Effects of NOS inhibitor, L-NAME, on PAR-1 mediated NO production in HCAECs. (a) HCAECs were pretreated with L-NAME (100 µmol/L) for 1 h prior to stimulation with thrombin (10 U/ml) for 0.5 min and 10 min. Phosphorylation of eNOS-Ser1177 was determined using Western blot analysis with phosphospecific antibody. Representative immunoblots are shown with densitometric analyses. Mean +/− SEM of measurements in cultures three of different passages; *P < 0.05 Thrombin 0.5 min versus Basal, **P < 0.01 L-NAME + Thrombin 0.5 min versus Basal. (b) NO production was determined indirectly by measuring total nitrate/nitrite using media from HCAECs pretreated with L-NAME (100 µmol/L) for 1 h prior to stimulation with thrombin (10 U/ml) for 0.5 min and 10 min ****P < 0.001 Thrombin 0.5 min versus Basal, ****P < 0.001 L-NAME + Thrombin 0.5 min versus Basal. (c) HCAECs were pretreated with L-NAME (100 µmol/L) for 1 h prior to stimulation with TFLLR (10 µmol/ml) for 0.5 min and 10 min. Phosphorylation of eNOS-Ser1177 was determined using Western blot analysis with phosphospecific antibody. Representative immunoblots are shown with densitometric analyses, n = 3, ****P < 0.0001 TFLLR 0.5 min and L-NAME + TFLLR 0.5 min versus Basal. (d) Total nitrate/nitrite production was measured using media from HCAECs pretreated with L-NAME (100 µmol/L) for 1 h prior to stimulation with TFLLR (10 µmol/L) for 0.5 min and 30 min, n = 3, TFLLR 0.5 min, no detection (nd) ****P < 0.0001 TFLLR 30 min and L-NAME + TFLLR 30 min versus basal