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DISTRIBUTION AND REGULATION OF AQUAPORINS IN INTESTINAL EPITHELIAL CELLS
EA Chow, WK MacNaughton
Inflammation Research Network, University of Calgary, Calgary, Alberta
BACKGROUND: Sodium absorption and chloride secretion create an osmotic gradient that drives water transport across intestinal epithelial cells. However, the mechanism of water movement across the epithelium is not well understood. Aquaporins (AQP) are tetrameric water-selective channels that may play an important role in facilitating transcellular water movement and maintaining physiological electrolyte balance. Furthermore, during intestinal inflammation, cAMP-dependent chloride secretion is reduced and altered AQP expression may be involved in the resulting decreased water transport. In the kidney, key AQPs are regulated by intracellular cAMP. Therefore, we hypothesized that the cellular distribution of AQPs in the intestinal epithelium is regulated by cAMP.
METHODS: RT-PCR and/or confocal imaging was used to analyze AQPs in three intestinal epithelial cell lines, SCBN (canine; polarized), T84 (human; carcinoma; polarized), and CMT93 (murine; carcinoma; polarized). To assess intracellular trafficking of AQP1, T84 cells were transfected with a GFP-linked human AQP1 gene construct (donated by Dr. N. Larusso). Real time confocal live cell fluorescence imaging was used to track the cellular movement of GFP-AQP1 after addition of 10 µM forskolin (FSK) to increase intracellular cAMP. Construction of z-stacks of 1 µm optical sections verified the intracellular localization of the GFP-AQP1. In addition, cellular fractionation by differential centrifugation was used to separate CMT93 whole cell lysate into plasma membrane and intracellular vesicle fractions. Western blot was used to detect AQP1 protein levels in the cellular fractions, before and 30 min after 10 µM FSK treatment.
Results/Discussion: RT-PCR demonstrated mRNA expression of: AQP1, 2, 3, and 5 in SCBN; AQP1, 2, 3, 5, 7, 8, and 9 in T84; and AQP1, 2, 3, 4, and 5 in CMT. Confocal imaging showed immunoreactivity for AQP1, 2, 3, 4, and 7 in SCBN and AQP1, 4, 6, and 9 in T84. Confocal live cell imaging suggested an increase in apical fluorescence of AQP1 within 20 min of FSK treatment. Densitometric analysis of AQP1 bands from the cellular fractionation process showed that there was no FSK-induced change in the intracellular vesicle fraction, but a significant increase in AQP1 in the plasma membrane fraction.
This study demonstrates that multiple AQPs are expressed in intestinal epithelial cell lines, and provides evidence for cAMP-dependent regulation of AQP1 trafficking.