LIPOPOLYSACCHARIDE-INDUCED BIPHASIC INOSITOL 1,4,5-TRISPHOSPHATE RESPONSE AND TYROSINE PHOSPHORYLATION OF 140-KILODALTON PROTEIN IN MOUSE PERITONEAL-MACROPHAGES

We previously showed that a relatively high dose of LPS induced the se lective translocation of protein kinase C-beta (PKC-beta) in LPS-respo nsive mouse macrophages. This result suggested that phosphatidylinosit ol-specific phospholipase C (PLC) might be activated in the upstream o f PKC-beta. Stimulation of C3H/HeN mouse macrophages by LPS induced th e characteristic phosphatidylinositol-1,4,5-trisphosphate (IP3) respon se, that is, a biphasic response consisting of a rapid increase occurr ing within the first 1 min, and another increase beginning at around 1 min after stimulation. Only the first response was disappeared when c ells were treated with a platelet-activating factor receptor antagonis t. LPS-inducible TNF-alpha gene activation, however, was not suppresse d by the same antagonist, but suppressed by PKC inhibitors, LPS-stimul ated macrophage lysates showed tyrosine phosphorylation of some protei ns, and the strongest phosphorylation was observed at molecular mass o f 140 kDa. The phosphorylation of this protein started at 40 s after L PS stimulation and continued to increase. Anti-PLC-gamma(2) Ab seemed to recognize the same protein as the tyrosine-phosphorylated 140-kDa p rotein, A low dose of LPS (1 ng/ml) could not induce the tyrosine phos phorylation of this protein. Furthermore, LPS induced only the first p hase change, but not the second phase increase in LPS-hyporesponsive C 3H/HeJ mouse macrophages. These results indicate that the first phase rapid IP3 change, which is also seen in HeJ macrophages, is mediated v ia a platelet-activating factor receptor, and is not responsible for T NF-alpha production, while the second phase change mediated by a molec ule other than CD14 is responsible for PKC-beta translocation and TNF- alpha production, The results also suggest that the later IP3 change i s considered to be mediated through a gamma(2), type of phosphatidyl-i nositol-specific PLC.