Referenced in: none

Automatically associated channels: TRP , TRPV , TRPV4

Title: [Role of TRPV4 channels in regulation of eNOS expression in brain microvascular endothelial cells under the condition of mechanical stretch].

Authors: Jiujun Pu, Zhiming Wang, Hui Zhou, Ailing Zhong, Lunliang Ruan, Kai Jin, Gang Yang

Journal, date & volume: Zhong Nan Da Xue Xue Bao Yi Xue Ban, 2015 Sep 28 , 40, 960-6

PubMed link: http://www.ncbi.nlm.nih.gov/pubmed/26408614

Abstract
To identify the subtype of transient receptor potential (TRPs) channel involved in stretch-induced injury of human brain microvascular endothelial cells (HBMEC) and to explore the mechanism responsible for eNOS expression.
TRPs expression was examined by Western blot and immunocytofluoresence in the cultured HBMEC. Mechanical stretch was performed by mini-type multi-functional bio-impact machine. The levels of free calcium ion in cells were examined by the flow cytometry. The eNOS expression was detected by Western blot.
The mRNA and protein expression of TRPV4 was detected in HBMEC by qRT-PCR, Western blot and immunocytofluoresence. The levels of free calcium ion in the stretch-treated HBMEC was significantly decreased in the presence of TRPV4 specific inhibitor (P<0.001), but there was no difference in calcium levels between the stretch and the control or unspecific inhibitor group (P=0.072 or 0.308). The levels of eNOS protein in the stretch-treated HBMEC were reduced in the presence of TRPV4 specific inhibitor or NOS inhibitor (P<0.05), but it was not changed compared with that in the control group (P>0.05).
The eNOS expression is up-regulated under the condition of mechanic stretch, which is related to the activation of TRPV4, resulting in the influx of calcium.目的：筛选人脑微血管内皮细胞(human brain microvascular endothelial cells，HBMEC)中瞬时感受器电位(transient receptor potential，TRP)通道亚型，检测该通道亚型在机械牵张刺激中对内皮型一氧化氮合酶(endothelial nitric oxide synthase，eNOS)蛋白表达的影响及探讨相关机制。方法：荧光定量RT-PCR (qRT-PCR)、Western印迹及细胞免疫荧光筛选HBMEC上TRP通道亚型。根据筛选的TRP通道亚型将细胞分组，应用多功能小型生物撞击机给予机械牵张刺激；流式细胞仪检测细胞牵张前后胞内Ca2+荧光强度变化；Western印迹检测eNOS蛋白表达情况。结果：qRT-PCR结果表明：TRPV4及TRPC1的mRNA相对表达量较多；细胞免疫荧光及Western印迹显示TRPV4通道蛋白表达；流式细胞仪检测结果显示，特异性TRPV4通道抑制组较单纯牵张组Ca2+相对浓度低(P<0.001)，较未牵张组(P=0.072)、非特异性TRP通道抑制组(P=0.308)无明显差异。Western印迹检测结果显示：TRPV4通道抑制组和内皮型一氧化氮合酶抑制组eNOS蛋白表达水平较单纯牵张组明显降低(P<0.05)，较未牵张组无明显差异(P>0.05)。结论：TRPV4在HBMEC上有表达，机械牵张将其激活后，可以引起eNOS蛋白表达增多，其机制可能与胞外Ca2+的内流有关。.