Measuring Activity of Endothelial Nitric Oxide Synthase and Nanodisc Complex through Nitrate Production

Nitric oxide is an important bioregulator generated in various regions throughout the body by a family of isozymes referred to as Nitric Oxide Synthases (NOS). Within vascular endothelial cells, nitric oxide is generated from oxygen and arginine (amino acid) by endothelial nitric oxide synthases (eNOS). Within this environment nitric oxide plays a critical paracrine role, mainly anithrombotic and anti-atherosclerotic. This is accomplished by vessel dilation and prevention of platelet and leukocyte aggregation and adherence to the vessel wall. The activity of the eNOS enzyme has been studied within solution and is well understood. However, the impact that the lipid bilayer of endothelial cells has on the activity is not known. To better understand this interaction, we have formed “nanodiscs” to bind to the eNOS. Nanodiscs have two components that combine and self-assemble when added to solution, POPC (a lipid) and MSP1E3D1 (Membrane Scaffold Protein). The nanodiscs help provide a better microenvironment to study the enzyme and its activity. Through reaction with an indicator dye in the Griess reagent system, activity levels, as calculated by nitrate production, reduced dramatically. Over a 50% reduction was seen when calculating specific activity of the eNOS enzyme when bound to nanodiscs. A possible indication that a lipid bilayer restricts activity of the eNOS enzyme.https://engagedscholarship.csuohio.edu/u_poster_2014/1036/thumbnail.jp

Endothelial Nitric Oxide Synthase Oxygenase on Lipid Nanodiscs: A Nano-Assembly Reflecting Native-Like Function of eNOS

© 2017 Elsevier Inc. Endothelial nitric oxide synthase (eNOS) is a membrane-anchored enzyme. To highlight the potential role and effect of membrane phospholipids on the structure and activity of eNOS, we have incorporated the recombinant oxygenase subunit of eNOS into lipid nanodiscs. Two different size distribution modes were detected by multi-angle dynamic light scattering both for empty nanodiscs, and nanodiscs-bound eNOSoxy. The calculated hydrodynamic diameter for mode 1 species was 9.0 nm for empty nanodiscs and 9.8 nm for nanodisc bound eNOSoxy. Spectroscopic Griess assay was used to measure the enzymatic activity. Remarkably, the specific activity of nanodisc-bound eNOSoxy is ∼65% lower than the activity of free enzyme. The data shows that the nano-membrane environment affects the catalytic properties of eNOS heme domain

Measuring Activity of Endothelial Nitric Oxide Synthase and Nanodisc Complex through Nitrate Production

Nitric oxide is an important bioregulator generated in various regions throughout the body by a family of isozymes referred to as Nitric Oxide Synthases (NOS). Within vascular endothelial cells, nitric oxide is generated from oxygen and arginine (amino acid) by endothelial nitric oxide synthases (eNOS). Within this environment nitric oxide plays a critical paracrine role, mainly anithrombotic and anti-atherosclerotic. This is accomplished by vessel dilation and prevention of platelet and leukocyte aggregation and adherence to the vessel wall. The activity of the eNOS enzyme has been studied within solution and is well understood. However, the impact that the lipid bilayer of endothelial cells has on the activity is not known. To better understand this interaction, we have formed “nanodiscs” to bind to the eNOS. Nanodiscs have two components that combine and self-assemble when added to solution, POPC (a lipid) and MSP1E3D1 (Membrane Scaffold Protein). The nanodiscs help provide a better microenvironment to study the enzyme and its activity. Through reaction with an indicator dye in the Griess reagent system, activity levels, as calculated by nitrate production, reduced dramatically. Over a 50% reduction was seen when calculating specific activity of the eNOS enzyme when bound to nanodiscs. A possible indication that a lipid bilayer restricts activity of the eNOS enzyme.https://engagedscholarship.csuohio.edu/u_poster_2014/1036/thumbnail.jp

Methods of Peroxynitrite Synthesis in the Context of the Development and Validation of Peroxynitrite Sensors

Peroxynitrite has emerged as a major cytotoxic agent implicated in a host of pathophysiological conditions triggered by its multifaceted nitroxidative reactions. Peroxynitrite\u27s very short half-life under physiological conditions reflects both its fast intrinsic decomposition and its notorious reactivity with many cellular targets. Sensitive and accurate measurement of peroxynitrite is crucial to investigations that aim at shedding light on the illusive pathophysiologic roles of this metabolite. The development process of probes and sensors capable of selectively detecting and measuring any analyte depends primarily on the availability of pure authentic amounts of it in order to evaluate sensor performance. There are numerous methods of peroxynitrite synthesis or generation in situ. However, they all come with varying levels of byproducts that may interfere with the characterization of performance of peroxynitrite sensing probes and devices. This chapter gives the background and a brief review of major methods of peroxynitrite synthesis and in situ generation. Particularly, we discuss advantages and drawbacks of the various methods in the context of the development and characterization of peroxynitrite sensors and probes

P101 – Endothelial Nitric Oxide Synthase (eNOS) on Lipid Nanodiscs: Toward A Soluble Assembly Reflecting Native-Like Function of eNOS

Cardiovascular disease (CVD) is the leading cause of death worldwide. Approximately 30% of all global deaths in 2008 were due to CVD. Endothelial cells cover the blood vessels lumen and provide a barrier against vascular disease. Nitric oxide is a unique bio-regulator with important signaling roles in cardiovascular as well as other physiologic systems. Nitric oxide synthases (NOSs) are a family of enzymes that generate nitric oxide from arginine and oxygen. Endothelial nitric oxide synthase (eNOS) is one member of this family, and is the dominant isoform in the inner walls of blood vessels. It regulates numerous essential cardiovascular functions including vasodilation (blood pressure), inhibition of platelet aggregation and adhesion to the vascular wall, which prevents atherosclerosis and unwanted blood clots. To determine the influence of the phospholipid bilayer on the structure and activity of eNOS in a defined system, we have incorporated the recombinant oxygenase subunit of the enzyme into miniature lipid membranes called nanodiscs which are 12.9 nm in diameter. These nanodiscs based on membrane scaffold proteins provide a unique system that mimics the enzyme\u27s native microenvironment, yet the prepared enzyme/nanodisc assemblies can be conveniently studied in solution like any soluble enzyme preparation. Homogenous eNOS/nanodisc samples are purified using size exclusion chromatography. The average size of nanodisc diameter was confirmed by particle analysis based on dynamic light scattering. Griess assay is used to measure activity of free and nanodisc-bound enzymes. As compared to the free enzyme, the specific activity of nanodisc-bound eNOS oxygenase appears to be much lower. These data suggest that the membrane environment affects the catalytic properties of eNOS oxygenase

Functional Layer-by-Layer Thin Films of Inducible Nitric Oxide (NO) Synthase Oxygenase and Polyethylenimine: Modulation of Enzyme Loading and NO-Release Activity

Nitric oxide (NO) release counteracts platelet aggregation and prevents the thrombosis cascade in the inner walls of blood vessels. NO-release coatings also prevent thrombus formation on the surface of blood-contacting medical devices. Our previous work has shown that inducible nitric oxide synthase (iNOS) films release NO fluxes upon enzymatic conversion of the substrate L-arginine. In this work, we report on the modulation of enzyme loading in layer-bylayer (LbL) thin films of inducible nitric oxide synthase oxygenase (iNOSoxy) on polyethylenimine (PEI). The layer of iNOSoxy is electrostatically adsorbed onto the PEI layer. The pH of the iNOSoxy solution affects the amount of enzyme adsorbed. The overall negative surface charge of iNOSoxy in solution depends on the pH and hence determines the density of adsorbed protein on the positively charged PEI layer. We used buffered iNOSoxy solutions adjusted to pHs 8.6 and 7.0, while saline PEI solution was used at pH 7.0. Atomic force microscopy imaging of the outermost layer shows higher protein adsorption with iNOSoxy at pH 8.6 than with a solution of iNOSoxy at pH 7.0. Graphite electrodes with PEI/iNOSoxy films show higher catalytic currents for nitric oxide reduction mediated by iNOSoxy. The higher enzyme loading translates into higher NO flux when the enzyme-modified surface is exposed to a solution containing the substrate and a source of electrons. Spectrophotometric assays showed higher NO fluxes with iNOSoxy/PEI films built at pH 8.6 than with films built at pH 7.0. Fourier transform infrared analysis of iNOSoxy adsorbed on PEI at pH 8.6 and 7.0 shows structural differences of iNOSoxy in films, which explains the observed changes in enzymatic activity. Our findings show that pH provides a strategy to optimize the NOS loading and enzyme activity in NOS-based LbL thin films, which enables improved NO release with minimum layers of PEI/NOS

cAMP-dependent activation of protein kinase A attenuates respiratory syncytial virus-induced human airway epithelial barrier disruption.

Airway epithelium forms a barrier to the outside world and has a crucial role in susceptibility to viral infections. Cyclic adenosine monophosphate (cAMP) is an important second messenger acting via two intracellular signaling molecules: protein kinase A (PKA) and the guanidine nucleotide exchange factor, Epac. We sought to investigate effects of increased cAMP level on the disruption of model airway epithelial barrier caused by RSV infection and the molecular mechanisms underlying cAMP actions. Human bronchial epithelial cells were infected with RSV-A2 and treated with either cAMP releasing agent, forskolin, or cAMP analogs. Structure and functions of the Apical Junctional Complex (AJC) were evaluated by measuring transepithelial electrical resistance and permeability to FITC-dextran, and determining localization of AJC proteins by confocal microscopy. Increased intracellular cAMP level significantly attenuated RSV-induced disassembly of AJC. These barrier-protective effects of cAMP were due to the activation of PKA signaling and did not involve Epac activity. Increased cAMP level reduced RSV-induced reorganization of the actin cytoskeleton, including apical accumulation of an essential actin-binding protein, cortactin, and inhibited expression of the RSV F protein. These barrier-protective and antiviral-function of cAMP signaling were evident even when cAMP level was increased after the onset of RSV infection. Taken together, our study demonstrates that cAMP/PKA signaling attenuated RSV-induced disruption of structure and functions of the model airway epithelial barrier by mechanisms involving the stabilization of epithelial junctions and inhibition of viral biogenesis. Improving our understanding of the mechanisms involved in RSV-induced epithelial dysfunction and viral pathogenesis will help to develop novel anti-viral therapeutic approaches

Increase in intracellular cAMP inhibits decreased RSV A2 F mRNA in epithelial monolayers.

<p>(A) Polarized epithelial cells were infected with RSV or rgRSV (MOI, 0.5), for 48 h in the presence of the vehicle, forskolin, or cAMP analogs. RSV A2 F mRNA in epithelial cell monolayers was determined by RT-PCR analysis. (B, C) GFP-positive rgRSV-infected cells were visualized and counted by immunofluorescence microscopy. Each image is representative of at least 3 independent experiments. Data is presented as mean ± SEM (n = 3) **, <i>P<</i> 0.01 and ***, <i>P<</i> 0.001 as compared to RSV-infected vehicle-treated cells.</p

Forskolin attenuates RSV-induced epithelial tight junction disassembly by increasing the intracellular cAMP level.

<p>(A) Confluent epithelial cell monolayers were infected with RSV (MOI, 0.5) for 48 h in the presence or absence of forskolin (10–50 μM,). TEER was measured at indicated time points. (B) Confluent epithelial cell monolayers were infected with RSV (MOI, 0.5) for 48 h in the presence or absence of forskolin (20 μM). Tight junction protein, ZO-1 (green), was visualized by immunofluorescence labeling and confocal microscopy. The nuclei were counterstained with DAPI (blue). Note the characteristic “chicken wire” appearance of ZO-1 in control non-infected cells (arrows), and ZO-1 translocation into cytoplasmic dot-like structures in RSV-infected cells (thin arrowheads). Also, note the syncytia formation in RSV-infected cells (thick arrowheads). Scale bar, 40 μm. (C) Primary human bronchial epithelial cells were infected with RSV (MOI, 2) for 48 h in the presence or absence of forskolin followed by visualizing ZO-1 by immunofluorescence labeling and confocal microscopy. Arrows demonstrate normal junction formation, and thin arrowheads indicate the disappearance of TJ ZO-1 staining in the RSV-infected cell monolayer and thick arrowheads shows syncytia formation. Scale bar, 40 μm (D) Cells were infected with RSV (MOI, 0.5) for 24 h, followed by forskolin treatment (20 μM) for 15 min and subsequent measurement of the cAMP concentration in the total cell lysates. Each image and graph is representative of at least 3 independent experiments. Data is presented as mean ± SEM **, <i>P<</i> 0.01 and ***, <i>P<</i> 0.001 as compared to RSV-infected vehicle-treated cells.</p

Forskolin prevents RSV-induced remodeling of the perijunctional actin cytoskeleton.

<p>Confluent airway epithelial cell monolayers were either left untreated or infected with RSV for 48 h in the presence of either vehicle or forskolin (20 μM). Cells were fixed and labeled for F-actin or cortactin. Note that RSV infection caused the appearance of disorganized apical actin filaments and increased apical cortactin labeling (arrowheads). All these cytoskeletal alterations were attenuated by forskolin treatment (arrows). Scale bar, 40 μm. Image is representative of at least 3 independent experiments.</p