Carbonic Anhydrase VI in the Mouse Nasal Gland
Departments of Oral Pathology (MK,YO), Oral and Maxillofacial Surgery II (MK,SI,YY), and Oral Radiology (TM), Osaka University Graduate School of Dentistry, Osaka, Japan, and CSIRO Health Science and Nutrition, Victoria, Australia (RTF)
Correspondence to: Yuzo Ogawa, DDS, PhD, Dept. of Oral Pathology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: ogawa{at}dent.osaka-u.ac.jp
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Summary |
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(J Histochem Cytochem 52:10571062, 2004)
Key Words: carbonic anhydrase VI nasal gland mouse immunohistochemistry Western analysis RT-PCR
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Introduction |
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A protein named gustin that is present in saliva and thought to be involved in the mechanism of taste has been described and has been shown to be identical to CA VI (Thatcher et al. 1998). Henkin et al. (1971)
(1975
) described a syndrome characterized by hypogeusia (loss of taste) with or without dysgeusia (distortion of taste) and hyposmia (loss of smell) with or without dysosmia (distortion of smell) after an influenza-like illness. The patients' saliva and nasal mucus characteristically showed decreased concentrations of CA (Doherty et al. 1997
; Henkin et al. 1999a
). After treatment with zinc, the enzyme concentration returned to normal and the taste and smell acuity were improved (Henkin et al. 1999b
). These facts suggest that CA VI is implicated in oral and nasal sensory functions. In fact, not only systemic but also topical application of a CA inhibitor, such as acetazolamide, alters taste sensation and eliminates the pungency or prickly sensation of carbonated drinks (Hansson, as quoted in Graber and Kelleher 1988
).
CA VI in tears is discharged through the nasolacrimal ducts into the nasal vestibule and may contribute to the nasal CA VI. Hansson's histochemistry has demonstrated CA activity in nasal glands of the guinea pig and bullfrog (Okamura et al. 1996; Coates et al. 1998
). A molecular analysis of the mucosal scrapings has suggested that CA VI is produced in the human nose (Tarun et al. 2003
). In the present study, using IHC in combination with biochemical and molecular techniques, we have localized CA VI in the mouse nasal gland. Therefore, CA VI is secreted by four exocrine glands: salivary, lacrimal, mammary, and nasal glands.
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Materials and Methods |
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For IHC, anesthetized mice were perfused through the left ventricle with 4% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2). The rostrum was removed, cleared of the surrounding soft tissues, and immersed in the same fixative overnight at 4C. The rostrum was then decalcified in 4.13% EDTA (pH 7.2) containing 0.2 M sucrose at 4C for a week, washed in 10 mM PBS, pH 7.2, containing 0.25 M sucrose, dehydrated, and embedded in paraffin. Coronal sections (6 µm thick) were cut and mounted on silane-coated glass slides.
Purification of Mouse CA and Production of Antibody to Mouse CA VI
Submandibular glands were homogenized on ice in 50 mM phosphate buffer (pH 7.4) containing 5 mM benzamidine and 1 mg/ml aprotinin. The homogenate was centrifuged (20,000 x g for 15 min at 4C). The supernatant was filtered through a 0.45-µm filter and loaded onto a sulfonamideSepharose affinity column. CA was isolated as described previously (Fernley et al. 1988). CA VI was purified from this salivary CA by anion exchange chromatography (Q Sepharose; Amersham Pharmacia Biotech, Uppsala, Sweden). The enzyme was eluted from the column with a linear salt gradient (00.5 M NaCl) in 50 mM Tris buffer (pH 8.0).
Rostrums were homogenized and the supernatant was prepared as described above. Nasal CA was isolated from the supernatant by the sulfonamide affinity chromatography, as was erythrocyte CA from erythrocyte lysate.
To raise a polyclonal antibody, 0.1 mg of purified CA VI was emulsified with an equal volume of Freund's complete adjuvant and injected SC into a male New Zealand rabbit. The rabbit was boosted with the same volume of the antigen in Freund's incomplete adjuvant according to the protocol by Fernley et al. (1988). Two weeks after the second booster, exsanguination was performed under anesthesia with pentobarbital sodium (50 mg/kg bw) and the antiserum was obtained. Immunoglobulin was prepared from the antiserum by protein G affinity chromatography (HiTrap Protein G HP; Amersham Pharmacia Biotech).
Western Analysis
Western analysis was performed as described previously (Ogawa et al. 2002). CA samples were run on a 10.5% SDS-polyacrylamide gel and electroblotted onto Immobilon (Millipore; Bedford, MA). The blot was blocked with 10% skimmed milk powder in 20 mM Tris-buffered saline (pH 7.5; TBS) and reacted with anti-CA VI antibody (1:5000 in 3% skimmed milk powder in TBS) or anti-rat erythrocyte CA antibody (1:10000; Ogawa et al. 1992
). The blot was then reacted with peroxidase-conjugated goat anti-rabbit IgG (1:2500; Bio-Rad Laboratories; Richmond, CA) and the complex was visualized by treatment with 3,3'-diaminobenzidine tetrahydrochloride-H2O2 solution (DAB-H2O2).
RT-PCR Analysis
Total RNA was isolated separately from nasal mucosa, salivary gland, and liver, and RT and PCR were performed as described previously (Ogawa et al. 2002). For PCR, 35 cycles of denaturation (94C, 30 sec), annealing (58C, 30 sec), and extension (72C, 40 sec) were performed in a DNA thermal cycler (Perkin-Elmer; Norwalk, CT). The primer sequences for mouse CA VI were 5'-CTGTGTTAGCCGTCTTGTTTA-3' and 5'-TTGAATGGTGTTGTTGTTGTG-3', which generate a 302-bp fragment (Sok et al. 1999
). The primers for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a constitutively expressed housekeeping gene, were 5'-AAGCAACATAGACGTTGTCGC-3' and 5'-AATCAACACCTTCTTCGCACC-3', which generate a 286-bp fragment (Tso et al. 1985
).
Immunohistochemistry
Deparaffinized sections were stained by an avidinbiotinperoxidase technique (Ogawa et al. 1999). After treatment with 0.3% H2O2 and then with normal swine serum (1:100 in PBS containing 1% bovine serum albumin; PBS-BSA), the sections were reacted with anti-CA VI antibody (1:5000 in PBS-BSA) or anti-rat erythrocyte CA antibody (1:10000). Then they were sequentially reacted with biotinylated swine anti-rabbit IgG (1:500 in PBS-BSA containing 1% normal mouse serum; DAKO, Glostrup, Denmark) and streptavidinbiotinylated peroxidase reagent (1:100 in PBS; DAKO). After incubation with DAB-H2O2, the sections were briefly counterstained with Mayer's hematoxylin, dehydrated, and coverslipped with Permount.
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Results |
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Discussion |
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CA VI is carried through the ducts of AGNS and LNG, which open into the nasal vestibule (Warshawsky 1963; Bojsen-Møller 1964
). CA VI immunoreactivity in the respiratory and olfactory mucus indicates that the enzyme has been dispersed throughout the nasal mucus, probably by atomization into the inhaled air current at a point of maximal air velocity and minimal air pressure (Bojsen-Møller 1964
) and by mucociliary transport (Jones 2001
). This mechanism has been proposed for the other mucous proteins such as OBP. OBP binds a variety of odorants with little specificity for chemical structures, and is believed to function in olfactory sensation (Pevsner and Snyder 1990
; Pelosi 1994
). Like CA VI, it is secreted by AGNS and LNG and is distributed throughout the nasal cavity (Pevsner and Snyder 1990
; Pelosi 1994
; Rama Krishna et al. 1994
).
CA VI is presumed to provide a greater buffering capacity to the nasal mucus and thus to maintain the integrity of the underlying tissues. Inhaled acids and acids generated by CO2 (see below) would be rapidly neutralized and, at the optimal pH, environmental toxins and microbes could be efficiently eliminated by proteins in the mucus such as cytochrome P-450, lysozyme, and lactoferrin (Anholt 1993; Shusterman and Avila 2003
). The CA VI activity should bring about the balanced ion concentration that is necessary for depolarization of the olfactory receptor neurons during olfactory transduction (Anholt 1993
; Paysan and Breer 2001
). Patients with gustatory and olfactory dysfunction after an influenza-like illness showed decreased CA in the saliva and the nasal mucus as well as apoptotic degeneration of the taste bud cells (Henkin et al. 1971
,1975
; Doherty et al. 1997
; Henkin et al. 1999a
). When the CA level was restored to normal by zinc treatment, the taste and smell acuity improved and the taste bud pathology disappeared (Henkin et al. 1999b
). CA VI has been detected in taste buds, taste pores, and in saliva secreted from von Ebner's gland in direct contact with taste buds in circumvallate papillae (Leinonen et al. 2001
). These results in saliva could be consistent with a role for CA VI in taste function through its action as a growth factor acting through stem cell stimulation, resulting in maturation of taste cell anatomy (Henkin et al. 1999b
). The present results suggest a similar action for nasal mucus in smell function through direct effects on olfactory epithelial cells.
CA is implicated in CO2 sensing by the oral mucosa (Komai and Bryant 1993). There appear to be two types of CO2 receptors in nasal cavity: trigeminal nerve endings stimulated by high concentrations of CO2 (noxious CO2) and olfactory neurons by low concentrations of CO2 (respiratory CO2). Both receptor responses are attenuated by acetazolamide (Coates 2001
), indicating that CA is also implicated in CO2 sensing by nasal mucosa. Because there is a 30-µm-thick mucus barrier between the nasal atmosphere and the nasal mucosa (Anholt 1993
), it is conceivable that CA VI, through hydration of CO2, facilitates CO2 diffusion into the mucus and thus detection by the mucosa of CO2 changes in the atmosphere. In the rat and guinea pig, a small subset of the olfactory neurons possessed CA activity (Brown et al. 1984
; Okamura et al. 1996
; Coates 2001
). The present study has demonstrated that this is also the case for the mouse, and the enzyme activity is attributed to CA II, the isozyme widely distributed throughout the body, including CO2 chemoreceptors (Lahiri 1991
, Neubauer 1991
, Yamamoto et al. 2003
). The high activity isozyme would enable the olfactory neurons to detect low levels of CO2. CO2 in the mucus readily diffuses across the cell membrane and is catalyzed by the intracellular CA II. This increases the intracellular H+, which elicits the signal transduction process (Lahiri 1991
; Neubauer 1991
). To our knowledge, CA has not been localized in the trigeminal nerve endings. Therefore, they may require a different mechanism. CO2 in the mucus should be hydrated by CA VI to raise the local concentration of H+ (Shusterman and Avila 2003
). For large amounts of CO2, the concentration would be sufficient to activate the nociceptors by gating of the acid-sensitive ion channels and/or H+ influx through the H+ or Na+ channel (Simons et al. 1999
).
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Footnotes |
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