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.
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| Civitelli, R; Beyer, E C; Warlow, P M et al. (1993) Connexin43 mediates direct intercellular communication in human osteoblastic cell networks. J Clin Invest 91:1888-96 |
