Histochemistry

Histochemistry59, 29-44 (1978)

9 by Springer-Verlag 1978

The Localization of Enzyme Activities in the Pancreatic Appendages of Sepia officinalis L. (Cephalopoda) '~ H.H. Donaubauer and R. Schipp** Institut fiir Allgemeineund SpezielleZoologieder Justus-Liebig-Universit/it, D-6300 Giessen, Federal Republic of Germany, and Station de Biologie Marine, Arcachon, France

Summary. In this study, enzyme activities of the pancreatic appendages of the ductus hepatopancreas (the so-called "Pancreas") in Sepia officinalis L. have been demonstrated by light and electron microscopical methods: Malate dehydrogenase, monoamlne oxidase, acid phosphatase, /%glucuronidase, adenosine triphosphatase and carbonic anhydrase were shown by the former, and monoamine oxidase, catalase, glutamic oxalacetic transaminase, choline esterase (non-specific), alkaline phosphatase, acid phosphatase and carbonic anhydrase by the latter technique. The correlation between enzyme activity and distribution, and the presumed function of the two pancreatic epithelia is discussed.

Introduction The pancreatic appendages in Sepioidea are composed of two epithelia which are separated by a blood sinus as has already been established (Schipp and v. Boletzky, 1976; Boucaud-Camou, 1972). Based on the appearence of the structure, Schipp and v. Boletzky (1976) suggested that the inner epithelium has a dual function serving both excretion and nutrient absorption, while the outer epithelium is associated with processes of osmoregulation and urine formation. To date there is little data available concerning the enzymatic activities of the pancreatic appendages in Sepioidea (Romijn, 1935; Boucaud-Camou, 1974). The present research represents an attempt to find further evidence for the above named functions by means of histochemical and cytochemical techniques. * This study was supported by the "Deutsche Forschungsgemeinschaft" ** Address for offprint requests: R. Schipp, Institut f/Jr Allgemeine und Spezielle Zoologie der Justus-Liebig-Universit~it,Stephanstrage24, D-6300 Giessen, Federal Republic of Germany

0301-5564/78/0059/0029/$03.20

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H.H. Donaubauer and R. Schipp

Material and Methods Light Microscopical Methods Pieces of fresh tissue of semi-adult/adult Sepia officinalis L. of the Bassin d'Arcachon/France were fixed in buffered formalin of formalin-cetylpyridinium (Williams and Jackson, 1956) and embedded in paraffin. Periodic acid-Schiff reaction (PAS) and staining with alcian blue, alcian blue/HC1, thionine and toluidine blue were performed to demonstrate mucosubstances. For the examination of enzymatical activities small pieces of tissue, either unfixed of prefixed in formalin or acetone, were frozen on the table of a freezing microtome (Leitz/Wetzlar, Type 1310). The following methods were applied to the sections: Dehydrogenases: Malate dehydrogenase (E.C. 1.1.1.37) (Pearse, 1961), glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) (Sasse, 1968). Oxidoreductases: Monoamine oxidase (E.C. 1.4.3.4) (Glenner, Burtner and Brown, 1957). Hydrolases: Alkaline phosphatase (E.C. 3.1.3.1) (Pearse, 1961), Acid phosphatase (E.C. 3.1.3.2) (Pearse, 1961),/3-glucuronidase (E.C. 3.2.1.31) (Seligman, Tsou, Rutenburg and Cohen, 1954), adenosine triphosphatase (E.C. 3.6.1.4) (von Deimling, 1964). Lyases: Carbonic anhydrase (E.C. 4.2.1.1) (Goebel and Puchtler, 1954b, modified after Burck, 1969). Cor~trols: Sections were incubated without substrate.

Electron Microscopical Methods Very small pieces of fresh tissue of semi-adult Sepia officinalis L. were incubated and, fixed in solutions of the following compositions:

a) Monoamine Oxidase (E.C. 1.4.3.4); (Boadle and Bloom, 1968). Fixation: 3,8% glutaraldehyde in 0.2 M phosphate buffer (pH 7.2) 1100 mOsm. Rinsing: Isotonic phosphate buffer. Incubation: Tryptamine hydrochloride 10 mg, tetranitroblue tetrazolium chloride 2 mg, Na2SO4 16 mg, 0.2 M sucrose in 8 ml 0.2 M phosphate buffer (pH 8.0) for 150 min at 37~ C. Rinsing: Isotonic phosphate buffer for 30 min. Post-fixation: t % OsO, in phosphate buffer for 1 h. - Controls: Incubation without substrate (tryptamine hydrochloride).

b) Catalase (E.C. 1.11.1.6); (Legg and Wood, 1970). Fixation: 3.8% glutaraldehyde in 0.2 M cacodylate buffer, 20 mM CaC12, 0.27 M NaC1 (pH 7.2) for 30 min at room temperature. Rinsing: Cooled 0.2 M cacodylate buffer. Incubation: After leaving the 30 gm chopper sections in 0.1% CuSO4 and 3% NaCI dissolved in 0.2 M tris-HC1 buffer (pH 9.0) for 12 h, the sections were incubated in 0.2 M tris-HC1 buffer, 6 x 10- 3 M 3,3'-diaminobenzidine tetrahydrochloride and 3% H202 for 1 h at 20 ~ Controls: Incubation without H202-

c) Glutamic Oxalacetic Transaminase (E.C.2.6.1.1); (Lee, 1970). Fixation: 3.8% glutaraldehyde with 0.25 M sucrose, 0.2 M imidazole and 0.25 M NaC1 adjusted with 0.1 N HC1 to pH 7.2-7.4; for 45 min at 4 ~ C. Rinsing: Overnight in cold imidazole-buffered sucrose. Incubation: 20 mM L-aspartic acid, 2 mM alpha ketoglutaric acid, 6 mM lead nitrate, 0.2 M imidazole and 0.25 M sucrose (pH 7.4-7.5) for 20 min at room temperature. Rinsing: Briefly in imidazole-buffered sucrose (0.2 M imidazole, 0.25 M sucrose) with 20 mM L-aspartic acid and 0.25 M NaC1. Postfixation: 1% OsO4 in veronal acetate buffer (pH 7.2) for 1 h. - Controls: Incubation without substrate. d) Choline Esternase (non-specific)(E.C. 3.I.1.8); (Kasa and Csillik, 1966, modified). Fixation: 4% glutaraldehyde in phosphate buffer 1050 mOsm (pH 7.4) for 2 h at 20 ~ C. Rinsing: 10% sucrose for 30 min. Incubation : Acetylthiocholine 70 rag, 0.1 M Na-acetate (22 ml), 0.1 N acetic acid (3 ml), 3.75% glycine (1 ml), 0.1 M CuSO4 (1 ml), 0.5% PbNO3 (1 ml) for 30 min at 20 ~ C. Rinsing: Sea-Water, followed by H2S-saturated sea-water and sea-water. Postfixation: 1% OsO4 in sea-water. - Controls: Before incubation rinsing in 10% sucrose and 10 -6 M Mipafox as inhibitor for 30 rain at 20 ~ C.

Enzyme Activities in the Pancreatic Appendages of Sepia

31

e) Alkaline Phosphatase (E.C.3.1.3.1) ; (Mayahara et al., 1967, modified). Fixation: 3.8% glutaraldehyde in 0.2 M cacodylate buffer and 1.4% NaC1, 920 mOsm (pH 7.4) for 2 h at 6~ C. Rinsing: Cycodylate buffer. Incubation: 0.2 M tris-HCl buffer pH 8.5 (1.4 ml), 0.1 M Na-fi-glycerophosphate (2 ml), 15 mM MgSO4 (2.6 ml), 0.5% alkaline lead citrate pH 10 (4 ml) and 0.8 g sucrose (final pH 9.2). Post-fixation: 1% OsO4 in isotonic sucrose solution. Controls: Incubation without substrate.

f) Acid Phosphatase (E.C. 3.1.3.2) ; (Ericsson and Trump, 1965, modified). Fixation: see e). Rinsing: 0.2 M cacodylate buffer. Incubation: 0.1 M acetate buffer pH 5.5 (500 ml), 0.6 g lead nitrate, 3% Na-/~-glycerophosphate (50 ml) for 13 h at 37~ C. Rinsing: 0.1 M acetate buffer and 2% acetic acid. Post-fixation: 1% OsOr in collidine buffer for 1 h. Controls: Incubation without substrate.

g) Carbonic Anhydrase (E.C.4.2.1.1); (Hansson, 1967, modification of Yokota, 1969). Fixation: 1% glutaraldehyde in 0.2 M cacodylate buffer (pH 7.2) for 3 h at 20 ~ C. Rinsing: Cacodylate buffer for 30 min. Incubation: 0.1 M COSO4, 0.5 M H2SO4, 1/15 M KHzPO4 and 0.22 M NaHCO 3. Rinsing: Cacodylate buffer. Post-fixation: 1% OsO4 in cacodylate buffer (pH 7.2) for 1 h at 4 ~ C. Controls: Pre-incubation in 10-4 M Diamox in 0.25 M sucrose for 20 min.

Results 1. Histochemical Findings Both epithelia of the pancreatic appendages of Sepia have a basal lamina lining t h e b l o o d space, a b a s a l f o l d e d l a b y r i n t h a n d a n a p i c a l b o r d e r o f m i c r o v i l l i . Basal lamina, basal labyrinth and microvilli show a positive PAS-reaction and, w i t h e x c e p t i o n o f t h e f o l d e d l a b y r i n t h , stain w i t h A l c i a n blue, t h u s i n d i c a t i n g neutral and acid mucopolysaccharids. The Alcian blue/HC1 method reveals a d i f f e r e n c e b e t w e e n t h e g l y c o c a l y x e s o f the e p i t h e l i a , i.e. o n l y t h e o u t e r e p i t h e l i u m c o n t a i n s s u l p h a t e d m u c o p o l y s a c c h a r i d s w h i c h stain specifically at p H 1.0 ( P e a r s e , 1968). T h e s e results are s u m m a r i z e d in T a b l e 1. A w e a k m e t a c h r o m a s i a c o u l d be f o u n d o n l y in t h e b a s a l l a m i n a ( T o l u i d i n e blue). T h e r e f o r e t h e a p i c a l g l y c o c a l y x d o e s n o t h a v e t h e n e c e s s a r y m i n i m u m

Table 1. Demonstration of Mucosubstances of the Pancreas of Sepia officinalis L. Method

Inner Epithelium

Outer Epithelium

microviIli

basal labyrinth

basal lamina

microvilli

basal labyrinth

basal lamina

+

+ +

+

+ +

+

+

Alcian blue

+

0

+

+ +

0

+

Alcian blue/ HCI, pH 1,0

0

0

+

++

0

+

PAS

Strongly positive + + ; positive + ; negative 0 Metachromasia with Thionine

no

no

no

no

no

no

Metachromasia with Toluidine

no

no

weak

no

no

weak

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H.H. Donaubauer and R. Schipp

Table 2. Histochemical (LM) and Cytochemical (EM) Enzyme Reactions of the Pancreas of Sepia

officinalis L. Class of enzyme

Enzyme

E.C.No.

Abbreviation

Kind of demonstration

Inner Epithe- Outer Epithelium lium apical basal

o~ t

Malate dehydro1.1.1.37 genase Glucose-61.1.1.49 phosphate dehydrogenase Monoamine oxidase 1.4.3.4

MDH

Pearse, 1960 (LM)

+ + + + + + + +

+ +

G-6-PD

Sasse, 1968 (LM)

0

0

0

0

MAO

Glenner et al., 1957 (LM) Boadle and Bloom, 1969 (EM) Legg and Wood, 1970 (EM)

(+)

+

+

+

+

(+)

+

(+)

+

+

(+)

0

(+)

(+)

+ +

(+)

0

(+)

+

(+)

0

0

0

0

o~

~=.~

Catalase

1.11.1.6

Catal

Glutamic oxalacetic transaminase

2.6.1.1

GOT

Lee, 1970 (EM)

Choline esterase

3.1.1.8

ChE

Alkaline phosphatase

3.1.3.1

al.Phos

Kasa and Csillik, 1966 (EM) Pearse, 196! (LM)

Acid phosphatase

3.1.3.2

ac. Phos

fi-glucuronidase

3.2.1.31

fl-Gluc

Adenosine triphosphatase

3.6.1.4

ATPase

o

,3

Carbonic anhydrase 4.2.1.1

apical basal

CAH

Pearse, 1961 (LM) Ericsson and Trump, 1965 (EM) Seligman et al., 1954 (LM) v. Deimling, 1964 (LM)

+ + (+) + +

+ + + +

(+) + + + +

+ + + + + + 0

Goebel and Puchtler, ( + ) 1954 (LM) Hansson, 1967 (EM) 0

0 (+) + + 0

(+)

0

0

0

+

0

Strongly positive + + + ; positive + + ; moderately positive + ; distinct but moderate coloration ( + ) ; negative 0

surface density of negative charges on the substrate (Pearse, 1968) to establish metachromasia. This is also the case with the glycocalyx of the outer epithelium although it contains sulphated mucopolysaccharides. The histochemical enzyme results are given in Table 2 and Fig. 1. Alkaline phosphatase and glucose-6-phosphate dehydrogenase could not be demonstrated by the methods applied. In comparing the histochemically detected enzyme activity of the pancreatic epithelia, it is apparent that the inner epithelium has a slightly higher enzyme

Enzyme Activities in the Pancreatic Appendages of Sepia

33

Fig. I. Adenosine triphosphatase reaction in the inner epithelium (ie) of the pancreas of Sepia

officinalis L. The outer epithelium (oe) reacts negatively, x 260

content. Adenosine triphosphatase in particular, and to a lesser extent acid phosphatase, lnalate dehydrogenase and carbonic anhydrase show some differences in the intensity of their reaction. A clearer illustration of these findings is presented in Fig. 2. With regard to the cytochemical results (Table 2), it is noteworthy that the both epithelia show almost identical reactions, but sometimes the intensity is higher in the outer layer (glutamic oxalacetic transminase, acid phosphatase).

2. Cytochemical Findings The ultrastructural localization of monoamine oxidase was not as distinct as could be expected from the positive results of the light-microscopic technique; but in accordance with the latter results, some dense bodies in the apical area of the inner and outer epithelium, as well as the outer membranes of vesicular and tubular structures of the endoplasmic reticulum and outer compartments of mitochondria gave a precipitation reaction (Fig. 3 a~z). The catalase reaction induces an increase in the electron density of some dense bodies, which are dispersed throughout the entire epithelium. This increase occurs particularly in stained dense bodies of the inner layer which are to be found among unstained dense bodies having the same structure and dimension (Fig. 4a-e).

H.H. Donaubauer and R. Schipp

34 r$

oe

bs

ie

Fig. 2. Diagram of enzyme distribution in the pancreas of Sepia officinalis L. demonstrated by histochemical methods. Acid phosphatase (Section 1), adenosine triphosphatase (2), carbonic anhydrase (3), fl-glucoronidase (4), monoamine oxidase (5), malate dehydrogenase (6). Outer epithelium (oe), inner epithelium (ie), blood sinus (bs), basal lamina (b/), dorsal renal sac (rs), ductus hepatopancreas (dh), dense bodies (db), ergastoplasm (er), nucleus (n)

Glutamic oxalacetic transarninase. In b o t h epithelia, the o u t e r c o m p a r t m e n t s o f the m i t o c h o n d r i a react positively. T h e e n z y m e can also be d e m o n s t r a t e d i n / o r on the m e m b r a n e o f vesicular a n d t y p i c a l r i n g - s h a p e d structures (Fig. 5) which c o m m u n i c a t e with the e n d o p l a s m i c reticulum. F u r t h e r m o r e , b a s a l - a p i c a l l y o r i e n t e d rows o f t u b u l i a n d vesicles, as well as d i c t y o s o m e s , also

Enzyme Activities in the Pancreatic Appendages of Sepia

35

Fig. 3a-c. MAO reaction in the outer epithelium. Reaction products can be seen in dense bodies (db), endoplasmic reticulum (er) and outer compartments of mitochondria (m0 (arrow). Unstained sections. Microvilli (my), lumen of dorsal renal sac (rs), nucleus (nu)

show a positive reaction (Fig. 6). Occasionally reaction products are located on the extracellular side of the plasmalemma, i.e. in the apical intercellular space and on the surface of the microvilli (Fig. 6).

Choline esterase (non-specific) is demonstrated in the outer epithelium at the surface of the microvilli and of the apical pits. The apical part of the intercellular space, as well as the basal lamina, show a high activity of this enzyme. Some precipitation also occurs within the infoldings of the basal labyrinth (Fig. 7 a-c). Alkaline phosphatase could not be detected with the method applied. Acid phosphatase. In the outer layer, this enzyme is localized on the microvilli and especially in the apical pits. The terminal part of the intercellular space shows some reaction product as, do parts of the basal labyrinth. In addition,

36

H.H. Donaubauer and R. Schipp

Fig. 4a-e. Catalase reaction in the outer epithelium (a and e) and the inner one (b). Positive reaction of microbodies (arrows). Unstained sections. Microvilli (my), basal lamina (b/), nucleus (nu), mitochondria (mi)

Enzyme Activities in the Pancreatic Appendages of Sepia

37

Fig. 5. GOT reaction in the inner epithelium. Outer compartments of mitochondria (arrows), dictyosome (go), vesicles (re) and ring-shaped structures (inset) show a positive reaction. Unstained section. Microvilli (mi), lumen of d. hepatopancreas (dh), nucleus (nu). inset: x 38,000

a few lysosomal dense bodies, particularly in the apical cell region, show enzyme activity (Fig. 8 a-b). In the inner epithelium, in particular the dense bodies (lysosomes) in the basal cell region show strong activity. Some precipitation can also be seen along and within the basal infoldings but n o n e at the microvilli (Fig. 8c d).

Carbonic anhydrase is present in the microvilli o f the outer layer and an intense reaction can be seen in the apical pits (Fig. 9a). Some precipitates are f o u n d in the intercellular space and the terminal web (Fig. 9 b), whereas n o n e occur within the cells of the two epithelia.

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H.H. Donaubauer and R. Schipp

Fig. 6. GOT reaction in the middle area of the outer epithelium. Reaction products are marked by arrows. Lateral interdigitations (x), Unstained sections. Further abbreviations as in Figure 5. inset: x38,000

Discussion The results presented here concerning the PAS-, alcian blue and toluidine blue reaction of the pancreatic epithelia of Sepia officinalis, coincide with those of Boucoud-Camou, 1968). The glycocalyx of the outer epithelium differs from that of the inner one, in its greater thickness and because it contains sulphated mucopolysaccharides. Furthermore, it should be mentioned that the apex of the inner epithelium bordering the fluid of the ductus hepatopancreas shows dynamic exo- and endocytotic processes to a greater extent than does the outer layer. Such processes, serving nutrient absorption, could be hindered by a too thick and stable glycocalyx like that of the outer epithelium. There, the glycocalyx may serve as protection from the high content of N H 2 and NH3 (505 ppm), the acid renal sac fluid (pH 5) (Schipp, 1976, unpublished) and perhaps also from the high a m o u n t of Dicyemida in this compartment.

Enzyme Activities in the Pancreatic Appendages of Sepia

39

Fig. 7a-e. ChE reaction in the outer epithelium. Microvilli (my), intercellular space (arrows) and the basal lamina (bl) react positively. Unstained sections

In considering the enzyme histo- and cytochemicalfindings, one can see that, from the general morphological conformity of the two epithelia (Schipp and von Boletzky, 1976), no distinct differences in the reactions o f the enzymes investigated are notable; i.e. if an enzyme can be demonstrated in the inner epithelium it can, generally, also be detected in the outer epithelium, or vice versa, but quite often differences in the intensity and of the sites of localization in the cell compartments are visible between the two layers. F r o m the class of oxidoreductases, malate dehydrogenase (MDH), glucose-6phosphate dehydrogenase (G-6-PD) and monoamine oxidase (MA O) were analyzed by histochemical or cytochemical methods (Table 2). The G-6-PD reaction was negative probably due to weaknesses in the research methods, since the enzyme was demonstrated to have a high activity in similarily organized epithelia of the branchial heart appendage (Schipp et al., 1971). The higher content of M D H in the inner epithelium is proportionate to the size and greater number of mitochondria of this layer. MAO could be demonstrated in both epithelia with the same intensity. The weaker cytochemical

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H.H. Donaubauer and R. Schipp

Fig. 8a-d. Ac. Phosphatase reaction in the outer epithelium (a and b) and inner epithelium (e and d). Reaction product is localized in the microvilli (inset), apical pits (ap) and intercellular space of the basal labyrinth (arrows in b) as well as in dense bodies (db) and sporadically in mitochondria (mi), cytoplasm (x) and intercellular gap (arrow). Unstained sections, inset: x 63,000

Enzyme Activities in the Pancreatic Appendages of Sepia

4t

Fig. 9a and b. CAH reaction in the outer epithelium. Electron dense deposits in microvilli (my), apical pits (arrows in a) and the intercellular gap (b). Unstained sections. Mitochondria (m0, lumen of dorsal renal sac (rs)

reaction can be explained by fixation with glutaraldehyde which induces a loss of enzyme activity (Boadle and Bloom, 1968). Nevertheless a cytochemical localization is possible in dense bodies, outer compartments of mitochondria and in the endoplasmic reticulum, which agrees with the findings of Shannon et al. (renal and hepatic tissues of the guinea pig, 1974), and Miiller and Da Lage (1977) (proximal renal tubules of the rat). Catalase shows a stronger reaction in the inner epithelium and is only localized in some dense bodies which can therefore be characterized as peroxisomes or microbodies (De Duve, 1965); (Veenhuis and Bonga, 1977). Glutamic oxalacetic transaminase (GOT) is primarily localized in the outer epithelium, in the outer compartments of mitochondria and membranes of vesicular and typical ring-shaped structures of the endoplasmic reticulum. This is in accordance with

42

H.H. Donaubauer and R. Schipp

the findings of Lee (1970) and Lolova and Dikov (1975) who could demonstrate this enzyme in the same organelles of the tubules epithelium of the rat kidney. Furthermore, the GOT was detected in the similarily organized epithelium of the renal appendages and the gill of Sepia officinalis. There the enzyme is localized in corresponding structures (Schipp, 1977, unpublished). We agree with Lee that a distinct group of vesicles with GOT-activity may be involved in the excretion of ammonia and hydrogen ions, or with the control of these processes. It is still open to question as to whether the passage of ammonia occurs - as in the tubule epithelium of the vertebrate kidney by means of reversible enzymatic transaminations of vesicular bound carrier substances such as glutamine or glutamate (Karnovsky and Himmelhoch, 1961; Lee, 1970; Lolova and Dikov, 1975) or if most of the NH3 of the blood is eliminated by diffusion via intercellular spaces into the renal sac fluid (Potts, 1965). Our agreement with Lee is supported by our morphological results (Schipp and yon Boletzky, 1976) and our measurements (unpublished) of the ammonia content of the dorsal renal sac which is bordered by the outer pancreatic epithelium; these results and measurements indicate an excretory function of this outer layer. Among the hydrolases examined, only alkaline phosphatase was negative in both epithelia. This is in contrast to the observations of Boucaud-Camou (1974), who found this enzyme, histochemically, in the microvillous border of the inner layer. This difference in findings may be explained by changes in enzyme activity, depending on the actual physiological conditions of the animal, since digestion shows different phases of activity (Bidder, 1966) and, as in vertebrates, it may be that the phosphatase activity changes rhythmically (Sch~ifer et al., 1974). The same possibility must also be considered for the acid phosphatase reaction. The enzyme is present in both epithelia (apical pits, dense bodies and basal labyrinth), but the intensity of the reaction seems to vary depending on the above mentioned phases of digestion. The cytochemical results show the lysosomal nature of some dense bodies (Stockinger, 1971), which are involved in the intracellular digestion of resorbed and reabsorbed organic materials respectively (DeDuve, 1965).

Non-specific choline esterase could be demonstrated cytochemically in the outer epithelium only, whereas the histochemically investigated 13-gtucuronidase reacted in both layers. Both enzymes are widespread in excretory and circulatory organs of cephalopods; choline esterase was localized in the excretory epithelium of the pericardial gland (Schipp et al., 1971) and also in the branchial heart of Sepia (Schipp, 1977). It is noteworthy that the basal lamina shows a strong activity in all cases, a result which is supported by the findings of Panula and Rechardt (1978) in vertebrate capillaries. The ATPase demonstrated with the method of von Deimling (1964) is the Mg § ---dependent (mitochondrial) enzyme. Therefore, the inner epithelium, containing a high amount of mitochondria (Schipp and von Boletzky, 1976), reacts very strongly, but we cannot explain why the outer epithelium does not react at all. Perhaps in this case, the (Na+-K+)-stimulated ATPase (currently under

Enzyme Activities in the Pancreatic Appendages of Sepia

43

investigation) is of greater importance for the urine formation and ional regulation of the body fluids. Among the enzyme class of lyases, we investigated carbonic anhydrase by histochemical (Goebel and Puchtler, 1954b) and cytochemical (Hansson, 1967) methods. As in the renal appendages, the enzyme may be involved in trasepithelial H30 + and HCO~-transport, i.e. the acidification of the urine in the dorsal renal sac. It is probable that these protons participate in the reaction: NH3+H+~-~NH2 as Potts described for the urine of Octopus dofleini (1965). Similar functions, as well as a possible supporting of the HCO~-stimulated ATPase, are discussed with regard to the vertebrate renal tubulus epithelium (Wistrand and Kinne, 1977). In conclusion, the results presented here support the opinion that the pancreatic appendages have a functional ambivalence in that they serve both for excretion and for nutrient absorption (Schipp and von Boletzky, 1976). However, there is no fundamental difference in the enzyme distribution in both epithelia. The excretory function of the outer layer is particularly emphasized by its higher GOT and CAH activities.

References Bidder, A.M.: Feeding and digestion in cephalopods. In: Physiology of Mollusca. Wilbur, K.M., Yonge, C.M., (eds.). New York, London: Academic Press 1966 Boadle, M.C., Bloom, F.E.: A method for the structural demonstration of monoamine oxidase activity. III. Internat. Congr. Histochem. Cytochem. New York 1968 Boucaud-Camou, E. : Etude histologique et histochimique de l'appareil digestif de Sepoila atlantica D'Orbigny et Sepia officinalis L. Bull Soc. Linn6 Normandie 9, 220-243 (1968) Boucaud-Camou, E.: Etude infrastructurale du pancreas de Sepia officinalis L. Bull. Soc. Zool. France 97, 197 203 (1972) Boucaud-Camou, E. : Localisation d'activit~s enzymatiques impliqu6es dans la digestion chez Sepia officinalis L. Arch. Zool. Exp. G~n. 115, 5-27 (1974) Buchwalow, I.B., Unger, E., Schulze, W., Sch6n, R., Raikhlin, N.T.: Hemmung und Stimulierung der ATPase-Aktivit/it und die nichtenzymatische Hydrolyse von ATP in der elektronenmikroskopischen Histochemie. Histochemistry 44, 1-11 (1975) Burck, H.C.: Histologische Technik. Stuttgart: Thieme 1969 DeDuve, C. : Function of microbodies (Peroxisomes). J. Cell Biol. 27, 25A (1965) von Deimling, O.: Die Darstellung phosphatfreisetzender Enzyme mittels Schwermetall-StimultanMethoden. Histochemie 4, 48-55 (1964) Ericcson, J.L.E., Trump, B.F.: Observations on the application of electron microscopy of the lead phosphate technique for the demonstration of acid phosphatase. Histochemie 4, 470-487 (1965) Glenner, G.G., Burtner, H.J., Brown, G.W.: The histochemical demonstration of monoamine oxidase activity by tetrazolium salts. J. Histochem. Cytochem. 5, 591-600 (1957) Goebel, A., Puchtler, H. : Zur Darstellung der Carboanhydrase im histologischen Schnitt. Naturwissenschaften 41, 531-532 (1954b) Hansson, H.P.J.: Histochemical demonstration of carbonic anhydrase activity. Histochemie 11, 112-128 (1967) Jokota, S.: Electron microscopic demonstration of carbonic anhydrase activity in mouse liver cells. Histochemie 19, 255-261 (1969) Karnovsky, M.J., Himmelhoch, S.R. : Histochemical localization of glutaminase I activity in kidney. Am. J. Physiol. 201, 786-790 (1961)

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Kasa, P., Csillik, B. : Electron microscopic Iocalization of choline esterase by a copper-lead-thiocholine technique. J. Neurochem. 13, 1345-1349 (1966) Lee, S.H. : The possible role of the vesicles in renal ammonia excretion. An implication of concentrated glutamic oxalacetic transaminase. J. Cell Biol. 45, 644-649 (1970) Legg, P.G., Wood, R.L. : Effects of catatase inhibitors on the ultrastructure and peroxidase activity of proliferating microbodies. Histochemie 22, 262-276 (1970) Lolova, I., Dikov, A. : HistochemicaI evidence of aminotransferase. IV. Histochemical and electrophoretical investigation of aminotranferases in rat organs. Acta Histochem. (Jena) 53, 12-27 (1975) Mayahara, H., Hirano, H., Saita, T., Ogawa, K. : The new lead citrate method for the ultracytochemical demonstration of activity of nonspecific alkaline phosphatase (orthophosphoric monoester phosphohydrolase). Histochemie 11, 88-96 (1967) M/iller, J., DaLage, C. : Ultracytochemical demonstration of monoamine oxidase activity in nervous and non-nervous tissues of the rat. J. Histochem. Cytochem. 25, 337 348 (1977) Panula, P., Rechardt, L. : Age-dependent increase in the non-specific cholinesterase activity of the capillaries in the rat neostriatum. Histochemistry 55, 49-54 (1978) Pearse, A.G.E.: Histochemistry. Theoretical and applied. 2nd and 3rd eds. London: Churchill 1961/1968 Potts, W.T.W.: Ammonia excretion in Octopus dofleini. Comp. Biochem. Physiol. 14, 339-355 (1965) Romijn, C.: Die Verdauungsenzyme bei einigen Cephalopoden. Arch. Neerl. Zool. 1, 373431 (1935) Sasse, D. : Glykogen in der Ontogenese des Verdauungstraktes - Chemomorphologische und stoffwechselhistochemische Analyse. Ergebn. Anat. Entwickl.-Gesch. 40/2, 1~68 (1968) SchS,fer, A., H6hn, P., Mika, H., Allbach, G. : Enzymhistochemische und elektronenmikroskopische Untersuchungen an der Darmschleimhaut der Ratte im Tagesrhythmus. Acta Histochem. (Jena) 48, 30l 319 (1974) Schipp, R., H6hn, P., SchS_fer, A. : Elektronenmikroskopische und histochemische Untersuchungen zur Funktion des Kiemenherzanhangs (Pericardialdriise) yon Sepia officinalis. Z. Zellforsch. 117, 252~74 (1971) Schipp, R., v. Boletzky, S., Doell, G.: Ultrastructural and cytochemicaI investigations on the renal appendages and their concrements in dibranchiate cephalopods (Mollusca, Cephalopoda). Z. Morphol. Tiere 81, 279-304 (1975) Schipp, R., v. Boletzky, S.: The pancreatic appendages of dibranchiate cephalopods. I. The fine structure of the "pancreas" in Sepioidea. Zoomorphologie 86, 81 98 (1976) Schipp, R.: An indirect demonstration of the substructure of the lamina basalis in the branchial heart of Sepia officinalis L. by means of cholinesterase reaction. Experientia 33, 74 (I977) Seligman, A.M., Tsou, K.C., Rutenburg, S.H., Cohen, R.B. : Histochemical demonstration of fl-glucuronidase with a synthetic substrate. J. Histochem. Cytochem. 2, 209-229 (1954) Shannon, W.A., Hannah, J., Wasserkrug, L., Seligman, A.M.: The ultrastructural localization of monoamine oxidase (MAO) with tryptamine and a new tetrazolium salt. 2-(2'-benzothiazolyl)5-styryl-3-(4'-phthalhydrazidyl) tetrazolium chloride. J. Histochem. Cytochem. 22, 170-182 (1974) Stockinger, L. : Fermentnachweise im Ultrastrukturbereich. In : Histochemie der Ultrastruktur. Acta Histochem. (Jena) Suppl. X, 27-63 (1971) Veenhuis, M., Bonga, S.D.W.: The cytochemical demonstration of catalase and D-amino acid oxidase in the microbodies of teleost kidney cells. Histochem. J. 9, 171 181 (1977) Williams, G., Jackson, D.S.: Two organic fixatives for acid mucopolysaccharids. Stain Technol. 31, 189 191 (1956)

Received September 19, 1978

The localization of enzyme activities in the pancreatic appendages of Sepia officinalis L. (Cephalopoda).

Histochemistry Histochemistry59, 29-44 (1978) 9 by Springer-Verlag 1978 The Localization of Enzyme Activities in the Pancreatic Appendages of Sepia...
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