Endothelial cell-surface tissue transglutaminase inhibits neutrophil adhesion by binding and releasing nitric oxide

Nitric oxide (NO) produced by endothelial cells in response to cytokines displays anti-inflammatory activity by preventing the adherence, migration and activation of neutrophils. The molecular mechanism by which NO operates at the blood-endothelium interface to exert anti-inflammatory properties is largely unknown. Here we show that on endothelial surfaces, NO is associated with the sulfhydryl-rich protein tissue transglutaminase (TG2), thereby endowing the membrane surfaces with anti-inflammatory properties. We find that tumor necrosis factor-α-stimulated neutrophil adherence is opposed by TG2 molecules that are bound to the endothelial surface. Alkylation of cysteine residues in TG2 or inhibition of endothelial NO synthesis renders the surface-bound TG2 inactive, whereas specific, high affinity binding of S-nitrosylated TG2 (SNO-TG2) to endothelial surfaces restores the anti-inflammatory properties of the endothelium, and reconstitutes the activity of endothelial-derived NO. We also show that SNO-TG2 is present in healthy tissues and that it forms on the membranes of shear-activated endothelial cells. Thus, the anti-inflammatory mechanism that prevents neutrophils from adhering to endothelial cells is identified with TG2 S-nitrosylation at the endothelial cell-blood interface

A pilot study on the kinetics of metabolites and microvascular cutaneous effects of nitric oxide inhalation in healthy volunteers

RATIONALE: Inhaled nitric oxide (NO) exerts a variety of effects through metabolites and these play an important role in regulation of hemodynamics in the body. A detailed investigation into the generation of these metabolites has been overlooked. OBJECTIVES: We investigated the kinetics of nitrite and S-nitrosothiol-hemoglobin (SNO-Hb) in plasma derived from inhaled NO subjects and how this modifies the cutaneous microvascular response. FINDINGS: We enrolled 15 healthy volunteers. Plasma nitrite levels at baseline and during NO inhalation (15 minutes at 40 ppm) were 102 (86-118) and 114 (87-129) nM, respectively. The nitrite peak occurred at 5 minutes of discontinuing NO (131 (104-170) nM). Plasma nitrate levels were not significantly different during the study. SNO-Hb molar ratio levels at baseline and during NO inhalation were 4.7E-3 (2.5E-3-5.8E-3) and 7.8E-3 (4.1E-3-13.0E-3), respectively. Levels of SNO-Hb continued to climb up to the last study time point (30 min: 10.6E-3 (5.3E-3-15.5E-3)). The response to acetylcholine iontophoresis both before and during NO inhalation was inversely associated with the SNO-Hb level (r: -0.57, p = 0.03, and r: -0.54, p = 0.04, respectively). CONCLUSIONS: Both nitrite and SNO-Hb increase during NO inhalation. Nitrite increases first, followed by a more sustained increase in Hb-SNO. Nitrite and Hb-SNO could be a mobile reservoir of NO with potential implications on the systemic microvasculature

Protocol for preparing Thiopropyl Sepharose resin used for capturing S-nitrosylated proteins

Summary: S-nitrosothiol (SNO)-Resin Assisted Capture (SNO-RAC) relies on a Thiopropyl Sepharose resin to identify S-nitrosylated proteins (SNO-proteins) and sites of S-nitrosylation. Here, we present a protocol for preparing Thiopropyl Sepharose resin with efficiency of SNO-protein capture comparable to the discontinued commercial version. We describe steps for amine coupling, disulfide reduction, and generation of thiol reactive resin. We then detail quality control procedures. This resin is also suitable for Acyl-RAC assays to capture palmitoylated proteins.For complete details on the use and execution of the SNO-RAC protocol, please refer to Forrester et al.,1 Fonseca et al.,2 and Seth et al.3 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

OxyR A Molecular Code for Redox-Related Signaling

AbstractRedox regulation has been perceived as a simple on-off switch in proteins (corresponding to reduced and oxidized states). Using the transcription factor OxyR as a model, we have generated, in vitro, several stable, posttranslational modifications of the single regulatory thiol (SH), including S-NO, S-OH, and S-SG, and shown that each occurs in vivo. These modified forms of OxyR are transcriptionally active but differ in structure, cooperative properties, DNA binding affinity, and promoter activities. OxyR can thus process different redox-related signals into distinct transcriptional responses. More generally, our data suggest a code for redox control through which allosteric proteins can subserve either graded (cooperative) or maximal (noncooperative) responses, and through which differential responsivity to redox-related signals can be achieved