|Budget Amount *help
¥15,000,000 (Direct Cost : ¥15,000,000)
Fiscal Year 1997 : ¥2,700,000 (Direct Cost : ¥2,700,000)
Fiscal Year 1996 : ¥5,000,000 (Direct Cost : ¥5,000,000)
Fiscal Year 1995 : ¥7,300,000 (Direct Cost : ¥7,300,000)
In this project, we have examined the regulation of arachidonic acid (AA) metabolism in various hematopoietic cells.
(1) AA metabolism in mast cells. Mouse and rat mast cells stimulated by FsepsilonRI crosslinking via IgE and antigen in the presence of specific accessory cytokines show two sequential prostaglandin (PG) D_2 biosynthetic responses over time. Immediate PGD_2 generation, occurring within a few minutes in response to a transient increase in cytoplasmic Ca^<2+> concentration, is associated with transient perinuclear translocation, phosphorylation and activation of cytosolic phospholipase A_2 (cPLA_2). The constitutively expressed cyclooxygenase (COX) isoform, COX-1, is the dominant enzyme involved in this rapid response, which converts arachidonic acid to PGH_2, which in turn is metabolized to PGD_2 via glutathione-dependent PGD_2 synthase. Delayd PGD_2 generation, occurring overseveral hours of culture, is associated with the de novo induction and function of COX-2. Delayd P
GD_2 generation is accompanied by the induction of type IIA secretory PLA_2 (sPLA_2), which is functionally linked to COX-2 through enhancing COX-2 expression. Another sPLA_2 isozyme, type VsPLA_2, is also expressedin mast cells and may compensate for type IIA sPLA_2. Furthermore, activation of mast cells elicits rapid and transient production of platelet-activating factor (PAF), which is inactivated by plasma-type PAF-acetylhydrolase exocytosed from mast cells, revealing an anti-inflammatory aspect of mast cells.
(2) AA metabolism in neutrophils. Release of AA and subsequent production of leukotriene B_4 and PAF in fMLP-orzymozan-stimulated rat neutrophils depend on cPLA_2. cPLA_2 activation is in part regulated by phospholipase D.
(3) AA metabolism in macrophages. Rat peritoneal macrophages stimulated with A23187 produce thromboxane (TX) B_2 in marked preference to PGE_2 within 30-60 min (constitutive immediate response), which is mediated by preexisting cPLA_2, COX-1, and TX synthase. Cells treated with LPS predominantly produce PGE_2 during culture for 3-24 h (delayd response), where cPLA_2 and type IIA sPLA_2 function cooperatively with inducible COX-2, which is in turn coupled with inducible PGE_2 synthase. Cells primed for 12h with LPS and stimulated for 30 min with A23187 produce PGE_2 in marked preference to TXB_2 (induced immediate response), in which three inducible enzymes, cPLA_2, COX2, and PGE_2 synthase are functionally linked.
[Conclusion] These results suggest that distinct PG-biosynthetic enzymes display segregated functional coupling following different transmembrane stimulation events even when enzymes that catalyze similar reactions in vitro coexist in the same cells. Less