Pulmonary endothelial barrier dysfunction mediated in part by Src-kinase activation plays a crucial role in acute inflammatory disease. Proinflammatory cytokines, such as tumor necrosis factor-α (TNFα), activate Src via phosphatidylinositide 3-kinase/Akt-dependent nitric oxide generation, a process initiated by recruitment of phosphatidylinositide 3-kinase regulatory subunit p85 to TNF-receptor-1. Because amide-linked local anesthetics have well-established anti-inflammatory effects, the authors hypothesized that ropivacaine and lidocaine attenuate inflammatory Src signaling by disrupting the phosphatidylinositide 3-kinase–Akt–nitric oxide pathway, thus blocking Src-dependent neutrophil adhesion and endothelial hyperpermeability.
Human lung microvascular endothelial cells, incubated with TNFα in the absence or presence of clinically relevant concentrations of ropivacaine and lidocaine, were analyzed by Western blot, probing for phosphorylated/activated Src, endothelial nitric oxide synthase, Akt, intercellular adhesion molecule-1, and caveolin-1. The effect of ropivacaine on TNFα-induced nitric oxide generation, co-immunoprecipitation of TNF-receptor-1 with p85, neutrophil adhesion, and endothelial barrier disruption were assessed.
Ropivacaine and lidocaine attenuated TNFα-induced Src activation (half-maximal inhibitory concentration [IC50] = 8.611 × 10−10 M for ropivacaine; IC50 = 5.864 × 10−10 M for lidocaine) and endothelial nitric oxide synthase phosphorylation (IC50 = 7.572 × 10−10 M for ropivacaine; IC50 = 6.377 × 10−10 M for lidocaine). Akt activation (n = 7; P = 0.006) and stimulus-dependent binding of TNF-receptor-1 and p85 (n = 6; P = 0.043) were blocked by 1 nM of ropivacaine. TNFα-induced neutrophil adhesion and disruption of endothelial monolayers via Src-dependent intercellular adhesion molecule-1- and caveolin-1-phosphorylation, respectively, were also attenuated.
Ropivacaine and lidocaine effectively blocked inflammatory TNFα signaling in endothelial cells by attenuating p85 recruitment to TNF-receptor-1. The resultant decrease in Akt, endothelial nitric oxide synthase, and Src phosphorylation reduced neutrophil adhesion and endothelial hyperpermeability. This novel anti-inflammatory “side-effect” of ropivacaine and lidocaine may provide therapeutic benefit in acute inflammatory disease.
The proinflammatory cytokine tumor necrosis factor-α activates Src tyrosine kinase and increases endothelial permeability. Whether some anti-inflammatory effects of amide local anesthetics (such as those observed in models of acute lung injury) are due to inhibition of signaling upstream or downstream of Src remains unknown.
Using human lung microvascular endothelial cells, it was shown that ropivacaine and lidocaine attenuated tumor necrosis factor-α–induced neutrophil adhesion and endothelial hyperpermeability via a reduction of Akt, endothelial nitric oxide synthase, and Src activation.
VASCULAR inflammation is thought to underlie the high morbidity and mortality of diseases such as acute lung injury, acute respiratory distress syndrome (ARDS), diabetes mellitus, and cancer.1–5 Therapeutic options are limited to the treatment of symptoms (e.g., lung-protective ventilation in patients with ARDS) rather than the cause of endothelial barrier dysfunction.6 Vascular hyperpermeability and endothelial barrier dysfunction play key roles in the pathogenesis of vascular injury due to inflammation.6,7 The proinflammatory cytokine tumor necrosis factor-α (TNFα) activates c-Src protein tyrosine kinase (Src) and increases endothelial permeability.8 Although the exact mechanism of TNFα-induced Src activation is not completely understood, it has been demonstrated that endothelial nitric oxide synthase (eNOS)–mediated nitric oxide production downstream of phosphatidylinositide 3-kinase (PI3K)-dependent activation of Akt9–11 and CaM kinase II induce Src activation.10,12 TNFα-induced PI3K activation is thought to be initiated by recruitment of PI3K regulatory subunit p85 to TNF-receptor-1 (TNF-R1) upon agonist binding.13 Downstream signaling of activated Src includes phosphorylation of intercellular adhesion molecule-1 (ICAM-1) and caveolin-1, which have both been shown to contribute to the development of the endothelial injury and inflammatory hyperpermeability.14–17
Experimental evidence indicates that amide-linked local anesthetics such as ropivacaine and lidocaine exhibit beneficial properties during the pathogenesis of acute lung injury: they were proven to attenuate experimental (endotoxin-induced) lung inflammation in vivo and in vitro18,19 as well as neutrophil-dependent acute lung injury induced by N-formyl-l-leucine-methionyl-l-phenylalanine20,21 In addition, we recently demonstrated inhibition of TNFα-induced Src activation by ropivacaine and lidocaine in a human lung cancer cell line in vitro.22 We therefore hypothesized that amide-linked local anesthetics, such as ropivacaine and lidocaine, may attenuate inflammatory Src signaling and subsequent ICAM-1 and caveolin-1 phosphorylation by disrupting the PI3K–Akt–nitric oxide signaling pathway, thus blocking Src-dependent vascular inflammation and hyperpermeability of the endothelium. The results of the current study indicate a novel anti-inflammatory mechanism by which ropivacaine and lidocaine may minimize endothelial injury during the pathogenesis of acute lung injury or ARDS.
Materials and Methods
Human lung microvascular endothelial cells (HLMVECs; Lonza, Walkersville, MD) were cultured on dishes coated with 0.2% gelatin (Sigma-Aldrich, St. Louis, MO) in VascuLife® Basal Medium (Lifeline Cell Technology, Walkersville, MD) supplemented with the VascuLife® VEGF-Mv LifeFactors Kit containing 5% fetal bovine serum, 10 mM of l-glutamine, 5 ng/ml of recombinant vascular endothelial growth factor, 5 ng/ml of recombinant epithelial growth factor, 5 ng/ml of fibroblast growth factor, 15 ng/ml of insuline-like growth factor-1, 1 μg/ml of hydrocortisone hemisuccinate, 0.75 U/ml of heparin, 50 μg/ml of ascorbic acid (Lifeline Cell Technology), and 1% penicillin–streptomycin (Gibco Invitrogen, Carlsbad, CA). Experiments were performed with 70 to 100% confluent cells from passages 5 to 11. Before the experiments, cells were grown in a serum-free media (VascuLife® Basal Medium) for 16 h. All cells were maintained in 5% carbon dioxide–95% room air in a water-jacketed 37°C incubator.
Cell monolayers were incubated with TNFα (Gibco Invitrogen) at a concentration of 20 ng/ml diluted in VascuLife® Basal Medium for 20 min for Western blot analysis or for 10 min for immunoprecipitation. Ropivacaine 0.5% (Naropin®; APP Pharmaceuticals, Schaumburg, IL) or lidocaine 2% (APP Pharmaceuticals) were diluted with VascuLife® Basal Medium (1 pM to 100 μM) and were incubated for the same time period as TNFα. If tested together on the same cell monolayer, ropivacaine and TNFα were added to the medium simultaneously.
Cell Harvest and Lysis
After the indicated incubation times, HLMVECs were washed once with ice-cold phosphate-buffered saline (Gibco Invitrogen), scraped from the bottom of the plate, collected, and lysed with radio-immunoprecipitation buffer (Boston Bioproducts, Ashland, MA) supplemented with protease inhibitor cocktail, 200 mM of phenylmethylsulfonyl fluoride, 1 mM of EDTA, 1 mM of sodium fluoride, and 1 mM of sodium orthovanadate (all from Sigma-Aldrich) as previously described.22,23
Determination of Total Protein Concentration
Whole cell lysates prepared as described above were subject to total protein concentration measurement using the DC Protein Assay Kit (BioRad, Hercules, CA) in accordance with the manufacturer’s instructions. Immunoglobulin G was used for the generation of a standard curve.
To assess the association of TNF-R1 and p85 subunit of PI3K, HLMVECs were washed once with ice-cold phosphate-buffered saline (Gibco Invitrogen) and lysed using a buffer consisting of 20 mM of Tris-HCl (pH 7.4), 150 mM of NaCl, 1.2% Triton X-100, protease inhibitor cocktail, 200 mM of phenylmethylsulfonyl fluoride, 1 mM of EDTA, 1 mM of sodium fluoride, and 1 mM of sodium orthovanadate for 20 min during gentle rocking of the dish at 4°C as previously described.13 Lysates were then incubated with magnetic sheep anti-mouse-IgG–coated Dynabeads (Gibco Invitrogen) for 1 h at 4°C that had previously been incubated for 30 min at 4°C with nonspecific mouse immunoglobulin G (the negative control) or anti-TNF-R1 monoclonal antibody (both from Santa Cruz Biotechnology). The lysate-bead suspensions were then placed in a magnetic particle concentrator (Invitrogen, Carlsbad, CA) and the pellet washed thrice with ice-cold (4°C) Ca2+- and Mg2+-free phosphate-buffered saline (Gibco Invitrogen) that also included the same supplements as the lysis buffer (see Materials and Methods, Cell harvest and lysis). Laemmli sample buffer (×2; BioRad) was added together with dithiothreitol (final concentration of 30 mM; Sigma-Aldrich). The suspension was then boiled for 5 min to elute the protein complexes from the beads. After removal of the beads by magnetic separation, samples were directly loaded on a 10% sodium dodecyl sulfate-polyacrylamide gel for electrophoresis and Western blot analysis or stored at −80°C until further use.
Western Blot Analysis
Western blotting of whole cell lysates was carried out as previously described.22,23 Antibodies against pY419 Src, total Src, and p85 were purchased from Cell Signaling Technologies (Danvers, MA), for pY512 ICAM-1, total ICAM-1, glyceraldehyde 3-phosphate dehydrogenase, and TNF-R1 (both rabbit and mouse origin) from Santa Cruz Biotechnology, and for pY14 caveolin-1, total caveolin-1, and β-actin from BD Biosciences (Franklin Lakes, NJ).
In Vitro Src Kinase Assay
Src kinase activity in the absence or presence of different concentrations of ropivacaine (1 nM to 1 mM) or the Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2, 10 μM; from Calbiochem Millipore, Billerica, MA) was assessed using an assay previously described by Hastie et al.,24 which uses the incorporation of radioactively labeled [γ-32P]-adenosine triphosphate (Perkin Elmer, Waltham, MA) into a specific substrate peptide. Both purified, active Src and its specific substrate peptide were purchased from Millipore. Radioactivity, expressed as counts per minute (cpm), was measured in a γ-scintillation counter (Beckman Coulter, Indianapolis, IN).
PI3K In Vitro Kinase Assay
Assessment of PI3K activity in vitro was carried out with a commercially available PI3K activity/inhibitor assay kit (Millipore) according to the manufacturer’s instructions. The assay is based on the principle of an enzyme-linked immunosorbent assay and detects biotinylated phosphatidylinositol (3,4,5)-triphosphate (B-PIP3) captured with general receptor for phosphoinositides-1 bound to plates coated with glutathione using a streptavidin-horse radish peroxidase conjugate and colorimetric readout at 450 nm. PI3K converts biotinylated phosphatidylinositol (4,5)-bisphosphate (PIP2) to PIP3. The generated PIP3 competes with added B-PIP3 for glutathione binding. A high signal in the colorimetric readout therefore indicates low levels of generated PIP3, thus low enzyme activity. We evaluated different concentrations of ropivacaine (Sigma-Aldrich; solved in dimethyl sulfoxide, final concentration of 1 nM, 1 μM, or 100 μM), as potential PI3K inhibitors and used wortmannin (100 nM), a known PI3K inhibitor, as a positive control. Four different isoforms of the p110 catalytic subunit of PI3K (α, β, γ, δ) were tested separately.
Measurement of Nitrite Ion Accumulation and Nitric Oxide Production in HLMVEC
Chemiluminescence measurement of nitrite ion accumulation in cell culture supernatant, which is directly proportional to nitric oxide production, was conducted with a Sievers® NOA 280i high-performance liquid chromatography with fluorescence detection (General Electric, Boulder, CO) as previously described.11 HLMVEC monolayers were treated with TNFα (20 ng/ml) in the absence or presence of different concentrations of ropivacaine (1 nM, 1 μM, or 100 μM). For some experiments, the cells were pretreated with the NOS inhibitor N5-(1-iminoethyl)-l-ornithine dihydrochloride (l-NIO; Cayman Chemical, Ann Arbor, MI) for 1 h before the application of TNFα.
Human lung microvascular endothelial cells were grown to confluence on goldfilm electrodes (ECIS cultureware; Applied Biophysics, Troy, NY) coated with 0.2% gelatin. Electrical impedance across the monolayer was measured at 1 V, 4,000 Hz with the Electric Cell-Substrate Impedance Sensing system (Applied Biophysics) over time as previously described.25 After an equilibration time of 30 min, TNFα was added with or without ropivacaine at a final concentration of 1 μM. For some experiments, cells were treated with the NOS inhibitor l-NIO (Cayman Chemical) before the addition of TNFα. Impedance values were used to calculate resistance using ECIS software.
Isolation of Human Polymorphonuclear Leukocytes
Whole blood samples were drawn from cubital veins from healthy donors and anticoagulated with 1 ml acid–citrate–dextrose (Sigma-Aldrich) per 5 ml whole blood according to approved Institutional Review Board (University of Illinois at Chicago, Chicago, IL) protocol. Samples were suspended in a three-fold volume of ice-cold lysis medium containing 155 mM of ammonium chloride and 10 mM of potassium bicarbonate (both from Sigma-Aldrich) and kept on ice for 10 min. After brief centrifugation at 1,500 rpm and 4°C (Eppendorf Centrifuge 5804 R; Eppendorf, Hamburg, Germany), the supernatant was discarded and the cell pellet was resuspended in 2 ml of ice-cold lysis medium supplemented with 0.5% bovine serum albumin. The suspension was then kept on ice 15 min followed by another centrifugation at 1,500 rpm and 4°C. Again, the supernatant was discarded. The pellet was resuspended in 2 ml of ice-cold Dulbecco phosphate-buffered saline (Gibco Invitrogen) and layered on 2 ml of Ficoll Histopaque 1077 (Sigma-Aldrich). After 30 min of centrifugation at 1,500 rpm and 4°C with low brake, the supernatant was discarded and the pellet consisting of neutrophils was resuspended in Hank balanced salt solution (Gibco Invitrogen). Viability was 95% or more as assessed with Trypan blue staining (Sigma-Aldrich).
Neutrophil Adhesion Assay
Neutrophil adhesion to a monolayer of HLMVECs was assessed by using the CytoSelect® Leukocyte-Endothelium Adhesion Assay (Cell Biolabs, San Diego, CA) in accordance with the manufacturer’s instructions.17 In brief, HLMVECs were grown in a 96-well plate (BD Biosciences) coated with 0.2% gelatin (Sigma-Aldrich). To increase ICAM-1 expression, all cells were subject to stimulation with Escherichia coli serotype 055:B5 lipopolysaccharide (Sigma-Aldrich) at a concentration of 4 μg/ml for 4 h. Cells were then stimulated with TNFα in the absence or presence of ropivacaine at different concentrations (1 nM, 1 μM, or 100 μM) for 20 min before addition of fluorescently labeled, freshly isolated human neutrophils (100,000 per well). After an incubation period of 30 min, nonadherent neutrophils were washed away and remaining cells were lysed; fluorescence of the lysate was measured at 485/535 nm (excitation/emission).
Determination of Half-maximal Inhibitory Concentration (IC50) and Maximum Effect Size
The half-maximal inhibitory concentration (IC50) is defined as the concentration of a drug where it exerts 50% of its maximum effect.26 To determine these values for the effect of ropivacaine and lidocaine on TNFα-induced Src and eNOS activation, densitometry data obtained from Western blot experiments (mean ± SD, normalized to values of TNFα alone, set as 1.0) were plotted against the (logarithmic) concentration of the local anesthetic used in the experiment. A concentration–response curve with a Hill slope of −1.0 was fitted by nonlinear regression analysis using the following equation:
Results are reported as values with 95% CI (for IC50, top, and bottom). Nonlinear regression curve fitting was computed with GraphPad Prism software for Mac, Version 6 (GraphPad Software, La Jolla, CA).
For Western blot, Src and PI3K activity assay, and transendothelial resistance (TER) data, normal distribution was assessed using a Shapiro–Wilk test. Normally distributed data were analyzed by one-way ANOVA with Bonferroni post hoc testing to correct for multiple testing and to keep the family-wise error rate less than 0.05. Not-normally distributed data were analyzed with nonparametric testing methods (Kruskal–Wallis tests) with Dunn post hoc correction.
Nitric oxide production and fluorescence from adhesion experiments were analyzed by using a two-way ANOVA with the local anesthetic and the absence or presence of TNFα as factors to be tested. Bonferroni post hoc testing was then used to detect significant differences between the different concentrations of ropivacaine in the absence or presence of TNFα (one family for all comparisons). Experimental data are presented as mean ± SD. All tests were performed two-sided and nonblinded using GraphPad Prism software for Mac, Version 6 (GraphPad Software). A P value of less than 0.05 was considered to be statistically significant. A few single values (approximately 5 to 7) were lost to observation due to unfortunate and unexpected technical difficulties during the experiment.
Ropivacaine and Lidocaine Inhibit the Activation of Src Tyrosine Kinase (Src) in HLMVEC but Do Not Act as Direct, Adenosine Triphosphate-competitive Src Inhibitors
We first assessed the effect of ropivacaine and lidocaine at a concentration of 1 nM on TNFα (20 ng/ml for 20 min)-induced activation of Src in HLMVEC. Western blot analysis of active-site Src phosphorylation (phospho-tyrosine 419; pY419 Src) and total Src protein level in whole cell lysates (fig. 1A) indicated that TNFα treatment of cells increased Src activity 2.3-fold compared with untreated cells (2.3 ± 0.93 [mean ± SD], P = 0.006, fig. 1Bi, and 2.3 ± 0.51, P = 0.004, fig. 1Bii). The TNFα-induced response was then reduced by 50 ± 9% in presence of 1 nM of ropivacaine (P = 0.035; fig. 1Bi) and by 49 ± 16% by lidocaine (1 nM; P = 0.034), which in both cases brought the level of Src activation nearly to baseline values. Neither ropivacaine nor lidocaine, even at concentrations of 100 μM, altered Src phosphorylation significantly compared with untreated cells (Kruskal–Wallis test; P = 0.534 for ropivacaine; P = 0.946 for lidocaine, data not shown).
To determine the IC50 of the observed inhibition of TNFα-induced Src activation by ropivacaine and lidocaine, another series of Western blot analyses treating HLMVEC with TNFα in the absence or presence of a broad range of concentrations of both ropivacaine and lidocaine (1 pM to 1 μM) was conducted. Figure 1C shows the ratio of pY419 Src over total Src (mean ± SD) plotted against the logarithmic concentration of either ropivacaine (fig. 1Ci) or lidocaine (fig. 1Cii) relative to TNFα alone. We then analyzed the data using nonlinear regression curve fitting. The results are summarized in table 1. The IC50 for TNFα-induced Src activation was 861 pM (95% CI, 79 pM to 9.4 nM) for ropivacaine and 586 pM (59.6 pM to 6.8 nM) for lidocaine. The maximum observed effect was 56% (95% CI, 68.9 to 43) for ropivacaine and 39% (55.8 to 23.3) for lidocaine.
To clarify whether ropivacaine blocks TNFα-induced Src activation as opposed to functioning as a competitive active-site Src inhibitor, we performed an in vitro Src kinase assay using radiolabeled [γ-32P] adenosine triphosphate (fig. 1D). Purified active Src kinase (15 ng) was incubated with a Src-specific substrate peptide (250 μM) in the absence or presence of either the Src inhibitor PP2 (10 μM) or different concentrations of ropivacaine (1 nM to 1 mM). Only the Src inhibitor PP2 reduced Src activity in this setting (P = 0.0008); ropivacaine did not alter Src activity at any of the concentrations tested (P = 1 for all groups compared with positive control substrate + enzyme, fig. 1D), and thus is not a direct inhibitor of c-Src.
Ropivacaine (and Lidocaine) Block TNFα-induced eNOS Phosphorylation and Nitric Oxide Production in HLMVECs
The generation of nitric oxide can promote Src activation in response to different stimuli.10 We therefore assessed eNOS activation by Western blot analyses of active-site serine 1177 phosphorylation in HLMVEC lysates treated with TNFα in the absence or presence of 1 nM of ropivacaine or lidocaine. As shown in figure 2, A and B, TNFα treatment induced a 2.76 ± 1.39-fold (P = 0.002) increase in eNOS phosphorylation (pS1177 eNOS) and this response to TNFα was reduced by 45 ± 15% in presence of 1 nM of ropivacaine (P = 0.022; fig. 2, Ai and Bi). Lidocaine at 1 nM concentration significantly reduced TNFα-induced eNOS phosphorylation (2.68 ± 0.66; P = 0.011; fig. 2, Aii and Bi) by 49 ± 32% (P = 0.022; fig. 2, Aii and Bii). The dose–response relationship for inhibition of TNFα-induced eNOS phosphorylation by both ropivacaine and lidocaine (fig. 2C) revealed an IC50 of 757 pM (95% CI, 117.7 pM to 4.9 nM; table 1) for ropivacaine, which had a maximal effect of 39.4% (58.9 to 19.8), whereas lidocaine showed an IC50 of 638 pM (92 pM to 44.2 nM) and a maximum effect of 54% (74.4 to 35.4; table 1) in these experiments. Neither ropivacaine nor lidocaine, even at concentrations of 100 μM, directly altered eNOS phosphorylation compared with untreated cells (Kruskal–Wallis test, P = 0.972 for ropivacaine; P = 0.359 for lidocaine, data not shown).
The effect of ropivacaine at different concentrations (1 nM, 1 μM, and 100 μM) on TNFα-induced (20 ng/ml) nitric oxide production in HLMVEC as compared with cells treated with the NOS-inhibitor l-NIO (20 μM) was examined. Data analysis using two-way ANOVA with the presence of TNFα and the different experimental treatments (l-NIO or ropivacaine) as the factors to be tested revealed a significant impact of the presence of TNFα (F(1, 31) = 40.35; P < 0.0001) and the different drugs and doses on nitric oxide production by the cells (F(4, 31) = 25.93; P< 0.0001). A significant interaction term between the two factors (F(4, 31) = 16.55; P< 0.0001) was also found. Post hoc testing revealed significant differences between l-NIO and the different concentrations of ropivacaine when incubated together with TNFα compared with TNFα alone (P < 0.0001 for all comparisons). As shown in figure 2D, the TNFα-induced increase was completely abolished by pretreating the cells with l-NIO and 1 nM of ropivacaine. However, the presence of neither ropivacaine nor l-NIO had an effect on basal nitric oxide production (P = 1 for all comparisons; fig. 2D).
Ropivacaine Blocks TNFα-induced Activation of Akt Upstream of eNOS but Does Not Directly Inhibit PI3K Activity Upstream of Akt
To establish the mechanism by which ropivacaine blocks TNFα-induced eNOS and Src activation, we determined whether ropivacaine blocks TNFα-induced activation of Akt, an upstream kinase known to activate eNOS11 via phosphorylation of serine 1177. HLMVECs exposed to ropivacaine (1 nM, 1 μM) in the absence or presence of TNFα (20 ng/ml) for 20 min were subject to Western blot analysis, probing for “activated” Akt, phosphorylated at threonine 308 (pT308 Akt, the PI3K-dependent phosphorylation site),27 as well as total Akt which served as a loading control (fig. 3A). Densitometry analysis revealed that ropivacaine alone had no effect on basal Akt phosphorylation (P = 0.878, data not shown), whereas the TNFα-induced increase in Akt phosphorylation (214 ± 44% of untreated cells; P = 0.013) was completely abolished by 1 nM of ropivacaine (P = 0.005; fig. 3B). As Akt activation is known to be induced by PI3K,11,28 we determined whether ropivacaine might act as a direct inhibitor of PI3K catalytic activity. PI3K activity in vitro was determined by enzyme-linked immunosorbent assay measurements of PIP3 generated by PI3K. In this particular assay, a high signal indicates poor competition with B-PIP3 and low enzyme activity. Different isoforms of p110 (α, β, γ, and δ), the catalytic subunit of PI3K, were incubated with PIP2 substrate in the absence or presence of different concentrations of either ropivacaine or wortmannin (100 nM), a known PI3K inhibitor.17 As shown in figure 3C, wortmannin decreased p110 activity, whereas ropivacaine had no significant effect on the activity of the four PI3K isoforms tested (fig. 3C).
To determine whether ropivacaine blocks Akt-dependent eNOS activation (and thereby prevents Src activation) specifically, Ca2+-dependent eNOS activation in the presence or absence of ropivacaine was assessed. HLMVECs were stimulated with the Ca2+ ionophore A23187 (5 μM) for 10 min to activate eNOS via Ca2+/calmodulin10 and subsequently Src via nitric oxide signaling rather than Akt signaling10 (fig. 4). A23187 treatment induced a 45 ± 34.8% increase in Src activity compared with control (P = 0.079; fig. 4, A and B); however, this effect was not blocked by 1 μM of ropivacaine (P = 1; fig. 4, A and B).
Ropivacaine Blocks Recruitment of p85 Regulatory Subunit of PI3K to TNF-R1
The recruitment of p85 regulatory subunit of PI3K to TNF-R1 is a key step leading to PI3K activation on binding of TNFα to TNF-R1.13 To detect the recruitment of p85, TNF-R1 was immunoprecipitated from HLMVEC lysates which were briefly (10 min) stimulated with TNFα in the absence or presence of ropivacaine at 1 nM. Western blots of the immunoprecipitated protein (fig. 5A) showed a statistically significant 4.46 ± 1.92-fold increase (P = 0.002) in co-immunoprecipitation of p85 and TNF-R1 compared with untreated cells (fig. 5B) which was attenuated by 64 ± 14.6% in presence of 1 nM ropivacaine (P = 0.04; fig. 5B).
Ropivacaine Inhibits Src-dependent Phosphorylation of ICAM-1 and Neutrophil Adhesion to Endothelial Cells
Src-dependent phosphorylation of ICAM-1 is known to increase leukocyte adhesion in endothelial cells.16 We therefore first assessed whether ropivacaine inhibits TNFα-induced ICAM-1 phosphorylation in our cell culture model. HLMVEC lysates, incubated with different concentrations of ropivacaine (1 nM, 1 μM) in the absence or presence of TNFα, were analyzed via Western blot, probing for phosphorylated ICAM-1 (at tyrosine 512, pY512 ICAM-1), and total ICAM-1 (fig. 6A). Densitometry analysis revealed that there was no alteration in basal pY512 ICAM-1 level in presence of ropivacaine alone (P = 0.537, data not shown). However, when applied in combination with TNFα, ICAM-1 phosphorylation observed in response to TNFα alone (175 ± 94.2% compared with untreated cells; P = 0.007) was reduced by 45 ± 23% in presence of 1 nM ropivacaine (P = 0.107) and was completely blocked by 1 μM (P = 0.006; fig. 6B). To assess the functional significance of the observed inhibition of ICAM-1 phosphorylation, we quantified neutrophil adhesion to HLMVEC monolayers. Before the addition of TNFα (20 ng/ml) ± ropivacaine (1 nM, 1 μM, 100 μM) for 20 min, HLMVECs were stimulated with lipopolysaccharide (4 μg/ml) for 4 h to increase ICAM-1 expression as shown in the Western blot in figure 6Ci. Quantification of adherent, fluorescently labeled human neutrophils revealed a 63 ± 27.6% increase in adhesion in cells treated with TNFα for 20 min. Analysis with two-way ANOVA revealed a significant effect of both TNFα (F(1, 56) = 14.14; P < 0.001) and ropivacaine (F(3, 56) = 6.765; P < 0.001) and a significant interaction term between these two factors (F(3, 56) = 5.177; P < 0.001). Post hoc comparison between the values obtained from incubation with TNFα alone and ropivacaine at different concentrations incubated together with TNFα revealed a dose-dependent inhibition of neutrophil adhesion by increasing ropivacaine concentrations (statistically significant decrease at a concentration of 1 nM ropivacaine [P = 0.01] and higher; fig. 6Cii).
Ropivacaine Inhibits Src-dependent Phosphorylation of Caveolin-1 and Disruption of Endothelial Monolayer Integrity
Src-dependent phosphorylation of caveolin-1 tyrosine 14 (pY14 Cav-1) leads to endothelial barrier dysfunction.8 Incubation of HLMVEC with ropivacaine at different concentrations for 20 min did not alter basal pY14 Cav-1 (P = 0.311, data not shown). As shown in figure 7, A and B, TNFα induced a 36 ± 46% increase in caveolin-1 phosphorylation, which was not considered to be statistically significant (P = 1). However, in the presence of 1 nM ropivacaine, we did not observe a significant increase in TNFα-induced caveolin-1 phosphorylation compared with untreated cells (P = 0.4522) but saw a reduction of 35 ± 18% compared with TNFα alone (P = 0.012; fig. 7, A and B).
Endothelial barrier disruption induced by TNFα (i.e., decrease in monolayer integrity which is thought to represent an increase in paracellular permeability)25 was evaluated by measuring TER of HLMVEC monolayers grown on gold-film electrodes. HLMVECs were treated with TNFα (20 ng/ml), ropivacaine (1 μM), TNFα + ropivacaine, TNFα + NOS-inhibitor l-NIO (20 μM, pretreatment for 1 h before addition of TNFα), TNFα + Src-inhibitor PP2 (10 μM, pretreatment for 30 min before addition of TNFα), or were left untreated (control). TER was measured over time and normalized to baseline values as shown in figure 7C. TNFα decreased monolayer resistance at 6 h (fig. 7D) by 57 ± 20% (P < 0.001) compared with untreated monolayers, respectively. Coincubation of cells with TNFα and 1 μM of ropivacaine significantly blocked the decrease in TER induced by TNFα alone by 42 ± 19.3% at 6 h (P = 0.006). As positive controls, pretreatment of monolayers with NOS and Src inhibitors l-NIO and PP2, respectively, was shown to also significantly attenuate the TNFα-induced decrease in TER at 6 h (P < 0.001 for l-NIO, P = 0.049 for PP2).
Inflammatory vascular hyperpermeability is mediated in part by Src-dependent signaling,8,14–17,29,30 and thus Src may be a therapeutic target in the pathogenesis of inflammatory diseases such as acute lung injury, ARDS, cancer, and diabetes mellitus.1,3,4,7,18,22 In the current study, we characterized the molecular mechanism by which the amide-linked local anesthetics ropivacaine and lidocaine block inflammatory Src signaling induced by TNFα. In functional studies in which human endothelial cells were used, we observed that TNFα-induced neutrophil binding and loss of endothelial monolayer integrity could be abolished in cells simultaneously treated with ropivacaine. The concentrations of ropivacaine and lidocaine that were effective (1 nM to 1 μM) are clinically relevant and consistent with a recent study which reported a well-tolerated, mean (total) maximal plasma concentration of 1.82 mg/l (5.5 μM) during continuous epidural infusions of 0.2% ropivacaine at a rate of 10 mg/h for 48 h after major abdominal or urologic surgery.31
Previous studies have also shown a protective effect of local anesthetics on endothelial cell viability and function.32,33 However, these studies primarily focused on the inhibition of effects of prolonged (>24 h) cytokine exposure and used concentrations of local anesthetics up to 100,000 times higher than that used in the current study32,33 although still much lower than concentrations required to achieve effective blockade of voltage-gated sodium channels (IC50 of 50 to 100 μM).34,35 Interestingly, in an earlier study, we observed that inhibition of TNFα-induced Src activation by local anesthetics occurs independently of sodium channel blockade.22 Due to their lipophilic nature, local anesthetics distribute into cell membranes, affect their dynamics,36 and interfere with the function of membrane-associated proteins like the G-protein–coupled m1 muscarinic acetylcholine receptor,37 also at a much lower concentration (the IC50 for lidocaine was 18 nM) than that which is necessary for sodium channel inhibition. Uniquely, the current report focused on the effect of amide-linked local anesthetics on rapidly appearing TNFα-induced signaling events known to be activated during the initial phase of vascular inflammation.16 We demonstrated specific inhibition of agonist (TNFα)-induced activation of signaling enzymes, but saw no effect on basal signaling, which might also be a possible explanation for the low IC50 values observed.
The mechanism(s) that account for the now well-established anti-inflammatory as well as anti-metastatic actions of local anesthetics requires further investigation.18,22 There are structural similarities between ropivacaine (and lidocaine) and commercially available Src kinase inhibitors such as the combined Bcr-Abl/Src inhibitor Dasatinib® (Bristol-Myers Squibb Company, Princeton, NJ).38,39 Both types of drugs share an amide-linked phenyl group with substitution at positions 2 and 6, whereas the phenyl ring in local anesthetics shows two methyl groups and Dasatinib® one methyl and one chloride.39,40 Interestingly, this particular part of Dasatinib® is important in the mechanism of inhibition, as it binds (and therefore blocks) to the hydrophobic pouch of the adenosine triphosphate binding pocket within the kinase.38 Our investigations, however, indicate that ropivacaine does not act as a competitive inhibitor of adenosine triphosphate binding in the Src kinase active site, that is, the common target of most kinase inhibitors.38 To address other possible targets of local anesthetics such as ropivacaine and lidocaine, we interrogated potential signaling intermediates known to play a role in TNFα-induced Src activation.
Src activity in cultured cells and tissues is often assessed by measuring the phosphorylation status of active-site Src tyrosine 419 and inhibitory tyrosine 529 residues.8 eNOS activation and nitric oxide generation play an important role in Src activation10 and were significantly inhibited by ropivacaine and lidocaine in our experiments. A reduction of (lipopolysaccharide-induced) nitric oxide production by lidocaine in bovine aortic endothelial cells has previously been reported, and the authors provided some evidence that the observed inhibition might (in part) have been due to a suppression of l-arginine uptake by the cells.41 Nitric oxide–dependent S-nitrosation of Src at cysteine 498 has also been reported not only to increase Src activity but also to lead to the disruption of cell–cell junctions.9
Akt protein kinase is known to activate eNOS via direct phosphorylation of serine 1177.42,43 Our studies demonstrate no effect of ropivacaine on Src activation induced by Ca2+ ionophore, a non–Akt-dependent activator of eNOS, but inhibited TNFα-induced Akt activation suggesting the site of ropivacaine action may be upstream of Akt, for example via inhibition of PI3K. PI3Ks are divided into three different classes, class I, II, and III, and class I PI3K consists of two subgroups, IA and IB.44 The class I PI3K isoforms function as heterodimers of the regulatory p85 subunit (of which there are two isoforms, p85α and p85β) and the catalytic p110 subunit (α, β, δ isoforms in class IA; γ isoform in class IB).45 PI3K phosphorylates the 3’-OH residue of the inositol ring of phosphatidylinositol-(4,5) phosphate (PIP2) to generate PIP3, a lipid second messenger that has been shown to activate Akt and phosphoinositide-dependent kinase-1.44,46 By using the principle of PIP3 generation by PI3K in an in vitro kinase assay, we showed that ropivacaine does not directly inhibit PI3K activity.
An alternative pathway leading to Src activation on stimulation with TNFα might be the generation of superoxide anions (O2−), for example by nicotinamide adenine dinucleotide phosphate-oxidase (NOX) via a pathway involving the activation of protein kinase C-ζ downstream of PI3K (fig. 8, gray arrows) as the stimulation of endothelial cells with TNFα was shown to induce a PI3K-dependent rapid increase in protein kinase C-ζ activation.16,47 Protein kinase C-ζ–mediated phosphorylation of p47phox is thought to initiate the assembly and activation of the NOX complex.48 The oxidants produced have been shown to activate Src as well.49,50
The results of the current study, however, indicate that ropivacaine blocks TNFα-induced Src activation upstream of PI3K. On the binding of TNFα to its primary receptor in endothelial cells, TNF-R1, p85 is recruited to the receptor which leads to the assembly of the p85 and p110 heterodimer and thereby to activation of PI3K13 (fig. 8). Interestingly, we observed a diminished recruitment of p85 to TNF-R1 by ropivacaine in co-immunoprecipitation experiments of TNF-R1 and p85 in HLMVEC suggesting that ropivacaine may block TNFα signaling by disrupting TNF-R1–mediated PI3K activation, Akt phosphorylation, and eNOS-dependent nitric oxide production.
The initiation of transmembrane signaling via TNF-R1 includes oligomerization of ligand–receptor complexes,51 and there is some evidence that certain anesthetics (as shown for the less common local anesthetic phenylethanol) are able to inhibit oligomerization of transmembrane proteins.52 In addition, it has been shown that amide-linked local anesthetics alter membrane fluidity as well as conformation of membrane proteins.53,54 Thus, on the basis of the results of the current study, we speculate that inhibition of p85 recruitment might be due to alteration in the conformation of TNF-R1 or attenuation of its ability to oligomerize to initiate signaling across the membrane.51
In endothelial cells, we previously demonstrated that TNFα-induced Src activation leads to ICAM-1 tyrosine 512 phosphorylation which, by promoting actin-mediated ICAM-1 clustering, increases the avidity of neutrophil binding during the rapid, initial phase of inflammation.16 Subsequently, this rapid increase in endothelial cell adhesiveness leads to accelerated recruitment of circulating neutrophils and development of inflammation.16 Ropivacaine was shown to inhibit TNFα-induced Src-dependent ICAM-1 phosphorylation as well as neutrophil adhesion to monolayers of HLMVECs, thus supporting the hypothesis that ropivacaine has beneficial anti-inflammatory effects by blunting TNFα-mediated endothelial cell activation.
Src-dependent caveolin-1 tyrosine 14 phosphorylation also contributes to the development of inflammatory vascular hyperpermeability.8,29,30 In response to thrombin or reactive oxygen species (H2O2), that is, agents that induce the disruption of endothelial monolayer integrity,29,55 phosphorylated caveolin-1 promotes disruption of vascular endothelial–cadherin/catenin interactions,29 perhaps by binding to catenins directly.56 Disruption of cadherin/catenin interactions thought to be essential in the regulation of adherens junction stability,57,58 leads to disassembly of these cell–cell junctions and thereby an increase in transcellular endothelial permeability.56 Alternatively, active Src may have directly modified vascular endothelial–cadherin in response to TNFα as it was shown that Src phosphorylation of vascular endothelial–cadherin tyrosine residues 658 and 685 in venous endothelial cells59 promotes vascular endothelial–cadherin internalization, ubiquitination, and degradation, ultimately resulting in endothelial barrier disruption and increased vascular permeability.58,59
Local anesthetic-mediated inhibition of Src-dependent ICAM-1 and caveolin-1 phosphorylation may be a potential mechanism for blocking endothelial barrier disruption during acute inflammation. In addition, inhibition of TNFα-induced PI3K activation by ropivacaine upstream of Src might also be clinically relevant. In a recent study, it was shown that the phosphatidylinositol (3,4,5)-trisphosphate-dependent Rac exchanger 1 (P-Rex1) mediates TNFα-induced endothelial barrier disruption.25 As ropivacaine inhibits TNF-R1-mediated PI3K activation, it should also decrease PIP3 generation and attenuate P-Rex1 activation60 and signaling.25
In conclusion, this study illustrates a novel anti-inflammatory mechanism of action of the local anesthetics ropivacaine and lidocaine. We demonstrate that in addition to the well-described action of Na+ channel blockade, local anesthetics also blocked TNFα-induced TNF-R1-dependent PI3K, Akt, and eNOS activation resulting in the inhibition of nitric oxide–driven Src signaling. We also showed that the inhibition of Src activation by the local anesthetics blocked TNFα-induced neutrophil adhesion and endothelial barrier disruption (inflammatory hyperpermeability). Thus, local anesthetics may have significant therapeutic benefit by attenuating vascular inflammation which we predict would minimize microvascular endothelial injury and inflammatory hyperpermeability associated with the pathogenesis of acute lung injury, ARDS, diabetes mellitus, and cancer.
This work was supported, in part, by the Society of Cardiovascular Anesthesiologists Foundation/International Anesthesia Research Society Starter Grant, Richmond, Virginia, (to Dr. Piegeler), U.S. Department of Defense research support W911NF-07-R-0003-04, Washington, D.C., and American Heart Association grant 09SDG2250933, Dallas, Texas (to Dr. Bonini), European Society of Anaesthesiology Research Grant, Brussels, Belgium (to Dr. Borgeat), and National Institutes of Health Heart Lung and Blood Institute grants R01 HL071626 and P01 HL060678, Bethesda, Maryland (to Dr. Minshall).
The authors declare no competing interests.