found in the lumen of capillaries which run parallel to the tubules. Very discrete deposits were sometimes seen in the cytoplasm of R-cells. Large granules in the F-cells showed a very dense, positive reaction (Figs. 1, 2, 4); however, in control sections, the brownish black color was only reduced, not eliminated. The Berlin Blue technique showed that these were Fe-granules (Ogura, 1959); it was concluded, therefore, that the background deposit was probably FeS or F%S a and, superimposed on it, was a positive enzyme reaction. Among the fourteen animals used in this study there was considerable variation in the extent of lipolytic activity throughout the gland. While in some animals cross sections through the hepatopancreas revealed that nearly all the tubules exhibited lipolytic activity, in other animals only a few tubules showed a reaction. There was also variation with respect to how many of the above-mentioned sites showed activity in the same gland; that is, some glands exhibited only striated border activity, others only luminal and B-cell vacuole activity, and others all three.
Effects of pH Serial sections of hepatopancreases from two animals were incubated in Tween 60 media at 14 different pH's ranging from 6.2 to 8-4. The relative lipolytic activities at five sites (tubule lumen, B-cell vacuole, Fe-granule, striated border and hemolymph) were all maximal in two pH regions: pH 7.1-7.3 and pH 8-1-8.4 (Table 1). The Fe-granule values were highest due to the fact that activity is superimposed on the natural density of the granule.
Effects of 10-3M sodium tauroeholate When sections were incubated in media containing sodium taurocholate, activity at all major sites was markedly inhibited. This was best demonstrated by examining sections at various intervals during incubation from 1½ to 16 hr. Sections incubated in normal media showed PbS deposits as early as 1½ hr, and the reaction had increased considerably by 5½ hr. Sections incubated in the media with bile salt exhibited little or no activity prior to 16 hr. FIG. 7. Low-power electron micrograph of hepatopancreatic B-cell. Cytoplasm (arrows) is almost totally obliterated due to engorgement of vacuole (vac). Interior of vacuole is electron-transparent except for a few small particles, vesicles, myelin forms and needle-like structures. A vesicle-loaded apical complex (ac) separates the vacuolar contents from the tubule lumen. FxG. 8. Low-power electron micrograph of hepatopancreatic F-cell with developing Fe-granule (Fe). The cytoplasm of this cell is filled with rough endoplasmic reticulum and several Golgi bodies (arrows), indicating its capacity for synthesizing exportable protein. R, adjacent R-cells.
Ftt;. 5. Electron m i c r o g r a p h of R-cell microvillar surfaces. Surface enteric c~mt (fuzz layer) is thickest ~m these cells, l,, lumen. Fro. 6. l : l e c m m m i c r o g r a p h of adjacent F-cell (F) a n d R-cell (R). A l t h o u g h tile microvilli of these cells are similar, the surface enteric coat a b r u p t l y decreases in thickness (arrow) over the F-cell. C o m p a r e with ~ap in e n z y m e activity s h o w n in Fig. 4.
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TABLEI~EFFECTS OF pH ON RELATIVELIPOLYTICACTIVITYIN FIVESITESOF HEPATOPANCREAS FROM Procambarusclarkii Site
6.23 6"45 6.55 6"70 6"99 7.13 7.23 7.40 7.51 7.66 7.87 8"13 8"28 8.37
Striated border B-cell vacuole Lumen Fe-granule Hemolymph
1 1 1 3 1
1 1 1 2 1
1 1 1 2 1
1 1 1 3 1
2 1 2 3 2
2 2 2 3 2
3 3 3 4 3
1 1 1 2 1
2 1 2 3 1
2 1 2 3 2
3 2 2 4 2
4 3 3 5 2
4 4 3 5 3
4 3 3 4 3
Sections (6/~) of hepatopancreas were incubated 24 hr in Tween 60 media (Gomori, 1950) at each of the above pH's. Relative activity was measured by estimating microscopically the amount of brown PbS deposit at each site and assigning a score of 0-5. Each value given is the average of three sections x two animals, rounded to the nearest whole number. All sites showed a first activity peak at approximately pH 7"1-7"3 and a second at pH 8.1-8"4.
Effects of
IO-SM
sodium fluoride
T h e presence of sodium fluoride in the incubation media slightly increased lipolytic activity on striated borders. It had no effect on activity in the tubule lumen or in B-cell vacuoles.
Effect of
IO-aM
quinine hydrobromide
Striated border activity was not affected by the presence of quinine in the incubation media. However, activity in the lumen and in B-cell vacuoles was markedly inhibited as demonstrated by examining sections at intervals during incubation.
Electron microscopy All cells lining the lumen of the hepatopancreas tubule possess a striated or microvillar border including B-cells (Figs. 5-7). Extending from the tips of the microvilli is a surface enteric coat or fuzz layer whose thickness varies with cell type. T h e coat is thickest on R-cells (Fig. 5), decreases abruptly over F-cells (Fig. 6), and is virtually absent on B-cells. T h e B-cell vacuole (Fig. 7) appears to have a watery interior in which a variety of small vesicles, granules and needle-like forms are suspended. Separating the vacuole from the lumen is an apical complex filled with pinocytic vesicles and mitochondria. T h e Fe-granule of the F-ceU (Fig. 8) also contains dense granules and vesicles similar to the B-cell vacuole. T h e quantity of these inclusions as well as the size of the Fe-granule are determined by the development of the F-cell. T h e cytoplasm of the F-cell is rich in rough endoplasmic reticulum, indicating high protein synthetic activity, and several Golgi bodies are present in a single cell.
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DISCUSSION Maximum Tween 60-esterase activity in these experiments was observed in two pH ranges: 7.1-7.3 and 8-1-8.4. The first peak coincides with the pH recommended for the Tween method (Gomori, 1949). The values are also in general agreement with the pH optima for lipolytic activity in crude gastric juice and hepatopancreas extracts, that of the former usually being more acidic than the latter (Vonk, 1960). With regard to enzymes of greater purity, four hepatopancreas esterase fractions, with pH optima between 8 and 9, have been separated electrophoretically from Astacus (Kleine, 1967), and one of these fractions has the same electrophoretic mobility as the single gastric esterase; however, maximal activity of the latter occurs at several pH's (viz. 6.0, 7.5, 8.0 and 8.5) depending on the substrate used. With tributyrin, two peaks (6.0 and 8.0-8.5) were observed. In view of the non-specificity of the Tween 60 technique with respect to lipases and esterases, it is likely that each major activity site observed in this study (striated border, lumen, Fe-granule, and B-cell vacuole) represents more than one lipolytic enzyme. The observance of lipolytic activity on the striated border is consistent with the results of many other studies (reviewed by Greenberger, 1969) indicating the presence of digestive enzymes associated with intestinal brush borders. It is thought that the microvillar network serves as an important site for the hydrolysis of numerous nutritive substrates within the intestine as well as a site for absorption into the ceil. Ugolev (1965) has suggested that the extracellular zone immediately luminal to the plasma membrane of the brush border is an important region for a terminal digestive process which he has termed "membrane or contact digestion". The latter is claimed to be more effective than "luminal or cavital digestion", since it results in a high concentration of hydrolyzed end products at the cell membrane, and thus, favors transport into the cell. The electron microscopic results of this study reveal the presence of an enteric surface coat or fuzz layer along the striated borders of R-cells within the crayfish hepatopancreas. This feature is present to a lesser extent on F-cells and virtually absent on B-cells. The surface enteric coat was first described by Ito (1965) on cat intestinal absorption cells and consists of a filamentous network originating mainly at the tips of the microvilli. Such an arrangement fits in well with Ugolev's concept of contact digestion. Results of the present study suggest that the enzymes responsible for high lipolytic activity on the R-cell striated borders are concentrated in the thick surface enteric coats of those cells and function in the final hydrolysis and absorption of lipid nutrients. Conversely, the reduced fuzz layer on F- and B-cells correlates with a lack of enzyme activity and suggests that these two cells are not major participants in contact digestion. Based on their responses to activators and inhibitors, hepatopancreatic lipolytic enzymes appear to fall into two categories. On the one hand, striated border activity was inhibited by bile salt, not affected by quinine, and slightly activated by fluoride. Conversely, both luminal and B-cell vacuole activities were inhibited by bile salt, inhibited by quinine, and not affected by fluoride. The conclusions,
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therefore, are (1) that the same enzyme or class of enzymes is represented by Tween 60-esterase activities in the lumen and B-cell vacuole, and (2) that the activity on the striated border represents an enzyme or class of enzymes different from that in the first two sites. Further identification of the two enzyme classes is limited by the data given. According to Gomori's classification (1952), most gastro-intestinal esterases have characteristics lying in between those of "true lipases" such as pancreatic lipase (favor fatty acids > C-12; inhibited by quinine; slightly inhibited by fluoride; and activated by bile salts), and those of "true esterases" such as liver esterase (favor fatty acids < C- 12; no effect by quinine; inhibited by fluoride; and inhibited by bile salt). Other differences in specificity were also described such as straight vs. iso-chains, aliphatic vs. aromatic fatty acids and glyceride vs. monohydric alcohol moieties. Although this classification does not allow as precise a differentiation between specific enzymes as does the classification based on using a series of related substrates (Pearse, 1960; George & Ambadkar, 1963), it does permit the grouping of different enzymes with similar physiological roles. Hence, in the present study, since all sites hydrolyzed Tween 60 (which contains an 18-carbon fatty acid), they would all be expected to play an important role in the hydrolysis of ingested fats. In addition, their responses to bile salt, quinine and fluoride suggest that the two enzyme classes observed in the hepatopancreas, like most gastrointestinal lipolytic enzymes, are intermediate between true lipases and true esterases. From the presence of lipolytic activity in the B-cell vacuole and the conclusion discussed above, that B-cell enzyme and luminal enzyme are probably one and the same, three possible mechanisms are suggested: First, B-cells themselves may synthesize the enzyme, sequester it in the B-cell vacuole, and later secrete it apically. However, based on the fine structural appearance of the B-cell, its rough endoplasmic reticulum is in a state of degeneration as a result of compaction by the enlarging B-cell vacuole and is probably no longer capable of synthesizing protein. A second possibility is that the enzyme is synthesized and stored in a precursor of the B-cell and secreted only after differentiation to the B-cell stage. In this regard, the F-cell has been suggested as a precursor of the B-cell (Hirsch & Jacobs, 1928, 1930). Its cytoplasm is rich in rough endoplasmic reticulum and its Fegranule has fine structural similarities to the B-cell vacuole (Loizzi, 1968). Finally, hepatopancreatic amylase content has been correlated with F-cell RNA (Fingerman et al., 1967). These characteristics suggest that F-cells synthesize digestive enzymes and sequester them in the supranuclear vacuole containing the Fegranule. The present study, for the first time, shows the presence of digestive enzyme activity in the Fe-granule, thus strongly supporting such a mechanism. Both of the above possibilities (synthesis by either B-cell or F-cell) define the B-cell as secretory in function. It is also conceivable that the enzyme is synthesized and also secreted into the lumen by the F-cell; later, it may be absorbed into the B-cell vacuole, along with partially digested nutrients, thus accounting for the presence of lipolytic activity in the B-cell vacuole. This interpretation favors the
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B-ceU as being primarily absorptive in nature. If this were the case, however, one might, in at least some animals, expect the lumen to be filled with lipase activity and protein, with a much lesser amount in the B-cell vacuole. In most cases, however, the reverse was true. Results of the present study alone, however, do not prove which of the above is the true mechanism because the experiments do not tell whether the enzyme has moved from B-cell to lumen or vice versa. Rather, the results provide a direct link between B-cells and digestive enzymes, a fact which was only assumed in previous studies where the secretory function of the B-cell was based on a correlation between B-cell number and enzyme activity in gland homogenate. Since this study shows that B-cells contain digestive enzymes, and that these are probably the same as the enzymes of the lumen, use of the correlative studies (Hirsch & Jacobs, 1928, 1930) to show enzyme movement from B-cell to lumen is now justified. Hence, it may be concluded that F-cells differentiate into B-cells, and that the latter are truly secretory. Finally, the presence of lipolytic activity in blood vessels and hemolymph is interesting in that it suggests the possibility of a circulating "clearing factor" in crustaceans such as the lipoprotein lipase found in mammalian serum (Engelberg, 1964) which functions in the removal of triglyceride from the bloodstream.
SUMMARY The Gomori Tween 60 technique was used to localize lipolytic enzyme activity in the hepatopancreas of Procambarus darkii. Regions showing activity were then examined with the electron microscope. Activity sites included the tubule lumen, B-cell vacuole, F-cell Fe-granule, R-cell striated border, and hemolymph bathing the tubules. All sites showed maximal activities in two pH regions: 7.1-7.3 and 8.1-8.4. Correlations between Tween 60-esterase results and fine structure indicates that the border activity is related morphologically to the surface enteric coat of the R-cell and functions in contact digestion. From their responses to quinine hydrobromide, sodium taruocholate and sodium fluoride, it was concluded that activity in the B-cell vacuole and in the lumen represent one class of esterases while that on the striated border constitutes a second class. The two classes occupy an intermediate position between "true lipases" and "true esterases" according to Gomori's classification; however, since both classes actively hydrolyzed Tween 60, an ester of stearic acid (C-18), all tubule sites localized in this study are probably involved in hepatopancreatic digestion of ingested fats. The presence of digestive enzyme activity in both the Fe-granule of the F-cell and in the B-cell vacuole supports the view that F-cells differentiate into B-cells. The similarities between luminal and B-cell enzyme responses to activators and inhibitors supports the view that B-cells secrete the digestive enzymes of the hepatopancreas.
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TRAVlS D. F, (1957) The molting cycle of the spiny lobster Panulirus argus latralle--IV. Post-ecdysial histochemical changes in the hepatopancreas and integumental tissues. Biol. Bull. 113, 451-479. U c o ~ v A. M. (1965) Membrane (contact) digestion. Physiol. Rev. 45, 555-595. VAN HAI~.WLD A. (1936) A physiological solution for fresh water crustaceans. Proc. Soc. exp. Biol. Med. 34, 428-432. VAN WFa~LP. B. (1955) Processes of secretion, restitution and resorption in gland of midgut of Atya spinipes newport. Physiol. Zool. 28, 40-54. VONK H. J. (1960) Digestion and metabolism. In Physiology of Crustacea--I : Metabolism and Growth (Edited by WATERMANT. H.), pp. 291-316. Academic Press, New York. WXSSlG S. L. & GRANEY D. O. (1968) Membrane modifications in the apical endocytic complex of ileal epithelial cells, ft. Cell Biol. 39, 564-579. ZWlLLING R., PFLEIDE~R G., SONN-EBOaNH., KRAFTV. & STUCKY I. (1969) The evolution of endopeptidases--V. Common and different traits of bovine and crayfish trypsin. Comp. Biochem. Physiol. 28, 1275-1287. Key Word lndex--Hepatopancreas; Procambarus clarkii; crayfish; lipase; esterase; Tween; histochemistry; fine structure; electron microscopy; cell differentiation; secretion; absorption; striated border; fuzz layer. While this article was in press, the following related reference was published: LoIzzI R. F. (1971) Interpretation of crayfish hepatopancreatic function based on fine structural analysis of epithelial cell lines and muscle network. Z. Zellforsch. 113, 420 n,~9.