Molecular Cloning of a New Aquaporin from Rat Pancreas and Liver*

A new water channel (aquaporin-8, gene symbol AQP8) was isolated from rat pancreas and liver by homology cloning. Ribonuclease protection assay showed intense expression of the gene in pancreas and liver, less intense in colon and salivary gland, and negligible in other organs. The full-length cDNA was obtained by ligation of ∼1.4-kilobase (kb) cDNA isolated from the rat liver cDNA library to ∼0.5 kb of the 5′-end fragment obtained by the rapid amplification of cDNA ends method. A major transcript of ∼1.45 kb was demonstrated in liver and colon by Northern blot analysis. Expression of the cRNA in Xenopusoocytes markedly enhanced osmotic water permeability in a mercury-sensitive manner, indicating a water channel function of this molecule. The open reading frame encoded a 263-amino acid protein with a predicted molecular size of 28 kDa. Hydropathy analysis represented six membrane-spanning domains and five connecting loops containing two sites of NPA motif as preserved in other aquaporins. Unlike other mammalian aquaporins, AQP8 has an unusual structure with a long N terminus and a short C terminus, which are found in plant aquaporin, γ-tonoplast intrinsic protein. By in situ hybridization,AQP8 mRNA expression was assumed in hepatocytes, acinal cells of pancreas and salivary gland, and absorptive colonic epithelial cells. The physiological role(s) of AQP8 remain to be elucidated.

The aquaporins are water-selective membrane channels found in many species of animals and plants as the family of major intrinsic protein (MIP) 1 (1)(2)(3). Aquaporin-1 (AQP1) is the first protein recognized as a channel-forming integral membrane protein of 28 kDa (CHIP-28) expressed in mammalian red blood cells (1,4) and then as a water channel expressed in renal proximal tubules (5)(6)(7) and other water-permeable epithelia (8,9). Thereafter, four other aquaporins have been cloned in mammals. AQP2 is the vasopressin-regulated water channel, exclusively present in apical membranes of principal cells of collecting ducts in the kidney (10 -13), whereas AQP3 is a water channel locating basolateral membranes of collecting duct cells and transporting water and small solutes such as glycerol and urea (14). AQP4 and AQP5 were cloned from brain and salivary gland, respectively, and were presumed to be implicated in the sensation of osmotic change in hypothalamus and secretion in exocrine glands (15,16). In plants, tonoplast intrinsic protein (TIP) is an integral membrane protein and belongs to the MIP family (17,18). ␥-TIP was found in the vegetative organs of plant and not in seeds, although ␣-TIP and ␤-TIP were seed-specific (19).
The previous studies showed the presence of unique aquaporins in various exocrine glands such as salivary gland and lacrimal gland (16). Since pancreas is a secretory organ, secreting various digestive enzymes such as lipase, amylase, and protease in a volume of about 2,000 ml/day in humans (20), it may be reasonable to speculate the presence of an aquaporin family in pancreas. In the present study, we attempted to isolate aquaporins expressed in pancreas by polymerase chain reaction (PCR) using degenerate primers for the aquaporin family (15,16). Through the study, AQP1 and AQP4 were cloned and coincidentally a new member of the aquaporin family, AQP8, was identified as a water channel prominently expressed in pancreas and liver.
Ribonuclease Protection Assays-The plasmids inserted with the ϳ360-bp PCR product and a partial sequence of the full-length cDNA described below (315 bp, 701-1015) were linearized with HindIII and EcoRI, respectively, and used as a template for in vitro transcription of 32 P-labeled antisense cRNA probe. To detect a housekeeping gene, a rat glyceraldehyde-3-phosphate dehydrogenase cDNA of 123 bp was used as reported previously (21). Total RNA was extracted from rat systemic organs by the acid/guanidinium/phenol/chloroform method, and 10 g * This work was supported in part by Grant-in-aid 09470237 for Science Research from the Ministry of Education, Science, Sports and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence reported in this paper has been submitted to the GenBank TM /EBL Data Bank with accession number AF007775 and the gene has been designated AQP8 by the HUGO/GDB Nomenclature Committee.
of total RNA were hybridized with the 32 P-labeled probes for 16 h at 48.5°C. The unhybridized probes were digested with ribonuclease A (4 g/ml) and ribonuclease T1 (120 units/ml) for 60 min at 30°C, and the ribonucleases were digested with proteinase K (0.5 g/ml) at 37°C for 30 min. After phenol/chloroform extraction, the hybridized probes were precipitated with ethanol and heat-denatured for electrophoresis on 6% polyacrylamide gels. The gels were exposed to x-ray films for 3 days.
Library Screening-Since mRNA expression was intense in liver and pancreas by a preliminary ribonuclease protection assay, ϳ2 ϫ 10 4 colonies of rat liver cDNA library (Life Technologies, Inc.) transferred to nylon membranes were hybridized with the 32 P-labeled antisense cRNA probe (10 6 cpm/ml) for 16 h at 50°C, and the membranes were washed at 65°C in 0.1 ϫ SSC containing 0.1% SDS for autoradiography. One positive clone of ϳ1.4 kb was isolated and sequenced. The 5Ј-end of the entire cDNA was isolated by rapid amplification of cDNA ends by heminested PCR. Two types of oligonucleotide primer for the 5Ј-end sequence of the 1.4-kb cDNA were synthesized: gene-specific primers GSP-1 (5Ј-CCTCCAACCAGTGTGACTGC-3Ј) and GSP-2 (5Ј-GTGTC-CACCGCTGATGTTCC-3Ј). Plasmids isolated from the rat liver cDNA library (100 pg) were amplified by PCR using 12.5 pmol of the SP6 promoter primer (5Ј-GATTTAGGTGACACTATAG-3Ј) and the GSP-1 primer for 40 cycles (1 min at 94°C, 1 min at 47°C, 1 min at 72°C). The products were re-amplified with 12.5 pmol of the SP6 promoter primer and the GSP-2 primer for 40 cycles (1 min at 94°C, 1 min at 47°C, 1 min at 72°C). The heminested PCR products were subcloned into pGEM-T vector (Promega Corp.) and sequenced. The longest cDNA obtained by 5Ј-RACE was ligated to the ϳ1.4-kb clone to construct the full length of ϳ1.45-kb cDNA. The hydrophobicity of the putative protein was analyzed by DNAsis software (Hitachi Software Engineering Co., Yokohama, Japan), identities to other aquaporins were searched using the FASTA program (GenomeNet), and phylogenesis was analyzed by PILEUP software (Genetic Computer Group, Madison, WI).
Expression in Vitro-In vitro protein synthesis from the cloned cDNA was performed in a cell-free translation system according to the manufacturer's protocol (Promega Corp.) with [ 35 S]methionine and canine pancreatic microsomes at 30°C for 90 min. The reaction products were electrophoresed on a 12.5% SDS-polyacrylamide gel and examined by autoradiography (15,16).
Oocyte Expression Studies-A 1.45-kb SalI/BamHI fragment containing the 5Ј-untranslated sequence and the entire open reading frame of the cloned cDNA was ligated into pSPORT2 vector (Life Technologies, Inc.). Capped cRNA was synthesized in vitro after linearization with NotI as described previously (22). Xenopus laevis oocytes (stage V-VI) were defolliculated by collagenase and microinjected with 50 nl of water or 10 ng of the synthesized cRNA. After incubation in 200 mosM modified Barth's buffer at 20°C for 36 h, the oocytes were transferred to 70 mosM modified Barth's buffer at 24°C, and their swelling was monitored by videomicroscopy. The coefficient of osmotic water permeability (Pf) was assessed by a real-time quantitative imaging method as described recently (4). The uptake of [ 14 C]urea or [ 14 C]glycerol was measured by incubating oocytes in 200 mosM modified Barth's buffer for 10 -20 min at 24°C followed by washing and SDS solubilization. No increase in ion conductance was found when control oocytes were compared with cRNA-injected oocytes by the two-electrode voltage clamp method (16). In Situ Hybridization-In situ hybridization was done by a procedure as described previously (23). In brief, 35 S-labeled antisense and sense RNA probes were synthesized with T7 and SP6 RNA polymerases using a linearized cDNA template encoding 81-889 nucleotides. Cryostat sections (10 m thickness) of rat liver, pancreas, salivary gland, and colon were fixed in 4% paraformaldehyde in phosphate-buffered saline and treated with 5 g/ml proteinase K (Promega Corp.) in 500 mM NaCl, 10 mM Tris-HCl, pH 8.0, for 10 min at room temperature and hybridized with each probe (1 ϫ 10 6 cpm/section) overnight at 55°C. After washing in 2 ϫ SSC, 1 mM EDTA, 10 mM ␤-mercaptoethanol at room temperature, the sections were treated with 20 g/ml ribonuclease A in 500 mM NaCl, 10 mM Tris-HCl, pH 8.0, for 30 min at room temperature, followed by washing in 0.1 ϫ SSC, 1 mM EDTA, 10 mM ␤-mercaptoethanol at 55°C for 4 h and washing in 0.5 ϫ SSC at room temperature. After dehydration, the sections were exposed to photographic emulsion for 5ϳ7 days in the dark at 4°C, developed, and counterstained with hematoxylin to observe under a bright or dark field microscope.

RESULTS AND DISCUSSION
Isolation and Distribution of the Cloned cDNA-AQP1 and AQP4 cDNAs were mainly amplified from rat pancreas cDNA by PCR using the degenerate primers. However, some were similar but completely different in nucleotide sequence to aquaporin family genes reported previously. Analysis of the PCR product revealed high nucleotide sequence homology to aquaporin family members: AQP1, 34%; AQP2, 40%; AQP3, 36%; AQP4, 34%; AQP5, 37%; and ␥-TIP, 41%. Preliminary ribonuclease protection assay using the PCR-cloned cDNA as a template showed intense expression of the mRNA in pancreas and liver and weak expression in colon and salivary gland. The expression profile of this gene in systemic organs was apparently distinct from those of other aquaporins.
Cloning of the Full-length cDNA-The ϳ1.4-kb cDNA was obtained after screening of the rat liver cDNA library at high stringency with the cRNA probe in vitro transcribed with the PCR-cloned cDNA. By the 5Ј-rapid amplification of cDNA ends method ϳ150 bp of the 5Ј-end of the cDNA was isolated and ligated to the ϳ1.4-kb cDNA. The cDNA sequence was com-posed of a 101-bp 5Ј-untranslated sequence preceding an initiation site (Kozak consensus) (24), a 789-bp open reading frame, and a 589-bp 3Ј-untranslated sequence containing a polyadenylation consensus (Fig. 1A).
Deduced Amino Acid Sequence and Structure-Analysis of the GenBank data base revealed that the cloned cDNA was a new member of the aquaporin family. The open reading frame encodes a polypeptide of 263 amino acids (M r ϭ ϳ28,000) with homology to aquaporins: ␥-TIP, 37%; AQP2, 37%; MIP, 37%; ␣-TIP, 35%; AQP1, 34%; AQP5, 33%; AQP4, 30%; AQP3, 26%, evaluated by identity using the FASTA program. Interestingly, the deduced amino acid sequence was highly homologous with the plant aquaporin family (␥-TIP and ␣-TIP). Kyte-Doolittle hydropathy analysis confirmed six putative membrane-spanning domains (I-VI) and five connecting loops containing two sites of NPA motif (Asn-Pro-Ala) preserved in other aquaporin family members (Fig. 1B). The predicted polypeptide has an unusual structure with a longer N terminus (37 residues) and a shorter C terminus (10 residues) compared with those of other mammalian aquaporins. The long N terminus and short C terminus have been observed in plant aquaporin, ␥-TIP. Near the NPA motifs, the deduced amino acid sequence contains a sequence highly conserved in other aquaporins. The gene has been designated aquaporin-8 (symbol AQP8) by the HUGO/GDB Nomenclature Committee (University College of London, London, UK) and registered in the GenBank data base (accession number AF007775). A phylogenetic analysis showed that AQP8 developed in a different branch from other aquaporins (Fig. 1C).
Expression and Water Transport Function-Cell-free translation of the cloned cDNA in the presence of microsomes yielded a major band at ϳ27 kDa ( Fig. 2A), corresponding to the pre- dicted mass (28 kDa) with slightly more rapid electrophoretic mobility as described previously (16,25).
Transmembrane water flow through the protein translated from the cloned cDNA was evaluated in Xenopus oocytes. After 36 h of incubation in 200 mosM buffer at 20°C, the oocytes were transferred in 70 mosM buffer, and swelling of the oocytes was monitored by videomicroscopy. Injection of oocytes with the cRNA synthesized from the cloned cDNA increased the Pf by approximately 14-fold, qualifying the characteristics of the translated protein as a new member of the aquaporin family (Fig. 2B).
The increase in Pf mediated by expression of AQP8 was blocked by treatment of oocytes with 1 mM HgCl 2 and restored by incubation in ␤-mercaptoethanol similar to most of the already known aquaporins (Fig. 2B). Likewise, oocytes expressing AQP8 exhibited no increase in the membrane transport of [ 14 C]urea or [ 14 C]glycerol and no increase in the membrane conductance measured by an electrophysiological method (data not shown).
mRNA Expression in Systemic Organs-Northern blot analysis using total RNA extracted from rat liver and colon showed a single band of ϳ1.5 kb (Fig. 3A), equivalent to the size of other aquaporin family members reported previously (10,14,16). No bands were detected in kidney and cerebrum RNA samples. The mRNA expression detected by Northern blot analysis was comparable with the finding obtained by the ribonuclease protection assay, confirming the predominant mRNA expression in pancreas and liver and weak expression in colon and salivary gland (Fig. 3B). The unique distribution of the expression suggested a crucial role(s) of this water channel in those organs.
In Situ Hybridization-Intense AQP8 mRNA expression was demonstrated in glandular lobules but not in islets of pancreas by in situ hybridization (Fig. 4A). No significant signals were observed in pancreas with the sense probe. No aquaporin family members except AQP1 have been shown in pancreas although the presence of water channels may be presumed as pancreas secretes a large volume of water. The predominant expression of AQP8 in pancreas suggested an important role of this aquaporin in the secretion of pancreatic juice.
In situ hybridization of the liver showed AQP8 mRNA expression in parenchymal cells and not in the portal area, predicting the expression in hepatocytes. Previous studies showed that osmotic flow of water across hepatocyte membranes occurred mainly via a water channel-independent pathway whereas cholangiocytes transported water probably through AQP1 (26,27). The observation may be consistent with negligible mRNA expression of aquaporin family members in hepatocyte (26). Contrasting with these studies, a new aquaporin family, AQP8, was isolated from liver cDNA, and its mRNA expression was shown to be intense in liver by ribonuclease protection assay and Northern blot analysis. Furthermore, in situ hybridization predicted the expression of AQP8 mRNA in hepatocytes. The role(s) of AQP8 gene products in liver may be apparent when the subcellular localization is elucidated by immunohistochemistry.
The mRNA expression of AQP8 was also localized in acinal cells in the salivary gland and absorptive columnar epithelial cell layer in colon, suggesting a cooperative role of this aquaporin with other aquaporins in the secretion of saliva and in water absorption.
The AQP8 mRNA expression of narrow tissue distribution may indicate important role(s) of AQP8 in these tissues. Further studies are required to elucidate the cellular and subcellular localization and function of this aquaporin.