THE ANATOMICAL RECORD 232:247-256 (1992)
Characterization of the Endocrine Cells in the Pancreatic-Bile Duct System of the Rat IN-SUN PARK AND MOISE BENDAYAN Department of Anatomy, Uniuersitk de Montrkal, Montreal, Quebec, Canada
ABSTRACT
Six types of endocrine cells showing immunolabelling against gut or pancreatic islet hormones were identified in the pancreatic-bile duct system of the normal adult rat at the light and electron microscopic levels. They were located within the epithelial lining of the duct system from the intercalated portion to its duodenal opening. However, the distribution and frequency of each endocrine cell varied along the length of the duct system. While insulin, glucagon, somatostatin, and pancreatic polypeptide cells were widely distributed along the entire duct system, small numbers of cholecystokinin and serotonin cells were confined to the terminal portion. A considerable number of somatostatin cells were concentrated in gland-like pouches of the terminal portion of the common pancreatic-bile duct. When the accessory pancreatic duct was present, insulin, glucagon, and somatostatin cells were also found in its epithelial lining. Electron microscopically, the specific content of the secretory granules of all endocrine cells was confirmed by immunolabelling or cytochemical staining. Further the characteristics of the secretory granules of each endocrine cell type corresponded to those present in the same kind of endocrine cells in gut or pancreatic islet. The duct endocrine cells displayed a particular ultrastructural appearance. The “open type cells” were highly polarized, with their apical cytoplasmic process reaching the duct lumen, whereas “closed type cells” showed long basal cytoplasmic processes with no connection with the duct lumen. In general, insulin, and somatostatin cells were of the “open type,” while no morphological connection with the duct lumen was found for glucagon and pancreatic polypeptide cells. The presence of various duct endocrine cells with their particular ultrastructural appearance implies that they may take part in modulating the function of the duct system.
In the 1930s the putative endocrine cells showing an systematic study on the duct endocrine cells not being argentaffin or argentophilic reaction were first de- carried out so far. scribed in the pancreatic-bile duct system by Erspamer In our previous study on insulin cells in the pancre(1937, 1938). With the help of immunohistochemistry, atic duct system (Bendayan, 19871, we raised the posLarsson et al. (1974) reported the occasional occurrence sibility that these cells exert a biological action upon of pancreatic polypeptide cells in the pancreatic duct surrounding tissues, such as epithelial, acinar, and istogether with pancreatic polypeptide cell rich islets. let endocrine cells and secrete endocrine peptides into This was followed by some reports on the existence of the pancreatic secretion for downstream paracrine horduct endocrine cells (Larsson et al., 1976; Heitz et al., monal regulation. 1977;Pelletier and Leclerc, 1977; Lee, 1988). In spite of The present study was designed to identify the diftheir distinct location, however, the duct endocrine ferent types of duct endocrine cells in the normal adult cells were regarded as part of the gut endocrine cells or rat pancreas using various antibodies against pancreof the endocrine pancreas. Moreover, these duct endo- atic and gut endocrine hormones in conjunction with crine cells have been considered as phylogenetic or em- cytochemical techniques. Together with the topographbryological remnants (Hellman, 1966; Pictet et al., ical mapping of the endocrine cells along the different 1972; Van Noorden and Falkmer, 1980) and pathoge- portions of the duct system according to their immunetically suspected to be at the origin of endocrine pan- noreaction, we have also attempted to reveal their morcreatic tumours (Pour 1978; Rutter, 1980; Chen et al., phological characteristics by applying conventional, 1988; Nonomura et al., 1989). On the other hand, the cytochemical, and immunocytochemical methods at the secretory function of the pancreatic duct system has been well established through intensive morphological, biochemical, and histochemical studies (Ekholm et al., 1962; Gemmell and Heath, 1973; Githens et al., 1980, Received July 23, 1991; accepted September 3, 1991. 1981,1987; Githens, 1989; Hubchak et al., 1990). HowAddress reprint requests to Dr. M. Bendayan, Departement danatever, most of the studies were focused on the non-en- omie, Universite de Montreal, C.P. 6128, Succ. A, Montreal (Quebec), docrine epithelial cells and their exocrine function; a Canada H3C 357. 0 1992 WILEY-LISS, INC.
248
I.-S. PARK AND M. BENDAYAN
TABLE 1. Source and optimal dilution of the primary antibodies' Antibody against Somatostatin Insulin Glucagon Pancreatic polypeptide Cholecystokinin-8 Serotonin Secretin
Dilution
LM 1:1,500 1:1,000 1:1,500
EM 1:lOO-200 1:lOO-200 1:200
Source INC Corp., Stillwater, MN ICN Immunobiologicals, IL INC Corp., Stillwater, MN
1:1,000 1:3,000 1:1,500 1:6,000
1:lOO-150 1:200-300
Dako Corp., Carpinteria, CA INC Corp., Stillwater, MN INC Corp., Stillwater, MN BioGenex Lab., San Ramon, CA
'All antisera were raised in rabbit except the insulin antiserum which was from guinea pig source.
electron microscopic level in order to account for their possible regulatory function on the neighbouring duct epithelial lining, exocrine parenchyma, and connective tissue. MATERIALS AND METHODS
then incubated with a protein A-gold complex formed by 15 nm gold particles and diluted to a n O.D. = 0.5, for 30 min a t room temperature. Control Test
In order to confirm the specificity of the immunolabellings, the antigen adsorption as well a s the omission of the primary antibody tests were carried out with the paraffin sections. For the antigen adsorption test, each antibody diluted a t its optimal concentration was preincubated with a n excess amount of its specific antigen (8 pg/ml) for 4 hours at 4°C. The sections were then Light Microscopic lrnrnunohistochernistry treated with these preincubated antibodies for 12 hours The tissue samples were fixed with Bouin's solution a t 4°C followed by the usual procedures. In addition, and embedded in paraffin, while others were fixed with cholecystokinin antiserum was preincubated with the 4% paraformaldehyde-1.5% picric acid in 0.1 M phos- gastrin (1-17) to assess any cross reactivity between phate buffer (pH 7.4) and embedded in Epon 812. Par- the anti-cholecystokinin and gastrin. Glucagon and affin sections (5 pm), and Epon semithin sections (1 pancreatic polypeptide antibodies were also reversely pm) were processed for immunohistochemistry with pretreated with pancreatic polypeptide and glucagon various primary antibodies. The Epon sections were for the same purpose. All the antigens used in the confirst treated with a saturated solution of potassium hy- trol tests were supplied by Sigma Chemical Co., (St. droxide in absolute alcohol for 30 min in order to re- Louis, MO). move the resin, while the paraffin sections were proElectron Microscopic Cytochernistry cessed after rehydration through decreasing ethanol The tissue was cut in small pieces and fixed with 1% solutions. The immunohistochemical method used for light microscopy was the avidin-biotin-peroxidase com- glutaraldehyde solution in 0.01 M phosphate buffer. plex technique (Hsu et al., 1981). The nature, source, After careful washing with the phosphate buffer and and optimal dilution of the primary antibodies are de- distilled water the tissue specimens were immersed in scribed in Table 1. Biotinylated anti-rabbit immuno- 1% silver nitrate-ammonia water for 1 hour a t 60°C globulins and the avidin-biotin-peroxidase complex according to the modification of Masson-Hamperl's were purchased from Vector Laboratories (Burlin- technique (Singh, 1964) for the argentaffin reaction. game, CA), while the biotinylated anti-guinea pig im- For the argyrophilic impregnation, after fixation the munoglobulins were obtained from Janssen Life Sci- tissue specimens were incubated in 0.03% silver nitrate solution in 0.2 M sodium acetate buffer for 4 ence (Olen, Belgium). hours at 60°C and developed with 5%hydroquinone-1% Electron Microscopic irnrnunocytochernistry sodium sulfite solution for 5 min a t 60°C (Grimelius, Small pieces of tissue specimens were fixed with 4% 1968). After silver impregnation all the specimens paraformaldehyde-1% glutaraldehyde in 0.1 M phos- were rinsed in distilled water and subsequently embedphate buffer for 2 hours at room temperature and em- ded in Epon. bedded in Lowicryl K4M (Bendayan, 1984) or Epon Conventional Electron Microscopy 812. Ultrathin sections were mounted on Parlodion and carbon coated nickel grids. Prior to immunolabelling, After fixation with the paraformaldehyde-glutaralthe sections were treated with a saturated solution of dehyde mixture, some tissue samples were post fixed sodium metaperiodate for 1hour a t room temperature. with 1% osmium tetroxide for 1 hour a t 4°C and emAfter brief washing with distilled water, the sections bedded in Epon. Several sets of serial sections were were laid on a drop of 1%ovalbumin in PBS for 30 min prepared for general ultrastructural examination and and incubated onto a drop of the primary antibody for determination of the open or close cell characterisbrought to optimal dilution (Table 1)for 12-14 hours a t tics. All of the ultrathin sections in the present study 4°C. The grids were rinsed with PBS and transferred were stained with uranyl acetate and lead citrate soonto the 1%ovalbumin solution for 30 min. They were lution and observed using the Phillips EM410. The
Pancreas and duodenum were sampled from seven normal male Sprague-Dawley rats (-25Og bw) under ether anaesthesia. The tissue specimens were processed for immunohisto- and cytochemistry according to the following procedures.
ENDOCRINE CELLS I N THE PANCREATIC-BILE DUCT
photographs of the same endocrine cell obtained from serial sections were integrated for their stereoscopic reconstruction. For morphometrical evaluations of the secretory granule sizes, their outer diameter delineated by a well-defined limiting membrane were measured. More than 100 secretory granules for each cell type were evaluated. RESULTS General Morphology -. of the Pancreatic Duct System
The pancreatic duct system of the rat consists of intra- and inter-lobular ducts leading to the common pancreatic-bile duct including its intramural opening portion into the duodenal wall. Of particular interest is the presence of several small interlobular ducts opening directly into the common pancreatic-bile duct in separate ways without forming an independent main pancreatic duct. On some occasions one relatively large duct does traverse the duodenal wall just beside the distal portion of the common pancreatic-bile duct and opens directly into the intestinal lumen thus constituting an accessory pancreatic duct. This was observed in four out of the seven rats studied. All parts of the pancreatic duct system were lined by a simple epithelium. In the common pancreatic-bile duct this epithelial lining invaginates into the surrounding connective tissue to make gland-like pouches. Distribution of the Endocrine Cells in the Pancreatic Duct
Endocrine cells displaying specific immunolabelling for insulin (Fig. l),glucagon (Figs. 1, 21, somatostatin (Figs. 3 , 4 , 5 ) , pancreatic polypeptide (Fig. 61, serotonin (Fig. 7) or cholecystokinin (Fig. 8) were found within the epithelial lining of the pancreatic-bile duct system. The endocrine cells exhibited differences both in frequency and topographical distribution according to the peptide or amine which they display and the portion of the duct system examined. They were scattered as single cells except in the terminal portion of the common pancreatic-bile duct where they showed remarkable accumulations. These endocrine cells were preferentially located in the gland like pouches. As presented in Table 2, the distribution pattern and frequency of the pancreatic duct endocrine cells differed with respect to the portions of the duct system. Somatostatin and pancreatic polypeptide cells showing similar distribution were the more numerous while insulin and glucagon cells, less numerous, followed the same locations. The latter were evenly scattered along the duct system including the accessory pancreatic duct where the glucagon cells outnumbered the other cell types (Fig. 2). The area of the duodenal opening of the duct was particularly rich in somatostatin cells (Fig. 3). A small number of the cholecystokinin and serotonin immunoreactive cells was found only in the distal portion of the duct system particularly in the common pancreatic bile duct (Figs. 7, 81, even though there was a large population of these cell types in the neighbouring duodenal mucosa. No secretin immunoreactive cells were found in any part of the pancreatic duct while they were numerous in the gut mucosa. Besides being located in duct epithelia or islets, a significant number of isolated insulin, glucagon, somatostatin, and pancreatic polypeptide cells were observed within the exocrine paren-
249
chyma of the pancreas. These cells were usually interposed between acinar cells (insets in Figs. 1, 6). Control Test
No immuno-positive cell was detected in sections incubated with any of the primary antibodies pretreated with their corresponding antigen. Cholecystokinin immunoreactive cells were identified in the section incubated with its primary antibody pretreated with excess amounts of gastrin 1-17. Glucagon and pancreatic polypeptide antibodies showed constant immunoreactive cells in spite the preincubation of the antibodies with the opposite antigen, pancreatic polypeptide, and glucagon, respectively. Electron Microscopical Structure of Duct Endocrine Cells
Endocrine cells of the pancreatic duct were distinguished from the surrounding epithelial cells by their pale cytoplasm and by the presence of secretory granules located mainly in the baso-lateral region of the cell. Besides these, the other organelles of the endocrine cell shared common features; the Golgi apparatus was located in a peri-nuclear portion, the rough endoplasmic reticulum, and the elongated mitochondria were well developed and intermingled with the secretory granules. Based on their general appearance, pancreatic duct endocrine cells can be sorted into two categories. One displayed a pyramidal or oval appearance and was found to be the “open type” having direct contact with the lumen of the duct. This was confirmed by examination of serial sections, which demonstrated that this kind of cell reaches the duct lumen with a slender apical cell process. The luminal surface of the cell is narrow and the apical plasma membrane is covered with a tuft of microvilli (Figs. 1, 5). The apical end of lateral plasma membranes was characterized by junctional complexes. These cells demonstrate distinct polarity, most of the secretory granules being located in the lateral and basal cytoplasm underneath the nucleus (Fig. 5). The other kind of cells, the “closed type,” manifested variable appearance with the cytoplasmic processes filled with secretory granules. These cells usually laid on the basal lamina and seemed to have no morphological connection with the duct lumen. Cell processes ran between the Saso-lateral border of surrounding epithelial cells and sometimes deeply invaginate into the cytoplasm near the nuclear region of the neighbouring cells (Fig. 6). The details and specific features of the secretory granules of the pancreatic duct endocrine cells together with their immunolabelling have allowed for the identification of each individual cell type. Consequently, the routine classification based on differences in the shape, size, electron density, and matrix texture of the secretory granules concurred with the cytochemical and immunocytochemical identification of cell types. We were also able to analyze the ultrastructural features of each endocrine cell type putting emphasis on the characteristics of their secretory granules using routine morphological examination as well as cytochemical and immunocytochemical approaches. Insulin(B) cell, a typical open type, contains large (outer mean diameter 400221 nm; range 310-570 nm) and round secretory granules in a somewhat darker
250
1 . 3 . PARK AND M. BENDAYAN
Fig. 1. Electron micrograph of part of the common pancreatic-bile duct from the non-osmicated Epon-embedded tissue section incubated with insulin antibody and protein A-gold. The insulin cell (B) is well identified by the specific labelling presented on the secretory granules. The apical cell surface is in contact with the duct lumen (DL) and displays and microvilli (arrowheads). Several secretory granules are present in the apical region of the cell. Another endocrine cell (A), assumed to be glucagon cell, shows its cytoplasmic process surrounded by the epithelial cells. x 7,000. Inset: Paraffin section of the pancreas
incubated with insulin antibody. An insulin cell is seen in the duct epithelial lining (arrow), while two isolated insulin cells (arrow heads) are located in the exocrine parenchyma. x 700. Fig. 2. Light micrograph of the accessory pancreatic duct from nonosmicated Epon semithin section incubated with glucagon antibody and the avidin-biotin-peroxidase technique. Several immunoreactive cells are formed in the epithelial lining. No luminal contact of the glucagon cell can be seen. DL, duct lumen x 2,000.
ENDOCRINE CELLS I N T H E PANCREATIC-BILE DUCT
Figs. 3 and 4. Paraffin sections of the terminal part of the common pancreatic-bile duct (Fig. 3) and its opening into the duodenal lumen (Fig. 4) incubated with somatostatin antibody. Figure 3 shows the duct interposed between duodenal mucosa (DM) and the muscle layers (ML). Numerous somatostatin cells are located in the base of glandlike pouches of the duct while no positive cell is observed in the duodenal mucosa. x 380. Figure 4 shows the luminal content of the duct draining through the connection between the duct and the duodenum. x 330. Inset: higher magnification of the boxed area of the duct demonstrating somatostatin positive cells (arrowheads). x 1,600.
251
Fig. 5. Non-osmicated Epon-embedded tissue. Ultrathin section of the common pancreatic-bile duct incubated with somatostatin antibody. A pyramidal shape of somatostatin cell shows distinct polarity, basally located secretory granules and a long slender cytoplasmic process reaching the duct lumen and displays a tuft of luminal microvilli (arrowheads). x 4,600.
252
1.3. PARK AND M. BENDAYAN
Fig. 6. Four consecutive electron micrographs of the same area of the common pancreatic-bile duct from routine electron microscopy. An endocrine cell (PP), assumed to be a “closed type” pancreatic polypeptide cell, displays long cytoplasmic process. The cell process invaginates into the cytoplasm and even comes in close contact with the
nucleus of adjacent epithelial cell (Ne). x 6,200. Inset in b: Pancreatic polypeptide immunoreactive cells in the paraffin section. One positive cell (arrow) is shown in the intralobular duct whereas two isolated cells (arrowheads) are intermingled with acinar parenchyma. x 900.
ENDOCRINE CELLS IN THE PANCREATIC-BILE DUCT
Figs. 7 and 8. Paraffin sections incubated with serotonin (Fig. 7) and cholecystokinin antibodies (Fig. 81, respectively. The positive cells are located in the gland-like pouches (GP) of the common pancreatic-bile duct. x 1,900, Fig. 9. Nan-osmicated Lowicryl embedded tissue. Ultrathin section
incubated with cholecystokinin antibody. A cholecystokinin cell is characterized by its medium sized round granules and bundles of mi-
253
crofilaments. x 5,300. b: higher magnification of the boxed area of a. Secretory granules are labelled with gold particles. x 24,000. Fig. 10. Electron micrographs of the argentafin cells in the common pancreatic-bile duct (a)and duodenal mucosa (b).Although the secretory granules of both cells are heavily impregnated with silver grains, the shape of the duct cell granules is somewhat different from that of the duodenal one. x 20,000.
1 . 3 PARK AND M. BENDAYAN
254
TABLE 2. Topographical distribution and relative frequency of the endocrine cells in the rat pancreatic duct system' Common
Endocrine cell Insulin Glucagon Somatostatin Pancreatic polypeptide Cholecystokinin Serotonin Secretin
Intra- Inter- pancreaticbile Accessory lobular lobular duct duct duct duct
+ + +-
+
+
+ ++
+ + ++++
+I++
+ +I+ + +
-
+I+
+I+
+
+ + +
+I+
~
?
-
-
-
none; ?, very few; + , few; + + , moderate; + + + , numerous; + + + + , very numerous.
l-,
cytoplasm than other endocrine cells. The secretory granules consisted of a homogenous, relatively dense inner core surrounded by a wide electron lucent halo with a n outer limiting membrane. The immunolabelling was restricted to the inner core of the granule. Occasionally some granules were observed in the apical cytoplasm close to the luminal surface (Fig. 1). Secretory granules of the glucagon (A) cell revealed round profiles (23026.2 nm; 190-250 nm) and were compactly filled with a homogenous and high electron dense matrix. The immunolabelling with the anti-glucagon antibody was present over the entire granule content. Although some cells showed a n oval shape, their contact with the duct lumen was never clearly defined (Figs. 1, 2). Somatostatin (D) cell displayed medium sized (21023.8 nm, 170-260 nm) round secretory granules loosely packed with variable electron-dense matrices. A narrow and clear halo intersected between the granule and its outer limiting membrane. The identity of the cell was confirmed by immunolabelling, with the gold particles being concentrated over the core of the granules. Somatostatin cells usually demonstrated a pyramidal or oval appearance and their luminal connection was frequently observed (Fig. 5). Pancreatic polypeptide immunoreactivity was identified in two cell types showing quite different granule morphology. One had rather small (180+2 nm, 150200 nm) round and light electron dense granules and the other showed relatively larger (200k5.7 nm, 190240 nm), round oval or rod-shaped granules with a homogenous and high electron-dense matrix. Both types of granules displayed immunoreactive pancreatic polypeptide as demonstrated by their specific labelling. Owing to the absence of luminal contact, both cell types were classified as closed (Fig. 6). Cholecystokinin cells had medium sized (220k6.3 nm, 180-240 nm), homogenous, and moderate-dense granules. These cells were further characterized by the presence of large bundles of microfilaments surrounding the nuclei. Their identity was confirmed by gold immunolabelling of the granule cores (Fig. 9). A few serotonin containing cells, identified by their argentaffin reaction, were recorded in the duct system. The secretory granules of these cells, displayed a relatively large (280k 11.4, 210-320 nm) and pleomorphic
configuration (Fig. 10). Some granules contained a small and electron-lucent core surrounded by dense granular matrix while others were rather homogenous. Except for the somatostatin cell, all the other cells exhibited strong silver staining of their granules with the argyrophilic silver impregnation method of Grimelius. In the case of somatostatin cell, in spite of the fact that some granules did reveal distinct argyrophilia, a large number of them were negative. DISCUSSION The pancreatic-bile duct system plays a n important role in producing the extra acinar components of the pancreatic secretion-bicarbonate, electrolytes, and mucous substance, as well as in modulating the flow of the luminal content (Case and Argent, 1986; Ryan, 1987; Shultz, 1987). These main functions of the duct system are controlled by neuronal and hormonal regulations (Solomon, 1987; Alder and Beglinger, 1990). The endocrine cells of the gastro-entero-pancreatic system secrete their regulatory peptide hormones following physiological stimuli. The primary target of these hormones is the neighbouring epithelial cells as well as the smooth muscle fibers of the vessels and muscle layers on which they exert a paracrine action. They may also control remote targets through the blood and possibly via the pancreatic secretion mediated pathway (Feyrter, 1953; Larsson, 1984; Bendayan, 1987; Solcia et al., 1987). From this point of view and in spite of the fact that they may represent phylogenetic and embryological remnants, the endocrine cells of the pancreatic-bile duct system should be considered as a n integral functional element that modulates ductal activities. In the present study we report the presence of six different types of endocrine cells displaying specific immunolabellings for insulin, glucagon, somatostatin, pancreatic polypeptide, cholecystokinin, and serotonin in the pancreatic duct of the rat. By combining routine electron microscopic examination with cytochemical and immunocytochemical electron microscopy, we have classified six types of cells according to their specific immunolabelling, cytochemical reaction, shape, and size of the secretory granules. Although there are some differences in interpreting the cell types, the A, B, D1-like, and EC cells previously identified by routine electron microscopy in the pancreatic duct of several mammals (Kodama, 1983) were also identified in the present study. We could not find ultrastructural differences in secretory granules between the duct endocrine cells and the same type of the cells present in the gut or in the pancreatic islet. The secretory granules of the duct insulin, glucagon, somatostatin, and pancreatic polypeptide cells shared common structural characteristics with the previously described pancreatic islet cell granules (Forssmann et al., 1969; Larsson et al., 1976; Erlandersen, 1980; Fiocca e t al., 1983; Orci, 1985; Solcia et al., 1987). The cholecystokinin and serotonin containing granules of the duct endocrine cells were similar to the intestinal I cell and EC cell granules (Grube and Forssmann, 1979; Solcia et al., 1975, 1987; Dobbins and Austin, 1991) respectively. The histochemical argyrophil silver impregnation method of Grimelius has been widely used to identify
ENDOCRINE CELLS IN THE PANCREATIC-BILE DUCT
the gut and pancreatic endocrine cells because of its broad spectrum of reaction (Grimelius, 1968; Solcia et al., 1987). In the case of the somatostatin cell, however, its argyrophilic reaction remains controversial. Although the somatostatin cell was described as the only cell type displaying negative reaction to the method of Grimelius (Solcia et al. 1975), we have shown argyrophilic impregnation and somatostatin immunolabelling in the same endocrine cell (Park, 1986). Consequently the argyrophilic silver impregnation was confirmed in all types of duct endocrine cells including the somatostatin cell which contains both positive and negative secretory granules. The function and mechanism of action of the endocrine cells are closely related to their morphological diversity. In the gut mucosa, it is generally accepted that most of the endocrine cells reach the lumen, their luminal membrane displays a receptor for the stimuli from the lumen (Fujita and Kobayashi, 1973; Grube and Forssmann, 1979; Solcia et al., 19871, whereas other endocrine cells characterized by the long basal cytoplasmic processes have a paracrine action on surrounding target cells (Alumets et al., 1979; Larsson et al., 1979; Larsson, 1984; Yamada, 1987). The present study has shown two categories of duct endocrine cells, the pyramidal-shaped “open cell type” and the pleomorphic “closed cell type.” In contrast to the gut endocrine cells, however, the manifestation of the “open” or “closed” feature of the duct endocrine cells is mainly relevant to their hormonal content regardless of the region in which they occurred. Almost all of the somatostatin and insulin cells were found to be of the “open type,” like the pyrolic and intestinal endocrine cells, while the glucagon and pancreatic polypeptide cells belong to the “closed type” like those of the fundic mucosa of the stomach (Grube and Forssmann, 1979; Larsson 1984;Solcia et al., 1987).This observation concurs with our previous findings on “open insulin cells” in the duct (Bendayan, 1987) but contradicts the report that all duct endocrine cells are of the “closed type” (Kodama, 1983).The ultrastructural findings of the present study support the concept that the duct endocrine cells have not only the possibility to display receptors for the stimuli originating from the duct lumen, but also the capability to secrete and to exert a paracrine influence on surrounding tissues. Although no clear evidence has been provided for duct secretion of peptides into the pancreatic secretion through the luminal cell membrane other than the presence of secretory granules close to the luminal membrane, we should not rule out the possibility that duct endocrine hormones are secreted into the duct lumen and affect the epithelial lining. The repeated reports on the presence of the pancreatic islet hormones in the duct lumen (Carr-Locke and Track, 1979; Colon, 1979; Lawrence et al., 1979; Prinz et al., 1980; Ertan et al., 1981; Sarfati et al., 1986) support that concept. Moreover, the recent identifications of hormone receptors in the gut epithelium (Pillon et al., 1985; Fernandez-Moreno et al., 1986, 1987, 1988) bring additional evidence along this line. The pancreatic duct endocrine cells display different patterns of distribution along the duct system. While the cholecystokinin and serotonin cells were confined to the terminal portion of the common pancreatic-bile duct in small numbers, the other endocrine cells,
255
namely, the pancreatic hormones secreting cells, were distributed all along the tract of the pancreatic-bile duct system, being concentrated in the terminal portion. The distribution of each cell type varied according to regional and functional differences of the duct. The intra- and inter-lobular ducts participate in secretion of the bicarbonate and electrolytes, while the main common duct is the secreting site of mucous substances and the final gate for delivery of the luminal flow into the duodenum (Kodama, 1983; Case and Argent 1986; Ryan, 1987; Dahlstrand et al., 1990). The main common duct is also characterized by numerous smooth muscle layers including the sphincter muscle in its terminal portion. This may imply that the duct endocrine cells as well as isolated endocrine cells in the pancreas form a separate endocrine unit with its proper biological function. Several peptide hormones applied exogenously have been shown to infuence the pancreatic-bile duct system; somatostatin, pancreatic polypeptide, and glucagon have an inhibitory effect, while cholecystokinin stimulates the secretory function of the duct (Case and Argent, 1986; Walsh, 1987). It is thus conceivable that the various hormones secreted by the duct endocrine cells exert a similar excitory or inhibitory effect on this same target tissue through endocrine or paracrine mode of action. ACKNOWLEDGMENTS
The authors are grateful to Diane Gingras, Jean Godbout, and Christiane Rondeau for excellent technical assistance. Dr. Park is a Fellow from the Catholic University Medical College, Seoul, Korea. This work was supported by a grant from the Medical Research Council of Canada. LITERATURE CITED Alder, G., and C. Beglinger 1990 Hormones as regulators of pancreatic secretion in man. Eur. J. Clin. Invest. 20, Suppl. ltS27S32. Alumets, J.,H.A. Ekelund, E. Munshid, R. Hikanson, I. Loren, and F. Sundler 1979 Topography of the somatostatin cells in the stomach of the rat: Possible functional significance. Cell Tiss. Res., 20.2: 177-188. Bendayan, M. 1984 Protein A-gold electron microscopic immunocytochemistry: Methods, applications and limitations. J. Electron. Microsc. Tech., lr243-270. Bendayan, M. 1987 Presence of endocrine cells in pancreatic ducts. Pancreas, 2:393-397. Carr-Locke, D.L., and N.S. Track 1979 Human pancreatic polypeptide in pancreatic juice. Lancet, 1:151-152. Case, R.M., and B.E. Argent 1986 Bicarbonate secretion by pancreatic duct cells: Mechanism and control. In: The Exocrine Pancreas: Biology, Pathology, and Diseases. Go, V.W.L., J.D Gardner, F.P. Brooks, E. Lebenthal, E.P. DiMagno and G.A. Scheele, eds. Raven Press, New York, pp. 213-244. Chen, J., S.I. Baithun, D.J. Pollock, and C.L. Berry 1988 Argyrophilic and hormone immunoreactive cells in normal and hyperplastic pancreatic ducts and exocrine pancreatic carcinoma. Virchows. Archiv. A Pathol. Anat., 413:399-405. Colon, J.M., D. Rouiller, G. Boden, and R.H. Unger 1979 Characterization of immunoreactive components of insulin and somatostatin in canine pancreatic juice. FEBS Lett., 105:23-26. Dahlstrand, C., A. Dahlstrom, E. Teodorsson, J. Rehfeld, and H. Ahlman 1990 Is the CCK-8 induced relaxation of the feline spincter of Oddi mediated by VIP neuron? J . Auton. Nerv. Syst., 31:75-84. Dobbins, W.O., and L.L. Austin 1991 Electron microscopic definition of intestinal endocrine cells: Immunogold localization and review. Ultrastruct. Pathol., 15t15-39. Ekholm, R., T. Zelander, and Y. Edlund 1962 The ultrastructural organization of the rat exocrine pancreas. 11. Centroacinar cells, intercalary and interlobular ducts. J . Ultrastruct. Res., 7t73-83. Erlandsen, S.L. 1980 Types of pancreatic islet cells and their immunocytochemical identification. In: The Pancreas. Fitzgerald P.J.,
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and A.B. Morrison, eds. International Academy of Pathology Monograph, Williams and Wilkins, Baltimore, London, pp. 140155. Erspamer, V. 1937 Cellule enterochromafini e cellule argentofile nel pancreas dell’uomo e dei mammiferi. Z. Anat. Entwickl-Gesch., 107r574-619. Erspamer, V. 1938 Sulla presenza di cellule argentofile (Preenterochromafhi) nelle vie biliari dell’uomo e di alcuni mammiferi. Anat. Anz., 85:272-284. Ertan, A,, T. Taminato, and K. Akdamar. 1981 Immunoreactive somatostatin in human pancreatic secretion. J. Clin. Endocrinol. Metab., 52t589-591. Feyrter, F. 1953 Uber die peripheren endokrinen (parakrinen). In: Drusen des Menschen. Maudrich, W. Wien-Dusseldorf, pp. 1-231. Fernandez-Moreno, M.D., E. Arilla, and J.C. Prieto 1986 Insulin binding to rat intestinal epithelial cells following partial small-bowel resection. Biosci. Rep., 6:445-450. Fernandez-Moreno, M.D., M.A. Fernandez-Gonzalez, J.L. Diaz-Juarez, M.P. Lopez-Luna, and J.C. Prieto 1988 Interaction of insulin with small intestinal cells from developing rats. Biol. Neonate, 54r289-293. Fernandez-Moreno, M.D., M. Serrano-Rios, and J.C. Prieto 1987 Identification of Insulin receptors in epithelial cells from duodenum, jejunum, ileum, caecum, colon and rectum in the rat. Diabete. Metab. 13t135-139. Fiocca, R., F. Sessa, P. Tenti, L. Usellini, C. Capella, M.M.T. OHare, and E. Solcia 1983 Pancreatic polypeptide (PP) cells in the PPrich lobe of the human pancreas are identified ultrastructurally and immunocytochemically as F cells. Histochemistry, 77511523. Forssmann, W.G., L. Orci, R. Pictet, A.E. Renold, and C. Rouiller 1969 The endocrine cells in the epithelium of the gastrointestinal mucosa of the rat. J . Cell Biol., 40:692-715. Fujita, T., and S. Kobayashi 1973 The cells and hormones of the GEP endocrine system: The current studies. In: Gastro-Entero-Pancreatic Endocrine System: A Cell-Biological Approach. Fujita, T. ed. Igaku Shoin, Tokyo, pp. 1-16. Gemmell R.T., and T. Heath 1973 Structure and function of the biliary and pancreatic tracts of the sheep. J. Anat., 115t221-236. Githens, S. 1989 Development of duct cells. In: Human Gastrointestinal Development. Lebenthal, E. ed. Raven Press, New York, pp. 669-683. Githens, S., J.J. Finley, C.L. Patke, J.A. Schexnayder, K.B. Fallon, and J.R. Rubby 1987 Biochemical and histochemical characterization of cultured rat and hamster pancreatic ducts. Pancreas, 2:427-438. Githens, S., D.R.G. Holmquist, J.F. Whelan, and J.R. Rubby 1980 Characterization of ducts isolated from the pancreas of the rat. J . Cell Biol., 85:122-135. Githens. S.. D.R.G. Holmauist, J.F. Whelan, and J.R. Rubbv 1981 Morphologic and biochemical characteristics of isolated and cultured pancreatic ducts. Cancer, 47t1505-1512. Grimelius, L. 1968 A silver nitrate stain for a2 cells in human pancreatic islets. Acta SOC.Med. Upsal., 73:243-270. Grube, D., and W.G. Forssmann 1979 Morphology and function of the entero-endocrine cells. Horm. Metab. Res., 1Ir589-606. Heitz, Ph., J.M. Polak, M. Kasper, C.M. Timson, and A.G.E. Pearse 1977 Immunoelectron cytochemical localization of motilin and substance P in rabbit bile duct enterochromafin (EC) cells. Histochemistry, 50t319-325. Hellman, B. 1966 The development of the mammalian endocrine pancreas. Biol. Neonate, 9:263-278. Hsu, S.M., L. Raine, and H. Fanger 1981 Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem., 29577-580. Hubchak, S., M.M. Mangino, M.K. Reddy, and D.G. Scarpelli 1990 Characterization of differentiated Syrian golden hamster pancreatic duct cells maintained in extended monolayer culture. In Vitro Cell Dev. Biol., 26:889-897. Kodama, T. 1983 A light and electron microscopic study on the pancreatic ductal system. Acta Pathol. Jpn., 33:297-321. Larsson, L.-I. 1984 Evidence for anterograde transport of secretory granules in processes of gastric paracrine (somatostatin) cells. Histochemistry, 80:323-326.
Larsson, L.-I., N. Goltermann, L. De Magistris, J.F. Rehfeld, and T.W. Schwartz 1979 Somatostatin cell processes as pathways for paracine secretion. Science, 205:1393-1395. Larsson, L.-I., F. Sundler, and R. HBkanson 1976 Pancreatic polypeptide- A postulated new hormone: Identification of its cellular storage site by light and electron microscopic immunocytochemistry. Diabetologia, 12211-226. Larsson, L.-I., F. Sundler, R. Hikanson, H.G. Polock, and J.R. Kimme11 1974 Localization of APP, a postulated new hormone in a pancreatic endocrine cell type. Histochemistry 43:377-381. Lawrence, A.M., R.A. Prinz, E. Paloyan, and W.A. Kokal 1979 Glucagon and insulin in pancreatic exocrine secretions. Lancet, 2: 1354-1355. Lee, M.Y. 1988 Presence of the endocrine cells in the bile and pancreatic ducts of the rabbit, rat and cat. J . Cathol. Med. Coll., 41 :787-797. Nonomura, A,, Y. Mizukami, F. Matsubara, N. Kono, and Y. Nakanuma 1989 Duct-islet cell tumor of the pancreas: A case report with immunohistochemical and electron microscopic findings. Acta. Pathol. Jpn., 39t328-335. Orci, L. 1985 The insulin factory: A tour of the plant surroundings and a visit to the assembly line. Diabetologia, 28528-546. Park, I.-% 1986 Distribution, histochemical properties and ultrastructure of the endocrine cells in the gastrointestinal tract of the ruminant. J . Cathol. Med. Coll., 39.1065-1090. Pelletier, G., and R. Leclerc 1977 Immunohistochemical localization of human pancreatic polypeptide (HPP) in the human endocrine pancreas. Gastroenterology, 72:569-571. Pjctet, R.L., W.R. Clark, R.H. Williams, and W.J. Rutter 1972 An ultrastructural analysis of the developing embryonic pancreas. Dev. Biol., 29t436-467. Pillon, D.J., V. Ganapathy, and F.H. Leibach 1985 Identification of insulin receptors on the mucosal surface of colon epithelial cells. J . Biol. Chem., 2605244-5247, Pour, P. 1978 Islet cells as a component of pancreatic ductal neoplasms. I. Experimental study: Ductular cells, including islet cell precursors, as primary progenitor cells of the tumors. Am. J . Pathol., got295-3 16. Prinz, R.A., L. Kirstein, E. Connick, E. Paloyan, and A.M. Lawrence 1980 Insulin and glucagon in human pacreatic exocrine fluid. Horm. Metab. Res., 12t38-39. Rutter, W.J. 1980 The development of the endocrine and exocrine pancreas. In: The Pancreas. Fitzgerald P.J. and A.B. Morrison, eds. Williams and Wilkins, Baltimore, London, pp. 30-38. Ryan, J.P. 1987 Motility of the gallbladder and biliary tree. In: Physiology of the Gastrointerstinal Tract. Johnson, L.R., ed. Raven Press, New York, pp. 665-694. Sarfati, P.D., G.M. Green, P. Brazeau, and J . Morisset 1986 Presence of somatostatin-like immunoreactivity in rat pancreatic juice: A physiological phenomenon. Can. J . Physiol. Pharmacol., 64539544. Schultz, I. 1987 Electrolyte and fluid secretion in the exocrine pancreas. In: Physiology of Gastrointestinal Tract. Johnson, L.R. ed. Raven Press, New York, pp. 1147-1171. Singh, I. 1964 A modification of the Masson-Hamper1 method for staining of argentafin cells. Anat. Anz., 115t81-82. Solcia, E., C. Capella, R. Buffa, L. Usellini, R. Fiocca, and F. Sessa 1987 Endocrine cell of the digestive system. In: Physiology of the Gastrointestinal Tract. Johnson L.R. ed. Raven Press, New York, pp. 111-130. Solcia, E., C. Capella, G. Vassallo, and R. Buffa 1975 Endocrine cells of the gastric mucosa. Int. Rev. Cytol. 42:223-286. Solomon, T.E. 1987 Control of exocrine pancreatic secretion. In: Physiology of the Gastrointestinal Tract. Johnson L.R. ed. Raven Press, New York, pp. 1173-1207. Van-Noorden, S., and S. Falkmer 1980 Gut-islet endocrinology: Some evolutionary aspects. Invest. Cell Pathol., 3.21-35. Yamada, T. 1987 Local regulatory actions of gastrointestinal peptides. In: Physiology of the Gastrointestinal Tract. Johnson, L.R. ed. Raven Press, New York, pp. 131-142. Walsh, J.H. 1987 Gastrointestinal hormones. In: Physiology of the Gastrointestinal Tract. Johnson, L.R. ed., Raven Press, New York, pp. 187-253.
Characterization of the Endocrine Cells in the Pancreatic-Bile Duct System of the Rat IN-SUN PARK AND MOISE BENDAYAN Department of Anatomy, Uniuersitk de Montrkal, Montreal, Quebec, Canada
ABSTRACT
Six types of endocrine cells showing immunolabelling against gut or pancreatic islet hormones were identified in the pancreatic-bile duct system of the normal adult rat at the light and electron microscopic levels. They were located within the epithelial lining of the duct system from the intercalated portion to its duodenal opening. However, the distribution and frequency of each endocrine cell varied along the length of the duct system. While insulin, glucagon, somatostatin, and pancreatic polypeptide cells were widely distributed along the entire duct system, small numbers of cholecystokinin and serotonin cells were confined to the terminal portion. A considerable number of somatostatin cells were concentrated in gland-like pouches of the terminal portion of the common pancreatic-bile duct. When the accessory pancreatic duct was present, insulin, glucagon, and somatostatin cells were also found in its epithelial lining. Electron microscopically, the specific content of the secretory granules of all endocrine cells was confirmed by immunolabelling or cytochemical staining. Further the characteristics of the secretory granules of each endocrine cell type corresponded to those present in the same kind of endocrine cells in gut or pancreatic islet. The duct endocrine cells displayed a particular ultrastructural appearance. The “open type cells” were highly polarized, with their apical cytoplasmic process reaching the duct lumen, whereas “closed type cells” showed long basal cytoplasmic processes with no connection with the duct lumen. In general, insulin, and somatostatin cells were of the “open type,” while no morphological connection with the duct lumen was found for glucagon and pancreatic polypeptide cells. The presence of various duct endocrine cells with their particular ultrastructural appearance implies that they may take part in modulating the function of the duct system.
In the 1930s the putative endocrine cells showing an systematic study on the duct endocrine cells not being argentaffin or argentophilic reaction were first de- carried out so far. scribed in the pancreatic-bile duct system by Erspamer In our previous study on insulin cells in the pancre(1937, 1938). With the help of immunohistochemistry, atic duct system (Bendayan, 19871, we raised the posLarsson et al. (1974) reported the occasional occurrence sibility that these cells exert a biological action upon of pancreatic polypeptide cells in the pancreatic duct surrounding tissues, such as epithelial, acinar, and istogether with pancreatic polypeptide cell rich islets. let endocrine cells and secrete endocrine peptides into This was followed by some reports on the existence of the pancreatic secretion for downstream paracrine horduct endocrine cells (Larsson et al., 1976; Heitz et al., monal regulation. 1977;Pelletier and Leclerc, 1977; Lee, 1988). In spite of The present study was designed to identify the diftheir distinct location, however, the duct endocrine ferent types of duct endocrine cells in the normal adult cells were regarded as part of the gut endocrine cells or rat pancreas using various antibodies against pancreof the endocrine pancreas. Moreover, these duct endo- atic and gut endocrine hormones in conjunction with crine cells have been considered as phylogenetic or em- cytochemical techniques. Together with the topographbryological remnants (Hellman, 1966; Pictet et al., ical mapping of the endocrine cells along the different 1972; Van Noorden and Falkmer, 1980) and pathoge- portions of the duct system according to their immunetically suspected to be at the origin of endocrine pan- noreaction, we have also attempted to reveal their morcreatic tumours (Pour 1978; Rutter, 1980; Chen et al., phological characteristics by applying conventional, 1988; Nonomura et al., 1989). On the other hand, the cytochemical, and immunocytochemical methods at the secretory function of the pancreatic duct system has been well established through intensive morphological, biochemical, and histochemical studies (Ekholm et al., 1962; Gemmell and Heath, 1973; Githens et al., 1980, Received July 23, 1991; accepted September 3, 1991. 1981,1987; Githens, 1989; Hubchak et al., 1990). HowAddress reprint requests to Dr. M. Bendayan, Departement danatever, most of the studies were focused on the non-en- omie, Universite de Montreal, C.P. 6128, Succ. A, Montreal (Quebec), docrine epithelial cells and their exocrine function; a Canada H3C 357. 0 1992 WILEY-LISS, INC.
248
I.-S. PARK AND M. BENDAYAN
TABLE 1. Source and optimal dilution of the primary antibodies' Antibody against Somatostatin Insulin Glucagon Pancreatic polypeptide Cholecystokinin-8 Serotonin Secretin
Dilution
LM 1:1,500 1:1,000 1:1,500
EM 1:lOO-200 1:lOO-200 1:200
Source INC Corp., Stillwater, MN ICN Immunobiologicals, IL INC Corp., Stillwater, MN
1:1,000 1:3,000 1:1,500 1:6,000
1:lOO-150 1:200-300
Dako Corp., Carpinteria, CA INC Corp., Stillwater, MN INC Corp., Stillwater, MN BioGenex Lab., San Ramon, CA
'All antisera were raised in rabbit except the insulin antiserum which was from guinea pig source.
electron microscopic level in order to account for their possible regulatory function on the neighbouring duct epithelial lining, exocrine parenchyma, and connective tissue. MATERIALS AND METHODS
then incubated with a protein A-gold complex formed by 15 nm gold particles and diluted to a n O.D. = 0.5, for 30 min a t room temperature. Control Test
In order to confirm the specificity of the immunolabellings, the antigen adsorption as well a s the omission of the primary antibody tests were carried out with the paraffin sections. For the antigen adsorption test, each antibody diluted a t its optimal concentration was preincubated with a n excess amount of its specific antigen (8 pg/ml) for 4 hours at 4°C. The sections were then Light Microscopic lrnrnunohistochernistry treated with these preincubated antibodies for 12 hours The tissue samples were fixed with Bouin's solution a t 4°C followed by the usual procedures. In addition, and embedded in paraffin, while others were fixed with cholecystokinin antiserum was preincubated with the 4% paraformaldehyde-1.5% picric acid in 0.1 M phos- gastrin (1-17) to assess any cross reactivity between phate buffer (pH 7.4) and embedded in Epon 812. Par- the anti-cholecystokinin and gastrin. Glucagon and affin sections (5 pm), and Epon semithin sections (1 pancreatic polypeptide antibodies were also reversely pm) were processed for immunohistochemistry with pretreated with pancreatic polypeptide and glucagon various primary antibodies. The Epon sections were for the same purpose. All the antigens used in the confirst treated with a saturated solution of potassium hy- trol tests were supplied by Sigma Chemical Co., (St. droxide in absolute alcohol for 30 min in order to re- Louis, MO). move the resin, while the paraffin sections were proElectron Microscopic Cytochernistry cessed after rehydration through decreasing ethanol The tissue was cut in small pieces and fixed with 1% solutions. The immunohistochemical method used for light microscopy was the avidin-biotin-peroxidase com- glutaraldehyde solution in 0.01 M phosphate buffer. plex technique (Hsu et al., 1981). The nature, source, After careful washing with the phosphate buffer and and optimal dilution of the primary antibodies are de- distilled water the tissue specimens were immersed in scribed in Table 1. Biotinylated anti-rabbit immuno- 1% silver nitrate-ammonia water for 1 hour a t 60°C globulins and the avidin-biotin-peroxidase complex according to the modification of Masson-Hamperl's were purchased from Vector Laboratories (Burlin- technique (Singh, 1964) for the argentaffin reaction. game, CA), while the biotinylated anti-guinea pig im- For the argyrophilic impregnation, after fixation the munoglobulins were obtained from Janssen Life Sci- tissue specimens were incubated in 0.03% silver nitrate solution in 0.2 M sodium acetate buffer for 4 ence (Olen, Belgium). hours at 60°C and developed with 5%hydroquinone-1% Electron Microscopic irnrnunocytochernistry sodium sulfite solution for 5 min a t 60°C (Grimelius, Small pieces of tissue specimens were fixed with 4% 1968). After silver impregnation all the specimens paraformaldehyde-1% glutaraldehyde in 0.1 M phos- were rinsed in distilled water and subsequently embedphate buffer for 2 hours at room temperature and em- ded in Epon. bedded in Lowicryl K4M (Bendayan, 1984) or Epon Conventional Electron Microscopy 812. Ultrathin sections were mounted on Parlodion and carbon coated nickel grids. Prior to immunolabelling, After fixation with the paraformaldehyde-glutaralthe sections were treated with a saturated solution of dehyde mixture, some tissue samples were post fixed sodium metaperiodate for 1hour a t room temperature. with 1% osmium tetroxide for 1 hour a t 4°C and emAfter brief washing with distilled water, the sections bedded in Epon. Several sets of serial sections were were laid on a drop of 1%ovalbumin in PBS for 30 min prepared for general ultrastructural examination and and incubated onto a drop of the primary antibody for determination of the open or close cell characterisbrought to optimal dilution (Table 1)for 12-14 hours a t tics. All of the ultrathin sections in the present study 4°C. The grids were rinsed with PBS and transferred were stained with uranyl acetate and lead citrate soonto the 1%ovalbumin solution for 30 min. They were lution and observed using the Phillips EM410. The
Pancreas and duodenum were sampled from seven normal male Sprague-Dawley rats (-25Og bw) under ether anaesthesia. The tissue specimens were processed for immunohisto- and cytochemistry according to the following procedures.
ENDOCRINE CELLS I N THE PANCREATIC-BILE DUCT
photographs of the same endocrine cell obtained from serial sections were integrated for their stereoscopic reconstruction. For morphometrical evaluations of the secretory granule sizes, their outer diameter delineated by a well-defined limiting membrane were measured. More than 100 secretory granules for each cell type were evaluated. RESULTS General Morphology -. of the Pancreatic Duct System
The pancreatic duct system of the rat consists of intra- and inter-lobular ducts leading to the common pancreatic-bile duct including its intramural opening portion into the duodenal wall. Of particular interest is the presence of several small interlobular ducts opening directly into the common pancreatic-bile duct in separate ways without forming an independent main pancreatic duct. On some occasions one relatively large duct does traverse the duodenal wall just beside the distal portion of the common pancreatic-bile duct and opens directly into the intestinal lumen thus constituting an accessory pancreatic duct. This was observed in four out of the seven rats studied. All parts of the pancreatic duct system were lined by a simple epithelium. In the common pancreatic-bile duct this epithelial lining invaginates into the surrounding connective tissue to make gland-like pouches. Distribution of the Endocrine Cells in the Pancreatic Duct
Endocrine cells displaying specific immunolabelling for insulin (Fig. l),glucagon (Figs. 1, 21, somatostatin (Figs. 3 , 4 , 5 ) , pancreatic polypeptide (Fig. 61, serotonin (Fig. 7) or cholecystokinin (Fig. 8) were found within the epithelial lining of the pancreatic-bile duct system. The endocrine cells exhibited differences both in frequency and topographical distribution according to the peptide or amine which they display and the portion of the duct system examined. They were scattered as single cells except in the terminal portion of the common pancreatic-bile duct where they showed remarkable accumulations. These endocrine cells were preferentially located in the gland like pouches. As presented in Table 2, the distribution pattern and frequency of the pancreatic duct endocrine cells differed with respect to the portions of the duct system. Somatostatin and pancreatic polypeptide cells showing similar distribution were the more numerous while insulin and glucagon cells, less numerous, followed the same locations. The latter were evenly scattered along the duct system including the accessory pancreatic duct where the glucagon cells outnumbered the other cell types (Fig. 2). The area of the duodenal opening of the duct was particularly rich in somatostatin cells (Fig. 3). A small number of the cholecystokinin and serotonin immunoreactive cells was found only in the distal portion of the duct system particularly in the common pancreatic bile duct (Figs. 7, 81, even though there was a large population of these cell types in the neighbouring duodenal mucosa. No secretin immunoreactive cells were found in any part of the pancreatic duct while they were numerous in the gut mucosa. Besides being located in duct epithelia or islets, a significant number of isolated insulin, glucagon, somatostatin, and pancreatic polypeptide cells were observed within the exocrine paren-
249
chyma of the pancreas. These cells were usually interposed between acinar cells (insets in Figs. 1, 6). Control Test
No immuno-positive cell was detected in sections incubated with any of the primary antibodies pretreated with their corresponding antigen. Cholecystokinin immunoreactive cells were identified in the section incubated with its primary antibody pretreated with excess amounts of gastrin 1-17. Glucagon and pancreatic polypeptide antibodies showed constant immunoreactive cells in spite the preincubation of the antibodies with the opposite antigen, pancreatic polypeptide, and glucagon, respectively. Electron Microscopical Structure of Duct Endocrine Cells
Endocrine cells of the pancreatic duct were distinguished from the surrounding epithelial cells by their pale cytoplasm and by the presence of secretory granules located mainly in the baso-lateral region of the cell. Besides these, the other organelles of the endocrine cell shared common features; the Golgi apparatus was located in a peri-nuclear portion, the rough endoplasmic reticulum, and the elongated mitochondria were well developed and intermingled with the secretory granules. Based on their general appearance, pancreatic duct endocrine cells can be sorted into two categories. One displayed a pyramidal or oval appearance and was found to be the “open type” having direct contact with the lumen of the duct. This was confirmed by examination of serial sections, which demonstrated that this kind of cell reaches the duct lumen with a slender apical cell process. The luminal surface of the cell is narrow and the apical plasma membrane is covered with a tuft of microvilli (Figs. 1, 5). The apical end of lateral plasma membranes was characterized by junctional complexes. These cells demonstrate distinct polarity, most of the secretory granules being located in the lateral and basal cytoplasm underneath the nucleus (Fig. 5). The other kind of cells, the “closed type,” manifested variable appearance with the cytoplasmic processes filled with secretory granules. These cells usually laid on the basal lamina and seemed to have no morphological connection with the duct lumen. Cell processes ran between the Saso-lateral border of surrounding epithelial cells and sometimes deeply invaginate into the cytoplasm near the nuclear region of the neighbouring cells (Fig. 6). The details and specific features of the secretory granules of the pancreatic duct endocrine cells together with their immunolabelling have allowed for the identification of each individual cell type. Consequently, the routine classification based on differences in the shape, size, electron density, and matrix texture of the secretory granules concurred with the cytochemical and immunocytochemical identification of cell types. We were also able to analyze the ultrastructural features of each endocrine cell type putting emphasis on the characteristics of their secretory granules using routine morphological examination as well as cytochemical and immunocytochemical approaches. Insulin(B) cell, a typical open type, contains large (outer mean diameter 400221 nm; range 310-570 nm) and round secretory granules in a somewhat darker
250
1 . 3 . PARK AND M. BENDAYAN
Fig. 1. Electron micrograph of part of the common pancreatic-bile duct from the non-osmicated Epon-embedded tissue section incubated with insulin antibody and protein A-gold. The insulin cell (B) is well identified by the specific labelling presented on the secretory granules. The apical cell surface is in contact with the duct lumen (DL) and displays and microvilli (arrowheads). Several secretory granules are present in the apical region of the cell. Another endocrine cell (A), assumed to be glucagon cell, shows its cytoplasmic process surrounded by the epithelial cells. x 7,000. Inset: Paraffin section of the pancreas
incubated with insulin antibody. An insulin cell is seen in the duct epithelial lining (arrow), while two isolated insulin cells (arrow heads) are located in the exocrine parenchyma. x 700. Fig. 2. Light micrograph of the accessory pancreatic duct from nonosmicated Epon semithin section incubated with glucagon antibody and the avidin-biotin-peroxidase technique. Several immunoreactive cells are formed in the epithelial lining. No luminal contact of the glucagon cell can be seen. DL, duct lumen x 2,000.
ENDOCRINE CELLS I N T H E PANCREATIC-BILE DUCT
Figs. 3 and 4. Paraffin sections of the terminal part of the common pancreatic-bile duct (Fig. 3) and its opening into the duodenal lumen (Fig. 4) incubated with somatostatin antibody. Figure 3 shows the duct interposed between duodenal mucosa (DM) and the muscle layers (ML). Numerous somatostatin cells are located in the base of glandlike pouches of the duct while no positive cell is observed in the duodenal mucosa. x 380. Figure 4 shows the luminal content of the duct draining through the connection between the duct and the duodenum. x 330. Inset: higher magnification of the boxed area of the duct demonstrating somatostatin positive cells (arrowheads). x 1,600.
251
Fig. 5. Non-osmicated Epon-embedded tissue. Ultrathin section of the common pancreatic-bile duct incubated with somatostatin antibody. A pyramidal shape of somatostatin cell shows distinct polarity, basally located secretory granules and a long slender cytoplasmic process reaching the duct lumen and displays a tuft of luminal microvilli (arrowheads). x 4,600.
252
1.3. PARK AND M. BENDAYAN
Fig. 6. Four consecutive electron micrographs of the same area of the common pancreatic-bile duct from routine electron microscopy. An endocrine cell (PP), assumed to be a “closed type” pancreatic polypeptide cell, displays long cytoplasmic process. The cell process invaginates into the cytoplasm and even comes in close contact with the
nucleus of adjacent epithelial cell (Ne). x 6,200. Inset in b: Pancreatic polypeptide immunoreactive cells in the paraffin section. One positive cell (arrow) is shown in the intralobular duct whereas two isolated cells (arrowheads) are intermingled with acinar parenchyma. x 900.
ENDOCRINE CELLS IN THE PANCREATIC-BILE DUCT
Figs. 7 and 8. Paraffin sections incubated with serotonin (Fig. 7) and cholecystokinin antibodies (Fig. 81, respectively. The positive cells are located in the gland-like pouches (GP) of the common pancreatic-bile duct. x 1,900, Fig. 9. Nan-osmicated Lowicryl embedded tissue. Ultrathin section
incubated with cholecystokinin antibody. A cholecystokinin cell is characterized by its medium sized round granules and bundles of mi-
253
crofilaments. x 5,300. b: higher magnification of the boxed area of a. Secretory granules are labelled with gold particles. x 24,000. Fig. 10. Electron micrographs of the argentafin cells in the common pancreatic-bile duct (a)and duodenal mucosa (b).Although the secretory granules of both cells are heavily impregnated with silver grains, the shape of the duct cell granules is somewhat different from that of the duodenal one. x 20,000.
1 . 3 PARK AND M. BENDAYAN
254
TABLE 2. Topographical distribution and relative frequency of the endocrine cells in the rat pancreatic duct system' Common
Endocrine cell Insulin Glucagon Somatostatin Pancreatic polypeptide Cholecystokinin Serotonin Secretin
Intra- Inter- pancreaticbile Accessory lobular lobular duct duct duct duct
+ + +-
+
+
+ ++
+ + ++++
+I++
+ +I+ + +
-
+I+
+I+
+
+ + +
+I+
~
?
-
-
-
none; ?, very few; + , few; + + , moderate; + + + , numerous; + + + + , very numerous.
l-,
cytoplasm than other endocrine cells. The secretory granules consisted of a homogenous, relatively dense inner core surrounded by a wide electron lucent halo with a n outer limiting membrane. The immunolabelling was restricted to the inner core of the granule. Occasionally some granules were observed in the apical cytoplasm close to the luminal surface (Fig. 1). Secretory granules of the glucagon (A) cell revealed round profiles (23026.2 nm; 190-250 nm) and were compactly filled with a homogenous and high electron dense matrix. The immunolabelling with the anti-glucagon antibody was present over the entire granule content. Although some cells showed a n oval shape, their contact with the duct lumen was never clearly defined (Figs. 1, 2). Somatostatin (D) cell displayed medium sized (21023.8 nm, 170-260 nm) round secretory granules loosely packed with variable electron-dense matrices. A narrow and clear halo intersected between the granule and its outer limiting membrane. The identity of the cell was confirmed by immunolabelling, with the gold particles being concentrated over the core of the granules. Somatostatin cells usually demonstrated a pyramidal or oval appearance and their luminal connection was frequently observed (Fig. 5). Pancreatic polypeptide immunoreactivity was identified in two cell types showing quite different granule morphology. One had rather small (180+2 nm, 150200 nm) round and light electron dense granules and the other showed relatively larger (200k5.7 nm, 190240 nm), round oval or rod-shaped granules with a homogenous and high electron-dense matrix. Both types of granules displayed immunoreactive pancreatic polypeptide as demonstrated by their specific labelling. Owing to the absence of luminal contact, both cell types were classified as closed (Fig. 6). Cholecystokinin cells had medium sized (220k6.3 nm, 180-240 nm), homogenous, and moderate-dense granules. These cells were further characterized by the presence of large bundles of microfilaments surrounding the nuclei. Their identity was confirmed by gold immunolabelling of the granule cores (Fig. 9). A few serotonin containing cells, identified by their argentaffin reaction, were recorded in the duct system. The secretory granules of these cells, displayed a relatively large (280k 11.4, 210-320 nm) and pleomorphic
configuration (Fig. 10). Some granules contained a small and electron-lucent core surrounded by dense granular matrix while others were rather homogenous. Except for the somatostatin cell, all the other cells exhibited strong silver staining of their granules with the argyrophilic silver impregnation method of Grimelius. In the case of somatostatin cell, in spite of the fact that some granules did reveal distinct argyrophilia, a large number of them were negative. DISCUSSION The pancreatic-bile duct system plays a n important role in producing the extra acinar components of the pancreatic secretion-bicarbonate, electrolytes, and mucous substance, as well as in modulating the flow of the luminal content (Case and Argent, 1986; Ryan, 1987; Shultz, 1987). These main functions of the duct system are controlled by neuronal and hormonal regulations (Solomon, 1987; Alder and Beglinger, 1990). The endocrine cells of the gastro-entero-pancreatic system secrete their regulatory peptide hormones following physiological stimuli. The primary target of these hormones is the neighbouring epithelial cells as well as the smooth muscle fibers of the vessels and muscle layers on which they exert a paracrine action. They may also control remote targets through the blood and possibly via the pancreatic secretion mediated pathway (Feyrter, 1953; Larsson, 1984; Bendayan, 1987; Solcia et al., 1987). From this point of view and in spite of the fact that they may represent phylogenetic and embryological remnants, the endocrine cells of the pancreatic-bile duct system should be considered as a n integral functional element that modulates ductal activities. In the present study we report the presence of six different types of endocrine cells displaying specific immunolabellings for insulin, glucagon, somatostatin, pancreatic polypeptide, cholecystokinin, and serotonin in the pancreatic duct of the rat. By combining routine electron microscopic examination with cytochemical and immunocytochemical electron microscopy, we have classified six types of cells according to their specific immunolabelling, cytochemical reaction, shape, and size of the secretory granules. Although there are some differences in interpreting the cell types, the A, B, D1-like, and EC cells previously identified by routine electron microscopy in the pancreatic duct of several mammals (Kodama, 1983) were also identified in the present study. We could not find ultrastructural differences in secretory granules between the duct endocrine cells and the same type of the cells present in the gut or in the pancreatic islet. The secretory granules of the duct insulin, glucagon, somatostatin, and pancreatic polypeptide cells shared common structural characteristics with the previously described pancreatic islet cell granules (Forssmann et al., 1969; Larsson et al., 1976; Erlandersen, 1980; Fiocca e t al., 1983; Orci, 1985; Solcia et al., 1987). The cholecystokinin and serotonin containing granules of the duct endocrine cells were similar to the intestinal I cell and EC cell granules (Grube and Forssmann, 1979; Solcia et al., 1975, 1987; Dobbins and Austin, 1991) respectively. The histochemical argyrophil silver impregnation method of Grimelius has been widely used to identify
ENDOCRINE CELLS IN THE PANCREATIC-BILE DUCT
the gut and pancreatic endocrine cells because of its broad spectrum of reaction (Grimelius, 1968; Solcia et al., 1987). In the case of the somatostatin cell, however, its argyrophilic reaction remains controversial. Although the somatostatin cell was described as the only cell type displaying negative reaction to the method of Grimelius (Solcia et al. 1975), we have shown argyrophilic impregnation and somatostatin immunolabelling in the same endocrine cell (Park, 1986). Consequently the argyrophilic silver impregnation was confirmed in all types of duct endocrine cells including the somatostatin cell which contains both positive and negative secretory granules. The function and mechanism of action of the endocrine cells are closely related to their morphological diversity. In the gut mucosa, it is generally accepted that most of the endocrine cells reach the lumen, their luminal membrane displays a receptor for the stimuli from the lumen (Fujita and Kobayashi, 1973; Grube and Forssmann, 1979; Solcia et al., 19871, whereas other endocrine cells characterized by the long basal cytoplasmic processes have a paracrine action on surrounding target cells (Alumets et al., 1979; Larsson et al., 1979; Larsson, 1984; Yamada, 1987). The present study has shown two categories of duct endocrine cells, the pyramidal-shaped “open cell type” and the pleomorphic “closed cell type.” In contrast to the gut endocrine cells, however, the manifestation of the “open” or “closed” feature of the duct endocrine cells is mainly relevant to their hormonal content regardless of the region in which they occurred. Almost all of the somatostatin and insulin cells were found to be of the “open type,” like the pyrolic and intestinal endocrine cells, while the glucagon and pancreatic polypeptide cells belong to the “closed type” like those of the fundic mucosa of the stomach (Grube and Forssmann, 1979; Larsson 1984;Solcia et al., 1987).This observation concurs with our previous findings on “open insulin cells” in the duct (Bendayan, 1987) but contradicts the report that all duct endocrine cells are of the “closed type” (Kodama, 1983).The ultrastructural findings of the present study support the concept that the duct endocrine cells have not only the possibility to display receptors for the stimuli originating from the duct lumen, but also the capability to secrete and to exert a paracrine influence on surrounding tissues. Although no clear evidence has been provided for duct secretion of peptides into the pancreatic secretion through the luminal cell membrane other than the presence of secretory granules close to the luminal membrane, we should not rule out the possibility that duct endocrine hormones are secreted into the duct lumen and affect the epithelial lining. The repeated reports on the presence of the pancreatic islet hormones in the duct lumen (Carr-Locke and Track, 1979; Colon, 1979; Lawrence et al., 1979; Prinz et al., 1980; Ertan et al., 1981; Sarfati et al., 1986) support that concept. Moreover, the recent identifications of hormone receptors in the gut epithelium (Pillon et al., 1985; Fernandez-Moreno et al., 1986, 1987, 1988) bring additional evidence along this line. The pancreatic duct endocrine cells display different patterns of distribution along the duct system. While the cholecystokinin and serotonin cells were confined to the terminal portion of the common pancreatic-bile duct in small numbers, the other endocrine cells,
255
namely, the pancreatic hormones secreting cells, were distributed all along the tract of the pancreatic-bile duct system, being concentrated in the terminal portion. The distribution of each cell type varied according to regional and functional differences of the duct. The intra- and inter-lobular ducts participate in secretion of the bicarbonate and electrolytes, while the main common duct is the secreting site of mucous substances and the final gate for delivery of the luminal flow into the duodenum (Kodama, 1983; Case and Argent 1986; Ryan, 1987; Dahlstrand et al., 1990). The main common duct is also characterized by numerous smooth muscle layers including the sphincter muscle in its terminal portion. This may imply that the duct endocrine cells as well as isolated endocrine cells in the pancreas form a separate endocrine unit with its proper biological function. Several peptide hormones applied exogenously have been shown to infuence the pancreatic-bile duct system; somatostatin, pancreatic polypeptide, and glucagon have an inhibitory effect, while cholecystokinin stimulates the secretory function of the duct (Case and Argent, 1986; Walsh, 1987). It is thus conceivable that the various hormones secreted by the duct endocrine cells exert a similar excitory or inhibitory effect on this same target tissue through endocrine or paracrine mode of action. ACKNOWLEDGMENTS
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