Thrombin induces NO release from cultured rat microglia via protein kinaseC, mitogen-activated protein kinase, and NF-kappa B

Microglia, brain resident macrophages, become activated in brains injured d ue to trauma, ischemia, or neurodegenerative diseases. In this study, we fo und that thrombin treatment of microglia induced NO release/ inducible nitr ic-oxide synthase expression, a prominent marker of activation. The effect of thrombin on NO release increased dose-dependently within the range of 5- 20 units/ml. In immunoblot analyses, inducible nitric-oxide synthase expres sion was detected within 9 h after thrombin treatment. This effect of throm bin was significantly reduced by protein kinase C inhibitors, such as Go697 6, bisindolylmaleimide, and Ro31-8220. Within 15 min, thrombin activated th ree subtypes of mitogen-activated protein kinases: extracellular signal-reg ulated kinase, p38, and c-Jun N-terminal kinase/stress-activated protein ki nase. Inhibition of the extracellular signal-regulated kinase pathway and p 38 reduced the NO release of thrombin-treated microglia. Thrombin also acti vated nuclear factor kappa B (NF-kappa KB) within 5 min, and N-acetyl cyste ine, an inhibitor of NF-kappa B, reduced NO release. However, thrombin rece ptor agonist peptide (an agonist of protease activated receptor-1 (PAR-1)), could not mimic the effect of thrombin, and cathepsin G, a PAR 1 inhibitor , did not reduce the effect of thrombin. These results suggest that thrombi n can activate microglia via protein kinase C, mitogen-activated protein ki nases, and NF-kappa B but that this occurs independently of PAR-1.