Thrombin and PAR-1 acitvating peptide increase iNOS expression in cytokine-stimulated C6 glioma cells

Thrombin (THR) plays a key role in the brain under physiological and pathol ogical conditions. Several of the biological activities of thrombin have be en shown to be mainly driven through activation of protease-activated recep tor-1 (PAR-1)-type thrombin receptor. Here we have studied the effect of TH R and PAR-1-activating peptide (PAR1-AP), SFLLRN, on cytokine-induced expre ssion of inducible nitric oxide (NOS), a prominent marker of astroglial act ivation using the rat C6 glioma cells. In this cell line, THR (1-10 U/mL) a nd PAR1-AP (1-100 mum) induced a significant concentration-dependent increa se both of IFN-gamma- (250 U/mL) or TNF-alpha-(500 U/mL) induced NO release . The observed increase of NO production was related to an enhancement of N OS expression as measured in cell lysates prepared from different treatment s by using SDS-PAGE followed by western blot analysis. The effect of THR, b ut not that of PAR1-AP, was significantly inhibited by hirulog (TM) (60 mug /mL), a specific and stochiometric THR inhibitor or by cathepsin-G (40 mU/m L), an inhibitor of PAR-1. In conclusion our data suggest a role for THR th rough activation of PAR-1 in the induction of astroglial NOS, and further s upport the hypothesis that THR may function as an important pathophysiologi cal modulator of the inflammatory response.