During vascular injury, nucleotides released from damaged cells and activated platelets stimulate neutrophils. Extracellular ATP has been shown to activate neutrophils resulting in chemotaxis, release of elastase, Mac-1 (alphaMbeta2 integrin) surface expression, and enhanced adhesion to endothelial cells, but the molecular mechanisms are yet to be elucidated. We have demonstrated that extracellular nucleotides stimulate surface expression of Mac-1 on neutrophils. We have cloned and localized LTB4 receptors on peripheral blood neutrophils and HL-60 cells. We hypothesize that leukotriene B4 is an essential intermediate in the nucleotide-induced neutrophil stimulation, leading to chemotaxis, release of elastase, and increased surface expression of adhesive proteins involved in the binding to endothelial cells. We propose to test this hypothesis by nucleotide-induced LTB4 generation in PMN and inhibiting the nucleotide-induced PMN stimulation using inhibitors of leukotriene biosynthesis, mice-deficient in leukotriene generation, LTB4 receptor antagonists, inhibitory receptor antibodies. In preliminary studies, we have shown that nucleotides generate LTB4 in peripheral blood leukocytes and the nucleotide-induced elastase release is abolished in isolated PMN by inhibitors of leukotriene biosynthesis or LTB4 receptor antagonists, and in mice lacking 5-lipoxygenase. We have also shown that polyclonal antibodies against the third extracellular loop of the LTB4 receptor specifically inhibit LTB4-, ATP- and ADP-, but not fMLP-, induced Mac-i expression on U937 cells. The differences in nucleotide- and LTB4 receptor mediated signaling will be evaluated to understand the requirement for LTB4 generation. The P2Y subtypes involved in PMN stimulation will be determined and the molecular mechanisms of P2Y2 receptor desensitization will be elucidated, using HL-60 cells as a model system. The studies proposed in this application will also help us understand the mechanisms of neutrophil activation and contribute to the knowledge of better treatments and to the design of therapeutics for inflammatory diseases.
| Murugappa, Swaminathan; Kunapuli, Satya P (2006) The role of ADP receptors in platelet function. Front Biosci 11:1977-86 |
| Ding, Zhongren; Tuluc, Florin; Bandivadekar, Kavita R et al. (2005) Arg333 and Arg334 in the COOH terminus of the human P2Y1 receptor are crucial for Gq coupling. Am J Physiol Cell Physiol 288:C559-67 |
| Meshki, John; Tuluc, Florin; Bredetean, Ovidiu et al. (2004) Molecular mechanism of nucleotide-induced primary granule release in human neutrophils: role for the P2Y2 receptor. Am J Physiol Cell Physiol 286:C264-71 |
| Tuluc, Florin; Garcia, Analia; Bredetean, Ovidiu et al. (2004) Primary granule release from human neutrophils is potentiated by soluble fibrinogen through a mechanism depending on multiple intracellular signaling pathways. Am J Physiol Cell Physiol 287:C1264-72 |
| Kunapuli, Satya P; Ding, Zhongren; Dorsam, Robert T et al. (2003) ADP receptors--targets for developing antithrombotic agents. Curr Pharm Des 9:2303-16 |
| Kunapuli, Satya P; Dorsam, Robert T; Kim, Soochong et al. (2003) Platelet purinergic receptors. Curr Opin Pharmacol 3:175-80 |
| Tuluc, Florin; Meshki, John; Kunapuli, Satya P (2003) Membrane lipid microdomains differentially regulate intracellular signaling events in human neutrophils. Int Immunopharmacol 3:1775-90 |
| Kunapuli, Satva P (2002) P2 receptors and platelet activation. ScientificWorldJournal 2:424-33 |
