Association of VASP with TRPC4 in PKG-mediated inhibition of the store-operated calcium response in mesangial cells

作者：Wang, Xiaoxia; Pluznick, Jennifer L; Settles, Deann C; Sansom, Steven C^*

来源：American Journal of Physiology - Renal Fluid and Electrolyte Physiology, 2007, 293(6): F1768-F1776.

DOI：10.1152/ajprenal.00365.2007

• cGMP
• SOC
• nitric oxide
• SNP

We tested the hypotheses that the NO-cGMP-PKG pathway mediates inhibition of the store-operated cation channel (SOC) in human glomerular mesangial cells (HMC) and that TRPC4, a molecular component of SOC in HMC, is associated with PKG-phosphorylated vasodilator-stimulated phosphoprotein (VASP). Using fura 2 ratiometry, we measured intracellular Ca(2 ) concentration [Ca(2 )](i) to determine whether sodium nitroprusside (SNP), an NO donor, and 8-Br-cGMP affected SOC-TRPC4 via PKG. We found that the SOC response in HMC was attenuated in the presence of 100 mu M SNP, an NO donor, or 100 mu M 8-Br-cGMP. Addition of DT-3 (2.5 mu M), a specific PKG-1 alpha inhibitor, reversed the effects of 8-Br-cGMP on the SOC response. Application of 100 mu M cAMP did not significantly inhibit the SOC response. RT-PCR and Western blotting revealed PKG-1 alpha transcript and protein in HMC. Immunocytochemical analysis localized PKG-1 alpha to the cytoplasm and plasma membrane of HMC. Previous studies have shown that PKG-mediated phosphorylation of VASP attenuates cellular Ca(2 ) entry, resulting in altered growth and proliferation. Therefore, we used Western blotting and immunocytochemistry to determine whether PKG-phosphorylated VASP associates with TRPC4. Western blot analysis revealed that 8-Br-cGMP enhanced the phosphorylation of VASP at serine 239 (Ser239), a known PKG phosphorylation site, in HMC within 5 min. Coimmunoprecipitation and coimmunostaining showed that P-Ser239-VASP associated with TRPC4. However, VASP that was unphosphorylated at Ser239 was not associated with TRPC4. These results indicate that VASP has a role in the NO/PKG-1 alpha-mediated inhibition of the TRPC4-SOC response in HMC.

• 出版日期2007-12
• 单位上海交通大学