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首页> 外文期刊>American Journal of Physiology >Vasoconstriction resulting from dynamic membrane trafficking of TRPM4 in vascular smooth muscle cells
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Vasoconstriction resulting from dynamic membrane trafficking of TRPM4 in vascular smooth muscle cells

机译：血管平滑肌细胞中TRPM4动态膜运输导致的血管收缩

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摘要

The melastatin (M) transient receptor potential (TRP) channel TRPM4 mediates pressure and protein kinase C (PKC)-induced smooth muscle cell depolarization and vasoconstriction of cerebral arteries. We hypothesized that PKC causes vasoconstriction by stimulating translocation of TRPM4 to the plasma membrane. Live-cell confocal imaging and fluorescence recovery after photobleaching (FRAP) analysis was performed using a green fluorescent protein (GFP)-tagged TRPM4 (TRPM4-GFP) construct expressed in A7r5 cells. The surface channel was mobile, demonstrating a FRAP time constant of 168 ± 19 s. In addition, mobile intracellular trafficking vesicles were readily detected. Using a cell surface biotinylation assay, we showed that PKC activation with phorbol 12-myristate 13-acetate (PMA) increased (～3-fold) cell surface levels of TRPM4-GFP protein in <10 min. Similarly, total internal reflection fluorescence microscopy demonstrated that stimulation of PKC activity increased (～3-fold) the surface fluorescence of TRPM4-GFP in A7r5 cells and primary cerebral artery smooth muscle cells. PMA also caused an elevation of cell surface TRPM4 protein levels in intact arteries. PMA-induced translocation of TRPM4 to the plasma membrane was independent of PKCα and PKCβ activity but was inhibited by blockade of PKCδ with rottlerin. Pressure-myograph studies of intact, small interfering RNA (siRNA)-treated cerebral arteries demonstrate that PKC-induced constriction of cerebral arteries requires expression of both TRPM4 and PKCδ. In addition, pressure-induced arterial myocyte depolarization and vasoconstriction was attenuated in arteries treated with siRNA against PKCδ. We conclude that PKCδ activity causes smooth muscle depolarization and vasoconstriction by increasing the number of TRPM4 channels in the sarcolemma.
机译：褪黑素（M）瞬态受体电位（TRP）通道TRPM4介导压力和蛋白激酶C（PKC）诱导的脑动脉平滑肌细胞去极化和血管收缩。我们假设PKC通过刺激TRPM4转移到质膜引起血管收缩。使用在A7r5细胞中表达的绿色荧光蛋白（GFP）标签的TRPM4（TRPM4-GFP）构建体进行光漂白（FRAP）分析后的活细胞共聚焦成像和荧光恢复。表面通道是可移动的，表明FRAP时间常数为168±19 s。另外，容易检测到移动的细胞内运输小泡。使用细胞表面生物素化测定法，我们发现用佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）激活PKC可以在10分钟内增加TRPM4-GFP蛋白的细胞表面水平（约3倍）。同样，全内反射荧光显微镜显示，PKC活性的刺激增加了A7r5细胞和原代脑动脉平滑肌细胞中TRPM4-GFP的表面荧光（〜3倍）。 PMA还引起完整动脉中细胞表面TRPM4蛋白水平的升高。 PMA诱导的TRPM4易位至质膜，与PKCα和PKCβ活性无关，但被rottlerin阻断PKCδ所抑制。完整的小干扰RNA（siRNA）处理的脑动脉的压力肌电图研究表明，PKC诱导的脑动脉收缩需要表达TRPM4和PKCδ。另外，在用针对PKCδ的siRNA处理的动脉中，压力诱导的动脉肌细胞去极化和血管收缩减弱。我们得出结论，PKCδ活性通过增加肌膜内TRPM4通道的数量而引起平滑肌去极化和血管收缩。

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