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TRPC6介导肺动脉高压大鼠肺动脉张力和肺动脉平滑肌细胞Ca2+ 浓度的升高

张明芳, 刘晓如, 杨娜, 林默君

福建医科大学基础医学院生理学与病理生理学系，福州350004

摘要

经典瞬时感受器电位通道6 (transient receptor potential channel 6, TRPC6)蛋白是受体操纵性Ca2+ 通道(ROCC)的分子基础。本文旨在研究TRPC6/ROCC 在野百合碱(monocrotaline, MCT)诱发的肺动脉高压大鼠模型中的作用。Sprague-Dawley大鼠随机分为正常对照组(CON 组)和MCT 组，CON 组正常饲养三周，而MCT 组按60 mg/kg 剂量一次性腹腔注射2% MCT，建立MCT 诱导的慢性肺动脉高压大鼠模型。通过测定右心室收缩压(RVSP)和右心室重量指数(RVMI)、HE 染色观察肺动脉血管形态，分析肺动脉结构重建。半定量RT-PCR 和Western blot 检测大鼠肺动脉TRPC6 mRNA 和蛋白表达水平。血管张力实验中用可特异性激活ROCC、可透膜的DAG 拟似物1-oleoyl-2-acetyl-sn-glycerol (OAG)检测大鼠离体肺动脉环的收缩效应。用荧光探针Fluo3-AM 测定OAG 诱导大鼠肺动脉平滑肌细胞(PASMCs)胞浆游离Ca2+ 浓度([Ca2+]i)。结果显示，与CON 组相比，MCT 组的RVSP、RVMI 均明显增高(P<0.01) ；形态学观察可见肺小动脉平滑肌层明显增厚，管腔减小；TRPC6 的mRNA 和蛋白质表达无明显变化。在CON 组，OAG 几乎不引起肺动脉环收缩，而在MCT 组，肺动脉环的收缩反应显著增强，差别有显著性意义(P<0.01)。相比较于CON 组，MCT 也可使OAG 触发的PASMCs[Ca2+]i 增量值显著升高(P<0.05)。上述结果提示，MCT 预处理对肺动脉TRPC6 mRNA 和蛋白质水平的表达无显著增强效应，但可促进TRPC6/ROCC 介导的PASMCs Ca2+ 内流和肺动脉张力升高，诱导大鼠产生肺动脉高压，并进一步诱发肺血管及右心室重构。

关键词： 经典瞬时感受器电位; 钙离子; 受体操纵性Ca2 + 通道; 野百合碱

分类号：R363

[TRPC6 mediates the enhancements of pulmonary arterial tone and intracellular Ca2+ concentration of pulmonary arterial smooth muscle cells in pulmonary hypertension rats.] [Ariticle in Chinese]

ZHANG Ming-Fang, LIU Xiao-Ru, YANG Na, LIN Mo-Jun

Department of Physiology and Pathophysiology, Fujian Medical University, Fuzhou 350004, China

Abstract

Pulmonary arterial hypertension is associated with profound vascular remodeling and alterations in Ca2+ homeostasis inpulmonary arterial smooth muscle cells (PASMCs). Recent studies show that canonical transient receptor potential channel 6 (TRPC6)genes, which encode receptor-operated cation channels (ROCC) in PASMCs, play an important role in Ca2+ regulation and cellproliferation. The aim of the present study was to investigate the role of TRPC6 in monocrotaline (MCT)-induced pulmonary arteryhypertension. Sprague-Dawley rats were randomly divided into normal control group and MCT group. In MCT group, pulmonaryarterial hypertension was induced by a single intraperitoneal injection of MCT at a dose of 60 mg/kg. After 3 weeks, the right ventricularsystolic pressure (RVSP) and the right ventricular mass index (RVMI) were measured. The lung sections were stained by HE andobserved under light microscope. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot wereperformed to detect the expression of TRPC6 in rat pulmonary arteries. The 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced contractiletension of pulmonary arteries were measured by vascular ring tension analysis and the intracellular Ca2+ concentration ([Ca2+]i)of PASMCs was monitored using Fluo3-AM assay. The results showed that RVSP and RVMI markedly elevated in MCT group (P<0.01)in comparison to CON group. The thickness of pulmonary vascular smooth muscles was increased and the inner diameter of pulmonaryarteries was diminished in MCT group. Though there was no significant difference in the levels of mRNA and protein of TRPC6between CON and MCT groups, the application of OAG, which can directly activate ROCC, induced greater contraction tension ofpulmonary arteries (P<0.01) and more Ca2+ entries in PASMCs (P<0.05) in MCT group compared to those in control group. Theseresults indicate that MCT induces pulmonary artery hypertension and thus remodeling of the right ventricle and pulmonary arteries inrats. The expression of mRNA and protein of TRPC6 is not potentiated by MCT, but the TRPC6/ROCC-mediated Ca2+ entry inPASMCs and vascular tone of pulmonary arteries are significantly increased with MCT treatment.

Key words: pulmonary arterial hypertension; transient receptor potential channels; Ca2+; receptor-operated Ca2+ channel

收稿日期：2009-09-22　　录用日期：2009-12-18

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引用本文：

张明芳, 刘晓如, 杨娜, 林默君. TRPC6介导肺动脉高压大鼠肺动脉张力和肺动脉平滑肌细胞Ca2+ 浓度的升高[J]. 生理学报 2010; 62 (1): 55-62.

ZHANG Ming-Fang, LIU Xiao-Ru, YANG Na, LIN Mo-Jun. [TRPC6 mediates the enhancements of pulmonary arterial tone and intracellular Ca2+ concentration of pulmonary arterial smooth muscle cells in pulmonary hypertension rats.] [Ariticle in Chinese] . Acta Physiol Sin 2010; 62 (1): 55-62 (in Chinese with English abstract).