GAP JUNCTIONS IN VASCULAR SMOOTH MUSCLE--GROWTH CONTROL

构成间隙连接的通道为相邻细胞之间的小分子扩散提供了通道。两个半通道，或连接子，包括每个间隙连接通道；每个连接子由六个称为连接蛋白（Cx）的亚基组成。哺乳动物基因组包含至少15种不同的连接蛋白，其中4种在心血管系统中表达（见表43、40、45和37）。间隙连接通过为电通信提供途径，使心脏和血管的协调收缩活动成为可能。它们在生长发育中也起着重要的作用。xs40和xs43形成同源/同型通道（通道中的所有连接蛋白都是相同的），具有非常不同的功能特性。然而，xs40和xs43很少单独表达。它们在心室和心房组织以及血管内皮和平滑肌中的共表达比例随发育（和衰老）、损伤和疾病的变化而变化。xs40和xs43形成功能“混合”通道（异质/异型），性质未知。拟议研究的长期目标是确定Cx40-43组成的连接如何促进心血管发育、损伤和疾病。我们的具体目标包括以下目标。1)确定Cx40: Cx43表达比的长期变化是否会引起可预测的连接通透性和细胞生长特性的变化。2)评估PDGF引起的脑结电导急性降低的幅度是否取决于Cx40: Cx43比值以及脑结通透性是否随之降低。3)研究Cx43磷酸化在PDGF急性效应中的作用。4)确定PDGF急性效应恢复的时间过程和潜在机制。通过基因操作诱导平滑肌细胞自然表达不同的Cx40: Cx43比率，转染这些基因的Rin细胞将作为这些研究的模型系统。间隙结渗透率和通道功能将使用双全电池和穿孔贴片电压钳技术进行评估。使用流式细胞术评估细胞增殖，以评估细胞周期行为。使用免疫染色细胞的三维成像和定量western来评估连接蛋白的表达和分布。这种强大的方法组合提供了一个独特的机会来发现间隙连接如何促进心脏和脉管系统的发育、损伤和疾病。

The channels that comprise gap junctions, provide a conduit for diffusion of small molecules between neighboring cells. Two hemichannels, or connexons, comprise each gap junction channel; each connexon is composed of six subunits called connexins (Cx). The mammalian genome contains at least fifteen different Connexins, four of which are expressed in the cardiovascular system (Cxs 43, 40, 45 and 37). Gap junctions make coordinated contractile activity of the heart and blood vessels possible by providing a pathway for electrical communication. They also play important roles in growth and development. Cxs 40 and 43 form homomeric/homotypic channels (all connexins in the channel are identical) with very different functional properties. However, Cxs 40 and 43 are rarely expressed in isolation. They are co- expressed at ratios that vary as a function of development (and aging), injury, and disease in ventricular and atrial tissue as well as in vascular endothelium and smooth muscle. Cxs 40 and 43 form functional "mixed" channels (heteromeric/heterotypic) with unknown properties. The long-term goal of the proposed studies is to determine how Cx40-43 comprised junctions contribute to cardiovascular development, injury and disease. Our specific aims include the following goals. 1) Determine whether long-term changes in Cx40: Cx43 expression ratio cause predictable changes in junctional permeability and cellular growth properties. 2) Assess whether the magnitude of the acute decrease in junctional conductance induced by PDGF depends on the Cx40: Cx43 ratio and whether junctional permeability decreases concomitantly. 3) Examine the role of Cx43 phosphorylation in these acute effects of PDGF. And 4) determine the time course and underlying mechanisms for recovery from the acute effects of PDGF. Smooth muscle cells induced to express varied Cx40: Cx43 ratios naturally and through genetic manipulation and Rin cells transfected with these genes will serve as model systems for these studies. Gap junction permeability and channel function will be assessed using dual whole cell and perforated patch voltage clamp techniques. Cell proliferation is evaluated using flow cytometry to assess cell cycle behavior of the cells. Connexin expression and distribution are evaluated using 3-D imaging of immumo-stained cells and quantitative Westerns. This powerful combination of approaches offers a unique opportunity to discover how gap junctions contribute to development, injury and disease in the heart and vasculature.