ARTICLE |
Correspondence to: Yuzo Ogawa, Dept. of Oral Pathology, Osaka U. Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: ogawa@dent.osaka-u.ac.jp
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Summary |
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We have previously demonstrated by immunohistochemistry the presence of secreted carbonic anhydrase (CA VI) in the acinar cells of the rat lacrimal glands. In this study we purified the sheep lacrimal gland CA VI to homogeneity and demonstrated by Western analysis that it has the same apparent subunit molecular weight (45 kD) as the enzyme isolated from saliva. RT-PCR analysis showed that CA VI mRNA from the lacrimal gland was identical to that of the parotid gland CA VI mRNA. An RIA specific for sheep CA VI showed the lacrimal gland tissue concentration of the enzyme to be 4.20 ± 2.60 ng/mg protein, or about 1/7000 of the level found in the parotid gland. Immunohistochemistry (IHC) and in situ hybridization (ISH) showed that lacrimal acinar cells expressed both immunoreactivity and mRNA for CA VI. Moreover, CA VI immunoreactivity was occasionally observed in the lumen of the ducts. Unlike the parotid gland, in which all acinar cells expressed CA VI immunoreactivity and mRNA, only some of the acinar cells of the lacrimal gland showed expression. These results indicate that the lacrimal gland synthesizes and secretes a very small amount of salivary CA VI. In tear fluid, CA VI is presumed to have a role in the maintenance of acid/base balance on the surface of the eye, akin to its role in the oral cavity.
(J Histochem Cytochem 50:821827, 2002)
Key Words: carbonic anhydrase VI, lacrimal gland, parotid gland, saliva, sheep, Western analysis, radioimmunoassay, RT-PCR, immunohistochemistry, in situ hybridization
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Introduction |
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CARBONIC ANHYDRASE (CA: EC. 4.2.1.1), a zinc metalloenzyme, catalyzes the reversible hydration of carbon dioxide and has widespread tissue and species distribution. As many as eleven isozymes of CA with defined enzyme activities have been reported in mammals (-class of carbonic anhydrases and share similar structures (
The secreted isozyme was first described in the saliva of sheep (
It was reported previously that, in a screening of some 18 different tissues in the sheep, the enzyme was found only in the parotid and submandibular salivary glands (
In this study we have demonstrated by a number of techniques, including Western analysis, RT-PCR, IHC, and ISH, that CA VI is indeed synthesized by, stored in, and secreted from the sheep lacrimal gland.
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Materials and Methods |
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Animals used were mature female sheep (Merino and Merino crossbred strains). CA VI was purified to homogeneity from parotid saliva as described previously (
Western Analysis
Glands from three sheep were homogenized in 50 mM phosphate buffer (pH 7.4) containing 5 mM benzamidine, 1 mM phenylmethanesulfonyl fluoride, and 1 mg/ml aprotinin. The homogenate was centrifuged (20,000 x g for 15 min at 4C). Partially purified CA VI was prepared by ammonium sulfate precipitation, followed by sulfonamideSepharose affinity chromatography (
Radioimmunoassay
Radioimmunoassay of sheep CA VI was carried out as described previously (
Immunohistochemistry
Deparaffinized sections were stained by an indirect immunoperoxidase technique (
RT-PCR Analysis
Total RNA was isolated from glands of two sheep using the Total RNA Isolation System (Promega; Madison, WI) and digested with RNase-free DNase I (Promega) for 15 min at 37C. Five µg of the denatured total RNA was reverse-transcribed in 20 µl of a reaction mixture containing 5 x RT buffer (375 mM KCl, 15 mM MgCl2, and 50 mM dithiothreitol in 250 mM Tris-HCl, pH 8.3), 0.5 mM each dNTP, 0.5 mg oligo(dT)15 primers, 100 U RNase inhibitor, and 200 U M-MLV reverse transcriptase (Wako Pure Chemicals; Osaka, Japan) for 1 hr at 37C and then heated for 10 min at 70C. After RT reaction, PCR was carried out in a total volume of 25 µl of a mixture containing 1 µl RT sample, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 0.2 mM each dNTP, 0.4 mM each primer and 1.0 U AmpliTaq Gold DNA polymerase (PerkinElmer; Norwalk, CT). Forty cycles of denaturation (94C, 30 sec), annealing (60C, 1 min) and extension (72C, 1 min) were carried out in a DNA thermal cycler (PerkinElmer). The primer sequences for CA VI were 5'-GATACATAATTGAGATTCACGTCGTCCACTAC-3' and 5'-GCTTGACAGTATCTGCTACCACAAACCA-3'. They were designed according to the nucleotide sequence of bovine submandibular CA VI (
Aliquots (10 µl) of the PCR products were run on in a 2% agarose gel. The amplified products were excised from the gel, purified using Qiaex II (Qiagen; Hilden, Germany), and sequenced. The purified parotid product was subcloned into pT7Blue vector (Novagen; Madison, WI) using the Perfectly BluntTM Cloning Kit (Novagen).
In Situ Hybridization
Two types of recombinant pT7Blue vector, each containing a 261-bp coding region of sheep parotid CA VI with different orientations, were obtained by screening the pT7 library and were used as templates of antisense and sense riboprobes. One mg of each plasmid was linearized with BamHI (BoehringerMannheim; Mannheim, Germany). In vitro transcription was performed with digoxygenin-11-UTP (BoehringerMannheim) using T7 RNA polymerase (Nippongene; Tokyo, Japan).
The following procedures were carried out at RT unless otherwise noted. Deparaffinized sections were washed in PBS, incubated in 1 µg/ml proteinase K (Nippongene) for 10 min, and washed in PBS. Nonspecific binding sites were blocked by acetylation with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 15 min. The sections were washed in 4 x SSC (600 mM NaCl in 60 mM sodium citrate) and prehybridized in 50% formamide in 2 x SSC for 30 min at 42C. Hybridization was carried out in 40 µl of a solution containing 50% formamide, 2 x SSC, 1 mg/ml tRNA, 1 mg/ml salmon sperm DNA, 1 mg/ml BSA, 10% dextran sulfate, and 1 µg/ml probe for 16 hr at 42C. The sections were washed three times in 50% formamide in 2 x SSC for 20 min each at 42C, treated for 30 min at 37C with 20 µg/ml RNase A (Nippongene) in 10 mM Tris-HCl (pH 8.0) containing 0.5 M NaCl and 1 mM EDTA, and washed three times in 0.1 x SSC for 20 min each at 42C. Detection of the labelled probe was carried out using the DIG Nucleic Acid Detection Kit (BoehringerMannheim) according to the manufacturer's protocol with modifications. The modifications were as follows: (a) maleic acid buffer and detection buffer were substituted with 0.1 M Tris-HCl (pH 7.5) containing 0.15 M NaCl and 0.1 M Tris-HCl (pH 9.5) containing 0.1 M NaCl and 0.05 M MgCl2, respectively; (b) incubation in blocking solution and that in antibody solution were performed for 1 hr each. After washing in 10 mM Tris-HCl (pH 8.0) with 1 mM EDTA, the sections were coverslipped with glycerol gelatin (Sigma Diagnostics; St Louis, MO), with or without methyl green counterstain.
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Results |
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Western Analysis and Radioimmunoassay
CA was isolated from lacrimal glands by affinity and ion-exchange chromatography, as for the parotid enzyme. However, the amount of enzyme recovered was very much smaller than that from the parotid. Fresh sulfonamide affinity resin was used for isolation of the lacrimal gland enzyme, and the Mono Q column was cleaned with 0.5 M NaOH and 1 M acetic acid before use to prevent contamination of the lacrimal gland preparation with the much more abundant salivary gland enzyme. Blank runs confirmed the absence of any contaminating salivary CA VI. Purified enzyme was run on a gel, blotted, and probed with the anti-CA VI antibody. The lacrimal gland CA had an apparent molecular weight of about 45 kD, the same as the salivary enzyme (Fig 1). The CA isolated from lacrimal glands appears to be the same as the salivary gland enzyme.
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To measure the amount of CA VI present in the lacrimal gland, a specific RIA for sheep CA VI was used. Lacrimal glands from 10 sheep were homogenized separately and centrifuged, and the CA VI concentration was measured by this RIA. The protein concentrations of the supernatants were measured and the amount of enzyme/mg total protein calculated. The individual values along with age of the sheep are shown in Table 1. The CA VI content of the lacrimal gland was 0.35 ± 0.22 µg/g tissue (mean ± SD) and the concentration was 4.20 ± 2.60 ng/mg protein with a range of 1.469.12 ng/mg. The content and concentration for the parotid gland from sheep #10 were 1.55 mg/g tissue and 29.9 µg/mg protein. There is about 7000 times the concentration of CA VI in the parotid tissue as there is in the lacrimal tissue.
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RT-PCR Analysis
RT-PCR was performed using 5'-primer and 3'-primer corresponding to positions 446477 and positions 739766, respectively, of the nucleotide sequence of bovine submandibular CA VI (
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A cDNA probe was obtained from the plasmid containing the 261-bp fragment of sheep CA VI and was used for Northern analysis. This probe detected CA VI mRNA in the parotid gland but not the lacrimal gland (data not shown).
Immunohistochemistry and In Situ Hybridization
The polyclonal antibody specific for highly purified sheep secreted CA was found to label only occasional acinar cells in occasional lobules in the sheep lacrimal gland (cf. Fig 4D). Therefore, the number of cells containing the enzyme was far less than in the parotid gland, in which all acinar cells appeared to contain CA VI (Fig 4B). The immunostaining pattern was basically similar between the indirect immunoperoxidase technique and the avidinbiotinperoxidase technique. Although the latter technique slightly enhanced the staining intensity and increased the number of stained cells, these were still a minority of the secretory cells. The avidinbiotinperoxidase technique sometimes generated nonspecific staining such as those in the nuclei (Fig 4A, inset). Lacrimal glands are of the compound serous tubuloalveolar type. The secretory cells, which we refer to as "acinar cells" in this manuscript, can be divided into alveolar secretory cells and tubular secretory cells (
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In the salivary gland, the acinar immunostaining often took a granular configuration and the duct lumen sometimes contained positive staining (arrow in Fig 4B), indicating that the enzyme was stored in the secretory granules in the acinar cells and secreted into the duct lumen from these cells. In contrast, the intensity of the immunostaining, even by the avidinbiotinperoxidase technique, was extremely weak in the lacrimal gland (compare Fig 4A and Fig 4B). The weak immunostaining made it difficult to judge whether the staining had a granular configuration (Fig 4A). However, the duct luminal content was occasionally labeled by the avidinbiotinperoxidase technique (Fig 4A, inset), indicating that the enzyme was secreted into the duct lumen. Such labeling was much less common in the lacrimal gland than in the salivary gland, and was not detected by the indirect immunoperoxidase technique.
The above specific staining was not seen when sections were reacted with normal rabbit IgG (Fig 4C), primary antibody pre-absorbed with CA VI, or a DIG-labeled sense probe (Fig 4F).
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Discussion |
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In this study, Western and RT-PCR analyses have confirmed that sheep lacrimal gland synthesizes CA VI and that this enzyme appears to be identical to the salivary enzyme. An RIA specific for sheep CA VI has demonstrated that the concentration of CA VI in the lacrimal gland is very much less than that in the salivary gland. This would explain why the relatively insensitive Northern analysis of lacrimal gland mRNA failed to detect CA VI mRNA. From the IHC and ISH results, it appears that the amount of CA VI in the lacrimal gland, as compared to the parotid gland, is due to the fact that only a small minority of the lacrimal acinar cells synthesizes CA VI. A similar result was observed in rat lacrimal glands by IHC using a monoclonal antibody specific for rat CA VI (
The role of CA VI in tear fluid is presumably akin to its proposed role in saliva, i.e., regulation of pH through the reversible hydration of CO2. On the exposed surface of ocular globe, the concentration of CA VI increases by evaporation of water from tear fluid into the surrounding air between consecutive blinks (
Previously, a range of tissues, including the pancreas, was examined for the presence of CA VI by Western analysis (
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Footnotes |
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1 Present address: CSIRO Health Sciences & Nutrition, Parkville, Victoria, Australia.
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Acknowledgments |
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Supported in part by an Institute grant from the National Health and Medical Research Council of Australia.
We thank Drs Haruhiko Takada, Yoshiaki Obara, and Takahiro Yamaguchi for their technical assistance.
Received for publication September 26, 2001; accepted January 16, 2002.
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