The application focuses on biologic mediators derivedfrom arachidonic acid metabolism (eicosanoids) and those formed via cell-cell interactions between two cells which synthesize a product that neither cell can produce alone. The direct cell metabolites are the leukotrienes (LT) and the transcellular metabolites are lipoxins (LX). Both classes can promote cell reactivity, but the LX can down-regulate multi-cellular events. Aspirin (ASA) is normally thought of as an inhibitor of eicosanoid production, but recent new findings concerning aspirin indicate that its inhibitory and/or stimulatory effects have a broader base. Aspirin appears to reduce the incidence of heart disease by mechanisms which are only partially understood and more recently aspirin has been found to decrease the incidence of colo-rectal cancer in a population of women ingesting the compound for relatively long periods of time. The long-term goals of the project are to understand mechanisms of aspirin-induced LX receptor activation and to examine the therapeutic potential of lipoxin receptor and lipoxin analogs in disease states. Many of the specific aims originate from the recent observation that aspirin promotes production of 15-epi-lipoxins and that recently synthesized lipoxin analogs can activate the myeloid LXA4 or a closely related receptor. Such interaction initiates leukocyte-directed regulatory signals which result in inhibition of vascular events initiated by neutrophils. They also promote macrophage anti-tumor activity. Therefore, the beneficial effects of aspirin could be mediated through activation of lipoxin receptors. To characterize leukocyte receptors for 15-epi-lipoxin production and new LXA4 analogs, synthetic precursors will be radiolabeled and binding ascertained in CHO cells which have been transfected with the lipoxin receptor CDNA. The monocyte LXA4 receptor will be cloned and sequenced and binding also studied in transfected cells. Probes will be generated for identification of lipoxin isoforms or subtypes of the receptors. In the next Specific Aim, actions of lipoxins and analogs on regulation of adhesion molecules in neutrophils and monocytes will be studied. The ability to generate oxygen radicals will be evaluated and also it will be determined whether lipoxin isoforms and/or subtypes expressed in transfected cells can transmit signals in response to epi-lipoxins or lipoxin analogs. In the third Aim, the contribution of each receptor domain to ligand binding and function will be evaluated. Chimeric LX receptors will be constructed for the mouse LXA4-R and human homologs. These have already been cloned in preliminary experiments. Lipoxin receptor deletion mutants and site-directed mutagenesis will be carried out to evaluate the affect of targeted domains in binding and signaling. Desensitization mechanisms will also be assessed. In the fourth Specific Aim, preliminary experiments which demonstrated that lipoxins can inhibit cell proliferation will be amplified. The action of analogs and their stereochemical requirements will be studied. If possible, the role of the LXA4-R in cell proliferation will be tested.
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