ARTICLE |
Correspondence to: Keith N. Christie, Dept. of Anatomy and Physiology, University of Dundee, Dundee DD1 4HN, Scotland, UK.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Carbonic anhydrase (CA) isoenzymes have been widely studied in the gastrointestinal tract, where they mediate membrane transport events and pH regulation. However, the esophagus has generally received scant attention. In an immunohistochemical study confirmed by Western blotting, we have detected four CA isoenzymes (CAI, II, III, and IV) in the epithelium of human esophagus. Isoenzymes I, III, and sometimes IV (<10%) were present in the cytoplasm of basal cells and II and IV in the cytoplasm and cell surface membranes, respectively, of suprabasal cells (prickle cells). The localization of CAIV to the plasma membranes was confirmed by electron microscopic immunocytochemistry. CA was effectively divided at the basal-suprabasal interface between low-activity CAI and III (basal) and high-activity CAII and IV (suprabasal). Carbonic anhydrase in esophageal epithelial cells may have several functions: elimination of CO2 and metabolites, participation in membrane transport events during active cell growth, and pH regulation as a protective mechanism against acidic gastric reflux. (J Histochem Cytochem 45:35-40, 1997)
Key Words: Esophagus, Epithelium, Human, Carbonic anhydrase isoenzymes, I, II, III, IV, Immunohistochemistry, Western blotting
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Carbonic anhydrase (CA; EC 4.2.1.1) is widely distributed throughout mammalian tissues, where it mediates the transfer of CO2, HCO3-, H+, and Cl- (
Immunohistochemistry has made a considerable contribution to the localization of CA beyond the erythrocyte. The first immunohistochemical attempts to localize CA isoenzymes were made by
Studies of the distribution and role of CA in the human gastrointestinal tract have seldom extended to the esophagus, the work of
The report by
In this report we describe the immunohistochemical localization of CA isoenzymes I, II, III, and IV in human esophageal epithelium in a study supported by Western blotting and immunoelectron microscopy. CA may have a role in provision of bicarbonate ions for maintenance of mucosal protection against gastric reflux (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue Samples
Pinch biopsies of endoscopically and histologically normal mid-third esophagus were obtained by informed consent from 15 patients undergoing endoscopic examination. The tissues were supported in Tryco-M-Bed (Aerosol Marketing and Chemical; London, UK) on cork disks and quenched in melting 2-methylbutane cooled with liquid nitrogen. Orientation of the tissues to produce transepithelial basal to luminal surface cells was often achieved in spite of the small size and nature of the samples. Sections (10 µm) were cut at -25°C, mounted on subbed (gelatin- and chromalum-coated) coverslips, and fixed in 3% formaldehyde (generated from paraformaldehyde) in 0.1 M phosphate buffer, pH 7.4, for 10 min at room temperature.
Immunohistochemistry of CAI, II, III, and IV
The sections on coverslips were washed in PBS (five times over 10 min); treated with 0.1 M ammonium chloride in PBS for 10 min; nonspecific sites blocked with 0.5% fish skin gelatin (FSG) (Sigma Chemical; Poole, Dorset, UK) in PBS for 10 min; reacted with the primary polyclonal antibodies raised in rabbits (diluted in the range 1:50-1:200 with FSG in PBS) (antibody sources are identified in the Acknowledgments; the specificity of the antibodies is well documented by their respective donors); washed in PBS (five times over 10 min); secondary antibody applied [goat anti-rabbit FITC (Sigma) 1:100 with FSG in PBS] for 20 min. After a final wash with PBS (five times over 10 min) the sections were mounted in Mowiol 40-88 (Aldrich Chemical; Gillingham, Dorset, UK) and examined on a Zeiss Axioskop epifluorescence microscope fitted with a 495-nm excitation filter. Specificity controls for the antibodies included application of nonimmune sera and reaction with tissues and cells known to give a positive (and negative) response. For example, positive controls included CAII in gastric parietal cells, CAIII in striated muscle, and CAIV in lung and kidney. Photomicrographs were taken on Ilford HP5 ISO 400/27° film.
Immunoelectron Microscopy of CAIV
Small fragments of epithelium were fixed in 4% paraformaldehyde containing 0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 48 hr at 4°C and soaked overnight in 2.3 M sucrose containing 20% (w/v) polyvinyl pyrrolidone (40,000 KD) in PBS. The tissue fragments were then cut and mounted on metal stubs, frozen in liquid nitrogen, and 100-nm sections cut at -100°C on a Reichert Ultracut E microtome with an FC4 cryoattachment. The sections were collected on 2.3 M sucrose PBS, mounted on pioloform-carbon-coated grids, and then incubated on 0.1 M NH4Cl in PBS (10 min), followed by 0.5% FSG in PBS (10 min). The grids were then floated on 20-µl drops of primary rabbit antisera, followed by washes in PBS (three times over 30 min) and 7-nm protein A-gold (
Western Blotting of Epithelial Cell Proteins
Because of their different cellular locations, CAIV membrane-associated and I, II, and III-soluble, different regimens for preparation of extracts from the biopsy specimens for Western blotting had to be adopted.
Protein Sample Preparation for CAIV.
Pinch biopsies of middle-third esophageal epithelium were chopped into small fragments and homogenized in a Dounce homogenizer in pH 7.4 isolation buffer (
Protein Sample Preparation for CAI, II, and III. Pinch biopsy samples were chopped into tiny fragments with scissors (0.5-0.25 mm) and added to 200 µl of cold lysis buffer (as above). They were left in the buffer on ice for 30 min, then centrifuged at 13,000 x g for 5 min at 4°C. The supernatant was extracted and the pellet discarded. Lysate protein (30 µg) was loaded onto SDS-PAGE gels and blotting carried out as above.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunohistochemistry of Carbonic Anhydrase Isoenzymes
The CAI isoenzyme was confined to the basal cell layer, and of all the antibodies gave the weakest immunolocalization (Figure 1). The CAII isoenzyme was present in the cytosol of suprabasal cells, extending luminally as far as the most superficial three or four cell layers. Large areas of epithelium were frequently devoid of CAII. Reactive cells tended to occur in clusters, with individual cells displaying markedly different levels of immunoreactivity (Figure 2). CAII was never observed in basal cells. CAIII was restricted to basal cells and consistently gave a strong immunolocalization (Figure 3 and Figure 4). CAIV was principally associated with the cell surface membranes of prickle and occasionally basal cells (<10%), where immunostaining appeared as a pericellular halo studded with intensely fluorescent particles, giving the cell peripheries a necklace-like appearance (Figure 5). Nucleus-associated elements were also identified by the CAIV antibody (Figure 5). Like CAII, CAIV tended to be localized to discrete clusters of cells, with large interstitial areas devoid of the isoenzyme. These findings were consistent for all of the biopsies.
|
Control and test tissue experiments gave results confirming the specificity of the antibodies.
Immunoelectron Microscopy of CAIV
In thawed ultrathin cryosections, immunogold labeling for the CAIV isoenzyme was located principally over the plasma membranes of suprabasal cells (Figure 6). Labeling appeared to be absent over membrane structures present within desmosomes. Plasma membranes of the most superficial epithelial cells were not labeled.
Figure 7 illustrates the different regions of the esophageal epithelium and distribution of the carbonic anhydrase isoenzymes.
|
Western Blots of CAI, II, III, and IV
All four isoenzymes were detected in the epithelial protein extracts. CAI, II, and III localized close to 30 KD, with CAIV at or about 35 KD (Figure 8). Additional weakly stained polypeptide bands were consistently seen in CAIV blots in the region of 74-60 KD and 27-20 KD. The former were assumed to be due to nonspecific crossreaction with other esophageal proteins, and the latter to proteolytic cleavage of intact CAIV (advice from Dr. A. Waheed, Edward A. Doisy Department of Biochemistry and Molecular Biology, St Louis University Health Sciences Center, St Louis, MO).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Four carbonic anhydrase isoenzymes (CAI, II, III, and IV) have been identified by immunohistochemistry and Western blotting in human esophageal epithelial cells. This supports an earlier study (
In our previous study the cell surface localization of the enzyme was the most prominent feature. We have now confirmed immunohistochemically at the light and electron microscopic levels that this is due to the presence of CAIV on the plasma membranes. However, we have not been able to identify at the electron microscopic level the CAIV-containing elements close to the nuclei that were apparent by light microscopy. The cytoplasmic reaction in the basal and suprabasal cells obtained previously can now be attributed to CAs I, II, and III.
The sharp demarcation at the basal cell layer between CAI and III and II and IV suggests that it is the location of the cells that largely dictates the isoenzymes expressed rather than the specific epithelial cell type. Both basal and parabasal compartments contain stem, committed progenitor, and dividing cells, yet CAI and III were never detected suprabasally, nor was CAII demonstrated in the basal cell layer. Apart from the occasional presence of CAIV in basal cells, the isoenzymes are divided at the basal-suprabasal interface between low-activity CAI and III (basal) and high-activity CAII and IV (suprabasal).
The role(s) of carbonic anhydrase in the esophageal epithelium is open to speculation. However, it is likely to be dictated to some extent by the anaerobic conditions in the epithelium and its need to rid itself of CO2 and H2O produced by oxidative metabolism. Accordingly, CAI and III may facilitate the efflux of CO2 and H2O from the metabolically active basal cells to blood vessels in the adjacent lamina propria. Similarly, as the basal cells mature and move suprabasally, they may require the more efficient CAII and IV to achieve the same end. Another conceivable role may be in cell volume regulation. When esophageal basal cells move into the prickle cell region they undergo a dramatic increase in volume (D. Hopwood, personal communication), and in this situation CAII may supply H+ and HCO3- for the Na+/H+ and Cl-/HCO3- exchange during electrolyte gain (
Finally, and perhaps most importantly, carbonic anhydrase probably plays a significant part in protecting the esophageal mucosa. Bicarbonate derived from saliva and esophageal submucosal glands has been demonstrated to protect against acidic gastric refluxate (
Carbonic anhydrase in esophageal epithelial cells may participate in CO2 and H2O elimination, ion exchange during growth, and may serve as an intrinsic source of bicarbonate, augmenting that of saliva and esophageal submucosal gland secretions in protecting the mucosa against corrosive gastric acid.
![]() |
Acknowledgments |
---|
We gratefully acknowledge the generosity of the following for their donations of carbonic anhydrase antibodies: Dr W.S. Sly, St Louis University Health Sciences Center, St Louis, MO (CAIV); Dr A.-K. Parkkila (CAI and CAII) and Dr H.K. Väänänen (CAIII), both of the University of Oulu, Oulu, Finland. We also thank Ms Jennifer Collie for excellent secretarial assistance in the preparation of the manuscript.
Received for publication April 26, 1996; accepted August 5, 1996.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254[Medline]
Brown CM, Snowdon CF, Slee B, Sandle LN, Rees WDW (1995) Effect of topical oesophageal acidification on human salivary and oesophageal alkali secretion. Gut 36:649-653[Abstract]
Brown CM, Snowdon CF, Slee B, Sandle LN, Rees WDW (1993) Measurement of bicarbonate output from the intact human oesophagus. Gut 34:872-878[Abstract]
Brown D, Zhu XL, Sly WS (1990) Localization of membrane-associated carbonic anhydrase type IV in kidney epithelial cells. Proc Natl Acad Sci USA 87:7457-7461[Abstract]
Christie KN, Thomson C, Morley S, Anderson J, Hopwood D (1995) Carbonic anhydrase is present in human oesophageal epithelium and submucosal glands. Histochem J 27:587-590[Medline]
Davidson HW, West MA, Watt C (1990) Endocytosis, intracellular trafficking, and processing of membrane IgG and monovalent antigen/membrane IgG complexes in B lymphocytes. J Immunol 144:4101-4109
Dodgson SJ (1991) Liver mitochondrial carbonic anhydrase (CAV), gluconeogenesis, and ureagenesis in the hepatocyte. In Dodgson SJ, Tashian RE, Gros G, Carter ND, eds. The Carbonic Anhydrases Cellular Physiology and Molecular Genetics. New York, Plenum Press, 297-306
Fernley RT (1991) Carbonic anhydrases secreted in saliva. In Dodgson SJ, Tashian RE, Gros G, Carter ND, eds. The Carbonic Anhydrases Cellular Physiology and Molecular Genetics. New York, Plenum Press, 365-373
Griffiths G, McDowall A, Back R, Dubochet J (1984) On the preparation of cryosections for immunocytochemistry. J Ultrastruct Res 89:65-78[Medline]
Hansson HPJ (1967) Histochemical demonstration of carbonic anhydrase activity. Histochemie 11:112-128[Medline]
Heider H, Hug C, Lucocq JM (1994) A 40-kDa myelin basic protein kinase, distinct from erk1 and erk2, is activated in mitotic HeLa cells. Eur J Biochem 219:513-520[Abstract]
Helm JF, Dodds WJ, Hogan WJ (1987) Salivary responses to esophageal acid in normal subjects and patients with reflux esophagitis. Gastroenterology 92:1393-1397
Lonnerholm G, Wistrand PJ (1991) Membrane-bound carbonic anhydrase IV in the human kidney. Acta Physiol Scand 141:231-234[Medline]
Lucocq JM (1993) Particulate markers for immunoelectron microscopy. In Griffiths G, ed. Fine Structure Immunocytochemistry. Berlin, Springer-Verlag, 279-302
Macknight ADC, Gordon LGM, Purves RD (1994) Problems in the understanding of cell volume regulation. J Exp Zool 268:80-89[Medline]
McDougall NI, Johnston BT, Kee F, Collins JSA, McFarland RJ, Love AHG (1996) Natural history of reflux oesophagitis: a 10 year follow up of its effects on patient symptomatology and quality of life. Gut 38:481-486[Abstract]
Parkkila S, Parkkila A-K, Juvonen T, Rajaniemi H (1994) Distribution of the carbonic anhydrase isoenzymes I, II and VI in the human alimentary tract. Gut 35:646-650[Abstract]
Seidler U, Carter K, Ito S, Silen W (1989) Effect of CO2 on pHi in rabbit parietal, chief and surface cells. Am J Physiol 256:G466-G475
Spicer SS (1993) Advantages of histochemistry for the study of cell biology. Histochem J 25:531-547[Medline]
Spicer SS, Stoward PJ, Tashian RE (1979) The immunohistolocalization of carbonic anhydrase in rodent tissues. J Histochem Cytochem 27:820-831[Abstract]
Strange K (1994) Are all cell volume changes the same? News Physiol Scis 9:223-228
Swenson ER (1991) Distribution and functions of carbonic anhydrase in the gastrointestinal tract. In Dodgson SJ, Tashian RE, Gros G, Carter ND, eds. The Carbonic Anhydrases Cellular Physiology and Molecular Genetics. New York, Plenum Press, 265-287
Tashian RE (1989) The carbonic anhydrases: widening perspectives on their evolution, expression and function. BioEssays 10:186-192[Medline]
Tobey NA, Reddy SP, Keku TO, Cragoe EJ, Orlando RC (1992) Studies on pHi in rabbit esophageal basal and squamous epithelial cells in culture. Gastroenterology 103:830-839[Medline]
Tobey NA, Powell DW, Schreiner VJ, Orlando RC (1989) Serosal bicarbonate protects against acid injury to rabbit esophagus. Gastroenterology 96:1466-1477[Medline]
Waheed A, Zhu XL, Sly WS (1992) Membrane-associated carbonic anhydrase from rat lung. Purification characterization, tissue distribution, and comparison with carbonic anhydrase IV's of other animals. J Biol Chem 267:3308-3311