Inhibition of TRPC1/TRPC3 by PKG contributes to NO-mediated vasorelaxation
Inhibition of TRPC1/TRPC3 by PKG contributes to NO-mediated vasorelaxation
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DOI：
10.1152/ajpheart.01130.2008
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发表时间：
2009-07-01
影响因子：
4.8
通讯作者：
Marrelli, Sean P.
中科院分区：
文献类型：
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作者：
Chen, Jie;Crossland, Randy F.;Marrelli, Sean P.
Chen J，Crossland RF，Noorani MM，Marrelli SP. PKG对TRPC 1/TRPC 3的抑制有助于NO介导的血管舒张。Am J Physiol Heart Circ Physiol 297：H417-H424，2009.首次发表于2009年6月5日; doi：10.1152/ajpheart.01130.2008。一氧化氮（NO）通过PKG依赖性机制抑制瞬时受体电位通道3（TRPC 3）通道。我们试图确定1）在新鲜分离的平滑肌细胞（SMC）中是否发生TRPC 3的NO抑制;和2）TRPC 3通道的NO抑制是否有助于NO介导的血管舒张。我们在新鲜分离的大鼠颈动脉（CA）平滑肌细胞膜片钳和完整的CA血管肌电图测试这些假设。我们证明了TRPC 3表达在整个CA（mRNA和蛋白质），定位于平滑肌层。TRPC 1蛋白也表达并与TRPC 3共免疫沉淀。全细胞膜片钳显示非选择性阳离子通道电流，激活UTP（60 μ M）和TRPC通道抑制剂，镧（100 μ M）完全抑制。抗TRPC 3或抗TRPC 1抗体的细胞内应用也抑制了UTP刺激电流（I-UTP），但不受抗TRPC 6或抗TRPC 4对照抗体的抑制。我们接下来评估了I-UTP上的NO信号通路。外源性NO [（Z）-1-{N-甲基-N-[ 6（N-methylammoniohexyl）-amino]}diazen-1-ium-1，2-diolate（MAHMA NONOate）]或细胞可渗透性cGMP类似物（8-bromo-cGMP）显著抑制I-UTP。预先应用PKG抑制剂（KT 5823）可逆转MAHMA NONOate或8-溴-cGMP的抑制作用，证明PKG在TRPC 1/TRPC 3的NO抑制中发挥关键作用。在存在或不存在La 3+（100 μ M）的情况下，用UTP（100 μ M）收缩完整的CA片段，然后评估对NO供体硝普钠（1 nM至1 μ M）的松弛。在La 3+治疗组中，硝普钠的松弛显著减少。我们的结论是，新鲜分离的SMC表达TRPC 1/TRPC 3通道，这些通道被NO/cGMP/PKG抑制。此外，NO通过抑制与TRPC 1/TRPC 3一致的La 3+敏感性通道来促进血管舒张。
Chen J, Crossland RF, Noorani MM, Marrelli SP. Inhibition of TRPC1/TRPC3 by PKG contributes to NO-mediated vasorelaxation. Am J Physiol Heart Circ Physiol 297: H417-H424, 2009. First published June 5, 2009; doi: 10.1152/ajpheart.01130.2008.-Nitric oxide (NO) inhibits transient receptor potential channel 3 (TRPC3) channels via a PKG-dependent mechanism. We sought to determine 1) whether NO inhibition of TRPC3 occurs in freshly isolated smooth muscle cells (SMC); and 2) whether NO inhibition of TRPC3 channels contributes to NO-mediated vasorelaxation. We tested these hypotheses in freshly isolated rat carotid artery (CA) SMC using patch clamp and in intact CA by vessel myograph. We demonstrated TRPC3 expression in whole CA (mRNA and protein) that was localized to the smooth muscle layers. TRPC1 protein was also expressed and coimmunoprecipitated with TRPC3. Whole cell patch clamp demonstrated nonselective cation channel currents that were activated by UTP (60 mu M) and completely inhibited by a TRPC channel inhibitor, La3+ (100 mu M). The UTP-stimulated current (I-UTP) was also inhibited by intracellular application of anti-TRPC3 or anti-TRPC1 antibody, but not by anti-TRPC6 or anti-TRPC4 control antibodies. We next evaluated the NO signaling pathway on I-UTP. Exogenous NO [(Z)-1-{N-methyl-N-[ 6(N-methylammoniohexyl)-amino]}diazen-1-ium-1,2-diolate (MAHMA NONOate)] or a cell-permeable cGMP analog (8-bromo-cGMP) significantly inhibited I-UTP. Preapplication of a PKG inhibitor (KT5823) reversed the inhibition of MAHMA NONOate or 8-bromo-cGMP, demonstrating the critical role of PKG in NO inhibition of TRPC1/TRPC3. Intact CA segments were contracted with UTP (100 mu M) in the presence or absence of La3+ (100 mu M) and then evaluated for relaxation to an NO donor, sodium nitroprusside (1 nM to 1 mu M). Relaxation to sodium nitroprusside was significantly reduced in the La3+ treatment group. We conclude that freshly isolated SMC express TRPC1/TRPC3 channels and that these channels are inhibited by NO/cGMP/PKG. Furthermore, NO contributes to vasorelaxation by inhibition of La3+-sensitive channels consistent with TRPC1/TRPC3.