Acute Insulin Stimulation Induces Phosphorylation of the Na-Cl Cotransporter in Cultured Distal mpkDCT Cells and Mouse Kidney

The NaCl cotransporter (NCC) is essential for sodium reabsorption at the distal convoluted tubules (DCT), and its phosphorylation increases its transport activity and apical membrane localization. Although insulin has been reported to increase sodium reabsorption in the kidney, the linkage between insulin and NCC phosphorylation has not yet been investigated. This study examined whether insulin regulates NCC phosphorylation. In cultured mpkDCT cells, insulin increased phosphorylation of STE20/SPS1-related proline-alanine-rich kinase (SPAK) and NCC in a dose-dependent manner. This insulin-induced phosphorylation of NCC was suppressed in WNK4 and SPAK knockdown cells. In addition, Ly294002, a PI3K inhibitor, decreased the insulin effect on SPAK and NCC phosphorylation, indicating that insulin induces phosphorylation of SPAK and NCC through PI3K and WNK4 in mpkDCT cells. Moreover, acute insulin administration to mice increased phosphorylation of oxidative stress-responsive kinase-1 (OSR1), SPAK and NCC in the kidney. Time-course experiments in mpkDCT cells and mice suggested that SPAK is upstream of NCC in this insulin-induced NCC phosphorylation mechanism, which was confirmed by the lack of insulin-induced NCC phosphorylation in SPAK knockout mice. Moreover, insulin administration to WNK4 hypomorphic mice did not increase phosphorylation of OSR1, SPAK and NCC in the kidney, suggesting that WNK4 is also involved in the insulin-induced OSR1, SPAK and NCC phosphorylation mechanism in vivo. The present results demonstrated that insulin is a potent regulator of NCC phosphorylation in the kidney, and that WNK4 and SPAK are involved in this mechanism of NCC phosphorylation by insulin

RGS2 inhibits the epithelial Ca2+ channel TRPV6.

Contains fulltext : 50327.pdf (Publisher’s version ) (Open Access)The epithelial Ca(2+) channels TRPV5 and TRPV6 constitute the apical Ca(2+) entry pathway in the process of active Ca(2+) (re)absorption. By yeast two-hybrid and glutathione S-transferase pulldown analysis we identified RGS2 as a novel TRPV6-associated protein. RGS proteins determine the inactivation kinetics of heterotrimeric G-protein-coupled receptor (GPCR) signaling by regulating the GTPase activity of G(alpha) subunits. Here we demonstrate that TRPV6 interacts with the NH(2)-terminal domain of RGS2 in a Ca(2+)-independent fashion and that overexpression of RGS2 reduces the Na(+) and Ca(2+) current of TRPV6 but not that of TRPV5-transfected human embryonic kidney 293 (HEK293) cells. In contrast, overexpression of the deletion mutant DeltaN-RGS2, lacking the NH(2)-terminal domain of RGS2, in TRPV6-expressing HEK293 cells did not show this inhibition. Furthermore, cell surface biotinylation indicated that the inhibitory effect of RGS2 on TRPV6 activity is not mediated by differences in trafficking or retrieval of TRPV6 from the plasma membrane. This effect probably results from the direct interaction between RGS2 and TRPV6, affecting the gating properties of the channel. Finally, the scaffolding protein spinophilin, shown to recruit RGS2 and regulate GPCR-signaling via G(alpha), did not affect RGS2 binding and electrophysiological properties of TRPV6, indicating a GPCR-independent mechanism of TRPV6 regulation by RGS2

Characterization of a Madin-Darby canine kidney cell line stably expressing TRPV5.

Contains fulltext : 48619.pdf (publisher's version ) (Closed access)To provide a cell model for studying specifically the regulation of Ca2+ entry by the epithelial calcium channel transient receptor potential-vanilloid-5 (TRPV5), green fluorescent protein (GFP)-tagged TRPV5 was expressed stably in Madin-Darby canine kidney type I (MDCK) cells. The localization of GFP-TRPV5 in this cell line showed an intracellular granular distribution. Ca2+ uptake in GFP-TRPV5-MDCK cells cultured on plastic supports was threefold higher than in non-transfected cells. Moreover, apical Ca2+ uptake in GFP-TRPV5-MDCK cells cultured on permeable supports was eightfold higher than basolateral Ca2+ uptake, indicating that GFP-TRPV5 is expressed predominantly in the apical membrane. Patch-clamp analysis showed the presence of typical electrophysiological features of GFP-TRPV5, such as inwardly rectifying currents, inhibition by divalent cations and Ca2+-dependent inactivation. Moreover, the TRPV5 inhibitor ruthenium red completely inhibited Ca2+ uptake in GFP-TRPV5-MDCK cells, whereas Ca2+ uptake in non-transfected cells was not inhibited. The characterized GFP-TRPV5-MDCK cell line was used to assess the regulation of TRPV5. The protein kinase C activator phorbol 12-myristate 13-acetate and the cAMP-elevating compounds forskolin/3-isobutyl-1-methylxanthine, 8-Br-cAMP and PGE2 stimulated TRPV5 activity in GFP-TRPV5-MDCK cells by 121+/-7, 79+/-5, 55+/-4 and 61+/-7%, respectively. These compounds did not affect Ca2+ uptake in non-transfected cells. In conclusion, the GFP-TRPV5-MDCK cell line provides a model to specifically study the regulation of TRPV5 activity