ARTICLE |
Correspondence to: Moise Bendayan, Dept. of Pathology and Cell Biology, Université de Montreal, CP 6128 Succ. Centre Ville, Montreal, Quebec, Canada H3C 3J7. E-mail: Moise.Bendayan@umontreal.ca
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Summary |
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By applying the highly sensitive cytochemical Gautron's technique, we were able to reveal AChE activity in rat pancreatic acinar cells, particularly at the level of a complex membrane-bound network formed by tubules with varicosities located around the nuclei and close to the basolateral membrane. The Golgi apparatus was devoid of cytochemical reaction beside the trans-Golgi network cisternae, which showed a positive reaction. The RER of some acinar cells also presented a signal, demonstrating their capability of synthesizing AChE. Immunogold using a specific anti-AChE antibody yielded similar results. Double-labeling experiments corroborated the presence of enzyme cytochemical and immunocytochemical signals in the same lysosomal tubular network. Biochemical sedimentation assays confirmed the presence of AChE in acinar cells, which exists as two globular molecular forms, G1 and G4. These results were obtained with pancreatic tissue in situ as well as with isolated acinar cells maintained in culture and devoid of neural elements. The existence of a continuous tubular lysosomal network containing AChE is in agreement with previous reports on acinar and other cell types, and supports a more general hypothesis on dynamic continuities among cell structures. Whether AChE is being secreted by the acinar cells or internalized through this endo-lysosomal system was not defined. However, the capability of the acinar cells to synthesize AChE and to channel it through a tubular system is a good indication that the cells can modulate their cholinergic stimulation for optimal secretion of digestive enzymes. (J Histochem Cytochem 49:2939, 2001)
Key Words: acetylcholinesterase, pancreas, acinar cells, immunocytochemistry, lysosomal system
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Introduction |
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AUTONOMIC INNERVATION plays a primordial and specific role in the regulation of exocrine pancreatic secretion, distinct from that fulfilled by intestinal factors such as pancreozymin and secretin (
Acetylcholinesterase (AChE) is an essential constituent of this autonomic innervation needed to prevent desensitization of cholinergic receptors and thereby preserve the efficiency of cholinergic regulation of pancreatic secretion. AChE exists in multiple molecular forms, a polymorphism that enables the cells to position AChE at different sites where it may fulfill specific roles (for review see
Lysosomes are polymorphic structures generated by the Golgi apparatus and belonging to the lysosomalendosomal system of the cell. Pancreatic acinar cells have been described as displaying two distinct populations of secondary lysosomes. One is represented by the classical Golgi-associated globular, acid phosphatase-positive structures, and the other, located in the perinuclear and basolateral regions, has been described as a tubular network (
Whereas most studies dealing with AChE in the pancreas were performed to understand the innervation of the organ and the neuronal control of pancreatic secretion, in the present study we took advantage of a highly sensitive cytochemical technique (
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Materials and Methods |
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Cytochemistry
For the cytochemical detection of AChE, the protocol developed by
For electron microscopy, pancreatic tissue from adult male SpragueDawley rats (100 g) was fixed in situ with 1% glutaraldehyde in 0.15 M sodium cacodylate buffer, pH 7.3. Tissue was then removed from the abdominal cavity and small samples were immersed in the same fixative for a total fixation time of 30 min. On thorough washing, the free aldehyde groups were quenched by adding 0.5 ml of 1 M NaOH to the cacodylate buffer (50 ml). The tissue samples were rinsed and immersed in 0.2 M Tris-maleateNaOH, pH 6.0, overnight. Incubation was carried out with a substrate medium composed of 0.05 ml acetyl sulfide (Aldrich; Milwaukee, WI) dissolved in 1 ml propylene glycol and diluted in 100 ml 0.2 M Tris-maleateNaOH, pH 6.0, complemented with 1 ml lead nitrate 3% (w/v) in aqueous solution and 50 mg of acetylthiocholine perchlorate or bromide. Two ml of 10-3 M aqueous solution of iso-OMPA (tetraisopropyl-pyrophosphoramide), a selective inhibitor of butyrylcholinesterase, was added to a final concentration of 2 x 10-5 M. The solution was filtered before adding the acetylthiocholine to remove any precipitate. For the incubation, the tissue samples were immersed in the substrate medium for 10 min at 4C and allowed to warm to room temperature. Incubation times were between 30 and 60 min. After rinsing in the Tris-maleateNaOH buffer, the tissue was transferred to the cacodylate buffer and postfixed for 60 min with 1% osmium tetroxide. The tissue samples were dehydrated and embedded in Epon according to standard techniques. Ultrathin sections were counterstained with uranyl acetate and lead citrate and examined with a Philips 410SL electron microscope. Thick sections (1 µm) were mounted on grids and examined, without any counterstaining, with a Philips CM30 at 300kV. In this case, most images were electronically registered on CD-ROM.
For light microscopy, rat pancreatic tissue was sampled and cryosections were made according to standard techniques. Five-µm-thick sections were processed for the cytochemical staining as described above. Prefixation with 1% glutaraldehyde was found to yield better results than non-fixed tissues.
Control assays were performed to assess the specificity of the cytochemical reaction. The participation of non-choline esterases to the final reaction product was estimated by inhibiting cholinesterases with eserine sulfate (5 x 10-4 M). The effect of omitting the substrate, acetylthiocholine, on the final reaction product was also tested.
Two types of double-labeling experiments were performed at the electron microscope level. In the first one, we combined pre-embedding cytochemical detection of AChE with postembedding immunocytochemical detection of a pancreatic secretory protein (amylase). The postfixation of the tissue with osmium tetroxide was omitted. For the postembedding immunocytochemical detection of amylase, the protein Agold technique (
Immunocytochemistry
For the postembedding immunocytochemical detection of AChE, a rabbit polyclonal antibody (a gift from J. Massoulié) raised against rat brain hydrophobic G4 AChE (
Acinar Cells in Culture
In addition to the work performed with rat pancreatic tissue fixed in situ, the cytochemical approach was also applied on rat acinar cells maintained in tissue culture. Rat pancreatic acinar cells were isolated and plated on Matrigel-coated Petri dishes as described previously (
Ganglionic Tissue
As an internal control, we used rat sympathetic superior cervical ganglion (SCG) in parallel with our pancreatic tissue. Neurons of these ganglia are known to contain large amounts of AChE, particularly in their Nissl bodies (
AChE Analysis
AChE was extracted from pancreas and primary cultures of pancreatic acinar cells, as well as from the SCG. The tissues were homogenized in 0.01 M Tris-HCl buffer (pH 7.0) containing 0.05 M MgCl2, 1% Triton X-100, 1 mg/ml bacitracin, and 0.1 mg/ml of soybean trypsin inhibitor, at a concentration of about 1:20 (w:v) in a Teflonglass Potter homogenizer. In the case of the SCG, the trypsin inhibitor was omitted.
Velocity sedimentation analyses of the AChE molecular forms were performed as described previously (
Control assays were performed with eserine (5 x 10-4 M) and anticholinesterase BW 284c51 (1,5-bis(4-allyldimethyl-ammoniumphenyl-pentan-3-one dibromide) (2 x 10-5 M).
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Results |
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By light microscopy (results not shown), the AChE cytochemical approach revealed a signal consisting of dark deposits over some acinar cells. These cells were scattered among the acinar parenchyma. Nerve paths were also delineated with a much more intense signal, in a pattern consistent with that reported previously (
By electron microscopy, the cytochemical reaction product was detected within the pancreatic cells, the neural elements, blood vessels, and in the extracellular space. The reaction product was characterized by small electron-dense spots of about 50 nm (Fig 1 and Fig 2). Within capillaries, the reaction was encountered in the lumen (Fig 1), consistent with the well-established presence of AChE in blood (
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In addition to the reaction product found in the lysosomal network, some acinar cells, in an isolated fashion, demonstrated a strong reaction in their rough endoplasmic reticulum (RER) (Fig 3). The reaction product was present within the lumen of the reticulum and displayed only a spotty appearance, resembling, again, that of the extracellular space. This reaction product was restricted to the RER and was not present in the Golgi apparatus and the secretory granules, although in many instances the trans-Golgi network did show a positive signal (Fig 5a). It was also noteworthy that cells with positive reaction in their RER were isolated: neighboring cells, even those of the same acinus, exhibited an RER free of any cytochemical reaction (Fig 3). In fact, the labeling for AChE within the RER displayed a mosaic-like pattern.
High-voltage electron microscopy of thick sections allowed the delineation of the tubular system in the acinar cells in a more extended fashion (Fig 6). Long tubular membranous structures, usually presenting various varicosities and bifurcations, were conspicuous throughout the cell cytoplasm, particularly in the basolateral and nuclear regions (Fig 6). They formed a complex 3D tubular network. Some of the tubules appeared to come in very close contact with the basolateral membrane (Fig 6, inset). In particularly well-oriented sections passing through the Golgi apparatus, a complex network of tubules with connected vesicles, as well as positively stained dense globular structures, were observed (Fig 7). This network could well represent the positively stained trans-Golgi network (TGN) as seen on thin sections (Fig 5a). It did display the classical TGN profile described in many secretory cells (
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In addition to the exocrine parenchyma, the endocrine cells in the islets of Langerhans also displayed AChE-positive tubular structures, although the lysosomal network appeared less well developed than in the acinar cells (result not shown).
The double-labeling experiment in which the AChE reaction was combined with the immunocytochemical detection of a secretory protein, i.e., amylase, demonstrated that the AChE-positive compartments, in addition to the RER, are in fact devoid of pancreatic secretory amylase (Fig 8).
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Under the control conditions tested, the reaction product was markedly reduced or eliminated. In the presence of eserine, the labeling found in the extracellular space was faint, whereas the globular and tubular lysosomes remained somewhat labeled, although with much reduced intensity. Removal of the acetylthiocholine from the incubation medium, on the other hand, abolished all labeling (not shown).
Overall, very similar results were obtained in acinar cells maintained in culture. The AChE reaction product was present in the globular and tubular lysosomal structures (results not shown).
Application of the immunocytochemical approach for detection of AChE on pancreatic tissue led to labeling at the level of the acinar cells. By light microscopy, the fluorescent staining within acinar cells displayed a punctate pattern. This was mainly located along the basolateral membrane and around the nuclei (not shown). The apical region of the cells, rich in secretory zymogen granules, was devoid of staining. On the other hand, strong signal was present along nerve paths. By electron microscopy, the labeling by gold particles, although of low intensity, was mainly detected in globular lysosomal structures, some of them displaying short tubular extensions, and in the RER (Fig 9a). In the extracellular space, the signal was mainly along the collagen fibers (Fig 9b). Double-labeling techniques combining enzyme cytochemistry and immunogold were able to confirm the location of the AChE (Fig 10). Globular positive structures with tubular extensions, as well as snake-like tubular structures, were simultaneously labeled by the enzyme cytochemical reaction product and the immunogold. However, the enzyme reaction product was much reduced in intensity compared to that obtained with the single-labeling protocol. This is probably due to extraction of the enzyme reaction product, particularly in areas containing small amounts of cytochemical reaction, during immunocytochemical processing. However, in spite of such extraction, it was possible to identify the globular and tubular AChE-positive network simultaneously labeled by enzyme and immunocytochemistry.
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Control experiments confirmed the specificity of the immunocytochemical results, because omitting the primary antibody or using normal rabbit serum led to absence of any labeling.
The parallel experiments performed on SCG supported our cytochemical and immunocytochemical results. The AChE cytochemical reaction was present at the level of the Nissl bodies, the entire Golgi apparatus, and in the extracellular space surrounding the ganglionic cells. These results were identical to those published previously (
To complete this study, we performed sedimentation analyses to examine the AChE molecular form content of the pancreas and of isolated acinar cells in comparison to that of the SCG (Fig 11). The sympathetic ganglion yielded its well-established profile of AChE molecular forms characterized by a largely predominant G4 peak and the presence of significant amounts of the asymmetric A12 form (
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Discussion |
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This study has revealed the presence of AChE in rat pancreatic tissue and, more specifically, in pancreatic acinar cells. That AChE is present in pancreas has been previously reported through cytochemical studies (
In the acinar cells, the AChE cytochemical reaction was observed in globular lysosomal structures located in the Golgi area and the basolateral region of the cell, as well as in a particular lysosomal compartment with a tubular snake-like appearance delineated by smooth membranes. Images of globular lysosomes extending into tubular projections, both displaying AChE activity, indicate continuity between the globular and tubular lysosomal structures. Examination of thick sections by high-voltage electron microscopy supported the existence of such a continuous complex network of tubular structures displaying varicosities and bifurcations. These snake-like tubular lysosomes form a complex 3D network within the acinar cell, intertwined between the cisternae of the ER, around the nuclei, and in the basolateral region, coming into very close contact with the plasma membrane itself. Similar networks were previously demonstrated in several types of cells, including pancreatic cells, and were shown to contain various lysosomal enzymes, such as adenine dinucleotide phosphatase, trimetaphosphatase, aryl sulfatase B, thiolacetic acid, and several other esterases (
In some acinar cells, AChE was also present within the cisternae of the RER, indicating that the acinar cells do synthesize this enzyme. Despite their presence in the RER, both the cytochemical and immunocytochemical reactions were absent from the main body of the Golgi apparatus and from the secretory zymogen granules, although the trans-Golgi network did display a positive reaction. The absence of AChE in the Golgi is further supported by the double-labeling protocol revealing AChE and amylase, a pancreatic secretory digestive enzyme, which showed that in addition to the RER where both molecules are present, AChE is absent from the classical regulated secretory pathway. The absence of AChE from the different cisternae of the Golgi could be related to the nature of the AChE molecules present in acinar cells. The A12 asymmetric molecules composed of globular catalytic tetramers and a collagen tail are assembled in the Golgi compartment (
That some cells, but not all, showed involvement in the synthesis of AChE may indicate that this cellular activity could be cyclic. The mosaic-like pattern detected for AChE in RER reflects differences in amounts of AChE among cells as related to the sensitivity of the technique and to the threshold levels of detection. A similar mosaic-like pattern of AChE was also reported for neural and muscle tissues (
As to the AChE signals obtained by enzyme cytochemistry and immunocytochemistry in the extracellular space, particularly those associated with the basement membranes and the collagen fibers, they are consistent with the literature showing interactions between AChE and various basement membrane components, including perlecan (
Recent progress has underlined the essential role of cholinergic receptors located on the acinar cells in the regulation of exocrine pancreatic secretion (
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Acknowledgments |
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Supported by a grant from the Medical Research Council of Canada.
We would like to thank Jean Gautron for advice and direction concerning the cytochemical technique, Jean Massoulié for kindly providing the specific antibody against AChE, and Bernard Jasmin for constructive discussion. We are also grateful to Gilles Lesperance for letting us use his high-voltage electron microscope facility. The technical assistance of Diane Gingras is gratefully acknowledged.
Received for publication May 22, 2000; accepted August 31, 2000.
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