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Vol. 40, No. 11, pp. 1627-1631, 1992 Printed in U X A .
The Journal of Histochemisuy and CytochemipUy Copyright 0 1992 by The Histochemical Society, Inc
Original Article
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Immunohistochemical and Immunoelectron Microscopic Analyses of a-Amylase Isozymes in Human Intrahepatic Biliary Epithelium and Hepatocytes TADASHI TERADA,' NAOKO KONO, and YASUNI NAKANUMA Second Department of Pathology, Kanazawa University School of Medicine, Kbnazawa, Japan. Received for publication February 10, 1992 and in revised form June 1, 1992; accepted June 12, 1992 (2A2587).
The expression and localization of the pancreatic and salivary isozymes of a-amylase in the intrahepatic biliary epithelium and hepatocytes were examined by the immunohistochemical method with polydonal and monoclonal antibodies in 45 normal autopsied human livers. Immunoelectron miaoscopicstudies with the protein A-gold method were performed with the monoclonal antibodies (MAb)on seven of the livers. The intrahepatic biliary system was divided into large ducts, septal ducts, interlobular ducts, bile ductules, and peribiliary glands. Immunohktochemidy, pancreatic isozyme was o b s e d in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, and peribiliary glands in almost all livers. Interlobular ducts expressed pancreatic isozyme in only four (3%)livers. Bile ductules and hepatocyteswere negative for pancreatic isozyme in all cases. Expression of salivary isozyme was observed in the supranudear cytoplasm of the epithelium of large ducts, septal ducts, interlobular ducts, bile ductules, and peribiliary
Introduction Human a-amylase (1,4-a-glucanohydrolase; EC 3.2.1.1) is mainly produced in the salivary glands and pancreas, and isozymes of a-amylase in these organs are called salivary and pancreatic a-amylase, respectively (46). Salivary a-amylase has also been noted in many other organs and tissues of humans (44), including the lungs (21), tonsils (3), thyroid (22), endometrium (24), prostate (29), and Fallopian tubes (1). Salivary a-amylase has also been observed in malignant tumors of several organs, such as ovarian cancers (41). However, to the best of our knowledge it is unknown whether a-amylase isozymes are present in the human liver, including the intrahepatic biliary epithelium. Our recent immunohistochemicalstudy with a polyclonal antibody demonstrated that pancreatic a-amylase is present in the intrahepatic large bile ducts and peribiliary glands in normal human adult livers (33). However, a-amylase isozymes, particularly
Correspondence to: Tadashi Terada, MD, Second Dept. of Pathology, Kanazawa Univ. School of Medicine, Kanazawa 920, Japan.
glands in almost all livers, although the expression in interlobular ducts and bile ductules was weak. Hepatocyteswere weakly positive for salivary isozyme. Immunoelectron microscopy revealed that both pancreatic and salivary isozymes were located in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, and peribiliary glands, and that hepatocytes had no pancreatic isozyme but contained salivary isozyme. These data suggest that pancreatic and salivary isozymes of a-amylaseare produced by the intrahepatic biliary epithelium and secreted into intrahepatic biliary lumens, and that they may play an important role in the physiology of the intrahepatic biliary tree and hepatic bile. It is also suggested that hepatocytes produce a small amount of salivary a-amylase that may be secreted into the biliary tree. ( J Hisrochem Cytochem 40:1627-1635, 1992) KEY WORDS: a-Amylase isozymes; Intrahepatic biliary tree; Immunohistochemistry; Immunoelectron microscopy; Protein A-gold; Hepatocytes.
the salivary isozyme of a-amylase, in the human liver have not been fully investigated. In addition, ultrastructural localization of the a-amylase isozymes in the epithelium of the intrahepatic biliary tree and hepatocytes is unclear. In this work we extended our previous study (33) to determine whether pancreatic and salivary isozymes of a-amylase are present in the intrahepatic biliary epithelium and hepatocytes. We investigated immunohistochemically the expression of pancreatic and salivary isozymes of a-amylase not only in the intrahepatic biliary epithelium but also in hepatocytes in normal human adult livers. We used both polyclonal and monoclonal antibodies against pancreatic or salivary a-amylase to strengthen the specificity of the a-amylase isozyme immunoreactivity. In addition, we investigated the ultrastructural localization of pancreatic and salivary isozymes of a-amylase in the bile duct epithelium and hepatocytes by immunoelectron microscopy with the protein A-gold method. In this study, the intrahepatic biliary system of humans was classified into intrahepatic bile ducts and peribiliary glands (39). Intrahepatic bile ducts were subclassified into large ducts, septal ducts, interlobular ducts, and bile ductules according to Masuko et al. (18) and Nakanuma and Ohta (20). Large ducts are grossly recog1627
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Figure 1. Immunoreactivity of the normal pancreas and salivary glands to (A.E) pancreatic and (C,D) salivary a-amylases. (A) The pancreatic acinar cells are positive for pancreatic a-amylase, whereas (E) acinar cells of the salivary glands are negative for pancreatic a-amylase. Conversely, (C) pancreatic acinar cells are negative for salivary a-amylase,whereas (D) acinar cells of the salivary glands are positivefor salivary a-amylase. (A$) lmmunostain(ABC method)for pannal antibody. (C,D) lmmunostain (ABC method) for sativary a-amylase using a monoclonal antibody. Or creatic a-amylase with a mon 300. Bars = 50 pm.
nizable. Septal ducts are identifiablemicroscopically and are composed of columnar epithelial cells and thick duct walls. Interlobular ducts are smallducts with cuboidal epithelium in the small portal tracts. Bile ductules are very small ducts composed of low cuboidal epithelial cells near the periportal hepatocytes. Intrahepatic periucous glands and are present around the intrahepatic large bile ducts (1039). The intrahepatic peribiliary glands have recently been demonstrated in our laboratory (10,39), and their normal histology and pathological features were previously reported (10,32-39).
Materials and Methods Tissue Prejmration and Processing. A total of 45 "normal" autopsied human adult livers (age range 29-68 years; 24 men and 21 women) were used. We used autopsy livers because the various ducts can be examined only in autopsied livers. At autopsy, the liver was cut longitudinally into several slices approximately 1 un thick. The slices were ftced in a4% formaldehyde solution. Then,according to the ptotocol of our laboratory(32-35,38,39), five tissue specimens were obtained from the followingsites: (a) both hepatic ducts; (b) left segment ducts (first major branches of the left hepatic duct); (c) right segment ducts (first major
branches of the right hepatic duct); (d) left area ducts (first major branches ofleft segment ducts); and (e) right area ducts (fust major branches ofright segment ducts). The nomenclature for intrahepatic large bile ducts was adapted from Healey and Schroy (8). Specimens of normal areas of surgid human salivary glands (five cases) and normal human pancases) that were surgically resected for gastric carcinoma were obtained and fixed in a 4% formaldehyde solution; these were used as controls. The specimens of1 ded in paraffin. Ten 5-pm embedded blodr; one of th Immunohistochemistry. Four serial sections from each paraffin block were stained for pancreatic and salivary isozymes of a-amylase by the avidin-biotin-peroxidase complex (ABC) method according to Hsu et al. (9),with a polyclonal antibody (sheep) to human pancreatic a-amylase, dilution L200 (Binding Site Ltd.; Birmingham, UK), a monoclonal antibody (mouse IgGl class) to human pancreatic a-amylase, dilution 1:200 (Chemicon; Temecula, CA), a polydonal antibody (sheep) to human salivary a-amylase, dilution 1:200 (BindingSite Ltd.), and a monoclonal antibody (mouse IgG1 class) to human salivary a-amylase, dilution L5 (Boehringer Mannheim Yamanouchi Co.; Tokyo, Japan). The monoclonal antibody (MAb) to pancreatic a-amylaseis specific for the pancreatic isozyme and does not crossreact with the salivary isozyme (25). The MAb to
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Figure 2. Pancreatic a-amylase immunoreactivity in the epithelium of (A) intrahepatic large bile duct, (6)septal bile ducts, (C) interlobular bile ducts and bile ductules, (D) peribiliary glands, and (C) hepatocytes. Pancreatic a-amylase immunoreactivityis strongly present in a granular pattern in the supranuclear cytoplasm of the epithelial cells of (A) intrahepatic large bile ducts, (6)septal bile ducts, and (D) peribiliary glands, while it is absent (C) in interlobular bile ducts, bile ductules, and hepatocytes. lmmunostain (ABC) method for pancreatic a-amylase using the monoclonal antibody. Original magnifications: A x 350; B x 200;C x 80; D x 120. Bars = 50 um.
salivary a-amylase is specific for salivary isozyme and crossreacts negligibly with the pancreatic isozyme (6). Endogenousperoxidase activity was eliminated by immersing the sectionsin absolute methanol containing 1% hydrogen peroxide for 20 min. The sections were treated with normal sera for 20 min, and then primary antibodies were applied to the sections at 4°C overnight. The sections were incubated with secondary biotinylated antibodies (anti-sheep IgG and anti-mouse IgG) (Vector Laboratories; Burlingame, CA) for 40 min. The sections were then treated with an avidin-biotin-peroxidase complex (Vectastain ABC Kit; Vector Laboratories) for 40 min. Reaction products were developed by immersing the sections in 3.3'diaminobenzidine tetrahydrochloridesolution (Sigma; St Louis, MO) containing hydrogen peroxide. Nuclei were lightly counterstained with hematoxylin. As negative controls, sections ofthe livers, pancreases, and salivary glands were treated with non-immuneserum (normal sheep or mouse serum)(Dako; Santa Barbara, CA) or PBS, followed by the procedure described above. Absorption tests were performed in selectedspecimens. T h e anti-pancreatic a-amylase and anti-salivary a-amylase antibody solutions were mixed with human pancreatic a-amylase (Athens Research and Technology; Athens, GA) and salivary a-amylase(Sigma), respectively. The mixed solutionswere incubated overnight at 4'C and centrifuged at 4000 rpm for 10 min. The supernatantswere collected and used as the primary antibodies in immunostainings. For cross-absorptionexperiments, performed on selected speci-
mens, the salivary isozyme antigen was mixed with antibodies against pancreatic a-amylaseor the pancreatic isozyme antigen was added to antibodies against the salivary enzyme. The mixed solutions were followed by the immunohistochemical procedure described above. Immunoelectron Microscopy. Fresh liver specimens of the hepatic hilus were obtained from five normal autopsied human adult livers as well as from two surgically resected human adult livers. Fresh specimens of human pancreases and salivary glands were also obtained from three autopsies as well as from five surgeries (salivary glands, three cases; pancreas, two cases). These specimenswere examined by immunoelectron microscopy for pancreatic and salivaryisozymes of a-amylaseby the post-embedding method (26). In brief, the specimens were dissected into cubes approximately 0.5 mm in size and fixed in 1%glutaraldehyde solution for 24 hr at room temperature. After an overnight buffer wash at 4'C. the specimens were dehydrated, and embedded in Lowicryl K4M according to the method reported by Carlemalm et al. (2). Roth et al. (27), and Lemanski et al. (16). The specimens were polymerized for 24-48 hr in a w radiation chamber at - 35°C. Semi-thin sections (1.0 pm) were stained with toluidine blue for selection of appropriate areas. Next, ultra-thin sections (80-100 nm) were mounted on carbon-coated nickel grids and were immersed for 20 min in 0.01 M PBS containing 1% bovine serum albumin. The sections were incubated for 1 hr in a drop of primary antibodies [MAb to pancreatic
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Figure 3. Salivary a-amylase immunoreactivityof the epithelium of (A) intrahepatic large bile ducts, (B) septal bile ducts, (C) interlobular bile ducts, bile ductules, and hepatocytes, and (D) peribiliary glands. Salivary a-amylase immunoreactivity is strongly present in a granular pattern in the supranuclear cytoplasm of the epithelial cells of (A) intrahepatic large bile ducts, (E) septal bile ducts, and (D) peribiliary glands. Salivary a-amylase immunoreactivity is weakly expressed in interlobular bile ducts and bile ductules (C). Hepatocytes are weakly positive for salivary a-amylase (C). lmmunostain(ABC method) for salivary a-amylase using the monoclonal antibody. Original magnifications: A x 350; B x 200;C x 80; D x 120. Bars = 50 bm.
a-amylase,dilution 1:lOO (Chemicon) or MAb to salivary a-amylase,dilution 1:20(BoehringerMannheim Yamanouchi)] rinsed in PBS, incubated in a drop of secondary antibody (goat anti-mouseIgG, dilution 1200) (Cappel; West Chester, PA) for 30 min, and then reacted with 15-nm protein A-gold particles for 30 min at room temperature. The sections were rinsed in PBS, fixed by 1% osmium tetroxide for 30 min, and stained with uranyl acetate and lead citrate. The sections were examined with a Hitachi H-300 electron microscope. As a negative control study, the primary antibodies were replaced by non-immune sera or PBS, followed by the immunoelectron microscopic procedure.
Results Specificity of Antibodies Pancreatic a-amylase (both polyclonal and monoclonal antibodies) was expressed in acinar cells of the normal pancreas but was not present in acinar cells of normal salivary glands (Figures 1A and 1B). Conversely, salivary a-amylase(both polydonal and monoclonal antibodies) was expressed in acinar cells of normal salivary glands but was not present in acinar cells of the normal pancreas (Figures 1C and ID). Positive staining was absent when non-immune
sera, PBS, or absorbed primary antibodies were used as the first layer. Cross-absorption tests resulted in positive staining.
Pancreatic a -Amyh e Immanohistochemistry Expression of pancreatic a-amylase with the polyclonal antibody was almost the same as that with the monoclonal antibody. Pancreatic isozyme immunoreactivitywas recognized in the epithelium of intrahepatic large ducts in 43 (96%) livers (Figure 2A) and in the epithelium of the septal ducts in all livers (Figure 2B). By contrast, it was discerned in the epithelium of interlobular ducts in only four (9%) livers and was not present in the bile ductule epithelium in any of the specimens (Figure 2C). Peribiliary glands were positive for pancreatic isozyme in all livers (Figure 2D). Pancreatic isozyme immunoreactivity was located in a granular pattem in the supranuclear cytoplasm of the biliary epithelium (Figures 2A-2C). Occasionally, pancreatic isozyme immunoreactivity was recognized in the lumens of bile ducts and peribiliary glands. The intensity of pancreatic isozyme immunoreactivity varied from case to case, as well as from area to area in the same case. Hepatocytes were negative for pancreatic isozyme in all cases (Figure 2C). Pan-
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Figure4. (A) Immunoelectron microscopicobsetvations (protein A-gold method) with the anti-pancreatic a-amylase monoclonal antibody reveal pancreatic a-amylase gold particles in the supranuclear cytoplasm of the septal bile duct. L,lumen of the septal bile duct; N, nucleus. (E) Negative control for immunoelectron microscopy (protein A-gold method) with non-immune sera instead of the anti-pancreatic a-amylase antibody reveals few gold particles in the epithelium of septal bile ducts. L, lumen of bile duct; N, nucleus. Original magnifications: A x 25,000; B x 18,000. Ears = 0.5 vm.
creatic isozyme immunoreactivity was negative in controls treated with non-immune sera, PBS. or absorbed primary antibodies. Crossabsorption tests showed positive immunostaining; absorption of primary antibodies with salivary isozyme resulted in positive immunostaining.
Salivary a -Amylase Immunohistochemistry Immunostaining of salivary a-amylase by polyclonal and monoclonal antibodies gave almost the same results. Salivary isozyme immunoreactivity was recognized in the epithelium of intrahepatic large ducts in 42 (93%) liven (Figure 3A) and in the epithelium of septal ducts in 43 (96%) liven (Figures 3B). It was also discerned in the epithelium of interlobular ducts and bile ductules in all livers (Figure 3C), although the salivary isozyme expression was weak in interlobular ducts and bile ductules (Figure 3C) relative to large ducts, septal ducts, and peribiliary glands. Peribiliary glands were positive for salivary isozyme in 43 (96%) liven (Figure 3D). Sali-
vary isozyme immunoreactivity was located in a granular pattern in the supranuclear cytoplasm of the biliary epithelium (Figures 3A-3D). Not infrequently, salivary isozyme immunoreactivity was recognized in the lumen of bile ducts and peribiliary glands. The intensity of salivary isozyme expression varied from case to case, as well as from area to area in the same case. Hepatocyteswere weakly and diffusely positive for salivary isozyme (Figure 3C) in all livers. Salivary isozyme immunoreactivity was negative in controls treated with non-immune sera, PBS, or absorbed primary antibodies. Crossabsorption tests showed positive immunostaining; absorption of primary antibodies with pancreatic isozyme resulted in positive immunostaining.
Immunoelectron Microscopic Findings Immunolabeling with monoclonal antibodies produced consistent labeling with negligible background, and individual gold particles were clearly identified. In immunostaining of pancreatic iso-
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Figure 5. lmmunoelectron microscopic observations(protein A-gold method) with the anti-salivary a-amylase monoclonalantibody reveal salivary a-amylase gold particles in the supranuclear cytoplasm of the intrahepatic peribiliaryglands. L, lumen of peribiliaryglands: N, nucleus. Original magnification x 30,000. Bar = 0.5 pm.
zyme, the epithelium of intrahepatic large ducts, septal ducts, and peribiliary glands had gold particles in the supranuclear cytoplasm (Figure 4A). Negative control specimens treated with non-immune sera or PBS showed few gold particles (Figure 4B). Hepatocytes had few pancreatic isozyme gold particles. In immunostaining of salivary isozyme, the epithelium of intrahepatic large ducts, septal ducts, and peribiliary glands had gold particles in the supranuclear cytoplasm (Figure 5 ) . Interlobular ducts, bile ductules, and hepatocytes had salivary isozyme gold particles (Figure 6A). Negative control specimens treated with non-immune sera or PBS showed few gold particles (Figure 6B). In normal pancreases, pancreatic isozyme gold particles were abundant in the supranuclear cytoplasm of acinar cells (Figure 7A), whereas salivary isozyme gold particles were not present. In normal salivary glands, pancreatic isozyme gold particles were not present, whereas salivary isozyme gold particles were abundant in the supranuclear cytoplasm of acinar cells (Figure 7B).
Discussion The results of the present study demonstrated that the pancreatic and salivary a-amylase immunoreactivities are specific for pancreatic and salivary isozymes, respectively. The present study demonstrated pancreatic a-amylase in the
epithelium of large ducts, septal ducts, and peribiliary glands. It was rarely expressed in the epithelium of interlobular ducts and was never expressed in bile ductules. By contrast, salivary a-amylase was expressed in the biliary epithelium from large ducts to bile ductules, although the intensity of the expression was weak in interlobular ducts and bile ductules. Immunoelectron microscopic observations confirmed these findings. These findings suggest that the epithelium of intrahepatic large ducts, septal ducts, and peribiliary glands contains pancreatic and salivary isozymes of a-amylase and that salivary isozyme is present in small amounts in the epithelium of interlobular ducts and bile ductules. Immunohistochemical and immunoelectron microscopic observations revealed that pancreatic and salivary isozyme immunoreactivity was located in a granular pattern in the supranuclear cytoplasm of the biliary epithelium, suggesting that both a-amylase isozymes are produced by the epithelium of intrahepatic large ducts, septal ducts, and peribiliary glands. Little is known about the functions of intrahepatic bile ducts and peribiliary glands. However, several investigators have speculated on their functions. For example, intrahepatic bile ducts have been shown to absorb and secrete water and electrolytes (31). Intrahepatic peribiliary glands may secrete seromucous fluid (39), IgA (30). secretory components (30). and several gut hormones (12). The present study demonstrated a-amylase isozymes not only in
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Figure 6. (A) lmmunoelectron microscopicobservations (protein A-gold method) with the anti-salivary a-amylase monoclonal antibody reveal salivary a-amylase gold particles in the cytoplasmof hepatocytes. BC, bile canaliculus. (E)Negative control for immunoelectronmicroscopy(proteinA-gold method) with non-immune sera instead of the anti-salivary a-amylase antibody reveals few gold particles in the hepatocytes. BC, bile canaliculus. Original magnification x 35,000.Bars = 0.5 vm.
the epithelium of the biliary tree but also within the biliary lumens, suggesting that pancreatic and salivary isozymes of a-amylase are secreted from the biliary epithelium into lumens of the intrahepatic bile ducts and peribiliary glands. A biochemical study that reported the presence of a-amylase activity in bile supports our suggestion (4). It has been reported that hepatic bile contains proteins, carbohydrates, and other organic substances (13J5). It seems likely that the secreted a-amylase isozymes play an important role in carbohydrate metabolism in hepatic bile. There was a difference in the distribution of the a-amylase isozymes in the intrahepatic biliary tree: pancreatic and salivary isozymes were observed in large ducts, septal ducts, and peribiliary glands, whereas they were absent (pancreatic isozyme) or only weakly expressed (salivary isozyme) in interlobular ducts and bile ductules. Although the reason for this difference in isoamylase distribution is not clear, these results indicate that intrahepatic large ducts, septal ducts, and peribiliary glands are phenotypically different from interlobular ducts and bile ductules. This distribution pattern of isozymes of a-amylase may reflect differences in the development of
the intrahepatic biliary tree. It seems possible that intrahepatic large ducts, septal ducts, and peribiliary glands, like the pancreas, develop from the embryonic foregut, while interlobular ducts and bile ductules arise from the periportal iepatocytes or the ductal plate, as previously reported (28,42,43). In fact, phenotypic characteristics of the exocrine pancreas have recently been reported in intrahepatic large bile ducts and peribiliary glands (38,44). Amylase activity in the human liver has been a matter of controversy (46). Some biochemical studies have demonstrated a low level of a-amylase activity in liver tissues (5,17). The present study did not reveal pancreatic a-amylase in the hepatocytes, suggesting that pancreatic a-amylase is not present in hepatocytes. By contrast, weak expression of salivary a-amylase immunoreactivity was found in hepatocytes, suggesting that a small amount of salivary a-amylase is present in hepatocytes. The salivary a-amylase in hepatocytes may be secreted into lumens of the intrahepatic biliary tree as well as into the circulation. However, the functional significance of the salivary isozyme in hepatocytes is unclear. It has been reported that the isozyme of a-amylase in organs
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Figure 7. lmmunoelectron microscopic findings (protein A-gold method) in positive control tissues. (A) The pancreatic acinar cell contains pancreatic a-amylase gold particles in the supranuclear cytoplasm, particularly in the zymogen granules. N. nucleus; L. lumen of the pancreatic acinus. (E) The acinar cell of the salivary gland contains salivary a-amylase gold particles in the supranuclear cytoplasm, particularly in the zymogen granules. N. nucleus; L, lumen of the acinus. Original magnifications: A x 24,000; B x 26.000. Bars = 0.5 pm.
other than the salivary glands and pancreas is of the salivary or nonpancreatic type (19.46). Of interest in this study is that pancreatic isozyme was expressed on the epithelium of intrahepatic large ducts, septal ducts, and peribiliary glands. The pancreas and intrahepatic bile ducts share a common developmental origin: both arise from the embryonic foregut. The potential of bile ducts to transdifferentiate to pancreatic acinar cells has been demonstrated in animal models fed certain chemicals (llJ4). Conversely, the exocrine pancreas can be transdifferentiated into hepatocytes and ductules in rats after copper depletion (23). The findings of the present study imply that the intrahepatic large ducts, septal ducts, and peribiliary glands share phenotypic characteristics with the exocrine pancreas. Recently, a-amylase isozymes have been analyzed at the molecular level. The genes of salivaryand pancreatic isozymes of a-amylase are named AMY1 and AMY2. respectively. More recently, a novel type of human amylase, which is encoded by the AMYZB, has been detected (40). This novel type of a-amylase has a high homology with pancreatic a-amylase in amino acid sequence (40,45). Recent studies have shown that the two different amylase-encoding genes,
AMY2A and AMYZB, were expressed in the normal human pancreas (7). It seems mandatory to analyze a-amylase isozymes in the intrahepatic biliary epithelium and hepatocytes by molecular analytical methods.
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35. Terada T, Nakanuma Y Pathological observations of intrahepatic peribiliary glands in 1,000 consecutive autopsy livers: 111. Survey of necroinflammation and cystic dilatation. Hepatology 12:1229, 1990 36. Terada T, Nakanuma Y Innervation of intrahepatic bile ducts and peribiliary glands in normal human livers, extrahepaticbiliary obstruction and hepatolithiasis: an immunohistochemical study.J Hepatol9:141, 1989 37. Terada T, Nakanuma Y Morphological examination of intrahepatic bile ducts in hepatolithiasis. Virchows Arch [A] 413:167, 1988 38. Terada T, Nakanuma Y, Kakita A: Pathologic observations of intrahepatic peribiliary glands in 1,000 consecutive autopsy livers: heterotopic pancreas in the liver. Gastroenterology 98:1333, 1990 39. Terada T, Nakanuma Y, Ohta G Glandular elements around the intrahepatic bile ducts in man: their morphology and distribution in normal livers. Liver 7:l. 1987 40. Tomita N, Horii A, Doi S, Yokouchi H, Shiosaki K, Higashiyama M, Matsuura N, Ogawa M, Mori T, Matsubara K: A novel type of human alpha-amylase produced in lung carcinoid tumor. Gene 76:11, 1989 41. Ueda G, Yamasaki M, Inoue M, Tanaka Y, Abe Y, Ogawa M: Immunohistochemical study of amylase in common epithelial tumors of the ovary. Int J Gynecol Pathol 4:240, 1985 42. Van Eyken P, Sciot R, Callea F, Van der Steen K, Moerman P, Desmet VJ: The development of the intrahepatic bile ducts in man: a keratin immunohistochemical study. Hepatology 83586, 1988 43. Van Eyken P, Sciot R, Desmet VJ: Intrahepatic bile duct development in the rat: a cytokeratin-immunohistochemical study. Lab Invest 59:52, 1988 44. Wolf HK, Burchette JL, Garcia MA, Michalopoulos G: Exocrine pancreatic tissue in human liver: a metaplastic process? Am J Surg Pathol 14:590, 1990 45. Yokouchi H, Horii A, Emi M. Tomita N, Doi S, Ogawa M, Mori T, Matsubara K: Cloning and characterization of a third type of human alpha-amylase gene. Gene 90:281, 1990 46. Zakowski JJ, Bruns DE: Biochemistry of human amylase isoenzymes. CRC Crit Rev Clin Lab Sci 21:283, 1984
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Vol. 40, No. 11, pp. 1627-1631, 1992 Printed in U X A .
The Journal of Histochemisuy and CytochemipUy Copyright 0 1992 by The Histochemical Society, Inc
Original Article
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Immunohistochemical and Immunoelectron Microscopic Analyses of a-Amylase Isozymes in Human Intrahepatic Biliary Epithelium and Hepatocytes TADASHI TERADA,' NAOKO KONO, and YASUNI NAKANUMA Second Department of Pathology, Kanazawa University School of Medicine, Kbnazawa, Japan. Received for publication February 10, 1992 and in revised form June 1, 1992; accepted June 12, 1992 (2A2587).
The expression and localization of the pancreatic and salivary isozymes of a-amylase in the intrahepatic biliary epithelium and hepatocytes were examined by the immunohistochemical method with polydonal and monoclonal antibodies in 45 normal autopsied human livers. Immunoelectron miaoscopicstudies with the protein A-gold method were performed with the monoclonal antibodies (MAb)on seven of the livers. The intrahepatic biliary system was divided into large ducts, septal ducts, interlobular ducts, bile ductules, and peribiliary glands. Immunohktochemidy, pancreatic isozyme was o b s e d in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, and peribiliary glands in almost all livers. Interlobular ducts expressed pancreatic isozyme in only four (3%)livers. Bile ductules and hepatocyteswere negative for pancreatic isozyme in all cases. Expression of salivary isozyme was observed in the supranudear cytoplasm of the epithelium of large ducts, septal ducts, interlobular ducts, bile ductules, and peribiliary
Introduction Human a-amylase (1,4-a-glucanohydrolase; EC 3.2.1.1) is mainly produced in the salivary glands and pancreas, and isozymes of a-amylase in these organs are called salivary and pancreatic a-amylase, respectively (46). Salivary a-amylase has also been noted in many other organs and tissues of humans (44), including the lungs (21), tonsils (3), thyroid (22), endometrium (24), prostate (29), and Fallopian tubes (1). Salivary a-amylase has also been observed in malignant tumors of several organs, such as ovarian cancers (41). However, to the best of our knowledge it is unknown whether a-amylase isozymes are present in the human liver, including the intrahepatic biliary epithelium. Our recent immunohistochemicalstudy with a polyclonal antibody demonstrated that pancreatic a-amylase is present in the intrahepatic large bile ducts and peribiliary glands in normal human adult livers (33). However, a-amylase isozymes, particularly
Correspondence to: Tadashi Terada, MD, Second Dept. of Pathology, Kanazawa Univ. School of Medicine, Kanazawa 920, Japan.
glands in almost all livers, although the expression in interlobular ducts and bile ductules was weak. Hepatocyteswere weakly positive for salivary isozyme. Immunoelectron microscopy revealed that both pancreatic and salivary isozymes were located in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, and peribiliary glands, and that hepatocytes had no pancreatic isozyme but contained salivary isozyme. These data suggest that pancreatic and salivary isozymes of a-amylaseare produced by the intrahepatic biliary epithelium and secreted into intrahepatic biliary lumens, and that they may play an important role in the physiology of the intrahepatic biliary tree and hepatic bile. It is also suggested that hepatocytes produce a small amount of salivary a-amylase that may be secreted into the biliary tree. ( J Hisrochem Cytochem 40:1627-1635, 1992) KEY WORDS: a-Amylase isozymes; Intrahepatic biliary tree; Immunohistochemistry; Immunoelectron microscopy; Protein A-gold; Hepatocytes.
the salivary isozyme of a-amylase, in the human liver have not been fully investigated. In addition, ultrastructural localization of the a-amylase isozymes in the epithelium of the intrahepatic biliary tree and hepatocytes is unclear. In this work we extended our previous study (33) to determine whether pancreatic and salivary isozymes of a-amylase are present in the intrahepatic biliary epithelium and hepatocytes. We investigated immunohistochemically the expression of pancreatic and salivary isozymes of a-amylase not only in the intrahepatic biliary epithelium but also in hepatocytes in normal human adult livers. We used both polyclonal and monoclonal antibodies against pancreatic or salivary a-amylase to strengthen the specificity of the a-amylase isozyme immunoreactivity. In addition, we investigated the ultrastructural localization of pancreatic and salivary isozymes of a-amylase in the bile duct epithelium and hepatocytes by immunoelectron microscopy with the protein A-gold method. In this study, the intrahepatic biliary system of humans was classified into intrahepatic bile ducts and peribiliary glands (39). Intrahepatic bile ducts were subclassified into large ducts, septal ducts, interlobular ducts, and bile ductules according to Masuko et al. (18) and Nakanuma and Ohta (20). Large ducts are grossly recog1627
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Figure 1. Immunoreactivity of the normal pancreas and salivary glands to (A.E) pancreatic and (C,D) salivary a-amylases. (A) The pancreatic acinar cells are positive for pancreatic a-amylase, whereas (E) acinar cells of the salivary glands are negative for pancreatic a-amylase. Conversely, (C) pancreatic acinar cells are negative for salivary a-amylase,whereas (D) acinar cells of the salivary glands are positivefor salivary a-amylase. (A$) lmmunostain(ABC method)for pannal antibody. (C,D) lmmunostain (ABC method) for sativary a-amylase using a monoclonal antibody. Or creatic a-amylase with a mon 300. Bars = 50 pm.
nizable. Septal ducts are identifiablemicroscopically and are composed of columnar epithelial cells and thick duct walls. Interlobular ducts are smallducts with cuboidal epithelium in the small portal tracts. Bile ductules are very small ducts composed of low cuboidal epithelial cells near the periportal hepatocytes. Intrahepatic periucous glands and are present around the intrahepatic large bile ducts (1039). The intrahepatic peribiliary glands have recently been demonstrated in our laboratory (10,39), and their normal histology and pathological features were previously reported (10,32-39).
Materials and Methods Tissue Prejmration and Processing. A total of 45 "normal" autopsied human adult livers (age range 29-68 years; 24 men and 21 women) were used. We used autopsy livers because the various ducts can be examined only in autopsied livers. At autopsy, the liver was cut longitudinally into several slices approximately 1 un thick. The slices were ftced in a4% formaldehyde solution. Then,according to the ptotocol of our laboratory(32-35,38,39), five tissue specimens were obtained from the followingsites: (a) both hepatic ducts; (b) left segment ducts (first major branches of the left hepatic duct); (c) right segment ducts (first major
branches of the right hepatic duct); (d) left area ducts (first major branches ofleft segment ducts); and (e) right area ducts (fust major branches ofright segment ducts). The nomenclature for intrahepatic large bile ducts was adapted from Healey and Schroy (8). Specimens of normal areas of surgid human salivary glands (five cases) and normal human pancases) that were surgically resected for gastric carcinoma were obtained and fixed in a 4% formaldehyde solution; these were used as controls. The specimens of1 ded in paraffin. Ten 5-pm embedded blodr; one of th Immunohistochemistry. Four serial sections from each paraffin block were stained for pancreatic and salivary isozymes of a-amylase by the avidin-biotin-peroxidase complex (ABC) method according to Hsu et al. (9),with a polyclonal antibody (sheep) to human pancreatic a-amylase, dilution L200 (Binding Site Ltd.; Birmingham, UK), a monoclonal antibody (mouse IgGl class) to human pancreatic a-amylase, dilution 1:200 (Chemicon; Temecula, CA), a polydonal antibody (sheep) to human salivary a-amylase, dilution 1:200 (BindingSite Ltd.), and a monoclonal antibody (mouse IgG1 class) to human salivary a-amylase, dilution L5 (Boehringer Mannheim Yamanouchi Co.; Tokyo, Japan). The monoclonal antibody (MAb) to pancreatic a-amylaseis specific for the pancreatic isozyme and does not crossreact with the salivary isozyme (25). The MAb to
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Figure 2. Pancreatic a-amylase immunoreactivity in the epithelium of (A) intrahepatic large bile duct, (6)septal bile ducts, (C) interlobular bile ducts and bile ductules, (D) peribiliary glands, and (C) hepatocytes. Pancreatic a-amylase immunoreactivityis strongly present in a granular pattern in the supranuclear cytoplasm of the epithelial cells of (A) intrahepatic large bile ducts, (6)septal bile ducts, and (D) peribiliary glands, while it is absent (C) in interlobular bile ducts, bile ductules, and hepatocytes. lmmunostain (ABC) method for pancreatic a-amylase using the monoclonal antibody. Original magnifications: A x 350; B x 200;C x 80; D x 120. Bars = 50 um.
salivary a-amylase is specific for salivary isozyme and crossreacts negligibly with the pancreatic isozyme (6). Endogenousperoxidase activity was eliminated by immersing the sectionsin absolute methanol containing 1% hydrogen peroxide for 20 min. The sections were treated with normal sera for 20 min, and then primary antibodies were applied to the sections at 4°C overnight. The sections were incubated with secondary biotinylated antibodies (anti-sheep IgG and anti-mouse IgG) (Vector Laboratories; Burlingame, CA) for 40 min. The sections were then treated with an avidin-biotin-peroxidase complex (Vectastain ABC Kit; Vector Laboratories) for 40 min. Reaction products were developed by immersing the sections in 3.3'diaminobenzidine tetrahydrochloridesolution (Sigma; St Louis, MO) containing hydrogen peroxide. Nuclei were lightly counterstained with hematoxylin. As negative controls, sections ofthe livers, pancreases, and salivary glands were treated with non-immuneserum (normal sheep or mouse serum)(Dako; Santa Barbara, CA) or PBS, followed by the procedure described above. Absorption tests were performed in selectedspecimens. T h e anti-pancreatic a-amylase and anti-salivary a-amylase antibody solutions were mixed with human pancreatic a-amylase (Athens Research and Technology; Athens, GA) and salivary a-amylase(Sigma), respectively. The mixed solutionswere incubated overnight at 4'C and centrifuged at 4000 rpm for 10 min. The supernatantswere collected and used as the primary antibodies in immunostainings. For cross-absorptionexperiments, performed on selected speci-
mens, the salivary isozyme antigen was mixed with antibodies against pancreatic a-amylaseor the pancreatic isozyme antigen was added to antibodies against the salivary enzyme. The mixed solutions were followed by the immunohistochemical procedure described above. Immunoelectron Microscopy. Fresh liver specimens of the hepatic hilus were obtained from five normal autopsied human adult livers as well as from two surgically resected human adult livers. Fresh specimens of human pancreases and salivary glands were also obtained from three autopsies as well as from five surgeries (salivary glands, three cases; pancreas, two cases). These specimenswere examined by immunoelectron microscopy for pancreatic and salivaryisozymes of a-amylaseby the post-embedding method (26). In brief, the specimens were dissected into cubes approximately 0.5 mm in size and fixed in 1%glutaraldehyde solution for 24 hr at room temperature. After an overnight buffer wash at 4'C. the specimens were dehydrated, and embedded in Lowicryl K4M according to the method reported by Carlemalm et al. (2). Roth et al. (27), and Lemanski et al. (16). The specimens were polymerized for 24-48 hr in a w radiation chamber at - 35°C. Semi-thin sections (1.0 pm) were stained with toluidine blue for selection of appropriate areas. Next, ultra-thin sections (80-100 nm) were mounted on carbon-coated nickel grids and were immersed for 20 min in 0.01 M PBS containing 1% bovine serum albumin. The sections were incubated for 1 hr in a drop of primary antibodies [MAb to pancreatic
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Figure 3. Salivary a-amylase immunoreactivityof the epithelium of (A) intrahepatic large bile ducts, (B) septal bile ducts, (C) interlobular bile ducts, bile ductules, and hepatocytes, and (D) peribiliary glands. Salivary a-amylase immunoreactivity is strongly present in a granular pattern in the supranuclear cytoplasm of the epithelial cells of (A) intrahepatic large bile ducts, (E) septal bile ducts, and (D) peribiliary glands. Salivary a-amylase immunoreactivity is weakly expressed in interlobular bile ducts and bile ductules (C). Hepatocytes are weakly positive for salivary a-amylase (C). lmmunostain(ABC method) for salivary a-amylase using the monoclonal antibody. Original magnifications: A x 350; B x 200;C x 80; D x 120. Bars = 50 bm.
a-amylase,dilution 1:lOO (Chemicon) or MAb to salivary a-amylase,dilution 1:20(BoehringerMannheim Yamanouchi)] rinsed in PBS, incubated in a drop of secondary antibody (goat anti-mouseIgG, dilution 1200) (Cappel; West Chester, PA) for 30 min, and then reacted with 15-nm protein A-gold particles for 30 min at room temperature. The sections were rinsed in PBS, fixed by 1% osmium tetroxide for 30 min, and stained with uranyl acetate and lead citrate. The sections were examined with a Hitachi H-300 electron microscope. As a negative control study, the primary antibodies were replaced by non-immune sera or PBS, followed by the immunoelectron microscopic procedure.
Results Specificity of Antibodies Pancreatic a-amylase (both polyclonal and monoclonal antibodies) was expressed in acinar cells of the normal pancreas but was not present in acinar cells of normal salivary glands (Figures 1A and 1B). Conversely, salivary a-amylase(both polydonal and monoclonal antibodies) was expressed in acinar cells of normal salivary glands but was not present in acinar cells of the normal pancreas (Figures 1C and ID). Positive staining was absent when non-immune
sera, PBS, or absorbed primary antibodies were used as the first layer. Cross-absorption tests resulted in positive staining.
Pancreatic a -Amyh e Immanohistochemistry Expression of pancreatic a-amylase with the polyclonal antibody was almost the same as that with the monoclonal antibody. Pancreatic isozyme immunoreactivitywas recognized in the epithelium of intrahepatic large ducts in 43 (96%) livers (Figure 2A) and in the epithelium of the septal ducts in all livers (Figure 2B). By contrast, it was discerned in the epithelium of interlobular ducts in only four (9%) livers and was not present in the bile ductule epithelium in any of the specimens (Figure 2C). Peribiliary glands were positive for pancreatic isozyme in all livers (Figure 2D). Pancreatic isozyme immunoreactivity was located in a granular pattem in the supranuclear cytoplasm of the biliary epithelium (Figures 2A-2C). Occasionally, pancreatic isozyme immunoreactivity was recognized in the lumens of bile ducts and peribiliary glands. The intensity of pancreatic isozyme immunoreactivity varied from case to case, as well as from area to area in the same case. Hepatocytes were negative for pancreatic isozyme in all cases (Figure 2C). Pan-
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Figure4. (A) Immunoelectron microscopicobsetvations (protein A-gold method) with the anti-pancreatic a-amylase monoclonal antibody reveal pancreatic a-amylase gold particles in the supranuclear cytoplasm of the septal bile duct. L,lumen of the septal bile duct; N, nucleus. (E) Negative control for immunoelectron microscopy (protein A-gold method) with non-immune sera instead of the anti-pancreatic a-amylase antibody reveals few gold particles in the epithelium of septal bile ducts. L, lumen of bile duct; N, nucleus. Original magnifications: A x 25,000; B x 18,000. Ears = 0.5 vm.
creatic isozyme immunoreactivity was negative in controls treated with non-immune sera, PBS. or absorbed primary antibodies. Crossabsorption tests showed positive immunostaining; absorption of primary antibodies with salivary isozyme resulted in positive immunostaining.
Salivary a -Amylase Immunohistochemistry Immunostaining of salivary a-amylase by polyclonal and monoclonal antibodies gave almost the same results. Salivary isozyme immunoreactivity was recognized in the epithelium of intrahepatic large ducts in 42 (93%) liven (Figure 3A) and in the epithelium of septal ducts in 43 (96%) liven (Figures 3B). It was also discerned in the epithelium of interlobular ducts and bile ductules in all livers (Figure 3C), although the salivary isozyme expression was weak in interlobular ducts and bile ductules (Figure 3C) relative to large ducts, septal ducts, and peribiliary glands. Peribiliary glands were positive for salivary isozyme in 43 (96%) liven (Figure 3D). Sali-
vary isozyme immunoreactivity was located in a granular pattern in the supranuclear cytoplasm of the biliary epithelium (Figures 3A-3D). Not infrequently, salivary isozyme immunoreactivity was recognized in the lumen of bile ducts and peribiliary glands. The intensity of salivary isozyme expression varied from case to case, as well as from area to area in the same case. Hepatocyteswere weakly and diffusely positive for salivary isozyme (Figure 3C) in all livers. Salivary isozyme immunoreactivity was negative in controls treated with non-immune sera, PBS, or absorbed primary antibodies. Crossabsorption tests showed positive immunostaining; absorption of primary antibodies with pancreatic isozyme resulted in positive immunostaining.
Immunoelectron Microscopic Findings Immunolabeling with monoclonal antibodies produced consistent labeling with negligible background, and individual gold particles were clearly identified. In immunostaining of pancreatic iso-
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Figure 5. lmmunoelectron microscopic observations(protein A-gold method) with the anti-salivary a-amylase monoclonalantibody reveal salivary a-amylase gold particles in the supranuclear cytoplasm of the intrahepatic peribiliaryglands. L, lumen of peribiliaryglands: N, nucleus. Original magnification x 30,000. Bar = 0.5 pm.
zyme, the epithelium of intrahepatic large ducts, septal ducts, and peribiliary glands had gold particles in the supranuclear cytoplasm (Figure 4A). Negative control specimens treated with non-immune sera or PBS showed few gold particles (Figure 4B). Hepatocytes had few pancreatic isozyme gold particles. In immunostaining of salivary isozyme, the epithelium of intrahepatic large ducts, septal ducts, and peribiliary glands had gold particles in the supranuclear cytoplasm (Figure 5 ) . Interlobular ducts, bile ductules, and hepatocytes had salivary isozyme gold particles (Figure 6A). Negative control specimens treated with non-immune sera or PBS showed few gold particles (Figure 6B). In normal pancreases, pancreatic isozyme gold particles were abundant in the supranuclear cytoplasm of acinar cells (Figure 7A), whereas salivary isozyme gold particles were not present. In normal salivary glands, pancreatic isozyme gold particles were not present, whereas salivary isozyme gold particles were abundant in the supranuclear cytoplasm of acinar cells (Figure 7B).
Discussion The results of the present study demonstrated that the pancreatic and salivary a-amylase immunoreactivities are specific for pancreatic and salivary isozymes, respectively. The present study demonstrated pancreatic a-amylase in the
epithelium of large ducts, septal ducts, and peribiliary glands. It was rarely expressed in the epithelium of interlobular ducts and was never expressed in bile ductules. By contrast, salivary a-amylase was expressed in the biliary epithelium from large ducts to bile ductules, although the intensity of the expression was weak in interlobular ducts and bile ductules. Immunoelectron microscopic observations confirmed these findings. These findings suggest that the epithelium of intrahepatic large ducts, septal ducts, and peribiliary glands contains pancreatic and salivary isozymes of a-amylase and that salivary isozyme is present in small amounts in the epithelium of interlobular ducts and bile ductules. Immunohistochemical and immunoelectron microscopic observations revealed that pancreatic and salivary isozyme immunoreactivity was located in a granular pattern in the supranuclear cytoplasm of the biliary epithelium, suggesting that both a-amylase isozymes are produced by the epithelium of intrahepatic large ducts, septal ducts, and peribiliary glands. Little is known about the functions of intrahepatic bile ducts and peribiliary glands. However, several investigators have speculated on their functions. For example, intrahepatic bile ducts have been shown to absorb and secrete water and electrolytes (31). Intrahepatic peribiliary glands may secrete seromucous fluid (39), IgA (30). secretory components (30). and several gut hormones (12). The present study demonstrated a-amylase isozymes not only in
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Figure 6. (A) lmmunoelectron microscopicobservations (protein A-gold method) with the anti-salivary a-amylase monoclonal antibody reveal salivary a-amylase gold particles in the cytoplasmof hepatocytes. BC, bile canaliculus. (E)Negative control for immunoelectronmicroscopy(proteinA-gold method) with non-immune sera instead of the anti-salivary a-amylase antibody reveals few gold particles in the hepatocytes. BC, bile canaliculus. Original magnification x 35,000.Bars = 0.5 vm.
the epithelium of the biliary tree but also within the biliary lumens, suggesting that pancreatic and salivary isozymes of a-amylase are secreted from the biliary epithelium into lumens of the intrahepatic bile ducts and peribiliary glands. A biochemical study that reported the presence of a-amylase activity in bile supports our suggestion (4). It has been reported that hepatic bile contains proteins, carbohydrates, and other organic substances (13J5). It seems likely that the secreted a-amylase isozymes play an important role in carbohydrate metabolism in hepatic bile. There was a difference in the distribution of the a-amylase isozymes in the intrahepatic biliary tree: pancreatic and salivary isozymes were observed in large ducts, septal ducts, and peribiliary glands, whereas they were absent (pancreatic isozyme) or only weakly expressed (salivary isozyme) in interlobular ducts and bile ductules. Although the reason for this difference in isoamylase distribution is not clear, these results indicate that intrahepatic large ducts, septal ducts, and peribiliary glands are phenotypically different from interlobular ducts and bile ductules. This distribution pattern of isozymes of a-amylase may reflect differences in the development of
the intrahepatic biliary tree. It seems possible that intrahepatic large ducts, septal ducts, and peribiliary glands, like the pancreas, develop from the embryonic foregut, while interlobular ducts and bile ductules arise from the periportal iepatocytes or the ductal plate, as previously reported (28,42,43). In fact, phenotypic characteristics of the exocrine pancreas have recently been reported in intrahepatic large bile ducts and peribiliary glands (38,44). Amylase activity in the human liver has been a matter of controversy (46). Some biochemical studies have demonstrated a low level of a-amylase activity in liver tissues (5,17). The present study did not reveal pancreatic a-amylase in the hepatocytes, suggesting that pancreatic a-amylase is not present in hepatocytes. By contrast, weak expression of salivary a-amylase immunoreactivity was found in hepatocytes, suggesting that a small amount of salivary a-amylase is present in hepatocytes. The salivary a-amylase in hepatocytes may be secreted into lumens of the intrahepatic biliary tree as well as into the circulation. However, the functional significance of the salivary isozyme in hepatocytes is unclear. It has been reported that the isozyme of a-amylase in organs
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Figure 7. lmmunoelectron microscopic findings (protein A-gold method) in positive control tissues. (A) The pancreatic acinar cell contains pancreatic a-amylase gold particles in the supranuclear cytoplasm, particularly in the zymogen granules. N. nucleus; L. lumen of the pancreatic acinus. (E) The acinar cell of the salivary gland contains salivary a-amylase gold particles in the supranuclear cytoplasm, particularly in the zymogen granules. N. nucleus; L, lumen of the acinus. Original magnifications: A x 24,000; B x 26.000. Bars = 0.5 pm.
other than the salivary glands and pancreas is of the salivary or nonpancreatic type (19.46). Of interest in this study is that pancreatic isozyme was expressed on the epithelium of intrahepatic large ducts, septal ducts, and peribiliary glands. The pancreas and intrahepatic bile ducts share a common developmental origin: both arise from the embryonic foregut. The potential of bile ducts to transdifferentiate to pancreatic acinar cells has been demonstrated in animal models fed certain chemicals (llJ4). Conversely, the exocrine pancreas can be transdifferentiated into hepatocytes and ductules in rats after copper depletion (23). The findings of the present study imply that the intrahepatic large ducts, septal ducts, and peribiliary glands share phenotypic characteristics with the exocrine pancreas. Recently, a-amylase isozymes have been analyzed at the molecular level. The genes of salivaryand pancreatic isozymes of a-amylase are named AMY1 and AMY2. respectively. More recently, a novel type of human amylase, which is encoded by the AMYZB, has been detected (40). This novel type of a-amylase has a high homology with pancreatic a-amylase in amino acid sequence (40,45). Recent studies have shown that the two different amylase-encoding genes,
AMY2A and AMYZB, were expressed in the normal human pancreas (7). It seems mandatory to analyze a-amylase isozymes in the intrahepatic biliary epithelium and hepatocytes by molecular analytical methods.
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