Pathological and physiological tissue responses commonly involve phospholipid (PL) and arachidonic acid (AA) metabolites. For example, activated neutrophils release AA from PL; acetylate resulting lyso-PL to platelet-activating factor (PAF) analogs; oxygenate AA to leukotriene (LT) B4 and 5-hydroxyicosa-tetraenoate (HETE), and otherwise metabolize resident PL. These metabolites are bioactive and may stimulate their parent cell or exit the cell to stimulate, e.g., lung contractile and inflammatory reactions. Many agonists that induce formation of these metabolites, as well as the metabolites themselves, operate through rhodopsin superfamily receptors (RSR). RSR interact with G proteins to stimulate cells to raise cytosolic Ca2+ ([Ca2+]i) which, it is widely maintained, then triggers protein kinase C (PKC) mobilization, PL metabolism, and consequently cell function. Our progress studies, however, suggest that RSR agonists (LTB4, PAF, N-formyl-MET-LEU-PHE, C5a, etc.) induce cells to form signals (e.g., AA) that activate these transduction pathways (e.g., PKC mobilization) regardless of Ca2+ while [Ca2+]i rises and 5-HETE up-regulate RSR to enhance cell excitation. Agonists that normally do not alter [Ca2+]i may also use these Ca2+-independent reactions to activate cells. Our application test this model in experiments that: determine the Ca2+ requirements for RSR and non-RSR agonists to stimulate PKC mobilization, PL metabolism, and cell function; examine the receptor-regulating actions of cell Ca2+; define the [Ca2+]i transient-independent signals that mobilize PKC and/or activate PL metabolism; and analyze the unique receptors and post-receptor mechanisms involved in the bioactions of 5-HETE. Studies are done with human polymorphonuclear neutrophils, HL-60 granulocytes, and cell-free systems that reconstitute metabolic pathways with purified components. Experiments employ various ligand binding techniques to quantitate and localize receptors; thin-layer, high-performance, and gas chromatographies, and mass spectroscopy, radiolabel precursor tracking, and immunoassays to measure AA and PL metabolism; radiolabeled GTP binding and hydrolysis to enumerate and classify G proteins; novel assays of phorbol diester binding and histone phosphorylation to quantitate and localize PKC; and various methods to measure neutrophil (Ca2+]i, degranulation, and oxidative metabolism responses. We hope to redefine the RSR and non-RSR response strategies that are used by virtually mammalian tissues. Results will pertain to a broad range of inflammatory, allergic, immunologic, cardiovascular, pulmonary, and hormonal responses of humans in health and disease.
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