Analysis of the signal transduction in the induction of nitric oxide synthase by lipoteichoic acid in macrophages.

1. This study investigates the signal transduction mechanisms leading to the enhanced formation of nitric oxide (NO) due to the induction of NO synthase (iNOS) in murine J774.2 macrophages in culture activated with lipoteichoic acid (LTA), a cell wall component of the gram-positive bacterium Staphylococcus aureus. 2. LTA (10 microgram ml-1) caused within 24 h an enhanced accumulation of nitrite (an indicator of NO biosynthesis) in the supernatant of J774.2 macrophages which was prevented by the non-selective NOS inhibitor NG-monomethyl-L-arginine (L-NMMA; IC50: 35 microM) or by the iNOS-selective NOS inhibitor, aminoethyl-isothiourea (AE-ITU; IC50: 6 microM). The inhibition of nitrite formation afforded by these agents was prevented by excess L-arginine (3-30 mM), but not by D-arginine (3-30 mM). Furthermore, the degree of iNOS inhibition was similar when these NOS inhibitors were added to the macrophages 10 h after LTA. 3. Pretreatment of J774.2 macrophages with cyclohexamide or dexamethasone prevented the enhanced formation of nitrite caused by LTA. This inhibition did not occur when dexamethasone or cyclohexamide were added to the cells 10 h after LTA. The increase in nitrite formation stimulated by LTA (10 micrograms ml-1) was not affected by polymyxin B (0.05-0.5 microgram ml-1), an agent which binds and inactivates endotoxin. 4. A specific inhibitor of phosphatidylcholine-phospholipase C (PC-PLC), D609, prevented the increase in nitrite formation (IC50 = 20 micrograms ml-1) caused by LTA. The inhibition afforded by D609 was significantly smaller when this agent was added to the cells 10 h after LTA. 5. The structurally distinct tyrosine kinase inhibitors, erbstatin, genistein, and tyrphostin AG126 prevented the formation of nitrite caused by LTA. The inhibition afforded by these compounds was significantly attenuated when they were added to the cells 10 h after LTA. In contrast, daidzein or tyrphostin A-1, which are inactive analogues of genistein and tyrphostin (up to a concentration of 10 microM) did not affect the nitrite formation caused by LTA. 6. Inhibitors of the activation of the nuclear transcription factor NF-kappa B such as pyrrolidine dithiocarbamate (PDTC; an antioxidant and a metal chelator), butylated hydroxyanisole (BHA; an antioxidant), L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), calpain inhibitor I (both I kappa B-protease inhibitors), or rotenone (an antioxidant which inhibits electron transport) prevented the nitrite formation stimulated by LTA. The inhibition afforded by these agents was significantly smaller when they were added to the macrophages 10 h after LTA. 7. Incubation of J774.2 cells with LTA over 24 h resulted in the expression of iNOS protein (130 kDa) as identified by Western blot analysis. The expression of iNOS protein by LTA was significantly attenuated by cyclohexamide, D609, tyrphostin AG126, PDTC or by TPCK. 8. Thus, the signal transduction leading to the expression of iNOS protein and activity caused by LTA in murine J774.2 macrophages involves (i) the activation of PC-PLC, (ii) phosphorylation of tyrosine kinase, and (iii) the activation of the transcription factor NF-kappa B.