Association of VASP with TRPC4 in PKG-mediated inhibition of the store-operated calcium response in mesangial cells.
Association of VASP with TRPC4 in PKG-mediated inhibition of the store-operated calcium response in mesangial cells.
复制标题
DOI：
10.1152/ajprenal.00365.2007
复制
发表时间：
2007-12
期刊：
影响因子：
--
通讯作者：
Xiaoxia Wang;J. Pluznick;D. Settles;S. Sansom
中科院分区：
文献类型：
--
作者：
Xiaoxia Wang;J. Pluznick;D. Settles;S. Sansom
我们检验了以下假设：NO-cGMP-PKG通路介导人肾小球系膜细胞（HMC）中储存操纵阳离子通道（SOC）的抑制，以及TRPC 4（HMC中SOC的分子组分）与PKG磷酸化血管舒张刺激磷蛋白（VASP）相关。使用Fura 2比率法，我们测量细胞内Ca（2+）浓度[Ca（2+）]（i）以确定硝普钠（SNP）（一种NO供体）和8-Br-cGMP是否通过PKG影响SOC-TRPC 4。我们发现HMC中的SOC反应在100 μ M SNP、NO供体或100 μ M 8-Br-cGMP存在下减弱。加入DT-3（2.5 μ M），一种特异性PKG-1 α抑制剂，逆转了8-Br-cGMP对SOC反应的影响。应用100 μ M cAMP并不显著抑制SOC反应。RT-PCR和Western blotting显示HMC中PKG-1 alpha的转录本和蛋白质。免疫细胞化学分析显示PKG-1 α定位于HMC的胞浆和质膜。以前的研究表明PKG介导的VASP磷酸化减弱细胞内Ca（2+）的进入，导致生长和增殖的改变。因此，我们使用蛋白质印迹和免疫细胞化学来确定PKG磷酸化的VASP是否与TRPC 4相关。Western印迹分析显示，8-Br-cGMP增强VASP的磷酸化丝氨酸239（Ser 239），一个已知的PKG磷酸化位点，在HMC在5分钟内。共免疫沉淀和共免疫染色显示，P-Ser 239-VASP与TRPC 4。然而，在Ser 239未磷酸化的VASP与TRPC 4无关。这些结果表明，VASP在NO/PKG-1 α介导的抑制HMC中TRPC 4-SOC反应中起作用。
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.