Molecular Cloning of a New Aquaporin from Rat Pancreas and Liver

Abstract

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 ϳ1.4-kilobase (kb) cDNA isolated from the rat liver cDNA library to ϳ0.5 kb of the 5-end fragment obtained by the rapid amplification of cDNA ends method. A major transcript of ϳ1.45 kb was demonstrated in liver and colon by Northern blot analysis. Expression of the cRNA in Xenopus oocytes 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 membranespanning 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, ␥-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 (1-3). 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 (5-7) 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...

The Water Channel Aquaporin-8 Is Mainly Intracellular in Rat Hepatocytes, and Its Plasma Membrane Insertion Is Stimulated by Cyclic AMP

“…1A, immunoblotting analysis for AQP8 detected a band of ϳ34 kDa in total liver and hepatocyte membranes. An identical band in location was obtained for colon mucosa (another rat AQP8 message-expression tissue) (7,8), whereas no band was detected for kidney or lung tissues (negative controls for rat AQP8 expression) (7,8). The 34 kDa band was absent when AQP8 antibody was preabsorbed with a molar excess of the immunizing peptide (not shown).…”

“…AQP8 has been identified recently (7)(8)(9), and its transcript has been found to be present in rat liver (7,8). In situ hybridization studies suggest that AQP8 mRNA is present in hepatocytes; nevertheless, studies on protein expression were not undertaken (7).…”

Regulation of blastocyst formation

“…AQP 9 is the most recently cloned water channel and is most similar to AQP 3 and 7 in that it also permits glycerol transport. It is expressed in leukocytes and liver and is weakly expressed in the heart, kidney and small intestine (224,237,238). The blastocyst provides a comparatively simple system from which fundamental information on AQP gene expression and function can be obtained.…”

The Aquaporins, Blueprints for Cellular Plumbing Systems

“…AQP6 is expressed in kidney but exhibits extremely low water permeability, and its distribution has not been defined (35). The cDNA encoding AQP8 was recently isolated from testis, pancreas, liver, and other tissues (36).…”