ATP-INDUCED GAP JUNCTION PORES IN MACROPHAGES

胞外ATP4-诱导质膜上的气孔形成 小鼠巨噬细胞和J774巨噬细胞样细胞系。这些三磷酸腺苷- 诱导的毛孔由缝隙连接蛋白Cx43组成，它 最初是从大鼠心脏克隆的，通过鉴定 连接蛋白43在J774细胞中表达，但在ATP中不表达。 来源于J774细胞的耐药细胞系。这些研究表明， 巨噬细胞可能使用连接蛋白43作为细胞间通讯的一种方式， 这种缝隙连接蛋白可能会形成“半缝隙”孔以及 两个细胞之间连接，且可以调节缝隙连接的通畅性 通过配对细胞胞质基质中的三磷酸腺苷。 拟议研究的长期目标有两个。第一，学习 ATP诱导的缝隙连接孔的结构和功能。独一无二的 该模型的特点是能够研究功能缝隙连接 蛋白质在单个细胞中而不是在细胞对中，以及获得 连接蛋白43六聚体的胞外面。第二，到 确定这些缝隙连接蛋白在细胞中的生理作用 巨噬细胞。在这些细胞中还没有描述缝隙连接。 连接蛋白可能允许巨噬细胞和 内皮细胞、淋巴细胞或其他细胞，或可替代新的 功能。 将使用各种技术来实现这些目标，包括 连接蛋白43对三磷酸腺苷抗性J774变异体等细胞的转染 不表达ATP诱导的毛孔、染料转移和免疫细胞化学 连接蛋白43在巨噬细胞间作用的研究 通讯，以及评估连接蛋白43是否结合的生化研究 如果巨噬细胞中的连接蛋白43被磷酸化，以及是否 细胞间隙连接蛋白43在单核巨噬细胞发育中的表达 受监管的。这些研究可能会产生关于 巨噬细胞的细胞间通讯，适用于各种 感染性、免疫性和炎症性反应；它们还可能揭示 缝隙连接的调节机制，增加我们对 许多生理和病理过程，如心脏的反应 缺血时的心肌细胞。

Extracellular ATP4- induces the formation of pores in the plasma membrane of mouse macrophages and the J774 macrophage-like cell line. These ATP- induced pores are comprised of the gap junction protein connexin43, which was originally cloned from rat heart, as demonstrated by identifying the presence of connexin43 mRNA and protein in J774 cells but not in ATP- resistant cell lines derived from J774 cells. These studies suggested that macrophages may use connexin43 as a means of intercellular communication, that gap junction proteins may be able to form "half gap" pores as well as junctions between two cells, and that gap junction patency may be regulated by ATP in the cytoplasmic matrix of the partner cell. The long term goals of the proposed studies are twofold. First, to study the structure and function of ATP-induced gap junction pores. The unique properties of this model are the ability to study functional gap junction proteins in single cells instead of in cell pairs, and the ability to gain access to the extracellular face of the connexin43 hexamer. Second, to determine the physiologic role of these gap junction proteins in macrophages. Gap junctions have not been described in these cells. Connexins may allow intercellular communication between macrophages and endothelial cells, lymphocytes, or other cells, or may subserve novel functions. A variety of techniques will be used to pursue these goals, including transfection of connexin43 into ATP-resistant J774 variants and other cells that do not express ATP-induced pores, dye transfer and immunocytochemical studies to assess the role of connexin43 in macrophage intercellular communication, and biochemical studies to assess whether connexin43 binds ATP directly, if connexin43 in macrophages is phosphorylated, and whether expression of connexin43 in mononuclear phagocytes is developmentally regulated. These studies may yield important information regarding intercellular communication in macrophages, applicable to a variety of infectious, immune, and inflammatory responses; they may also shed light on mechanisms of gap junction regulation, and increase our understanding of many physiologic and pathologic processes, such as the response of cardiac myocytes in ischemia.