Fig. 7.
Ropivacaine inhibits tumor necrosis factor-α (TNFα)–induced Src-dependent phosphorylation of caveolin-1 and attenuates endothelial barrier disruption. (A) Representative Western blots of caveolin-1 phosphorylated at tyrosine 14 (pY14 Cav-1, row 1), total Cav-1 (row 2), and β-actin (row 3) after treatment with TNFα (20 ng/ml) in the absence or presence of ropivacaine (1 nM, 1 μM) for 20 min. (B) Quantitative analysis of densitometry of Western blots showing the ratio of pY14 Cav-1 over total Cav-1 in human lung microvascular endothelial cells treated with TNFα (20 ng/ml) in the absence (black bar) or presence (striped gray bars) of ropivacaine compared with untreated cells (white bar, set as 1.0). Data shown are mean ± SD (n = 8); *P < 0.05 compared with TNFα alone. (C) Transendothelial electrical resistance of endothelial monolayers over time (normalized to baseline values). After 30-min equilibration, cells were incubated (arrow indicates beginning of treatment) with either TNFα (20 ng/ml, red), ropivacaine (1 μM, purple), TNFα + ropivacaine (blue), TNFα + nitric oxide synthase Inhibitor N5-(1-iminoethyl)-l-ornithine dihydrochloride (l-NIO) (yellow, pretreatment for 1 h before addition of TNFα), TNFα + Src-inhibitor PP2 (gray, pretreatment for 30 min before addition of TNFα), or vehicle (control, green). Data shown are mean ± SD of normalized resistance (n = 7 per group). (D) Quantification of decrease in normalized resistance at 6 h. Data shown are mean ± SD; untreated cells (white bars), ropivacaine at 1 μM (light gray bars), TNFα alone (black bar), TNFα + ropivacaine 1 μM (dark gray bars), and TNFα + l-NIO (striped bars), TNFα + PP2 (checkered bars); n = 7 per group; #P < 0.05 versus untreated cells, *P < 0.05 compared with TNFα alone.