Platelet activating factor (PAF) and thromboxane A2 (TxA2) are potent lipid chemical mediators of acute allergic and inflammatory responses. Recently, we observed that AGEPC (synthetic PAF) and U-46619 (a TxAs analog expert powerful stimulatory effects on glycogenolysis in perfused rat livers by an indirect mechanism involving constriction of the hepatic vasculature. Constriction of the hepatic vasculature occurs during acute allergic reactions and PAF and TxA2 are produced by inflammatory cells and live during immune challenge. Thus, PAF and TxA2 may play a potentially important role in regulating hepatic blood flow and supplying extrahepatic tissues with glucose during pathophysiological states. The major objectives of this study are to elucidate the roe of PAF and TxA2 on hepatic hemodynamics and glycogenolysis in vivo and to provide insight into the molecular basis of action of these lipid mediators in hepatic endothelial and Kupffer cells, which regulate flow through the hepatic sinusoids. Experiments in vivo will characterize the effects of the se mediators on hepatic hemodynamics by measurements of hepatic portal pressure and hepatic portal and arterial flows. Simultaneous measurements will be performed to evaluate hepatic glycogenolysis and blood glucose concentrations under these experimental conditions. finally, we will assess the role of PAF and TxA2 as mediators of hemodynamic and glycogenolytic changes in liver during pathophysiologic situations. Experiments to characterize the hepatic receptors involved in hemodynamic responses to these lipid agonists will be performed in perfused livers and hepatic endothelial and Kupffer cells. Radioligand binding to PAF ad TxA2 receptors and mechanisms involved in receptor signal transduction in endothelial and Kupffer cells will be explored. Our preliminary experiments suggest these receptors are coupled to changes in cytosolic free calcium concentrations ([Ca2+]i) in Kupffer cells and, with AGEPC receptors, stimulation of phosphoinositide-specific phospholipase C. Effects of these lipid agonists on [Ca2+]i will be characterized by digital image analysis of Fura 2-loaded cells and compared to effects on activation of phospholipase C and production of Ca2+-regulating second messengers. Roles of protein kinase C and guanine nucleotide regulatory proteins in receptor-mediated biosignalling will be explored. Correlations will be made between rank order of potencies of agonists and antagonists on hepatic vasoconstriction, receptor binding, [Ca2=]i and phospholipase C. The proposed study will provide new insights into the mechanisms of action of PAF and TxA2 in liver and hopefully will contribute to a more precise understanding of their actions in other non-hepatic cells and tissues.
