生理和缺血再灌注状态下的冠脉内皮功能 - - 内皮离子通道间信号关联的研究

Endothelial cells release vasoactive agents to regulate vascular tone. The normal function of endothelium is controlled by a number of ion channels that include calcium-activated potassium channels of intermediate- and small- conductance (IKCa and SKCa). Previous studies have demonstrated that the activation of endothelial IKCa and SKCa underlies the action of endothelium-derived hyperpolarizing factor (EDHF) that requires a rise of intracellular Ca2+ concentration ([Ca2+]i). Canonical transient receptor potential channels (TRPC) are Ca2+-permeable channels that are important in endothelial Ca2+ regulation. However, it remains unknown whether TRPC channels may associate with KCa channels in endothelial function. In this study, we will first investigate the role of TRPC3 in the channel activity of endothelial KCa and the role of TRPC3 channel in endothelial KCa channel-dependent EDHF-responses. Whether the functional association between TRPC3 and KCa involves physical interactions will be further studied. ..Endothelial dysfunction occurs during ischemia-reperfusion (I-R) / hypoxia-reoxygenation (H-R) injury. Our previous studies have demonstrated that the impairment of EDHF-type responses in coronary arteries under H-R conditions is attributable to the inhibition of channel activity of IKCa and SKCa in endothelial cells. We also demonstrated that H-R inhibits TRPC3 channels that results in a decrease of [Ca2+]i in endothelial cells accompanied by a marked reduction of coronary vasorelaxation. Nevertheless, whether inhibition of TRPC3 contributes to the suppression of IKCa and SKCa as well as the IKCa and SKCa-dependent endothelial function under H-R conditions remains unknown, and this therefore forms the second aim of the present study. ..This study will be conducted with multiple approaches at different levels that include recording of channel currents by patch-clamp and measurements of Ca2+ concentration with fluorescence dyes in endothelial cells; microelectrode measurements of membrane potential in both endothelial cells and in smooth muscle cells of endothelium-intact arteries; and myograph studies of relaxation in coronary arteries. Coimmunoprecipitation and double-labeling imunofluorescence will be used to detect the expression and localization of the channels. ..The proposed study shall advance our understanding of TRPC channels in endothelium physiology with new information added to EDHF signaling cascade and endothelial dysfunction under I-R/H-R conditions. This study may reveal new molecular targets and shed light on future therapeutic interventions of endothelial dysfunction in ischemic / hypoxic states, such as in ischemic heart disease.

内皮细胞释放血管活性物质调节血管张力。中小电导钙激活钾通道(IKCa和SKCa)的激活是内皮源性超极化因子EDHF的作用机制，而内皮细胞内Ca2+浓度升高是通道激活的先决条件。规范瞬时受体电位通道TRPC在内皮Ca2+调节中起重要作用。然而TRPC是否与KCa关联参与内皮功能未见报道。本研究将首先明确TRPC3在内皮KCa活性及在KCa依赖性EDHF功能中的作用。TRPC3和KCa是否存在物理联系也将被研究。缺血再灌注/缺氧再复氧(H-R)时内皮功能失调。我们发现H-R时EDHF损伤是由于内皮IKCa和SKCa的抑制。我们也证实H-R可抑制TRPC3而致内皮Ca2+信号障碍。然H-R时TRPC3抑制会否导致内皮KCa及KCa依赖性EDHF功能抑制尚不明确。本项目通过对内皮离子通道关联的研究可为EDHF信号串联和H-R时内皮功能失调提供新信息，有助于发现新的靶点防治缺血缺氧时的内皮功能障碍。

血管内皮功能失调参与多种心血管疾病的发生和发展。尽管以往在内皮保护的研究工作方面取得了一定成果，但如何能更有效地预防和治疗内皮功能失调仍是现代医学所面临的挑战。从细胞和分子层面更深入地理解内皮功能的调控机制无疑有助于发展新的内皮保护措施。本项目通过对内皮细胞中瞬时受体电位通道和钙激活钾通道间相互关联的研究对缺血再灌注/缺氧再复氧时的内皮损伤机制进行了探讨。研究发现在冠状动脉内皮细胞中，TRPC3通道与KCa通道相互关联参与内皮EDHF功能的调控。此两种通道间的关联发生在功能层面而非物理层面。缺血再灌注/缺氧再复氧抑制内皮TRPC3通道的活性，进而引致KCa通道的抑制以及KCa依赖性的EDHF功能的损伤。通过调控TRPC3通道活性可有效防止缺血再灌注/缺氧再复氧时的内皮功能障碍。本项目将不同离子通道在内皮功能调控中的作用机制联系在一起，对与内皮功能有关的不同离子通道之间的相互作用作出了系统研究，首次将缺血再灌注/缺氧再复氧状态下的内皮功能失调从不同离子通道与EDHF相互关系的角度出发进行了探讨。研究结果为发展缺血缺氧状态下，如缺血性心脏病或心脏手术时以内皮离子通道作为靶点干预血管损伤的新策略奠定了基础。