Carbonic Anhydrase Isoenzymes I,II,III,and IV Are Present in Human Esophageal Epithelium

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 (Lucocq, 1993). Both the antibodies and the gold complexes were diluted in 0.5% FSG. After further washes in PBS (three times over 30 min) and distilled water (four times over 4 min), the grids were embedded and contrasted in 1.8% (w/v) methyl cellulose/0.3% (w/v) uranyl acetate as described in Griffiths et al. (1984). Sections were examined in a Jeol 1200EX electron microscope in transmission mode.

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 (Davidson et al., 1990) for 5 min on ice. This was followed by centrifugation at 1000 × g for 10 min and the supernatant extracted. The pellet was then resuspended in isolation buffer and homogenized again, followed by centrifugation at 1000 × g for 10 min. The first and second postnuclear supernatants were combined and centrifuged with an SW40Ti rotor at 36,000 rpm for 110 min. The supernatant was discarded and the pellet resuspended in 200 μl of lysis buffer (Heider et al., 1994) and left at 0°C for 10 min. The lysates were centrifuged at 13,000 X g for 5 min and the supernatant prepared for running on SDS-PAGE gels after protein concentration was determined by the Bradford method (Bradford, 1976). Samples containing 30 μg protein were loaded on a 12% gel and run at 200 V for 1 hr. Transfer was carried out overnight at 30 V onto nitrocellulose membranes (Sigma; 0.45-μm pore). This was followed by immunolabeling with the appropriate primary and secondary (goat anti-rabbit IgG; Sigma) antibodies. Primary antibody dilutions were in the range 1:1000–1:2000; the secondary antibody was used at 1:1500. Bands were revealed by the ECL reaction (Amersham Life Science; Little Chalfont, Bucks, UK).

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.

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 (Figures 3 and 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).