Association of VASP with TRPC4 in PKG-mediated inhibition of the store-operated calcium response in mesangial cells.
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 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.
Wang
Xiaoxia
X
Pluznick
Jennifer L
JL
Settles
Deann C
DC
Sansom
Steven C
SC
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D015220:Calcium Channels / Q000502:physiology*; D020013:Calcium Signaling / Q000502:physiology*; D015815:Cell Adhesion Molecules / Q000502:physiology*; D002462:Cell Membrane / Q000502:physiology; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000502:physiology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D005456:Fluorescent Dyes; D016257:Fura-2; D005920:Glomerular Mesangium / Q000166:cytology / Q000201:enzymology / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D051379:Mice; D008840:Microfilament Proteins / Q000502:physiology*; D009569:Nitric Oxide / Q000502:physiology; D010750:Phosphoproteins / Q000502:physiology*; D010766:Phosphorylation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D050052:TRPC Cation Channels / Q000502:physiology*; D000098662:Vasodilator-Stimulated Phosphoprotein
D015220:Calcium Channels; D015815:Cell Adhesion Molecules; D005456:Fluorescent Dyes; D008840:Microfilament Proteins; D010750:Phosphoproteins; D050052:TRPC Cation Channels; C489571:TRPC4 ion channel; D000098662:Vasodilator-Stimulated Phosphoprotein; D009569:Nitric Oxide; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP; D002118:Calcium; D016257:Fura-2