Polymorphonuclear neutrophils (PMN) produce the important lipid inflammatory mediators arachidonic acid (AA), 5-hydroxyeicosatetraenoic acid (5-HETE), leukotriene B4 (LTB4), and platelet activating factor (PAF). Pharmacologic activation of human PMN with calcium ionophore has demonstrated that the initial step in formation of these molecules involves phospholipase A2 (PLA2) catalyzed release of AA from membrane phospholipids; however, progress in studying formation of these lipid mediators has been limited by lack of information on the physiological control of PLA2. In the proposed studies we will employ a newly developed model of permeabilized PMN to study PLA2 activation initiated with soluble stimuli and will test the hypothesis that receptor-mediated PLA2 activation involves a G-protein mediated event which alters enzyme requirements for specific cofactors, in particular, Ca2+. We also will define the optimum intracellular concentrations of cofactors and/or ions necessary for PLA2 activation in stimulated cells. Requirements for PLA2 activity will be assessed in PMN permeabilized with the plasma membrane specific agent, Staphylococcus aureus alpha-toxin. This model allows manipulation of intracellular concentration of small molecules or ions which are potential cofactors for PLA2 activation while keeping enzymes, proteins and other intracellular organelles intact. PLA2 activation will be assayed by determining the release and metabolism of radiolabeled AA from membrane phospholipids and by monitoring radiolabeled lyso-PAF and PAF formation. Thin layer chromatographic techniques and high pressure liquid chromatography (HPLC) will be utilized for lipid identification. Mass determinations of AA, LTB4, 5-HETE, lyso-PAF and PAF will be performed using techniques of gas chromatography, radioimmunoassay, HPLC and coomassie blue staining followed by densitometry. While stimulation of human PMN with a single physiologic agonist evokes only limited PLA2 activity, receptor-mediated activation of PLA2 is greatly enhanced during """"""""primed-stimulation"""""""". In this process, neutrophils are sequentially treated with non-stimulatory concentrations of agonists; in primed PMN exposed to a second agonist, PLA2 activity is initiated and release of AA is much greater than expected from an additive response to the individual stimulus. After investigating the mechanism(s) involved in direct PLA2 activation in permeabilized PMN, the molecular basis leading to enzyme activation during """"""""primed-stimulation"""""""" will be determined.
