The mechanism of caveolin-1–dependent eNOS inactivation is not clear. These studies reveal that NO-mediated Src kinase activation and caveolin-1 phosphorylation promote eNOS binding and inactivation, that is, eNOS negative feedback regulation.

In the present study, we tested the hypothesis that chronic inflammation and oxidative/nitrosative stress induce caveolin 1 (Cav-1) degradation, providing an underlying mechanism of endothelial cell activation/dysfunction and pulmonary vascular remodeling in patients with idiopathic pulmonary arterial hypertension (IPAH). We observed reduced Cav-1 protein despite increased Cav-1 messenger RNA expression and also endothelial nitric oxide synthase (eNOS) hyperphosphorylation in human pulmonary artery endothelial cells (PAECs) from patients with IPAH. In control human lung endothelial cell cultures, tumor necrosis factor α-induced nitric oxide (NO) production and S-nitrosation (SNO) of Cav-1 Cys-156 were associated with Src displacement and activation, Cav-1 Tyr-14 phosphorylation, and destabilization of Cav-1 oligomers within 5 minutes that could be blocked by eNOS or Src inhibition. Prolonged stimulation (72 hours) with NO donor DETANONOate reduced oligomerized and total Cav-1 levels by 40%-80%, similar to that observed in IPAH patient-derived PAECs. NO donor stimulation of endothelial cells for >72 hours, which was associated with sustained Src activation and Cav-1 phosphorylation, ubiquitination, and degradation, was blocked by NOS inhibitor L-NAME, Src inhibitor PP2, and proteosomal inhibitor MG132. Thus, chronic inflammation, sustained eNOS and Src signaling, and Cav-1 degradation may be important causal factors in the development of IPAH by promoting PAEC dysfunction/activation via sustained oxidative/nitrosative stress.

NOS1−/− mice show reduced inflammatory responses and tissue damage in experimental sepsis models. Baig et al show that NOS1-derived NO production in macrophages leads to proteolysis of SOCS1 to alleviate its repression of NFkB transcriptional activity in response to TLR4-mediated responses.

In the present study, we assessed the cooperative roles of C-terminal Src kinase (Csk) binding protein (Cbp) and Caveolin-1 (Cav-1) in the mechanism of Src family tyrosine kinase (SFK) inhibition by Csk. SFKs are inactivated by phosphorylation of their C-terminal tyrosine by Csk. Whereas SFKs are membrane-associated, Csk is a cytoplasmic protein and therefore requires membrane adaptors such as Cbp or Cav-1 for recruitment to the plasma membrane to mediate SFK inhibition. To determine the specific role of Cav-1 and Cbp in SFK inhibition, we measured c-Src activity in the absence of each membrane adaptor. It is noteworthy that in lungs and fibroblasts from Cav-1(Ϫ/Ϫ) mice, we observed increased expression of Cbp compared with wild-type (WT) controls. However, both c-Src activity and Csk localization at the membrane were similar between Cav-1(Ϫ/Ϫ) fibroblasts and WT cells. Likewise, Cbp depletion by small interfering RNA (siRNA) treatment of WT cells had no effect on basal c-Src activity, but it increased the phosphorylation state of Cav-1. Immunoprecipitation then confirmed increased association of Csk with phosphomimicking Cav-1. Knockdown of Cbp by siRNA in Cav-1(Ϫ/Ϫ) cells revealed increased basal c-Src activity, and re-expression of WT Cav-1 in the same cells reduced basal c-Src activity. Taken together, these results indicate that Cav-1 and Cbp cooperatively regulate c-Src activity by recruiting Csk to the membrane where it phosphorylates c-Src inhibitory tyrosine 529. Furthermore, when either Cav-1 or Cbp expression is reduced or absent, there is a compensatory increase in the phosphorylation state or expression level of the other membrane-associated Csk adaptor to maintain SFK inhibition.

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.

Key Points Gα12 interaction with α-SNAP regulates basal EC vWF secretion. PAR-1 activation-dependent signaling via Gα12/RhoA and Gαq/11 enhances vWF secretion.

Background Genome wide association studies (GWAS) have revealed significant association of caveolin-1 (Cav1) gene variants with increased risk of cardiac arrhythmias. Nevertheless, the mechanism for this linkage is unclear. Methods and Results Using adult Cav1−/− mice, we revealed a marked reduction in the left ventricular (LV) conduction velocity in the absence of myocardial Cav1, which is accompanied with increased inducibility of ventricular arrhythmias. Further studies demonstrated that loss of Cav1 leads to the activation of cSrc tyrosine kinase, resulting in the downregulation of connexin 43 (Cx43) and subsequent electrical abnormalities. Pharmacological inhibition of cSrc mitigates Cx43 downregulation, slow conduction and arrhythmia inducibility in Cav1−/− animals. Using a transgenic mouse model with cardiac-specific overexpression of angiotensin converting enzyme (ACE8/8), we demonstrated that, upon enhanced cardiac RAS activity, Cav1 dissociated from cSrc because of increased Cav1 S-nitrosation (SNO) at Cys156, leading to c-Src activation, Cx43 reduction, impaired gap junction function, and subsequent increase in the propensity for ventricular arrhythmias and sudden cardiac death. RAS-induced Cav1 SNO was associated with increased Cav1-eNOS binding in response to increased mitochondrial reactive oxidative species (ROS) generation. Conclusions The present studies reveal the critical role of Cav1 in modulating cSrc activation, gap junction remodeling and ventricular arrhythmias. These data provide a mechanistic explanation for the observed genetic link between Cav1 and cardiac arrhythmias in humans and suggest that targeted regulation of Cav1 may reduce arrhythmic risk in cardiac diseases associated with RAS activation.

The NF-B pathway is central to the regulation of inflammation. Here, we demonstrate that the low-output nitric oxide (NO) synthase 1 (NOS1 or nNOS) plays a critical role in the inflammatory response by promoting the activity of NF-B. Specifically, NOS1-derived NO production in macrophages leads to proteolysis of suppressor of cytokine signaling 1 (SOCS1), alleviating its repression of NF-B transcriptional activity. As a result, NOS1 / mice demonstrate reduced cytokine production, lung injury, and mortality when subjected to two different models of sepsis. Isolated NOS1 / macrophages demonstrate similar defects in proinflammatory transcription on challenge with Gram-negative bacterial LPS. Consistently, we found that activated NOS1 / macrophages contain increased SOCS1 protein and decreased levels of p65 protein compared with wild-type cells. NOS1-dependent S-nitrosation of SOCS1 impairs its binding to p65 and targets SOCS1 for proteolysis. Treatment of NOS1 / cells with exogenous NO rescues both SOCS1 degradation and stabilization of p65 protein. Point mutation analysis demonstrated that both Cys147 and Cys179 on SOCS1 are required for its NO-dependent degradation. These findings demonstrate a fundamental role for NOS1-derived NO in regulating TLR4-mediated inflammatory gene transcription, as well as the intensity and duration of the resulting host immune response.