ARTICLE |
Correspondence to: Jukka Leinonen, Dept. of Anatomy and Cell Biology, PO Box 5000, 90014 University of Oulu, Finland. Fax: 358-8-537 5172
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Salivary carbonic anhydrase VI (CA VI) appears to contribute to taste function by protecting taste receptor cells (TRCs) from apoptosis. The serous von Ebner's glands locating in the posterior tongue deliver their saliva into the bottom of the trenches surrounding the TRC-rich circumvallate and foliate papillae. Because these glands deliver their saliva directly into the immediate vicinity of TRCs, we investigated whether CA VI is secreted by the von Ebner's glands, using immunochemical techniques. The immunohistochemical results showed that CA VI is present in the serous acinar cells, ductal cells, and ductal content of von Ebner's glands and in the demilune and ductal cells plus ductal content of rat lingual mucous glands. More importantly, CA VI was also detected in taste buds and in the taste pores. Western blotting of saliva collected from the orifices of human von Ebner's glands and CAs purified from rat von Ebner's glands confirmed that CA VI is expressed in these glands and secreted to the bottom of the trenches surrounding the circumvallate and foliate papillae. These findings are consistent with the hypothesis that locally secreted CA VI is implicated in the paracrine modulation of taste function and TRC apoptosis. (J Histochem Cytochem 49:657662, 2001)
Key Words: carbonic anhydrase, human, immunohistochemistry, rat, saliva, taste, von Ebner's glands
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Carbonic anhydrases (EC 4.2.1.1: CA) catalyze the reversible reaction H++ HCO3-H2O + CO2 and are involved in ion, fluid, and acidbase balance in various tissues (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibodies and Chemicals
Peroxidase-conjugated goat anti-rabbit IgG, biotin-conjugated swine anti-rabbit IgG, and streptavidin-conjugated peroxidase were obtained from Sigma Chemicals (St Louis, MO). Freund's adjuvants were obtained from Gibco (Grand Island, NY). CM Bio-Gel A, p-aminomethyl benzenesulfonamide, and all reagents for SDS-PAGE and transblotting were purchased from Bio-Rad Laboratories (Richmond, CA). Complete protein inhibitor tablets were purchased from BoehringerMannheim (Mannheim, Germany). Pilocarpin was a product of Santen (Napa, CA).
Tissue and Saliva Samples
Ten male SpragueDawley rats were sacrificed and their tongues, kidneys, and parotid glands were dissected out. Rat saliva was collected from anesthetized animals. Secretion of saliva was stimulated by injecting 6 mg/kg of pilocarpin IP. The rats were bled from the heart after collection of saliva. Human tongue and parotid gland specimens were obtained from the Department of Forensic Medicine, University of Oulu, and from patients undergoing surgery for lingual carcinoma at the Clinic of Otorhinolaryngology, Oulu University Hospital. Saliva from human von Ebner's glands was collected from healthy volunteers' circumvallate papillae with a micropipette and from foliate papillae with nitrocellulose sheets (
Production of Anti-CA VI Sera
Rat CA VI was purified from saliva using inhibitor-affinity chromatography. The saliva samples were centrifuged for 10 min at 13,000 x g at 4C and the supernatant was used for purification. An affinity chromatography matrix was prepared by coupling the CA inhibitor p-aminomethyl benzenesulfonamide to CM Bio-Gel A according to
The production and characterization of rabbit anti-human CA VI serum have been described by
Immunohistochemistry and Western Blotting
Tissue samples for immunohistochemistry were fixed in Carnoy's fluid or 4% paraformaldehyde for 18 hr and embedded in paraffin. Thick sections of 5 µm were subjected to immunohistochemistry. Immunohistochemical staining was performed using the immunoperoxidase procedure as described by
Parotid glands, rat von Ebner's glands, and kidneys were homogenized in 0.1 M Tris-SO4, pH 8.7, containing Complete protease inhibitors dissolved and diluted according to the manufacturer's instructions. The homogenized parotid glands, rat von Ebner's glands, and saliva samples were then centrifuged for 10 min at 13,000 x g at 4C. The resulting supernatants were used for the experiments. CAs were purified from the homogenized von Ebner's glands using inhibitor-affinity chromatography as described in detail by
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Characterization of Anti-rat CA VI Serum
Rabbit antiserum raised against rat CA VI identified two major polypeptides of 41 and 37 kD in rat saliva, purified rat CA VI, and homogenized parotid gland plus a minor 56-kD polypeptide in rat whole saliva (Fig 1, Lanes 13, respectively). The 56-kD polypeptide band is probably attributable to protein complex formation because its intensity increased during storage of the sample (data not shown). The 37-kD band most probably represents a partially deglycosylated form of CA VI reported earlier in the human enzyme (
|
Immunohistochemistry
Immunohistochemical staining shows that CA VI is abundantly expressed in von Ebner's glands, which empty into the bottom of the trenches of the papillae (Fig 2A). An intense peroxidase reaction is seen in the serous acinar cells of both human (Fig 2B) and rat (Fig 2C) von Ebner's glands. In addition, CA VI was found in the demilune cells of rat lingual mucous glands (Fig 2D). Ductal content and cells in rat von Ebner's glands and mucous glands also stained for CA VI (Fig 2C and Fig 2D). Interestingly, taste pores and some cells in rat taste buds stained specifically for CA VI (Fig 2E and Fig 2F). Control sections stained with preimmune serum remained unstained (Fig 2G and Fig 2H). Sections from rat parotid and submandibular glands stained for positive control with anti-CA VI serum yielded a specific reaction in serous acinar cells (data not shown).
|
Western Blotting
Immunoblotting experiments confirmed that the immunohistochemical stainings represent CA VI staining. Antiserum to human CA VI recognized a strong 40-kD polypeptide band from circumvallate and foliate papillae saliva (Fig 3, Lanes 1 and 2, respectively). A similar 40-kD band was also recognized in human whole saliva, purified human salivary CA VI, and homogenized human parotid gland (Fig 3, Lanes 35, respectively). In addition, a weak 56-kD polypeptide band was seen in the human von Ebner's gland saliva collected from the foliate papilla. Antiserum to rat CA VI recognized 41-kD and 37-kD polypeptide bands in the rat CA VI purified from von Ebner's glands (Fig 3, Lane 6). Preimmune serum did not recognize any polypeptide bands in the control immunoblots (data not shown).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recently,
Although we provide no functional data on the linkage of CA VI with taste function here, at least three possible roles for CA VI can be proposed on the basis of previous studies. First, CA VI is an enzymatically active member of the -CA gene family and, accordingly, the enzyme present in von Ebner's gland saliva may be linked to pH in the immediate proximity of the taste buds. Acid stimuli are known to inactivate the proton-gated potassium channels that result in depolarization of TRCs (
Second, CA VI present in von Ebner's gland saliva may also affect the apoptosis of TRCs in the taste buds of the circumvallate and foliate papillae (
Third, CA VI may function as a paracrine growth factor in TRCs. This hypothesis is supported by the present finding that CA VI was indeed located in the taste pores and in some cells within the taste buds. The CA VI-positive cells in the taste buds were not identified here. Nevertheless, they do not appear to represent CA II-producing cells (Type I) because these cells are more abundant than the CA VI-positive cells (
![]() |
Acknowledgments |
---|
Supported by grants from the Finnish Cultural Foundation and the Finnish Dental Association to Jukka Leinonen.
We gratefully thank Ms Lissu Hukkanen, Ms Pirkko Peronius, and Mr Eero Oja for technical assistance.
Received for publication August 28, 2000; accepted December 20, 2000.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aldred 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]
Brown D, GarciaSegura LM, Orci L (1984) Carbonic anhydrase is associated with taste buds in rat tongue. Brain Res 324:346-348[Medline]
Calvert R, Millane G, Pothier P, Beaulieu JF (1993) An intestinal secretory protein is found in most glands associated with the gastrointestinal tract: von Ebner's and salivary glands, gallbladder, and pancreas. J Histochem Cytochem 41:1223-1231
Chandrashekar J, Mueller KL, Hoon MA, Adler E, Feng L, Guo W, Zuker CS, Ryba NJ (2000) T2Rs function as bitter taste receptors. Cell 100:703-711[Medline]
Daikoku H, Morisaki I, Ogawa Y, Maeda T, Kurisu K, Wakisaka S (1999) Immunohistochemical localization of carbonic anhydrase isozyme II in the gustatory epithelium of the adult rat. Chem Senses 24:255-261
Feldstein JB, Silverman DN (1984) Purification and characterization of carbonic anhydrase from the saliva of the rat. J Biol Chem 259:5447-5453
Fernley RT, Wright RD, Coghlan JP (1979) A novel carbonic anhydrase from the ovine parotid gland. FEBS Lett 105:299-302[Medline]
Graber M, Kelleher S (1988) Side effects of acetazolamide: the champagne blues. Am J Med 84:979-980[Medline]
Gurkan S, Bradley RM (1988) Secretions of von Ebner's glands influence responses from taste buds in rat circumvallate papilla. Chem Senses 13:655-661
Hamilton SR, Farber JL, Rubin E (1998) The gastrointestinal tract. In Rubin E, Farber JL eds. Pathology. 3rd ed. Philadelphia, LippincotRaven, 676693
Henkin RI, Law JS, Nelson NR (1988) The role of zinc in on the trophic growth factors nerve growth factor and gustin. In Hurley LS, Keen CL, Lonnerdal B, Rucker RB, eds. Trace Elements in Man and Animals. Vol 6. New York, Plenum Press, 385-388
Henkin RI, Martin BM, Agarwal RP (1999a) Decreased parotid saliva gustin/carbonic anhydrase VI secretion: an enzyme disorder manifested by gustatory and olfactory dysfunction. Am J Med Sci 318:380-391[Medline]
Henkin RI, Martin BM, Agarwal RP (1999b) Efficacy of exogenous oral zinc in treatment of patients with carbonic anhydrase VI deficiency. Am J Med Sci 318:392-405[Medline]
Jiang W, Woitach JT, Gupta D (1996) Sequence of bovine carbonic anhydrase VI: potential recognition sites for N-acetylgalactosaminyltransferase. Biochem J 318:291-296[Medline]
Karhumaa P, Parkkila S, Waheed A, Parkkila A-K, Kaunisto K, Tucker PW, Huang C-J, Sly WS, Rajaniemi H (2000) Nuclear NonO/p54nrb protein is a nonclassical carbonic anhydrase. J Biol Chem 275:16044-16049
Khalifah RG, Strader DJ, Bryant SH, Gibson SM (1977) Carbon-13 nuclear magnetic resonance probe of active-site ionizations in human carbonic anhydrase B. Biochemistry 16:2241-2247[Medline]
Kinnamon SC, Dionne VE, Beam KG (1988) Apical localization of K+ channels in taste cells provides the basis for sour taste transduction. Proc Natl Acad Sci USA 85:7023-7027[Abstract]
Kinnamon SC, Roper SD (1987) Passive and active membrane properties of mudpuppy taste receptor cells. J Physiol (Lond) 383:601-614[Abstract]
Kinnamon SC, Roper SD (1988) Membrane properties of isolated mudpuppy taste cells. J Gen Physiol 91:351-371[Abstract]
Kivelä A, Parkkila S, Saarnio J, Karttunen TJ, Kivelä J, Parkkila AK, Waheed A, Sly WS, Grubb JH, Shah G, Tureci O, Rajaniemi H (2000) Expression of a novel transmembrane carbonic anhydrase isozyme XII in normal human gut and colorectal tumors. Am J Pathol 156:577-584
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685[Medline]
Leinonen J, Kivela J, Parkkila S, Parkkila AK, Rajaniemi H (1999) Salivary carbonic anhydrase isoenzyme VI is located in the human enamel pellicle. Caries Res 33:185-190[Medline]
Li XJ, Snyder SH (1995) Molecular cloning of ebnerin, a von Ebner's gland protein associated with taste buds. J Biol Chem 270:17674-17679
Lyall V, Feldman GM, Heck GL, DeSimone JA (1997) Effects of extracellular pH, PCO2, and HCO3- on intracellular pH in isolated rat taste buds. Am J Physiol 273:C1008-1019
MorrisWiman J, Sego R, Brinkley L, Dolce C (2000) The effects of sialoadenectomy and exogenous EGF on taste bud morphology and maintenance. Chem Senses 25:9-19
MossSalentijn L, Klyvert M (1980) Dental and Oral Tissues. 1st ed London, Henry Kimpton Publishers, pp. 4851
Murakami H, Sly WS (1987) Purification and characterization of human salivary carbonic anhydrase. J Biol Chem 262:1382-1388
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]
Parkkila S, Parkkila AK, Vierjoki T, Stahlberg T, Rajaniemi H (1993) Competitive time-resolved immunofluorometric assay for quantifying carbonic anhydrase VI in saliva. Clin Chem 39:2154-2157
Simons CT, Dessirier JM, Carstens MI, O'Mahony M, Carstens E (1999) Neurobiological and psychophysical mechanisms underlying the oral sensation produced by carbonated water. J Neurosci 19:8134-8144
Sly WS, Hu PY (1995) Human carbonic anhydrases and carbonic anhydrase deficiencies (review). Annu Rev Biochem 64:375-401[Medline]
Spielman AI, D'Abundo S, Field RB, Schmale H (1993) Protein analysis of human von Ebner saliva and a method for its collection from the foliate papillae. J Dent Res 72:1331-1335[Abstract]
Thatcher BJ, Doherty AE, Orvisky E, Martin BM, Henkin RI (1998) Gustin from human parotid saliva is carbonic anhydrase VI. Biochem Biophys Res Commun 250:635-641[Medline]
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350-4354[Abstract]
Wong GT, Gannon KS, Margolskee RF (1996) Transduction of bitter and sweet taste by gustducin. Nature 381:796-800[Medline]