Although the molecular signaling mechanisms underlying macrophage endotoxin (LPS) responsiveness are not fully understood, alterations in intracellular phosphatidylinositol (PI) metabolism appear to contribute. We evaluated the effects of endotoxin tolerance (ET) induction upon peritoneal macrophage (PM) expression of the principal PI enzymes phospholipase PLC-gamma 1 (PLC-gamma 1) and phosphatidylinositol 3'-kinase (PI-3-K). Rats received either 5 mg/kg LPS (ET) or phosphate-buffered saline (nontolerant, NT) which enabled 88% of ET and 25% of NT to survive a 25 mg/kg LPS dose 3 days later. PM were harvested by lavage on Day 3 from both ET and NT rats. Following overnight culture, 5 x 10(6) PM in serum-free media were stimulated with 5 ng/ml LPS for 0 to 30 min. Cell lysates fractionated by SDS-PAGE were transferred to nitro-cellulose and blotted with PLC-gamma 1, PI-3'-K, and phosphotyrosine (4G10) monoclonal antibodies. Western immunoblots were developed by enhanced chemiluminescence and quantitated by densitometry. Unlike NT cells in which PLC-gamma 1 was expressed constitutively and increased with LPS stimulation, PLC-gamma 1 expression in ET cells stimulated with LPS was markedly reduced in three separate experiments. In contrast, ET cells expressed considerably higher concentrations of PI-3'-K to NT cells. Patterns of protein tyrosine phosphorylation were similar in both NT and ET cells regardless of LPS stimulation. The development of endotoxin tolerance decreased PLC-gamma 1 expression and markedly amplified PI-3'-K expression in macrophages. PI-3'-K-generated second messengers may contribute to unique signaling pathways responsible for tempered cellular responses to LPS.