Endothelial Barrier Protection by Local Anesthetics

Abstract

Background 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. Methods 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. Results 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. Conclusions 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.

Insulin Signaling in Bupivacaine-induced Cardiac Toxicity

“…We quantified concentrations of Akt phosphorylated at S473 & T308, and the downstream targets GSK-3β at S9, p70s6k at T421, ribosomal protein s6 at S235, and feedback phosphorylation of IRS1 at S612 (Figure 1D). Consistent with previous reports 7–9 , bupivacaine reduced signaling in the Akt pathway with treatment accounting for 48% of the effect (Figure 1E, 2-way matched sample ANOVA interaction p=0.0035, kinase effect p=0.0035, treatment effect p=0.0083, matching p<0.0001). Phosphorylation was decreased on Akt at S473 to 63 ± 5% (mean ± SEM, Sidak post-test p=0.017), p70s6k at T421 to 50 ± 17% (p=0.0043), ribosomal protein s6 at S235 to 44 ± 11% (p=0.0015) and IRS1 at S612 to 31 ± 8% (p=0.0004) compared to baseline values.…”

“…AMPK, GSK-3β) occur during induction of toxicity and recovery from it. Our results agree with previous in vitro studies wherein lethal or cytotoxic concentrations of bupivacaine induce de-phosphorylation of Akt 7–9 and phosphorylation of AMPK 11,12 . Both of these kinases integrate signaling at mTORC1 to modulate sensitivity to endogenous insulin 30 during recovery from toxicity.…”

“…Recent reports demonstrate that bupivacaine disrupts targets of classical insulin signaling 6 including protein kinase B (Akt) and ribosomal protein s6 kinase 1, in cellular models 7,8 . Further, the amide-linked local anesthetics, ropivacaine and lidocaine, disrupt assembly of phosphoinositide-3-kinase (Pi3k) thereby blocking phosphorylation of Akt 9 , an effect that is independent of classical sodium-channel blockade 10 . Beyond Akt, bupivacaine activates other controllers of glucose homeostasis, including 5’ adenosine monophosphate activated protein kinase (AMPK) 11,12 , an effect which may provide cyto-protection during toxicity 13 .…”

Multi-modal contributions to detoxification of acute pharmacotoxicity by a triglyceride micro-emulsion

“…We have elucidated an integrated model that can explain the mechanism of lipid resuscitation and provide insight into the need for multi-modal biodetoxification. Like other cardiotoxic drugs, bupivacaine exerts a number of adverse effects that can lead to cardiovascular collapse; these include blockage of key ionotropic channels [32,34], interference with mitochondrial processing [42,43], and blocking intracellular kinase signaling [44] (Fig 5A,B). We have shown that treatment with a triglyceride micro-emulsion provides a rapid scavenging effect, removing drugs from key organs including the heart and brain (Fig 5C).…”

What′s new in critical illness and injury science? State of the art in management of ARDS.

“…[4546] In the former, Piegeler et al ., studied the addition of ropivacaine, lidocaine, and chloroprocaine to NCI-H838 lung cancer cells incubated with tumor necrosis factor-α (TNFα). Ropivacaine and lidocaine significantly decreased Src -activation and intercellular adhesion molecule-1 phosphorylation.…”