Functions of polymorphonuclear granulocytes (PMN) are altered during inflammatory lung diseases. PMN can be also primed by exposure to inflammatory mediators in vitro, and stimulation of primed PMN results in enhanced activation of PLA2's, which release arachidonic acid (AA), and products such as leukotriene B4. The goal of this project is to determine mechanisms of priming of PMN. Our main hypotheses are: (i) PMN contain at least two types of PLA2 enzymes, one cytosolic (cPLA2) and one granule-associated (or secretory"""""""", sPLA20. (ii) With stimulated mobilization of cytosolic Ca++ to the high nanomolar range, the cytosolic cPLA2 is translocated to cell membrane(s) and activated. This translocation might be effected during priming, to increase efficiency of PLA2 activation with primed-stimulation. (iii) In addition, during primed- stimulation, granule-associated sPLA2 is moved to the cell surface by fusion of granule and plasma membranes; at the external surface, millimolar extracellular Ca++ activates sPLA2 to hydrolyze adjacent membrane to release fatty acids which tend to remain as free fatty acids. (IV) Delayed (cytokine-mediated) priming also involves increased mass of both PLA2's, suggesting specific molecular regulation of PLA2's during priming. Studies will initially compare normal PMN and PMN primed in vitro. Increased protein mass and/or altered localization (translocation of PLA2's will be analyzed by enzymatic assays, by Western blots of cell fractions, by immune electron microscopy. Whether priming of cPLA2 requires phosphorylation of the enzyme will be assessed. Determining the level(s) of regulation of protein mass will include studies of protein stability, mRNA transcription and translation, and message stability. Preliminary data indicate the sPLA2 of granulocyte to be a novel protein; this will be cloned; sequence data will be used to select full length clones for expression, design probes for Northerns and RNAse protection experiments for study of sPLA2 expression in cells, and predict protein sequence which will be confirmed by amino acid sequencing of both cloned proteins and immunoaffinity purified granulocyte sPLA2. Expression of the active enzyme will allow direct studies of the effect of extracellular granulocyte sPLA2on PMN AA release and other PMN functions. It will also facilitate direct studies of a possible receptor for the protein. We will determine whether similar events occur during lung diseases in vivo by using PMN obtained from blood and BAL of patients with asthma and patients with burns/ARDS. Release of AA (and other fatty acids) will be accurately quantitated by negative ion chemical ionization mass spectrometry.
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