Figure 2

Effect of endothelin-1 (ET-1) on nitric oxide (NO) release and inducible nitric oxide synthase (iNOS) expression by human osteoarthritis (OA) chondrocytes. NO was measured in the culture media, and iNOS protein was detected in cell extracts and revealed by western blot using specific antiserum, as described in Materials and methods. (a) Concentration-dependent ET-1-induced NO accumulation in the culture media from confluent human OA chondrocytes treated with ET-1 (0–100 nM) at 37°C for 24 hours. (b) Effect of protein kinase inhibitors and of guanylate cyclase inhibitor on ET-1-induced NO release in OA chondrocytes. Confluent monolayer chondrocytes were preincubated with SB 202190 (1 μM), PD98059 (10 μM), Wortmannin (100 nM), KT5720 (4 μM) or LY83583 (2 μM) for 30 min at 37°C and then challenged with ET-1 for 24 hours, and NO was determined in the culture media. (c) Effect of iNOS inhibition on NO release induced by ET-1 in human OA chondrocytes. The chondrocytes were pretreated with the allosteric inhibitor of iNOS, L-N⁶ (1-iminoethyl)lysine (L-NIL) (0–50 μM), for 30 min and were then incubated with ET-1 (10 nM) for an additional 24 hours. The NO level was measured in the culture media. (d) Effect of protein kinase inhibitors and LY83583 on ET-1-induced iNOS in human OA chondrocytes. Chondrocytes were preincubated with SB 202190 (1 μM), PD98059 (10 μM), Wortmannin (100 nM), KT5720 (4 μM) or LY83583 (2 μM) for 30 min at 37°C and then challenged with ET-1 for 24 hours, and iNOS was then quantified. M.W., molecular weight. (a)–(c) Values are the mean ± standard error of the mean of six independent experiments performed in duplicate. (d) Representative blot of three independent experiments. P values indicate the significant difference between ET-1 treated cells and cells treated with indicated inhibitors + ET-1 (*) and versus control (^#). Significant differences: ^#, * P < 0.05, ^###, *** P < 0.005.