| IA Induce the Expression of AQP1 in Cultured BALB/c FibroblastsPrefaceThe aquaporins(AQPs) are a recently described family of water channels. The prototypic AQP, AQP1, is a 28 kDa protein which was first isolated from red blood cells. It is a water channel protein expressed in many tissues for kidney, lung, brain and eye, whose regulation is known to be complex. Incubation of BALB/c fibroblasts spontaneously express AQP1. Here BALB/c fibroblasts in culture was used to examine the changes of AQP1 expression in iodacetamide-induced hypoxia both in isotonic condition and hypertonic condition.Materials and Methods1. materials(1)BALB/c fibroblasts(2)iodacetamide(3)rabbit polyclonal IgG for AQP1(4)horseradish peroxidase-couple secondary antibody(5)actin(6)FBS(7)modified Eagle's medium(8)sorbitol2. methods(1)Cell Culture and Harvest. BALB/c fibroblasts were cultured in MEM supplemented with 10%(vol/vol)FBS, at 37℃in 5% CO2. Where indicated, medium was made hypertonic by addition of sorbitol. Iodacetamide was added to isotonic or hypertonic medium at the specified concentration (30umol/L) for the duration of the experiment. At the time of harvest, ceils were washed with ice-cold PBS, scraped pelleted (12, 000, 5 min, 4℃), and frozen on dry ice. Cell pellets were resuspended in ice-cold homogenization buffer, and subjected to an additional cycle of freeze-thawing. Cell lysate protein concentrations were determined by the BCA assay, using BSA as standard.(2)SDS/PAGE and Western blotting. Cell lysates were subjected to SDS/PAGE using 12% acrylamide gels (anti-AQP1 immunoblots) and transferred to poly membrance for protein immunoblotting as described. Blots were visualized by enhanced chemiluminescence and autoradiography. Relative band intensities were determined by densitometry using MACBAS bioimaging analyzer.(3)Statistics. Densitometric analysis of protein immunoblots and autoradiography from metabolic labeling studies are expressed as mean±SEM for each group. Unpaired t-tests were performed to assess the effect of different interventions.Results1. The morphology changes of BALB/c 3T3 in isotonic are obvious.Normally BALB/c fibroblasts(BALB/c 3T3) are mainly in fusiform shape, ceiod, cells in dividing phase are round like, and grow in monolayer and stick to the wall. After incubation with IA for 4 hours and 6 hours the cell became larger and larger and fragementation, cytolysis and dead cells increased.2. AQP1 expression increased after incubation with IA measured by Western blotting.AQP1 expression in BALB/c fibroblasts cultured in isotonic medium was determined by using quantitative immunoblot analysis. After 4 h incubation with 30umol/l IA , AQP1 expression was increased by 24.4%(n=6, P＞0.05), it was significantly increased by 193.8％(n=6, P＜0.05) after 6h incubation with IA.AQP1 expression in BALB/c fibroblasts cultured in 0.1M hypertonic medium was increased by 84.7％(n=2, P＞0.05) after 4h incubation with 30umol/l IA. It was increased by 63.8％after 6h(n=2, P＞0.05).AQP1 expression in BALB/c fibroblasts cultured in 0.4M hypertonic medium was at high level. After 4h incubation with 30umol/l IA it was attenuate to 91.1％(n=1). After 6h it was increased by 26.2％(n=1).Compared with isotonic medium, the increasing peak of AQP1 expression is advance in hypertonic stress.DiscussionSeveral lines of evidence reveal that aquaporins are expressed in lung epithelia and endothelia; water permeability is high in epithelia and endothelia where aquaporins are expressed; aquaporins expression increased near the time of birth; the osmotic water permeability of lung microvessels was decreased in AQP1 knockout mice compared with wild-type mice; AQP1 expression is induced by hypertonic stress in kidney cells lines, as well as in BALB/c fibroblasts in culture and in rat in vivo.Our results indicated that iodacetamide contributed to induction of AQP1 both in isotonic condition and in hypertonic condition, furthermore the indution of AQP1 in 0.1M htpertonic condition was more rapid than in isotonic condition, the increasing peak was advance. We speculated that AQP1 mediates osmotically-driven transmembrane water permeability in hypoxic condition.ConclusionIt was suggested that AQP1 mediates osmotically-driven transmembrane water permeability in hypoxic condition induced by IA.
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