Ropivacaine blocks activation of Akt kinase (Akt) upstream of endothelial nitric oxide synthase (eNOS) but does not directly inhibit phosphatidylinositide 3-kinase (PI3K) upstream of Akt. (A) Representative Western blot of Akt phosphorylated at threonine 308 (pT308 Akt, row 1), total Akt (row 2), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, row 3) after treatment with tumor necrosis factor α (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 pT308 Akt over total Akt in human lung microvascular endothelial cells treated with TNFα (20 ng/ml) in the absence (black bar) or presence (striped gray bars) of ropivacaine (1 nM, 1 μM) compared with untreated cells (set as 1.0). Data shown are mean ± SD (n = 7 per group); #P < 0.05 versus untreated cells, *P< 0.05 compared with TNF-α alone. (C) PI3K activity measured by enzyme-linked immunosorbent assay of biotinylated phosphatidylinositol (3,4,5)-triphosphate (B-PIP3) after incubation of different isoforms of p110 catalytic subunit of PI3K (α, β, γ, δ) with phosphatidylinositol (4,5)-bisphosphate (PIP2) substrate and ropivacaine (1 nM, 1 μM, 100 μM) or wortmannin (100 nM). Generated PIP3 competes with added B-PIP3 for binding, thus a high signal indicates low enzyme activity. Data shown are mean ± SD (n = 4 per group); #P < 0.05 versus all other groups.