The objectives of this Physician Scientist Award proposal are: 1) to foster the transition of a fully trained veterinarian into an independent investigator; 2) to test the hypothesis that the monocyte thromboxane A2 (TXA2) receptor represents a unique subtype; and 3) to test the hypothesis that activation of the monocyte TXA2 receptor affects its function. Our studies demonstrated of a TXA2 receptor on equine peripheral blood monocytes utilizing a new 125I-labeled TXA2 receptor agonist (125I-BOP). 125-BOP bound to a single class of binding sites (Kd=1.0+0.3nM;Bmax=383+180 fmoles/mg protein; n=5). 125I-BOP binding was saturable and displaceable by TXA2 receptor agonists and antagonists. Further classification of the TXA2 receptor subtype will require competition binding assays performed with TXA2 receptor agonists and antagonists that have been used previously to classify platelet and vascular TXA2 receptors. Potential second messenger system(s) responsible for TXA2 receptor signal transduction will be investigated with initial emphasis on cAMP. In contrast to the decrease in cAMP synthesis by platelets in response to TXA2 agonists, we found that I-BOP significantly increased cAMP formation in a dose-dependent fashion from 5.4+0.5 to 68.5+11.1fmoles cAMP/mug protein (P<0.05; N=5) in monocytes. The TXA2 receptor antagonists SQ29548 and L657,925 blocked I- BOP did not increase intracellular-free calcium in monocytes. These findings indicate that TXA2 receptors exist on monocytes, and that the second messenger system(s) for this receptor is novel. This proposal will further characterize this TXA2 receptor and identify the second messenger system(s). The second hypothesis will explore the effect of activation of the TXA2 receptor on monocyte function. I-BOP inhibited chemotaxis and E. coli endotoxin-induced tumor necrosis factor (TNF) formation in monocytes (n=6; P<.002). To further characterize the effects of activation of the monocyte TXA2 receptor, our studies will focus on the mechanism by which TXA2 receptor activation reduces TNF secretion. Combined, these studies will contribute to our understanding of 1) the fundamental biology of a unique monocyte TXA2 receptor and its signal transduction events and 2) TXA2 autocrine control of monocyte function. Furthermore, studies may allow the development of selective therapeutic agents.
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| Simmons, T R; Cook, J A; Moore, J N et al. (1993) Thromboxane A2 receptors in equine monocytes: identification of a new subclass of TXA2 receptors. J Leukoc Biol 53:173-8 |
