MODULATION OF SUPEROXIDE ANION GENERATION BY MANOALIDE, ARACHIDONIC-ACID AND STAUROSPORINE IN LIVER INFILTRATED NEUTROPHILS IN A RAT MODEL OF ENDOTOXEMIA

Early accumulation of polymorphonuclear leukocytes (PMN) in the liver after in vivo exposure to Escherichia coli lipopolysaccharide (LPS) an d concomitant in vitro phorbol myristate acetate (PMA)-stimulated supe roxide anion (O2-) generation has recently been described in a rat mod el of endotoxemia. The purpose of this investigation was to study the role of phospholipase A2 (PLA2), arachidonic acid (AA), its metabolite s, and protein kinase C (PKC) in the mechanism of PMA-stimulated O2- g eneration of liver infiltrated PMN as compared to circulating blood PM N. Rat PMN were isolated after a 1.5-h infusion of saline or LPS from the blood (SAL-PMN) or the liver (LPS-PMN), respectively. The followin g results were observed in both SAL-PMN and LPS-PMN: 1) Inhibitors of cyclooxygenase (indomethacin) and 5-lipoxygenase [eicosatetraynoic aci d, WY 50,295 tromethamine and VZ 65, 4-(11-hydroxy-1,9-undecadiin)-bre nzcatechin] pathways did not inhibit O2- generation; 2) the potent mar ine PLA2 inhibitor Manoalide inhibited O2- generation in a dose-depend ent manner (IC50 = 0.5 muM); 3) exogenously added AA enhanced PMA-stim ulated O2 generation in a time- and dose-dependent manner and partiall y reversed the effect of Manoalide in LPS-PMN; 4) staurosporine, a put ative PKC inhibitor, blocked PMA-stimulated O2- generation completely in the absence of AA and 79% in the presence of AA. It was concluded t hat LPS-induced liver sequestration of PMN does not alter the role PLA 2, AA and PKC play in PMA-stimulated O2- generation. These findings sh ould have implications on the design of novel therapeutic approaches f or the modulation of O2- release in the pathogenesis of LPS hepatotoxi city.