ARTICLE |
Correspondence to: Hiro-oki Okamura, Dept. of Pathology and Laboratory Medicine, Medical U. of South Carolina, 165 Ashlet Avenue, Suite 309, PO Box 250908, Charleston, SC 29425.
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Summary |
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We examined the histochemical localization of carbonic anhydrase (CA) in Bowman's glands by light and electron microscopy. Neither CAI nor CAII was detected immunohistochemically in the duct cells. However, by enzyme histochemistry the duct cells revealed electron-dense precipitates demonstrative of CA in the microvilli and intercellular digitations. The reaction product was also noted in small vesicles in the cytoplasm of duct cells. In cells of the acini, the well-developed short microvilli, basolateral cell membrane, and mitochondria along the basolateral membrane showed strong deposits indicating CA activity. Dense reaction product of CA was also detected in a small core within the electron-lucent granules of the secretory cells, although CAI and CAII were not detected by immunostaining in the secretory granules. Although the functional significance of CA in Bowman's glands is obscure, the enzyme may play a role in regulation of pH and ion balance in the mucous layer covering the olfactory epithelium. The presence of CA activity in the ducts suggests that these structures are not simple tubes serving as a conduit for secretory substances but participate in modifying the luminal content by secreting CA. (J Histochem Cytochem 47:15251531, 1999)
Key Words: carbonic anhydrase, immunohistochemistry, enzyme histochemistry, electron microscopy, Bowman's gland, guinea pig
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Introduction |
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Carbonic anhydrase (CA; E.C.4.2.1.1) is a zinc-containing metalloenzyme which catalyzes the reversible hydration of carbon dioxide and dehydration of carbonic acid (H2O + CO2 HCO3- + H+). In terrestrial vertebrates, seven CA isozymes have been identified, but they differ in their tissue distribution and subcellular localization. CAI-III and VII are cytoplasmic, IV is membrane-associated, V is mitochondrial, and VI is secretory (
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Materials and Methods |
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Immunohistochemistry
The experimental protocol was approved by the Animal Care and Experimentation Committee of our institutes. Under general urethane anesthesia (sodium pentobarbital, 1.5 g/kg body weight), female albino guinea pigs (approximately 300 g body weight) were perfused with physiological saline followed by perfusion with 0.5% zinc10% formalin. After perfusion, the rostrum containing the nose was quickly removed and immersed in the same fixative for another 2 hr. The specimens were decalcified with 5% ethylenediamine tetraacetic acid (EDTA) (pH 7.3) for 14 days at 4C. The dehydrated specimens were embedded in paraffin and cut at 20 µm. After dissolving the paraffin, the sections were incubated with 1% H2O2, 5% normal horse serum (NHS), and then incubated overnight at room temperature with one of the primary antibodies. The primary antibody against anti-CAI (anti-human CAI-IgG, Code PC046; Binding Site, Birmingham, UK) was used at a dilution of 1:100, whereas CAII (anti-human CAIIIgG, Code PC076; Binding Site) was used at a 1:3000 dilution with 1% NHS0.01 M phosphate buffer, pH 7.2, containing 0.9% NaCl (PBS). The sections were rinsed in PBS and flooded for 1 hr with a 1:400 dilution of biotinylated anti-sheep IgG and incubated in Vectastain ABC reagent for 1 hr. Sites of bound primary antibodies were visualized by development in 3,3'-diaminobenzidineH2O2 substrate medium. We also tested the specificity of the immunohistochemical reaction with red blood cells in the nasal capillary, which are known to possess CAI and CAII.
Enzyme Histochemistry
Anesthetized guinea pigs were perfused with physiological saline followed by perfusion with 4% paraformaldehyde3% glutaraldehyde. After perfusion, the rostrum containing the nose was quickly removed and immersed in the same fixative for another 2 hr. The specimens were decalcified with 5% EDTA (pH 7.3) for 14 days at 4C and were frozen after treatment with a series of solutions of graded sucrose. The frozen tissues were cut into 20 µm sections and reacted in Hansson's medium
Electron Microscopic Histochemistry
For exact localization of CA activity in Bowman's gland, tissue sections showing distinct reaction products in the acini and ducts by light microscopy were selected for electron microscopy. The selected sections were rinsed in 0.01 M phosphate buffer (pH 9.4) and further fixed with 1% OsO4 buffered with 0.1 M phosphate buffer (pH 5.0) and treated with 1% uranyl acetate in 0.1 M maleate buffer (pH 5.2), based on the method of
Measurement of CA Activity in the Olfactory Mucus
The rostrum of anesthetized guinea pig nose as above mentioned was cut down and the olfactory epithelium was opened. The olfactory mucous layer was washed with distilled water and the washing solution was examined for CA activity (
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Results |
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Bowman's gland of guinea pig consisted of an intraepithelial portion in which the ducts were located and an extraepithelial portion in which mainly branched tubuloalveolar secretory acini were located (Figure 1a). The immunohistochemical reactions using CAI or CAII antibodies showed different results. CAII was detected in Bowman's glands but CAI was not (Figure 1b and Figure 1c). In particular, the cytoplasm near the basolateral membrane of the acinar cells showed localization of CAII, but the supranuclear region and ducts revealed no immunohistochemical reaction. CAI and CAII were not present in three types of olfactory epithelial cells, consisting of the receptor, sustentacular, and basal cells (Figure 1b and Figure 1c) except for some epithelial cells on the endoturbinate showing CAII (Figure 1d). Both CAI and CAII were detected on red blood cells in the nasal capillaries.
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Light microscopic examination of enzyme histochemistry showed the localization of CA in Bowman's glands, passing through the olfactory epithelium and opening into the lumen (Figure 2). No CA was present in the olfactory epithelium, including the receptor, sustentacular, and basal cells, except for some epithelial cells on the endoturbinate, as mentioned above (
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The electron microscopic examination defined the fine structures and showed definite electron-dense precipitates as reaction products for CA. The duct was composed of a simple cuboidal epithelium with electron-lucent and amorphous cytosol and with well-developed microvilli on the luminal surface (Figure 3). The cytoplasmic processes on the lateral cell membrane of duct cells showed several interdigitations. Flocculent, fine electron-dense reaction precipitates were detected as deposits covering the luminal surface of the microvilli, and reaction products were localized between apposed lateral cell membranes (Figure 3 and Figure 4). CA was also detected in small vesicles scattered throughout the cytoplasm (Figure 4).
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In the acini, well-developed short microvilli protruded through the secretory cell surface into the gland lumen, ovoid and flattened nuclei were located towards the basal side of secretory cells, and quite numerous rod-shaped mitochondria were present along the basolateral cell membrane. The apposed lateral cell membrane with very well-developed cytoplasmic processes showed intercellular digitations. The basal cell membrane showed well-developed basal invaginations. Dense reaction product of CA was detected in the cytoplasm of the basolateral cell membranes, showing interdigitations, and was found in mitochondria along the basolateral membrane (Figure 5 and Figure 6). Reaction precipitate was also detected on the luminal surface of microvilli. The cytoplasm of acinar cell was filled with abundant, densely distributed secretory granules (Figure 5). These granules were classified into two groups according to the texture and electron density of the secretory granule matrix. The first group consisted of electron-dense granules with compact matrix, and the second group consisted of electron-lucent granules with loose matrix. The reaction product was detected as electron-dense particles scattered on the loose matrix of the electron-lucent secretory granules (Figure 7).
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CA activity and hemoglobin concentration from the solution washed out of the olfactory mucous layer showed 3.91 U/ml and 0.003 mg/ml, respectively. Hemoglobin concentration was very low and near zero, demonstrating that this solution was not affected by the blood serum. These results showed that the olfactory mucous layer included CA activity.
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Discussion |
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Only a few studies have provided a detailed analysis of the histological features of Bowman's gland (
CA was detected in the cytoplasm, basal cell processes and lateral cell membrane of the acinar epithelial cells and was found in mitochondria along the basolateral membrane. The CA reaction produced in the cytoplasm can be attributed to CAII because mitochondrial CA is known to be CAV, and intense immunohistochemical reactivity for cytoplasmic CAII was detected in the cytoplasm near the basolateral membrane of acinar cell. Although the significance of CA enzyme in the cytoplasm is not clear, it is possible that the enzyme plays a role in ion transport across the basal cell membrane by supplying bicarbonate or hydrogen ions for exchange. In this regard, the cytoplasm of the acinar cells contains many mitochondria adjacent to the basolateral cell membrane. The mitochondria supply CO2 and H2O for generating H+ and HCO3-, which can be exchanged for K+ and Cl-, respectively, as in gastric parietal cells (
Two types of secretory granules have been detected in acinar cells, including secretory granules with an electron-dense matrix and others with an electron-lucent matrix.
Results of several studies have suggested that CA is involved in a number of biological functions, such as pH regulation, ion balance, and stabilization of macromolecules in secretions in the oral cavity, airway ducts, and conjunctiva (
Our results also showed accumulation of the enzyme histochemical reaction precipitates of CA on the luminal side of the microvilli at the apical surface of duct cells. These precipitates probably originate from one of two possible sources. First, the surface cell membrane of the duct cells contains the CA enzyme and shows histochemical reactivity on the luminal side of the plasmalemma. Second, enzyme secreted by acinar cells adheres to the microvilli at the surface of the ducts. Our results provide evidence for the latter explanation because stain deposits in duct cells appeared too sparse to account for the heavy deposits on the duct surface. Instead, the small enzyme-positive foci shown in acinar granules could provide the source of the CA at the duct surface. These findings suggest that the mature secretory granules contain CA enzyme, which is secreted into the gland lumen through a regulatory secretion mechanism.
Ion-transporting epithelia that show CA activity are present in several organs, including the pancreas (
Using routine neutral or alkaline-buffered OsO4 for postfixation after enzyme histochemistry for CA results in dissolving of the cobalt sulfide reaction product and its loss from the section (
Finally, we found that infrequent nasal epithelial cells possess CAII. We speculated that these cells represent specialized chemoreceptors, e.g., detecting changes of pH or CO2 concentration (
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Acknowledgments |
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We should like to thank Dr Samuel S. Spicer (MUSC) for kindly reading the manuscript and for his useful suggestions. This manuscript was also prepared to extend our congratulations on the tenth anniversary of Prof Naonori Sugai as Chairman of the Department of Anatomy and Histology.
Received for publication March 25, 1999; accepted June 29, 1999.
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Literature Cited |
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Alfred P, Fu P, Barrett G, Penschow JD, Wright RD, Coghlan JP, Fernley RT (1991) Human secreted carbonic anhydrase: cDNA cloning, nucleotide sequence, and hybridization histochemistry. Biochemistry 30:569-575[Medline]
Andres KH (1966) Der Feinbau der Regio Olfactoria von Markosmatikern. Z Zellforsch 69:140-154[Medline]
Andres KH (1969) Der olfaktorische Saum der Katze. Z Zellforsch 96:250-274[Medline]
Bloom W, Fawcett DW (1975) A Textbook of Histology. 10th ed Philadelphia, WB Saunders
Briggman JV, Tashian RE, Spicer SS (1983) Immunohistochemical localization of carbonic anhydrase I and II in eccrine sweat glands from control subjects and patients with cystic fibrosis. Am J Pathol 112:250-257[Abstract]
Buanes T, Grotmol T, Landsverk T, Ridderstråle Y, Ræder MG (1986) Histochemical localization of carbonic anhydrase in the pig's exocrine pancreas. Acta Physiol Scand 128:437-444[Medline]
Carter N, Wistrand PJ, Lönnerholm G (1989) Carbonic anhydrase localization to perivenous hepatocytes. Acta Physiol Scand 135:163-167[Medline]
Coates EL, Wells CM, Smith RP (1998) Identification of carbonic anhydrase activity in bullfrog olfactory receptor neurons: histochemical localization and role in CO2 chemoreception. J Comp Physiol 182:163-174. [A][Medline]
Crosby WH, Furth FW (1956) A modification of the benzidine method for measurement of hemoglobin in plasma and urine. Blood 11:380-383[Medline]
Deutsch HF (1987) Carbonic anhydrases. Int J Biochem 19:101-113[Medline]
Frings S, Benz S, Lindemann B (1991) Current recording from sensory cilia of olfactory receptor cells in situ. J Gen Physiol 97:725-747[Abstract]
Frisch D (1967) Ultrastructure of mouse olfactory mucosa. Am J Anat 121:87-120[Medline]
Getchell ML, Rafols JA, Getchell TV (1984) Histological and histochemical studies of the secretory components of the salamander olfactory mucosa: effects of isoproterenol and olfactory nerve section. Anat Res 208:553-565[Medline]
Hansson HPJ (1967) Histochemical demonstration of carbonic anhydrase activity. Histochemie 11:112-128[Medline]
Hennigar RA, Schulte BA, Spicer SS (1983) Immunolocalization of carbonic anhydrase isozymes in rat and mouse salivary and exorbital lacrimal glands. Anat Rec 207:605-614[Medline]
Kumpulainen T, Jalovaara P (1981) Immunohistochemical localization of carbonic anhydrase isoenzymes in the human pancreas. Gastroenterology 80:796-799[Medline]
Maren TH (1960) A simplified micromethod for the determination of carbonic anhydrase and its inhibitors. J Pharmacol Exp Ther 130:26-29
Murakami H, Sly WS (1987) Purification and characterization of human salivary carbonic anhydrase. J Biol Chem 262:1382-1388
Okamura H, Sugai N, Ohtani I (1996) Identification of nasal epithelial cells with carbonic anhydrase activity. Brain Res 728:263-266[Medline]
Parkkila S, Kaunisto K, Rajaniemi L, Kumpulainen T, Jokinen K, Rajaniemi H (1990) Immunohistochemical localization of carbonic anhydrase isoenzymes VI, II, and I in human parotid and submandibular glands. J Histochem Cytochem 38:941-947[Abstract]
Spicer SS, Sens MA, Hennigar RA, Stoward PJ (1984) Implications of the immunohistochemical localization of the carbonic anhydrase isozymes for their function in normal and pathologic cells. Ann NY Acad Sci 429:382-397[Abstract]
Spicer SS, Sens MA, Tashian RE (1982) Immunocytochemical demonstration of carbonic anhydrase in human epithelial cells. J Histochem Cytochem 30:864-873[Abstract]
Storey BT, Dodgson SJ, Forster RE, II (1984) Mitochondrial carbonic anhydrase: the purified enzyme. Ann NY Acad Sci 429:210-211
Sugai N, Ito S (1980) Carbonic anhydrase, ultrastructual localization in the mouse gastric mucosa and improvements in the technique. J Histochem Cytochem 28:311-325[Abstract]
Tashian RE (1989) The carbonic anhydrases: widening perspectives on their evolution, expression and function. Bioessays 10:186-192[Medline]
Whitney PL, Briggle TV (1982) Membrane-associated carbonic anhydrase purified from bovine lung. J Biol Chem 257:12056-12059
Yamamoto M (1976) An electron microscopic study of the olfactory mucosa in the bat and rabbit. Arch Histol Jpn 38:359-412[Medline]
Yokota S (1969) Electron microscopic demonstration of carbonic anhydrase activity in mouse liver cells. Histochemie 19:255-261[Medline]
Zhu XL, Sly WS (1990) Carbonic anhydrase IV from human lung: purification. characterization and comparison with membrane carbonic anhydrase from human kidney. J Biol Chem 265:8795-8801