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MUCOSAL BIOLOGY
Department of Molecular Oral Physiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
Submitted 30 September 2005 ; accepted in final form 15 July 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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water channel; salivary gland
In a previous study (11), we found that the AQP5 expression level in the SMG was divergent among individual rats of the Sprague-Dawley (SD) strain, allowing us to classify them into two groups, i.e., high AQP5 producers and low AQP5 producers (they will be referred hereafter as "high producers" and "low producers," respectively). The offspring of high producers expressed a significantly higher level of SMG AQP5 than those from low producers, suggesting that the different phenotype in the SMG AQP5 expression is a transmitted hereditary characteristic. The hybrid offspring between high and low producers showed either a high or an intermediate level of AQP5 expression, implying that the high level of AQP5 expression may be dominant. Because the result of our previous study suggested the existence of genetic diversity, in the present study, therefore, we sought to explore the cause of this diversity and to study the effects of such a difference on salivary secretion.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Animals. The SD rats were obtained from Japan SLC (Shizuoka, Japan) and maintained in our animal facility. The protocol applied for the present animal experiment was approved by the Institutional Review Board of the Animal Committee of the University of Tokushima. Rats expressing high-level AQP5 and low-level AQP5 were determined as described previously (11), and breeding between brothers and sisters was repeated after the AQP5 level in their SMG had been confirmed by Western blotting. The 5th generation progenies of high and low producers were obtained in this way. These rats were killed by cervical dislocation, and the SMG, parotid gland, lacrimal gland, and lungs were excised. These tissues were analyzed for their levels of AQP5 proteins by Western blotting and AQP5 mRNA by Northern blotting. AQP5 cDNAs were PCR cloned from total RNA of the SMG, and their sequences were determined. Rats of the 3rd, 6th, and 11th generations were also used in an in vivo study for determination of salivary secretion.
Preparation of membrane fraction and Western blotting. Total membrane fraction was prepared as described previously (13). Briefly, tissue specimens were homogenized in 9 vol (wt/vol) of ice-cold homogenization buffer [5 mM HEPES buffer (pH 7.5), 50 mM mannitol, 10 mM MgCl2, 1 mM PMSF, 1 µg/ml aprotinin, 2 µg/ml peptstatin A, 2 µg/ml leupeptin, and 1 tablet of Complete EDTA-free protease inhibitor cocktail per 25 ml of buffer], and cell debris and nuclei were removed by centrifugation at 800 g for 5 min at 2°C. The supernatant thus obtained was centrifuged at 105,000 g for 1 h at 4°C to obtain the total membrane fraction. Protein concentrations were determined by a Bio-Rad protein assay kit using BSA as a standard (2).
Tissue protein samples (5 µg each) from 5th-generation rats were subjected to Western blotting (12% polyacrylamide gels were employed for SDS-PAGE). The proteins separated on the gel were transferred onto a nitrocellulose filter according to Towbin et al. (20) and immunoreacted with 1:1,000-diluted anti-AQP5 antiserum and 1:3,000-diluted peroxidase-labeled anti-rabbit IgG according to a standard procedure. Filters were reacted with chemical luminescence reagents (ECL detection kit) and subsequently exposed to X-ray films. Anti-AQP5 antiserum was prepared in our laboratory, and its specificity had been verified earlier (13, 14, 19); i.e., a 27-kDa band was specifically detected in the sample of the SMG and other tissues, and this band completely disappeared when the antibody was preabsorbed with AQP5 COOH-terminal peptide used for immunization.
RNA isolation and Northern blotting. Total RNA was isolated from the SMG of 5th-generation high- and low-producer adult male SD rats by using TRI reagent according to the manufacturer’s protocol. Total RNA (10 µg) was mixed with 10–15 µl of sample buffer composed of 1x MOPS solution (20 mM MOPS, 5 mM sodium acetate, and 1 mM EDTA; pH 7.0), 50% formamide, 6.4% formaldehyde, and 5.3% autoclaved glycerol prepared freshly, resolved by electrophoresis in a 1% agarose gel containing 2% formaldehyde, and transferred onto a nylon membrane (Hybond-N+). The membrane was prehybridized at 42°C for at least 3 h and then hybridized at 42°C for more than 15 h in hybridization buffer composed of 5x SSPE (5-fold higher concentration of 1x SSPE, which was composed of 150 mM NaCl, 10 mM sodium phosphate, and 1 mM EDTA; pH 7.4), 50% formamide, 2.5x Denhard’s solution, 10% SDS, 0.2 mg/ml salmon sperm DNA, 10% dextran sulfate, and 50 ng/ml DIG-labeled cDNA probe specific for AQP5. DIG-labeled cDNA probe was prepared as described below. The membranes were washed with 2x SSPE at room temperature for 5 min, at 42°C for 20 min, and then twice with 1x SSPE at 42°C for 20 min each time. Before the membrane was incubated in 1% blocking solution at room temperature for 30–60 min, it was washed with washing buffer for 5 min. The membrane was reacted with 10,000 times-diluted anti-DIG-Fab conjugated with alkaline phosphatase in 1% blocking solution at room temperature for 30–60 min and then washed in washing buffer for 15 min twice. It was next equilibrated with buffer III, which consisted of 0.1 M Tris·HCl (pH 9.5), 0.1 M NaCl, and 50 mM MgCl2, and then treated with 100 times diluted CSPD in buffer III for 5 min. The excess liquid was blotted onto Whatmann 3 MM filter paper, and the membrane was then placed in a hybridization bag, preincubated at 37°C for 15 min in an air oven, and finally exposed to Fuji RX X-ray film.
Synthesis of DIG-labeled cDNA probes. The DIG-labeled cDNA probe was prepared by PCR using DIG labeling mix and AQP5 cDNA as a template. Namely, 2.5 µl of PCR DIG-Labeling Mix, 5 pmol of each primer, 2 units of Taq DNA polymerase, 50 pg of template AQP5 cDNA, and 1x PCR buffer (supplied with Ex Taq polymerase, Takara) were mixed in a total volume of 25 µl to make the reaction mixture. The sequences of sense and antisense primers, as well as the PCR thermal conditions used in this labeling experiment, were the same as those shown below (see RT-PCR and PCR cloning and DNA sequencing). cDNA was prepared from total RNA of high-producer SMG by RT-PCR as described in the same section below and purified by the Qiagen spin column method.
RT-PCR and PCR cloning. Full-length AQP5 cDNA was synthesized by RT-PCR using a SuperScript One-Step RT-PCR system; i.e., 0.5 µg of template RNA, 5 pmol of each primer, 0.5 µl of RT/Taq mix, and 12.5 µl of 2x buffer were mixed to make a 25-µl reaction mixture. The RT reaction was carried out at 45°C for 30 min followed by DNA amplification by PCR for 35 cycles, each consisting of denaturation at 94°C for 15 s, primer annealing at 55°C for 30 s, and extension at 72°C for 1.5 min. The RT-PCRs were conducted in a TaKaRa PCR Thermal Cycler MP model TP 3000. The primer sets used in the present study were the following: rAQP5–1 (sense; 5'-CCCCAAGGCACCATGAAAAA-3') and Clo-2A (antisense; 5'-TCACGAATCTCTGAGGTCTG-3'), which were same as those reported previously (1, 20). The AQP5 cDNA (1.073 kb) thus synthesized was used for preparation of the DIG-cDNA probe described above (see Synthesis of DIG-labeled cDNA probes), and sequence analysis was carried out as described below.
The above cDNA products were ligated into pGEMR-T Easy Vectors. The constructs were then introduced into competent E. coli. The bacterial colonies transfected with a plasmid having a 1.073-kb insert were grown in 2 ml of Super Broth medium containing 100 µg/ml ampicillin, and plasmid DNAs were extracted by the alkaline extraction method (21).
For analysis of the sequence around the Kozak area of AQP5 mRNA, total RNA from the SMG from high- and low-producer (15th and 10th generations, respectively) rats was prepared. The region from nt –36 to +204 of AQP5 cDNA was synthesized by RT-PCR using 5'-GCAACCCTCCCGCTGCCA-3' (sense; corresponding to nt –36 to –19) and 5'-GATGTGGCCACCACTCACAG-3' (antisense; corresponding to nt +185 to +204) primers under the same RT-PCR thermal conditions as described above. The reaction mixture was treated with ExoSAP to remove single-stranded DNA, and the cDNA synthesized was subjected to direct sequencing as described below.
Extraction of genomic DNA and PCR amplification. For analysis of the sequence of exon 1 of the AQP5 gene, genomic DNA from the SMG, liver, and lungs from the 8th generation of low-producer rats was extracted following the standard procedures. Briefly, 100-mg tissues were cut into small pieces and digested with 100 µg/ml proteinase K in 10 mM Tris·HCl (pH 8.0) buffer containing 150 mM NaCl, 10 mM EDTA, and 0.1% SDS by incubation at 55°C for 1 h with occasional mixing and then at 37°C overnight (16 h). After the solution had been extracted with neutral phenol, the aqueous phase was separated and mixed with a mixture of phenol, chloroform, and isoamylalcohol (25:24:1) to precipitate the DNA. Exon 1 of the AQP5 gene was PCR amplified by using a set of primers, 5'-GGCACCATGAAAAAGGAGGT-3' (sense) and 5'-TGTTGTTCAGCGCATTGACG-3' (antisense) under the thermal conditions consisting of denaturation at 95°C for 30 s, primer annealing at 55°C for 30 s, and extension at 72°C for 1 min. The reaction mixture after amplification was treated with ExoSAP to remove single-stranded DNA, and cDNA synthesized was subjected to direct sequencing as described below.
DNA sequencing. The purified plasmids (150–300 ng) described above were subjected to cycle sequencing by using Big Dye Terminator V3.1 and either one of two specific primers, 5'-TCTTGACTTTCCAGCTAGCC-3' (sense) or 5'-AAAGATCGGGCTGGGTTCAT-3' (antisense). The product was analyzed with an ABI Prism 3100 genetic analyzer, and the sequencing result was confirmed again according to the reported sequence of the AQP5 gene (15). Similarly, direct sequencing of the Kozak area of AQP5 gene was carried out by using the same primers used for RT-PCR amplification (i.e., sense, 5'-GCAACCCTCCCGCTGCCA-3', and antisense, 5'-GATGTGGCCACCACTCACAG-3'). For analysis of exon 1 of AQP5 genomic DNA, primers 5'-GGCACCATGAAAAAGGAGGT-3' (sense) and 5'-TGTTGTTCAGCGCATTGACG-3' (antisense) were employed for cycle sequencing.
Immunohistochemistry and hematoxylin-eosin staining. Fixative (3% formaldehyde in 0.1 M sodium phosphate buffer, pH 7.4) was circulated through the entire rat body from the left ventricle, after which SMG was removed. The SMG tissue was cut at the size of 5 mm3 for immunohistochemistry and 1 mm3 for hematoxylin-eosin staining. Both specimens were fixed further at 4°C for 3 h, followed by a wash with PBS containing either 20% or 6.8% sucrose (for immunohistochemistry and hematoxylin-eosin staining, respectively) at 4°C overnight. The specimens for immunohistochemistry were then embedded in Tissue-Tec optimal cutting temperature compound and rapidly frozen in liquid nitrogen. AQP5 in the SMG tissue was examined as described previously (11, 14). Briefly, frozen sections of 5 µm thickness were cut and fixed further in ethanol at –20°C for 1 min. The sections were washed in PBS, blocked with 1.5% goat serum in PBS, and immunoreacted with 1,000 times diluted rabbit anti-AQP5 antiserum (primary antibody). After having been washed with PBS, the sections were reacted with 200 times diluted FITC-conjugated affinity purified goat anti-rabbit IgG (H+L) (second antibody) and washed with PBS. For control staining, some sections were incubated with the same concentration of antibody preabsorbed with the peptide used for immunogen. All sections were next incubated for 15 min at room temperature with PBS containing 0.1 µg/ml of propidium iodide and 20 µg/ml of RNase A and then washed with PBS to allow the nucleus to become stained. The stained specimens were examined under a fluorescent microscope equipped with a DXM 1200 digital camera (Nikon, Tokyo, Japan) with excitation at 450–490 nm (for FITC) and 510–560 nm (for propidium iodide).
The fixed tissue pieces cut at 1 mm3 were embedded into Technovit 8100 following the manufacturer’s instructions. Sections at 2 µm thickness were cut and stained with hematoxylin and eosin following the standard procedure.
Measurement of salivary secretion.
Secretion of saliva in high- and low-AQP5 producers was measured under both nonstimulated and stimulated conditions. For the former condition progenies of 3rd-generation rats were used, and spontaneously secreted saliva was collected every 5 min under urethane anesthesia; secretion rates were then compared between high and low producers. For measurement of salivary secretion, preweighed small cotton balls (weighing 
20 mg) were inserted into the animal’s mouth under the tongue. The balls were removed, and their weights were immediately weighed on a precision balance (cotton ball procedure; Refs. 16 and 17).
The 11th-generation high- and 6th-generation low-producer rats were used to measure the salivary secretion under the stimulated condition. These rats were anesthetized with Nembutal (50 mg/kg body wt ip), and saliva production was stimulated by an injection of pilocarpine (1 mg/kg body wt ip). The saliva secreted before and after pilocarpine stimulation was collected by the cotton ball procedure as described above. The dose of pilocarpine was selected so as to induce the maximum response of salivary secretion in rats. In this experiment, daily water intake during 7 days and the body weight of both groups were also measured.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Detection of AQP5 mRNA in the SMG by Northern blotting. To confirm the expression level of AQP5 mRNA, we performed Northern blotting using the DIG-labeled cDNA probe. Total RNA of the SMG from 5th-generation male SD rats (consisting of 3 high producers and 3 low producers) were analyzed for their AQP5 mRNA levels. By Northern blotting, a 1.6-kb band was specifically detected in all samples by using the DIG-labeled cDNA probe for AQP5 mRNA. The size of this band was the same as that reported previously (1, 6, 15). The mRNA levels for AQP5 were the same between high (lanes 1–3) and low (lanes 4–6) producers (Fig. 2). These data suggest that AQP5 protein expression in the low producers may have been impaired posttranscriptionally or that there may have been some structural defect in the AQP5 cDNA sequence that made this channel protein fail to express in the cell membrane. Therefore we next determined the sequences of AQP5 cDNA from low and high producers.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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To determine whether such difference might be based on a difference in transcription activity, we examined the mRNA level for AQP5 in the SMG of the two groups of rats. Northern blot analysis demonstrated the presence of a 1.6-kb mRNA for AQP5 in the SMG, parotid gland, lacrimal gland, and lungs (1, 6, 15). Even though the protein level of AQP5 in the 5th-generation high producers was significantly higher than that in the same generation of low producers, the Northern blot analysis showed that the AQP5 mRNA level in the two groups was almost same (Fig. 2), indicating that the decreased expression of AQP5 protein in the membrane fraction is not a consequence of a change in the mRNA level. The sequence immediately before the AUG translation initiation codon (nt –18 to –1), which is known as the Kozak sequence (5), was also the same between the two groups. However, by analyzing the sequence of cDNA and genomic DNA (exon 1) for AQP5, we found a point mutation in the third transmembrane domain that led to the replacement of 103Gly with 103Asp (Fig. 3). Probably this mutation may not affect the water flow (oximotic water permeability coefficient value) of the molecule since the mutated amino acid is located at the third transmembrane domain, which is distant from the aqueous pore of the AQP5 molecule. An experiment that explores this point is now being conducted using the Xenopus laevis oocyte.
On the other hand, AQP5 expression at lateral and basal plasma membrane was extremely diminished in the SMG of the mutant rat (low producers) compared with high producers (Fig. 4). In the mutant rat, only limited area of the apical membrane in very few acinar cells showed positive reaction. The appearance of irregularly shaped intercellular secretory canaliculi (omega-shaped structure) implies the AQP5 localization in the intracellular vesicles or secretory granules and their trafficking toward the plasma membrane. The meshlike structure sometimes appeared inside of the acinar cells, which implies that they may be the granular membrane or the structure conveying the AQP5 vesicles (e.g., microtubules). Thus, in the SMG, the membrane of intracellular vesicles or secretory granules appeared to bear AQP5. On the other hand, in the low producer (mutant rats), the aspect of AQP5 localization in the intracellular structure and cell membrane was not clear because of the extreme reduction of its expression, although very weak or poor AQP5 expression was evident in the apical and lateral areas.
The mutation may have affected the expression of AQP5 protein in the plasma membrane, probably because either the insertion of AQP5 into the vesicle membrane would be diminished or trafficking of AQP5 toward the apical membrane would be ineffective. In our preliminary experiments using MDCKII cells, expression of mutant AQP5 in the cell membrane was decreased compared with that of normal AQP5 (M. R. Karabasil, unpublished observations). Therefore, the mutation appears to have caused the decreased expression of AQP5 in the plasma membrane (Fig. 4), resulting in decreased water secretion from the salivary gland.
The basolateral membrane of acinar cells in the salivary glands is believed to have high water permeability, and it has been implied that AQP5 in the luminal area serves as a rate-limiting step in the water transport across the acinar cells (3). We noticed that the rate of spontaneous salivary secretion was different between high and low producers, although there was no difference in the gland weight (Fig. 5). Under Nembutal anesthesia and pilocarpine stimulation, the rate of salivary secretion of low producers was also low compared with that of the high producers (Fig. 6). There was an approximately threefold difference between the two stimulated groups, although there was no difference in the SMG weight between the high and low producers. Ma et al. (8) prepared AQP5 knockout mice and measured the rate of salivary secretion after pilocarpine stimulation. The results indicated that the salivary secretion was reduced to a half of that found for normal mice. This fact clearly indicates that AQP5 is strongly involved in the secretion of saliva.
In the salivary gland, the doctrine of the presence of two pathways for water transport in acinar cells, i.e., transcellular and paracellular (12), is generally accepted; and the study by Ma et al. (8) clearly showed that AQP5 is responsible for the water transport in the transcellular pathway. Our data well agree with the data of their AQP5 knockout experiment. The results of our time-course study suggest that transcellular transport activity is very low in low producers. The lack of the initial secretion in the low producers agrees with the earlier finding that the transcellular transport pathway is involved in the initial phase of salivary secretion (18). We found that the daily water intake in low producers was higher than that in high producers. This result suggests that the decreased salivary secretion may have induced thirst or dryness appreciably in the oral cavity and triggered an increase in water drinking in the low producers.
The AQP5 mutant rat showed a low level of AQP5 expression in the salivary gland cell membrane, as well as a low level of salivary secretion, suggesting the existence of a strong linkage between this AQP5 mutation and some type of xerostomia. Because this AQP5 mutation was found in rats, a study should be undertaken to learn whether it also occurs in humans.
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GRANTS |
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K. Murdiastuti is a Ronpaku fellow supported by the Japan Society for the Promotion of Science; Department of Periodontology, Faculty of Dentistry, Gadjah Mada University, Jogjakarta, Indonesia. N. Purwanti and M. R. Karabasil are supported by a Scholarship from the Ministry of Education, Culture, Sports, Science and Technology of Japan. X. Li is supported by a Scholarship from the Rotary Yoneyama Foundation, Japan.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Nucleotide numbering: the first nucleotide, adenine at the translation initiation site (ATG), was denoted as nucleotide number 1 in this study. 
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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