Thrombin and PAR-1 activating peptide increase iNOS expression in cytokine-stimulated C6 glioma cells.
Thrombin (THR) plays a key role in the brain under physiological and pathological conditions. Several of the biological activities of thrombin have been shown to be mainly driven through activation of protease-activated receptor-1 (PAR-1)-type thrombin receptor. Here we have studied the effect of THR and PAR-1-activating peptide (PAR1-AP), SFLLRN, on cytokine-induced expression of inducible nitric oxide (iNOS), a prominent marker of astroglial activation using the rat C6 glioma cells. In this cell line, THR (1-10 U/mL) and PAR1-AP (1-100 microM) induced a significant concentration-dependent increase 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 iNOS expression as measured in cell lysates prepared from different treatments by using SDS-PAGE followed by western blot analysis. The effect of THR, but not that of PAR1-AP, was significantly inhibited by hirulog(TM) (60 microg/mL), a specific and stochiometric THR inhibitor or by cathepsin-G (40 mU/mL), an inhibitor of PAR-1. In conclusion our data suggest a role for THR through activation of PAR-1 in the induction of astroglial iNOS, and further support the hypothesis that THR may function as an important pathophysiological modulator of the inflammatory response.
Meli
R
R
Department of Experimental Pharmacology, University of Naples, 'Federico II', Naples, Italy. meli@unina.it
Raso
G M
GM
Cicala
C
C
Esposito
E
E
Fiorino
F
F
Cirino
G
G
D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D056649:Cathepsin G; D002403:Cathepsins / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D004357:Drug Synergism; D005910:Glioma; D006490:Hemostatics / Q000494:pharmacology*; D007371:Interferon-gamma / Q000494:pharmacology*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology*; D051381:Rats; D012697:Serine Endopeptidases; D013917:Thrombin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D006490:Hemostatics; D009573:Nitrites; D010446:Peptide Fragments; D014409:Tumor Necrosis Factor-alpha; C082835:thrombin receptor peptide (42-47); D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D002403:Cathepsins; D012697:Serine Endopeptidases; D056649:Cathepsin G; C534097:Ctsg protein, rat; D013917:Thrombin