Long-term objectives are: 1) to determine the mechanism and sites of action of lipoxygenase (LO)-derived eicosanoids and 2) to evaluate whether lipoxins and/or their receptors are targets for interventions in human vascular and inflammatory diseases. LO-derived products such as leukotrienes (LT) are important signals in inflammation. Interactions between major LO of human tissues (5-, 12-, and 15-LO) can lead to the generation of tetraene-containing eicosanoids that we termed lipoxins (LX). LX display bioactions in vivo and in vitro that include inhibition of some LT-mediated events. Recently, we found that LX are generated by novel routes during neutrophil (PMN)-platelet interactions and that they are formed in activated whole blood and within coronary arteries of patients undergoing angioplasty. However, detailed knowledge of LX action is lacking.
The specific aims extend studies in this laboratory that use human PMN and HL-60 cells to systematically investigate the mechanism(s) underlying the actions of LXA4 and LXB4.
The aims are to: 1) Characterize LXA4 and LXB4 recognition sites. We have prepared both 3H- LXA4 and a fluorescent derivative and studies in progress show that human PMN display stereospecific binding (Kd 0.5 nM). Focus will be to prepare labeled LXA4, LXB4 and their all-trans isomers for binding studies and to determine Kd, structural requirements, recovery and subcellular distribution of LX binding sites. 2) Establish the relationship between LX binding and PMN responses. Focus will be on documenting LX-induced changes in lipid remodeling by establishing sensitive methods employing ED-HPLC and NICI-GC-MS to monitor changes in mass of arachidonic acid (C20:4) and phosphatidic acid. Results will be used to determine fractional occupancies required to elicit responses. A second series of experiments will investigate the regulation of LX surface binding and LX modulation of PMN responses. 3) Cloning LX receptors: LXA4 binding sites are induced in HL-60 cells. Here, a multi-pronged cloning strategy will be undertaken to identify LXA4 and LXB4 receptors (r). LX-r cDNA will be coexpressed in CHO to investigate LX-triggered C20:4 release and test potential desensitization of other receptor classes (peptide and lipid mediators). These results are expected to give a better understanding of the actions of LX by identifying their targets. Moreover, they will supply the basis for evaluating the role of LO-derived eicosanoids and specifically the LX system in human disease.
Showing the most recent 10 out of 102 publications
