The applicant proposes to investigate the mechanism of mouse bone marrow derived mast cell adenosine receptor biosignaling. Adenosine is an important mediator and modulator of immediate hypersensitivity reactions. As a mediator, adenosine is released by stimulated mast cells in hypoxic lung tissue. When inhaled, it can induce bronchospasm in asthmatic subjects. As a modulator, adenosine enhances the release of histamine from activated mast cells. This enhancement can be blocked by adenosine receptor antagonists including theophylline, a potent anti-asthmatic drug. Adenosine is also of interest as a modulator of mast cell function in that it augments mast cell degranulation but not the generation of new mediators such as leukotriene C4. The biochemical processes involved in the effect of adenosine on mast cells and in signal transduction in general are only beginning to be understood. Recent evidence has supported the possibility that the ability of adenosine to markedly potentiate mast cell preformed, granule-associated mediator release may involve an activation of protein kinase C. Protein kinase C activators such as the phorbol esters or synthetic diacyl glycerol analogs exhibit varying effects on mast cell mediator release, depending on the secretagogue utilized, the dose of the agent, and the time of addition of the agent. However, the ability of adenosine to enhance preformed mediator release is uniformly suppressed by a preincubation with any of these agents, suggesting the possible requirement for adenosine to interact with protein kinase C to induce its mediator release augmentation. However, leukotriene generation is not altered under identical experimental conditions, again underscoring the potential for dissociation of the two types of mast cell mediator release and a possible lesser role for protein kinase C in mast cell leukotriene production. The key issues in this proposal include the effect of adenosine itself in the presence and absence of other pharmacologic agents on protein kinase C activation as well as specific protein phosphorylation, calcium fluxes, and inositol trisphosphate generation, the phospholipase A2 products of phosphatidylcholine metabolism, lysophosphotidylcholine and fatty acids (particularly arachidonic and oleic acids), will also be examined for their ability to alter mast cell secretion, modulate mast cell adenosine responsiveness, induce leukotriene production, activate or inhibit protein kinase C, and express a potentially unique pattern of protein phosphorylation. Inhibitors or stimulators of specific enzymes important in diacylglycerol metabolism will be studied to identify key areas of regulation of the mast cell secretory process. The overall objective of this project is to better understand receptor signal transduction by focusing on an agent, adenosine, known to positively influence the mast cell secretory process. By determining specific sites and mechanisms of action of adenosine on mast cells, one would hope to be able to develop agents that would prove efficacious in the treatment of asthma and allergic diseases.
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