Arachidonic acid (AA) is the precursor of various mediators of inflammatory and allergic reactions. AA metabolites of the 5-lipoxygenase pathway include leukotriene LTB4 which is a very potent chemotactic agent and airway muscle contractant, and LTC4, LTD4 and LTE4, which are the active components of SRS, an important mediator in anaphylaxis and immediate hypersensitivity reactions. Leukotrienes are produced in leukocytes, lymphocytes, macrophages, RBL-1 and certain mast and mastocytoma cells. The 15-lipoxygenase product 15-HETE inhibits the 5-lipoxygenase in PMNs and lymphocytes resulting in decreased formation of leukotrienes and 5-HETE. We have found that 15-HETE can activate a cryptic 5-lipoxygenase in the cultured mast/basophil cell line PT-18 forming LTB4 and 5-HETE from exogenously added AA. 15-HETE-induced activation of the 5-lipoxygenase was enhanced four-fold upon pretreatment of PT-18 cells with indomethacin. Similar induction of LT formation by 15-HETE was observed in RBL-1 cells pretreated with 1 MuM A23187. However, with higher amounts of this ionophore, inhibition of LT biosynthesis by 15-HETE was found. Finally, 15-HETE also potentiated SRS/LT formation in vivo in the rat peritoneum stimulated with A23187. This project is designed to investigate the mechanism of modulation of LT biosynthesis by 15-HETE. The in vitro studies will use PT-18 amd RBL-1 cells. We will determine whether a further metabolite of 15-HETE or a structurally related analog of 15-HETE is the physiological activator. We will also examine whether 15-HETE acts directly on the 5-lipoxygenase, via a receptor and/or via induction of Ca++ flux. We will attempt to identify the cyclooxygenase metabolite that inhibits the total activation of the 5-lipoxygenase in PT-18 cells by 15-HETE. To investigate the mechanism of in vivo regulation of SRS/LT formation in the rat peritoneum by 15-HETE, we will examine the in vitro effects of 15-HETE on isolated peritoneal macrophages, mast cells and neutrophils, determine how 15-HETE modulates in vivo SRS formation induced by different immunological and non-immunological stimuli and investigate how pretreatment with aspirin or indomethacin affects the in vivo regulation of SRS production by 15-HETE. The information obtained from these studies will provide new insights into regulation of the 5-lipoxygenase which is an essential component in the formation of leukotrienes. It is hoped that ultimately this will lead to new therapeutic approaches in the management of certain inflammatory diseases.
