The effects of altered extracellular pH on dDAVP‐induced phosphorylation (serine 256) and intracellular trafficking of AQP2 (1137.1)

Abstract

The effects of altered extracellular pH (pHe) on dDAVP‐induced phosphorylation (S256, p‐AQP2) and trafficking of AQP2 were examined in the rat kidney inner medullary collecting duct (IMCD) cells. Freshly prepared IMCD suspension was exposed to buffer with pH 6.4, 7.4, or 8.4 for 1 h. dDAVP (10‐10 M, 3 min)‐induced AQP2 phosphorylation was more prominent when tubule suspension was exposed to pH 7.4 or 8.4, compared to pH 6.4. When primary cultured IMCD cells were exposed to pHe 6.4, 7.4 or 8.4 for 1 h, intracellular pH was changed to 6.1, 7.2 and 8.1, respectively. IMCD cells cultured in transwell chamber were exposed to transepithelial pH gradient for 1 h (pH 6.4, 7.4 or 8.4 at the apical side vs. pH 7.4 at the basolateral side). Laser scanning confocal microscopy and cell surface biotinylation assay revealed that exposure to lower pH (pH 6.4, 1 h) significantly reduced dDAVP (10‐9M, 15 min, basolateral)‐induced AQP2 trafficking and surface expression of p‐AQP2. Importantly FRET analysis revealed that dDAVP (10‐10 M)‐induced increase of PKA activity was attenuated when cells were exposed to pHe 6.4, whereas forskolin (10‐7 M)‐induced PKA activation was not affected. Taken together, decreased pHe is likely to exert an inhibitory effect on dDAVP‐induced AQP2 phosphorylation and intracellular trafficking in IMCD cells, presumably via an inhibition of G‐protein‐cAMP‐PKA action.