title: Extracellular pH affects phosphorylation and intracellular trafficking of AQP2 in inner medullary collecting duct cells.
abstract: Kidney collecting duct cells are continuously exposed to the changes of extracellular pH (pHe). We aimed to study the effects of altered pHe on desmopressin (dDAVP)-induced phosphorylation (Ser(256), Ser(261), Ser(264), and Ser(269)) and apical targeting of aquaporin-2 (AQP2) in rat kidney inner medullary collecting duct (IMCD) cells. When freshly prepared IMCD tubule suspensions exposed to HEPES buffer with pH 5.4, 6.4, 7.4, or 8.4 for 1 h were stimulated with dDAVP (10(-10) M, 3 min), AQP2 phosphorylation at Ser(256), Ser(264), and Ser(269) was significantly attenuated under acidic conditions. Next, IMCD cells primary cultured in transwell chambers were exposed to a transepithelial pH gradient for 1 h (apical pH 6.4, 7.4, or 8.4 vs. basolateral pH 7.4 and vice versa). Immunocytochemistry and cell surface biotinylation assay revealed that exposure to either apical pH 6.4 or basolateral pH 6.4 for 1 h was associated with decreased dDAVP (10(-9) M, 15 min, basolateral)-induced apical targeting of AQP2 and surface expression of AQP2. Fluorescence resonance energy transfer analysis revealed that the dDAVP (10(-9) M)-induced increase of PKA activity was significantly attenuated when LLC-PK1 cells were exposed to pHe 6.4 compared with pHe 7.4 and 8.4. In contrast, forskolin (10(-7) M)-induced PKA activation and dDAVP (10(-9) M)-induced increases of intracellular Ca(2+) were not affected. Taken together, dDAVP-induced phosphorylation and apical targeting of AQP2 are attenuated in IMCD cells under acidic pHe, likely via an inhibition of vasopressin V2 receptor-G protein-cAMP-PKA actions.
authors: Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Taegu, Korea; and BK21 Plus KNU Biomedical Convergence Program, Department of Biomedical Science, Kyungpook National University, Taegu, Korea.
authors: Hyo-Jung
authors: HJ
authors: Choi
authors: Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Taegu, Korea; and.
authors: Hyun Jun
authors: HJ
authors: Jung
authors: Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Taegu, Korea; and BK21 Plus KNU Biomedical Convergence Program, Department of Biomedical Science, Kyungpook National University, Taegu, Korea thkwon@knu.ac.kr.
authors: Tae-Hwan
authors: TH
authors: Kwon
mesh_terms: D000818:Animals; D051399:Aquaporin 2 / Q000187:drug effects / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D003894:Deamino Arginine Vasopressin / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D042541:Intracellular Space / Q000378:metabolism; D007684:Kidney Tubules / Q000187:drug effects / Q000378:metabolism; D007685:Kidney Tubules, Collecting / Q000378:metabolism*; D018374:LLC-PK1 Cells; D008297:Male; D010766:Phosphorylation / Q000502:physiology; D021381:Protein Transport / Q000187:drug effects; D017207:Rats, Sprague-Dawley; D012076:Renal Agents / Q000494:pharmacology; D013552:Swine
chemical_list: D051399:Aquaporin 2; D012076:Renal Agents; D003894:Deamino Arginine Vasopressin
keywords: acidosis; collecting duct; fluorescence resonance energy transfer; vasopressin; vasopressin V2 receptor