Immunohistochemical Demonstration of Carbonic Anhydrase Isoenzyme VI (CA VI) Expression in Rat Lower Airways and Lung
Department of Anatomy and Cell Biology, University of Oulu, Oulu, Finland
Correspondence to: Jukka S. Leinonen, Dept. of Anatomy and Cell Biology, PO Box 5000, 90014 University of Oulu, Oulu, Finland. E-mail: juleinon{at}paju.oulu.fi
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Summary |
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(J Histochem Cytochem 52:11071112, 2004)
Key Words: carbonic anhydrase trachea lungs rat immunohistochemistry
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Introduction |
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Mucosa of the lower airways, similarly to the upper alimentary tract mucosa, is exposed to heavy microbial, physical, and chemical stress and is covered by protective mucus produced by the seromucous tracheobronchial glands and by the surface epithelium (Quinton 1979; Tam and Verdugo 1981
; Gail and Lenfant 1983
; Basbaum et al. 1990
). Expression of CA VI in the lower airways suggests that it has a general mucosa-protective and renewing role not only in the alimentary tract but also in the respiratory tract. CA activity has been demonstrated earlier in the tracheobronchial glands, goblet cells, and some other unidentified epithelial cells of the guinea pig trachea, but the only CA isoenzyme identified there to date is CA II in the tracheobronchial glands (Spicer et al. 1982
; Okamura et al. 1996
). Therefore, the present study aimed to elucidate whether CA VI is expressed in the lower airway epithelia.
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Materials and Methods |
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Tissue and Saliva Samples
Tracheal and lung samples were collected from three fetuses and 1-, 2-, 5-, 10-, 20-, 30-day-old and adult (60- to 90-day-old) male SpragueDawley rats, fixed in Carnoy's fluid or in 4% paraformaldehyde for 18 hr at 4C, and embedded in paraffin. Sections of 5 µm were subjected to immunohistochemical (IHC) stainings. To confirm that saliva contamination does not cause positive CA VI staining in the respiratory tract, samples from sialoadenectomized rats were also subjected to CA VI staining. The salivary glands were removed surgically from six adult male rats under fentanyl fluanisonemidazolam anesthesia. Three of them were sacrificed on the third postoperative day and another three on the seventh postoperative day. The tracheas and lungs were dissected out and subjected to IHC stainings as described above. Rat saliva was collected from anesthetized animals injected IP with 6 mg/kg of pilocarpine. All animal experiments described had the approval of the Animal Care and Use Committee of the University of Oulu.
Absorption of the Anti-rat CA VI Serum with CA VI
To further confirm the specificity of the CA VI staining, anti-rat CA VI serum was absorbed with purified rat CA VI. Rat saliva samples were centrifuged for 10 min at 13,000 x g at 4C. A volume of 500 µl of the supernatant was incubated in constant rotation for 1 hr at 20C with 100 µl of inhibitor affinity gel matrix supplemented to 1 ml with buffer containing 0.1 M Tris-SO4 and 0.2 M sodium sulfate and Complete protease inhibitors dissolved and diluted according to the manufacturer's instructions at pH 7.0. The affinity gel matrix was prepared by coupling the CA inhibitor p-aminomethyl benzenesulfonamide to CM Bio-Gel A according to Khalifah et al. (1977). After incubation the gel was washed three times with 1.5 ml of the above-mentioned buffer. The gel was then incubated for 1 hr with anti-rat serum diluted 1:200 in 1% (w/v) BSA-PBS to absorb the CA VI antibodies from the serum to the inhibitor-bound CA VI. The gel was then centrifuged and the supernatant containing the serum devoid of anti-rat CA VI serum was used for IHC stainings. To confirm that the absorbed anti-rat CA VI serum was devoid of antibodies to CA VI, the antibodies bound to CA VI were eluted from the inhibitor affinity gel matrix using 50 µl of specific elution buffer containing 0.4 M NaN3, 0.1 M Tris-SO4, 0.1 mM benzamidine, and 20% (v/v) glycerol, pH 7.0. Aliquots (1 µl) of the eluate were subjected to immunoblotting with either rabbit anti-rat CA VI serum, absorbed anti-rat CA VI serum, or normal serum using dilution 1:10,000 in 0.1% (v/v) PBSTween-20. Immunoblotting in conjunction with anti-rat CA VI serum revealed that the eluate from the inhibitor affinity matrix contained the expected CA VI polypeptide bands of 41 kD (glycosylated form) and 37 kD (partially glycolysated form) and a CA VI antibody band (IgG) (Figure 1). Only one band representing IgG was detected with the absorbed serum and normal serum, indicating that the absorbed and normal sera lacked CA VI antibodies (Figure 1).
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Results |
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Controls
No difference was detected between the sialoadenectomized and the normal rats in the distribution of staining for CA VI in the trachea or lung (results not shown). Polyclonal rabbit antisera to human salivary -amylase and rat CA II used as positive controls (Spicer et al. 1982
) yielded distinct staining in the tracheobronchial glands (results not shown). CA II staining was diffusely distributed in the cytoplasm of all glandular cells, whereas salivary
-amylase was, similarly to CA VI, located exclusively in the apical secretory granules of the serous acinar cells (results not shown). Sections stained with anti-rat CA VI serum absorbed with purified rat CA VI and normal rabbit serum did not show positive staining, confirming that staining is CA VI-specific (Figures 2B and 2G).
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Discussion |
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Recent studies have provided evidence that CA VI is a multifunctional protein possessing growth factor activity in addition to carbonic anhydrase activity (Henkin et al. 1999a,b
; Karhumaa et al. 2001
). Gustin, a salivary secretory protein identical to CA VI (Thatcher et al. 1998
), has been demonstrated to protect the taste receptor cells from apoptosis (Henkin et al. 1999a
,b
). The exact mechanisms, however, remain unidentified. An attractive possibility is that, as in the taste buds, CA VI could also promote the maintenance of the lining epithelium in the respiratory tract. The mechanism may be related to pH neutralization or growth factor function via a specific receptor protein (Hooper et al. 1995
). It is also possible that CA VI is implicated in the acidification of the cytoplasm which, in turn, triggers apoptosis, one of the responses of the respiratory tract epithelium to an injury, via a pH-dependent pro-apoptotic regulator called Bax (Basbaum et al. 1990
; Antonsson et al. 1997
; Sok et al. 1999
). The association of CA VI with the brush border of the ciliated epithelial cells in the trachea and bronchi is another interesting finding. Acidic pH has been shown to lower the beat frequency of bronchial cilia (Clary-Meinesz et al. 1998
). Our results suggest that CA VI binds from the airway surface liquid to cilia and may be implicated locally in the regulation of their motility via pH neutralization. This could be one mechanism by which CA VI is linked to the protection of the respiratory tract epithelium from microbial infections because cilia motility reduces susceptibility of the airway epithelia to bacterial infection (Afzelius 1976
; Whitelaw et al. 1981
).
Our results here demonstrate that CA VI is secreted by the seromucous tracheobronchial glands, the serous epithelial cells of the trachea and bronchi, and by the Clara cells of the bronchioli. The presence of CA VI in the airway surface liquid is consistent with the hypothesis that CA VI has a mucosa-protective role not only in the gastrointestinal tract but also in the respiratory tract, where CA VI may act as a pH neutralizer and/or growth factor.
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Footnotes |
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Literature Cited |
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