Fig. 2.
Ropivacaine and lidocaine inhibit endothelial nitric oxide synthase (eNOS) phosphorylation and ropivacaine blocks tumor necrosis factor-α (TNFα)–induced nitric oxide (NO) production in human lung microvascular endothelial cells (HLMVEC). (A) Representative Western blots of eNOS phosphorylated at serine 1177 (pS1177 eNOS, row 1), total eNOS (row 2), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, row 3) after treatment with TNFα (20 ng/ml) in the absence or presence of (i) ropivacaine (1 nM) or (ii) lidocaine (1 nM) for 20 min. (B) Quantitative analysis of densitometry of Western blots showing the ratio of pS1177 eNOS over total eNOS in HLMVEC treated with TNFα (20 ng/ml) in the absence or presence of (i) ropivacaine (1 nM) or (ii) lidocaine (1 nM) compared with untreated cells (set as 1.0). Data shown are mean ± SD (n = 6); #P < 0.05 versus untreated cells, *P < 0.05 compared with TNFα alone. (C) Dose–response curve of Western blot densitometry data showing the ratio of pS1177 eNOS over total eNOS in HLMVEC treated with TNFα (20 ng/ml) in the absence or presence of log concentrations of (i) ropivacaine (1 pM to 1 μM) or (ii) lidocaine (1 pM to 1 μM) compared with TNFα alone (set as 1.0). Data shown are mean ± SD (n = 6–8 for ropivacaine and n = 5–6 for lidocaine). (D) Assessment of cumulative NO production via chemiluminescent detection of nitrite ion (NO2–) directly proportional to NO in HLMVEC culture supernatants after treatment for 1 h with ropivacaine (1 nM, 1 μM, 100 μM) or the nonselective NO-synthase inhibitor N5-(1-iminoethyl)-l-ornithine dihydrochloride (l-NIO) (20 μM) in the absence (empty circles) or presence (black squares) of TNFα (20 ng/ml). NO production from untreated cells was set as 1.0. Data shown are mean ± SD; n = 3 per group, except for TNFα alone and data with l-NIO, where n = 6. #P < 0.05 versus untreated cells, *P < 0.05 compared with TNFα alone.