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Association of VASP with TRPC4 in PKG-mediated inhibition of the store-operated calcium response in mesangial cells Wang X; Pluznick JL; Settles DC; Sansom SCAm J Physiol Renal Physiol 2007[Dec]; 293 (6): F1768-76We 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 microM SNP, an NO donor, or 100 microM 8-Br-cGMP. Addition of DT-3 (2.5 microM), a specific PKG-1alpha inhibitor, reversed the effects of 8-Br-cGMP on the SOC response. Application of 100 microM cAMP did not significantly inhibit the SOC response. RT-PCR and Western blotting revealed PKG-1alpha transcript and protein in HMC. Immunocytochemical analysis localized PKG-1alpha 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-1alpha-mediated inhibition of the TRPC4-SOC response in HMC.|Animals[MESH]|Calcium Channels/*physiology[MESH]|Calcium Signaling/*physiology[MESH]|Calcium/metabolism/*physiology[MESH]|Cell Adhesion Molecules/*physiology[MESH]|Cell Membrane/physiology[MESH]|Cells, Cultured[MESH]|Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors/*physiology[MESH]|Cyclic GMP/physiology[MESH]|Fluorescent Dyes[MESH]|Fura-2[MESH]|Glomerular Mesangium/cytology/enzymology/*physiology[MESH]|Humans[MESH]|Immunohistochemistry[MESH]|Immunoprecipitation[MESH]|Mice[MESH]|Microfilament Proteins/*physiology[MESH]|Nitric Oxide/physiology[MESH]|Phosphoproteins/*physiology[MESH]|Phosphorylation[MESH]|Reverse Transcriptase Polymerase Chain Reaction[MESH]|Signal Transduction/physiology[MESH]|TRPC Cation Channels/*physiology[MESH]|Vasodilator-Stimulated Phosphoprotein[MESH] |
