Erythrocytic metabolism of ATLX-0199: An agent that increases minute ventilation.

Both L- and D-isomers of S-nitrosocysteine (CSNO) can bind to the intracellular domain of voltage-gated potassium channels in vitro. CSNO binding inhibits these channels in the carotid body, leading to increased minute ventilation in vivo. However, only the l-isomer is active in vivo because it requires the l-amino acid transporter (LAT) for transmembrane transport. In rodents and dogs, the esterified D-CSNO precursor-d-cystine dimethyl ester (ATLX-0199)-overcomes opioid- and benzodiazepine-induced respiratory depression while maintaining analgesia. Although ATLX-0199 can enter cells independently of LAT because it is an ester, its stability in plasma is limited by the presence of esterases. Here, we hypothesized that the drug could be sequestered in erythrocytes to avoid de-esterification in circulation. We developed a liquid chromatography-mass spectrometry method for detecting ATLX-0199 and characterized a new metabolite, S-nitroso-d-cysteine monomethyl ester (DNOCE), which is also a D-CSNO precursor. We found that both ATLX-0199 and DNOCE readily enter erythrocytes and neurons and remain stable over 20 min; thus ATLX-0199 can enter cells where the ester is stable, but the thiol is reduced. Depending on hemoglobin conformation, the reduced ester can be S-nitrosylated and enter carotid body neurons, where it then increases minute ventilation. These data may help explain the paradox that ATLX-0199, a dimethyl ester, can avoid de-esterification in plasma and exert its effects at the level of the carotid body.

Polymeric Amines Induce Nitric Oxide Release from S-Nitrosothiols.

Catalytic generation of nitric oxide (NO) from NO donors by nanomaterials has enabled prolonged NO delivery for various biomedical applications, but this approach requires laborious synthesis routes. In this study, a new class of materials, that is, polymeric amines including polyethyleneimine (PEI), poly-L-lysine, and poly(allylamine hydrochloride), is discovered to induce NO generation from S-nitrosothiols (RSNOs) at physiological conditions. Controlled NO generation can be readily achieved by tuning the concentration of the NO donors (RSNOs) and polymers, and the type and molecular weight of the polymers. Importantly, the mechanism of NO generation by these polymers is deciphered to be attributed to the nucleophilic reaction between primary amines on polymers and the SNO groups of RSNOs. The NO-releasing feature of the polymers can be integrated into a suite of materials, for example, simply by embedding PEI into poly(vinyl alcohol) (PVA) hydrogels. The functionality of the PVA/PEI hydrogels is demonstrated for Pseudomonas aeruginosa biofilm prevention with a ≈4 log reduction within 6 h. As NO has potential therapeutic implications in various diseases, the identification of polymeric amines to induce NO release will open new opportunities in NO-generating biomaterials for antibacterial, antiviral, anticancer, antithrombotic, and wound healing applications.

Reduction in blood pressure following acute dietary nitrate ingestion is correlated with increased red blood cell S-nitrosothiol concentrations.

Dietary nitrate (NO3-) supplementation can enhance nitric oxide (NO) bioavailability and lower blood pressure (BP) in humans. The nitrite concentration ([NO2-]) in the plasma is the most commonly used biomarker of increased NO availability. However, it is unknown to what extent changes in other NO congeners, such as S-nitrosothiols (RSNOs), and in other blood components, such as red blood cells (RBC), also contribute to the BP lowering effects of dietary NO3-. We investigated the correlations between changes in NO biomarkers in different blood compartments and changes in BP variables following acute NO3- ingestion. Resting BP was measured and blood samples were collected at baseline, and at 1, 2, 3, 4 and 24 h following acute beetroot juice (∼12.8 mmol NO3-, ∼11 mg NO3-/kg) ingestion in 20 healthy volunteers. Spearman rank correlation coefficients were determined between the peak individual increases in NO biomarkers (NO3-, NO2-, RSNOs) in plasma, RBC and whole blood, and corresponding decreases in resting BP variables. No significant correlation was observed between increased plasma [NO2-] and reduced BP, but increased RBC [NO2-] was correlated with decreased systolic BP (rs = -0.50, P = 0.03). Notably, increased RBC [RSNOs] was significantly correlated with decreases in systolic (rs = -0.68, P = 0.001), diastolic (rs = -0.59, P = 0.008) and mean arterial pressure (rs = -0.64, P = 0.003). Fisher's z transformation indicated no difference in the strength of the correlations between increases in RBC [NO2-] or [RSNOs] and decreased systolic blood pressure. In conclusion, increased RBC [RSNOs] may be an important mediator of the reduction in resting BP observed following dietary NO3- supplementation.

Photoactivation of tDodSNO induces localized vasodilation in rats: Metabolically stable S-nitrosothiols can act as targeted nitric oxide donors in vivo.

Nitric oxide (NO) is a key vasodilatory signalling molecule and NO releasing molecules (NO donors) are being examined as potential treatments for many pathologies. The photoresponsive NO donor tert-dodecane S-nitrosothiol (tDodSNO) has been designed to be highly resistant to metabolism; in principle photoactivation of tDodSNO should therefore enable the controlled release of NO in situ via light modulation. To investigate the therapeutic utility of tDodSNO, we tested drug efficacy in Sprague Dawley rats to assess systemic and localised hemodynamic responses under photoactivation, and to confirm drug safety. For comparison, drug action was evaluated alongside the existing NO donors sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO). Across a dosing range (0.1-3.0 mg/kg) tDodSNO exerted markedly reduced systemic hypotensive action compared to these standard NO donors, inducing a slight decrease in mean arterial pressure (maximum 14.2 ± 3.0%) without affecting heart rate. Target limb photoactivation of tDodSNO resulted in a substantial localized vasodilatory response, with increases to mean (26.0 ± 7.3%) and maximum (53.2 ± 10.4%) blood flow and decreases to vascular resistance (27.1 ± 3.9%) that were restricted to light exposed tissue. In comparison GSNO and SNP showed variable peripheral effects and were not responsive to photoactivation. tDodSNO did not induce met-Hb formation in blood, or display any signs of toxicity, and was rapidly cleared from the systemic circulation, with no hemodynamic effects detectable 5 min post administration. These data are the first demonstration that drugs based upon a metabolically stable S-nitrosothiol group can be photoactivated in vivo to release NO, and that such agents cause less systemic side effects than existing NO donors. Our data support the use of S-nitrosothiols to enable the spatiotemporal control of NO for therapeutic applications.

BIOFLAVONOIDS AS AGENTS FOR CORRECTING NITRO-OXIDATIVE STRESS AND SALIVARY GLAND FUNCTIONS IN RATS EXPOSED TO ALCOHOL DURING MODELED LIPOPOLYSACCHARIDE-INDUCED SYSTEMIC INFLAMMATORY RESPONSE.

OBJECTIVE: The aim: To investigate the effects of bioflavonoids (curcumin, epigallocatechin-3-gallate and quercetin) on nitro-oxidative stress and the functions of submandibular SGs in rats under alcohol exposure during SIR. PATIENTS AND METHODS: Materials and methods: The studies were conducted on 35 rats of the Wistar line weighing 205-220 g, divided into 5 groups of seven animals in each: the 1st group, control group I, included animals receiving isotonic sodium chloride solution intragastrically twice a day; the 2nd group, control group II, included rats exposed to alcohol (in a dose of 24 mg/kg intragastrically through gavage a twice a day) for last 2 weeks during lipopolysaccharide (LPS)-induced SIR; the rats of the 3rd, 4th and 5th groups exposed to alcohol during LPS-induced SIR, which also received bioflavonoids. The bioflavonoids ("Sigma-Aldrich, Inc.", USA) were as following: curcumin (in a daily dose of 200 mg/kg), epigallocatechin-3-gallate (in a daily dose of 40 mg/kg), and quercetin (in a daily dose of 200 mg/kg), respectively. SIR was induced by intraperitoneal administration of Salmonella typhi LPS (during the first week a dose of 0.4 µg/kg of body weight was administered 3 times a week; during the next 7 weeks of the experiment rats received 0.4 µg/kg of body weight once a week. The formation of superoxide anion radical (О2 -), activity of NO-synthase - total (NOS), its constitutive and inducible isoforms (cNOS, iNOS), and concentration of peroxynitrites and S-nitrosothiols were evaluated spectrophotometrically. To assess the functional status of submandibular SGs in their homogenate we determined α-amylase activity (spectrophotometrically) and the aquaporin-5 concentration (by enzyme-linked immunosorbent assay). through gav-age with orogastric cannul. RESULTS: Results: When applying bioflavonoids under the conditions of alcohol administration during SIR, NADH-induced .О2 - production decreased and yielded to the result in the control group II by 36.8% under administering curcumin, by 34.5% under administering epigallocatechin-3-gallate, and by 41.3% under administering quercetin. The total NOS activity in SGs tissues was inferior by 42.8% to the relevant data in the control group II (under curcumin administration), by 33.7% (under epigallocatechin-3-gallate administration) and by 46.6% (under quercetin administration); and the iNOS activity decreased by 47.0, 38.3 and 52.0%, respectively. Under the administration of bioflavonoids peroxynitrites concentration in the submandibular SGs tissues was inferior to the control group II by 35.6% (under curcumin administration), by 37.4% (under epigallocatechin-3-gallate administration), and by 39.3% (under quercetin administration); the content of S-nitrosothiols was lower by 34.5, 31.1 and 35.3%, respectively. The administration of bioflavonoids led to the changes in α-amylase activity in the submandibular SGs tissues: its values exceeded the relevant data in the control group II by 40.4% (under curcumin administration), by 38.2% (under epigallocatechin-3-gallate administration), and by 34.1% (under quercetin administration); under those conditions aquaporin-5 concentration grew in 2.66, 2.61 and 2.55 times, respectively. CONCLUSION: Conclusions: The use of bioflavonoids (curcumin, epigallocatechin-3-gallate, and quercetin) under the combined administration of 40% ethanol solution and LPS considerably limits the development of nitro-oxidative stress in the tissues of the submandibular SGs. The administration of the bioflavonoids increases the level of cNOS coupling, and improves the functional status of the submandibular SGs under the combined administration of alcohol and LPS enhancing the activity of α-amylase and concentration of aquaporin-5.

Extra-platelet low-molecular-mass thiols mediate the inhibitory action of S-nitrosoalbumin on human platelet aggregation via S-transnitrosylation of the platelet surface.

Nitrosylation of sulfhydryl (SH) groups of cysteine (Cys) moieties is an important post-translational modification (PTM), often on a par with phosphorylation. S-Nitrosoalbumin (ALB-Cys34SNO; SNALB) in plasma and S-nitrosohemoglobin (Hb-Cysß93SNO; HbSNO) in red blood cells are considered the most abundant high-molecular-mass pools of nitric oxide (NO) bioactivity in the human circulation. SNALB per se is not an NO donor. Yet, it acts as a vasodilator and an inhibitor of platelet aggregation. SNALB can be formed by nitrosation of the sole reduced Cys group of albumin (Cys34) by nitrosating species such as nitrous acid (HONO) and nitrous anhydride (N2O3), two unstable intermediates of NO autoxidation. SNALB can also be formed by the transfer (S-transnitrosylation) of the nitrosyl group (NO+) of a low-molecular-mass (LMM) S-nitrosothiol (RSNO) to ALB-Cys34SH. In the present study, the effects of LMM thiols on the inhibitory potential of ALB-Cys34SNO on human washed platelets were investigated. ALB-Cys34SNO was prepared by reacting n-butylnitrite with albumin after selective extraction from plasma of a healthy donor on HiTrapBlue Sepharose cartridges. ALB-Cys34SNO was used in platelet aggregation measurements after extended purification on HiTrapBlue Sepharose and enrichment by ultrafiltration (cutoff, 20 kDa). All tested LMM cysteinyl thiols (R-CysSH) including L-cysteine and L-homocysteine (at 10 µM) were found to mediate the collagen-induced (1 µg/mL) aggregation of human washed platelets by SNALB (range, 0-10 µM) by cGMP-dependent and cGMP-independent mechanisms. The LMM thiols themselves did not affect platelet aggregation. It is assumed that the underlying mechanism involves S-transnitrosylation of SH groups of the platelet surface by LMM RSNO formed through the reaction of SNALB with the thiols: ALB-Cys34SNO + R-CysSH ↔ ALB-Cys34SH + R-CysSNO. Such S-transnitrosylation reactions may be accompanied by release of NO finally resulting in cGMP-dependent and cGMP-independent mechanisms.

Enhanced Drug Delivery by Nanoscale Integration of a Nitric Oxide Donor To Induce Tumor Collagen Depletion.

Delivery of therapeutics into the solid tumor microenvironment is a major challenge for cancer nanomedicine. Administration of certain exogenous enzymes which deplete tumor stromal components has been proposed as a method to improve drug delivery. Here we present a protein-free collagen depletion strategy for drug delivery into solid tumors, based on activating endogenous matrix metalloproteinases (MMP-1 and -2) using nitric oxide (NO). Mesoporous silica nanoparticles (MSN) were loaded with a chemotherapeutic agent, doxorubicin (DOX) as well as a NO donor ( S-nitrosothiol) to create DN@MSN. The loaded NO results in activation of MMPs which degrade collagen in the tumor extracellular matrix. Administration of DN@MSN resulted in enhanced tumor penetration of both the nanovehicle and cargo (DOX), leading to significantly improved antitumor efficacy with no overt toxicity observed.

Attenuation of Macrophage Migration Inhibitory Factor-Stimulated Signaling via S-Nitrosylation.

Nitric oxide (NO) is a key signaling molecule that has various effects via S-nitrosylation, a reversible post-translational modification that affects the enzymatic activity, localization, and metabolism of target proteins. As chronic nitrosative stress correlates with neurodegeneration, the targets have received focused attention. Macrophage migration inhibitory factor (MIF) plays a pivotal role in the induction of gene expression to control inflammatory responses. MIF acts as a ligand for CD74 receptor and activates the Src-p38 mitogen-activated protein kinase (MAPK) cascade. MIF also elevates the expression of brain-derived neurotrophic factor (BDNF), which contributes to the viability of neurons. Here, we show that MIF is S-nitrosylated by a physiological NO donor. Interestingly, the induction of S-nitrosylation resulted in a loss of MIF activity following stimulation of the Src and p38 MAPK signaling pathways and the induction of BDNF expression. Our results shed light on the pathogenic mechanisms of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease.

Wavelength-dependence of vasodilation and NO release from S-nitrosothiols and dinitrosyl iron complexes by far red/near infrared light.

Far red/near infrared (R/NIR) energy is a novel therapy, but its mechanism of action is poorly characterized. Cytochrome c oxidase (Cco) of the mitochondrial electron transport chain is considered the primary photoacceptor for R/NIR to photolyze a putative heme nitrosyl in Cco to liberate free nitric oxide (NO). We previously observed R/NIR light directly liberates NO from nitrosylated hemoglobin and myoglobin, and recently suggested S-nitrosothiols (RSNO) and dinitrosyl iron complexes (DNIC) may be primary sources of R/NIR-mediated NO. Here we indicate R/NIR light exposure induces wavelength dependent dilation of murine facial artery, with longer wavelengths (740, and 830 nm) exhibiting reduced potency when compared to 670 nm. R/NIR also stimulated NO release from pure solutions of low molecular weight RSNO (GSNO and SNAP) and glutathione dinitrosyl iron complex (GSH-DNIC) in a power- and wavelength-dependent manner, with the greatest effect at 670 nm. NO release from SNAP using 670 was nearly ten-fold more than GSNO or GSH-DNIC, with no substantial difference in NO production at 740 nm and 830 nm. Thermal effects of irradiation on vasodilation or NO release from S-nitrosothiols and DNIC was minimal. Our results suggest 670 nm is the optimal wavelength for R/NIR treatment of certain vascular-related diseases.

Antitumor Potential of S-Nitrosothiol-Containing Polymeric Nanoparticles against Melanoma.

Melanoma is a malignant proliferative disease originated from melanocyte transformations, which are characterized by a high metastatic rate and mortality. Advances in Nanotechnology have provided useful new approaches and tools for antitumor chemotherapy. The aim of this study was to investigate the molecular mechanisms underlying chitosan nanoparticles containing S-nitrosomercaptosuccinic acid ( S-nitroso-MSA-CS) induced cytotoxicity in melanoma cells. S-Nitroso-MSA-CS induced concentration-dependent cell death against B16-F10 tumor cells, whereas non-nitroso nanoparticles (CS or MSA-CS) did not induce significant cytotoxicity. Additionally, melanoma cells were more sensitive to cell death than normal melanocytes. S-Nitroso-MSA-CS-induced cytotoxicity exhibited features of caspase-dependent apoptosis, and it was associated with oxidative stress, characterized by increased mitochondrial superoxide production and oxidation of protein thiol groups. In addition, tyrosine nitration and cysteine S-nitrosylation of amino acid residues in cellular proteins were observed. The potential use of these nanoparticles in antitumor chemotherapy of melanoma is discussed.

Nitrite potentiates the vasodilatory signaling of S-nitrosothiols.

Nitrite and S-nitrosothiols (SNOs) are both byproducts of nitric oxide (NO) metabolism and are proposed to cause vasodilation via activation of soluble guanylate cyclase (sGC). We have previously reported that while SNOs are potent vasodilators at physiological concentrations, nitrite itself only produces vasodilation at supraphysiological concentrations. Here, we tested the hypothesis that sub-vasoactive concentrations of nitrite potentiate the vasodilatory effects of SNOs. Multiple exposures of isolated sheep arteries to S-nitroso-glutathione (GSNO) resulted in a tachyphylactic decreased vasodilatory response to GSNO but not to NO, suggesting attenuation of signaling steps upstream from sGC. Exposure of arteries to 1 µM nitrite potentiated the vasodilatory effects of GSNO in naive arteries and abrogated the tachyphylactic response to GSNO in pre-exposed arteries, suggesting that nitrite facilitates GSNO-mediated activation of sGC. In intact anesthetized sheep and rats, inhibition of NO synthases to decrease plasma nitrite levels attenuated vasodilatory responses to exogenous infusions of GSNO, an effect that was reversed by exogenous infusion of nitrite at sub-vasodilating levels. This study suggests nitrite potentiates SNO-mediated vasodilation via a mechanism that lies upstream from activation of sGC.

Balance between S-nitrosylation and denitrosylation modulates myoblast proliferation independently of soluble guanylyl cyclase activation.

Nitric oxide (NO) contributes to myogenesis by regulating the transition between myoblast proliferation and fusion through cGMP signaling. NO can form S-nitrosothiols (RSNO), which control signaling pathways in many different cell types. However, neither the role of RSNO content nor its regulation by the denitrosylase activity of S-nitrosoglutathione reductase (GSNOR) during myogenesis is understood. Here, we used primary cultures of chick embryonic skeletal muscle cells to investigate whether changes in intracellular RSNO alter proliferation and fusion of myoblasts in the presence and absence of cGMP. Cultures were grown to fuse most of the myoblasts into myotubes, with and without S-nitrosocysteine (CysNO), 8-Br-cGMP, DETA-NO, or inhibitors for NO synthase (NOS), GSNOR, soluble guanylyl cyclase (sGC), or a combination of these, followed by analysis of GSNOR activity, protein expression, RSNO, cGMP, and cell morphology. Although the activity of GSNOR increased progressively over 72 h, inhibiting GSNOR (by GSNOR inhibitor - GSNORi - or by knocking down GSNOR with siRNA) produced an increase in RSNO and in the number of myoblasts and fibroblasts, accompanied by a decrease in myoblast fusion index. This was also detected with CysNO supplementation. Enhanced myoblast number was proportional to GSNOR inhibition. Effects of the GSNORi and GSNOR knockdown were blunted by NOS inhibition, suggesting their dependence on NO synthesis. Interestingly, GSNORi and GSNOR knockdown reversed the attenuated proliferation obtained with sGC inhibition in myoblasts, but not in fibroblasts. Hence myoblast proliferation is enhanced by increasing RSNO, and regulated by GSNOR activity, independently of cGMP production and signaling.

S-nitrosylation of the IGF-1 receptor disrupts the cell proliferative action of IGF-1.

The insulin-like growth factor 1 receptor (IGF-1R) is a disulfide-linked heterotetramer containing two α-subunits and two ß-subunits. Earlier studies demonstrate that nitric oxide (NO) can adversely affect IGF-1 action in the central nervous system. It is known that NO can induce S-nitrosylation of the cysteine residues in proteins, thereby partly contributing to the regulation of protein function. In the present study, we sought to determine whether S-nitrosylation of the cysteine residues in IGF-1R is an important post-translational modification that regulates its response to IGF-1. Using cultured SH-SY5Y human neuroblastoma cells as an in vitro model, we found that treatment of cells with S-nitroso-cysteine (SNOC), a NO donor that can nitrosylate the cysteine residues in proteins, induces S-nitrosylation of the ß subunit of IGF-1R but not its α-subunit. IGF-1Rß S-nitrosylation by SNOC is coupled with increased dissociation of the IGF-1R protein complex. In addition, disruption of the IGF-1R function resulting from S-nitrosylation of the IGF-1Rß subunit is associated with disruption of the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways. Further, we observed that SNOC-induced IGF-1Rß S-nitrosylation results in a dose-dependent inhibition of cell proliferation and survival. Together, these results suggest that elevated nitrosative stress may result in dysfunction of cellular IGF-1R signaling through S-nitrosylation of the cysteine residues in the IGF-1Rß subunit, thereby disrupting the downstream PI3K and MAPK signaling functions and ultimately resulting in inhibition of cell proliferation and survival.

Antidepressant action of ketamine via mTOR is mediated by inhibition of nitrergic Rheb degradation.

As traditional antidepressants act only after weeks/months, the discovery that ketamine, an antagonist of glutamate/N-methyl-D-aspartate (NMDA) receptors, elicits antidepressant actions in hours has been transformative. Its mechanism of action has been elusive, though enhanced mammalian target of rapamycin (mTOR) signaling is a major feature. We report a novel signaling pathway wherein NMDA receptor activation stimulates generation of nitric oxide (NO), which S-nitrosylates glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Nitrosylated GAPDH complexes with the ubiquitin-E3-ligase Siah1 and Rheb, a small G protein that activates mTOR. Siah1 degrades Rheb leading to reduced mTOR signaling, while ketamine, conversely, stabilizes Rheb that enhances mTOR signaling. Drugs selectively targeting components of this pathway may offer novel approaches to the treatment of depression.

Thiolate-based dinitrosyl iron complexes: Decomposition and detection and differentiation from S-nitrosothiols.

Dinitrosyl iron complexes (DNIC) spontaneously form in aqueous solutions of Fe(II), nitric oxide (NO), and various anions. They exist as an equilibrium between diamagnetic, dimeric (bi-DNIC) and paramagnetic, monomeric (mono-DNIC) forms. Thiolate groups (e.g., on glutathione or protein cysteine residues) are the most biologically relevant anions to coordinate to Fe(II). Low molecular weight DNIC have previously been suggested to be important mediators of NO biology in cells, and emerging literature supports their role in the control of iron-dependent cellular processes. Recently, it was shown that DNIC may be one of the most abundant NO-derived products in cells and may serve as intermediates in the cellular formation of S-nitrosothiols. In this work, we examined the stability of low molecular weight DNIC and investigated issues with their detection in the presence of other NO-dependent metabolites such as S-nitrosothiols. By using spectrophotometric, Electron Paramagnetic Resonance, ozone-based chemiluminesence, and HPLC techniques we established that at neutral pH, bi-DNIC remain stable for hours, whereas excess thiol results in decomposition to form nitrite. NO was also detected during the decomposition, but no S-nitrosothiol formation was observed. Importantly, mercury chloride accelerated the degradation of DNIC; thus, the implications of this finding for the diagnostic use of mercury chloride in the detection of S-nitrosothiols were determined in simple and complex biological systems. We conclude S-nitrosothiol levels may have been substantially overestimated in all methods where mercury chloride has been used.

S-nitrosothiols dilate the mesenteric artery more potently than the femoral artery by a cGMP and L-type calcium channel-dependent mechanism.

S-nitrosothiols (SNOs) are metabolites of NO with potent vasodilatory activity. Our previous studies in sheep indicated that intra-arterially infused SNOs dilate the mesenteric vasculature more than the femoral vasculature. We hypothesized that the mesenteric artery is more responsive to SNO-mediated vasodilation, and investigated various steps along the NO/cGMP pathway to determine the mechanism for this difference. In anesthetized adult sheep, we monitored the conductance of mesenteric and femoral arteries during infusion of S-nitroso-l-cysteine (L-cysNO), and found mesenteric vascular conductance increased (137 ± 3%) significantly more than femoral conductance (26 ± 25%). Similar results were found in wire myography studies of isolated sheep mesenteric and femoral arteries. Vasodilation by SNOs was attenuated in both vessel types by the presence of ODQ (sGC inhibitor), and both YC-1 (sGC agonist) and 8-Br-cGMP (cGMP analog) mediated more potent relaxation in mesenteric arteries than femoral arteries. The vasodilatory difference between mesenteric and femoral arteries was eliminated by antagonists of either protein kinase G or L-type Ca(2+) channels. Western immunoblots showed a larger L-type Ca(2+)/sGC abundance ratio in mesenteric arteries than in femoral arteries. Fetal sheep mesenteric arteries were more responsive to SNOs than adult mesenteric arteries, and had a greater L-Ca(2+)/sGC ratio (p = 0.047 and r = -0.906 for correlation between Emax and L-Ca(2+)/sGC). These results suggest that mesenteric arteries, especially those in fetus, are more responsive to SNO-mediated vasodilation than femoral arteries due to a greater role of the L-type calcium channel in the NO/cGMP pathway.

In vivo pharmacological activity and biodistribution of S-nitrosophytochelatins after intravenous and intranasal administration in mice.

S-nitrosophytochelatins (SNOPCs) are novel analogues of S-nitrosoglutathione (GSNO) with the advantage of carrying varying ratios of S-nitrosothiol (SNO) moieties per molecule. Our aim was to investigate the in vivo pharmacological potency and biodistribution of these new GSNO analogues after intravenous (i.v.) and intranasal (i.n.) administration in mice. SNOPCs with either two or six SNO groups and GSNO were synthesized and characterized for purity. Compounds were administered i.v. or i.n. at 1 µmol NO/kg body weight to CD-1 mice. Blood pressure was measured and biodistribution studies of total nitrate and nitrite species (NOx) and phytochelatins were performed after i.v. administration. At equivalent doses of NO, it was observed that SNOPC-6 generated a rapid and significantly greater reduction in blood pressure (∼60% reduction compared to saline) whereas GSNO and SNOPC-2 only achieved a 30-35% decrease. The reduction in blood pressure was transient and recovered to baseline levels within ∼2 min for all compounds. NOx species were transiently elevated (over 5 min) in the plasma, lung, heart and liver. Interestingly, a size-dependent phytochelatin accumulation was observed in several tissues including the heart, lungs, kidney, brain and liver. Biodistribution profiles of NOx were also obtained after i.n. administration, showing significant lung retention of NOx over 15 min with minor systemic increases observed from 5 to 15 min. In summary, this study has revealed interesting in vivo pharmacological properties of SNOPCs, with regard to their dramatic hypotensive effects and differing biodistribution patterns following two different routes of administration.

A substrate trapping approach identifies proteins regulated by reversible S-nitrosylation.

Protein S-nitrosylation, the nitric oxide-mediated posttranslational modification of cysteine residues, has emerged as an important regulatory mechanism in diverse cellular processes. Yet, knowledge about the S-nitrosoproteome in different cell types and cellular contexts is still limited and many questions remain regarding the precise roles of protein S-nitrosylation and denitrosylation. Here we present a novel strategy to identify reversibly nitrosylated proteins. Our approach is based on nitrosothiol capture and enrichment using a thioredoxin trap mutant, followed by protein identification by mass spectrometry. Employing this approach, we identified more than 400 putative nitroso-proteins in S-nitrosocysteine-treated human monocytes and about 200 nitrosylation substrates in endotoxin and cytokine-stimulated mouse macrophages. The large majority of these represent novel nitrosylation targets and they include many proteins with key functions in cellular homeostasis and signaling. Biochemical and functional experiments in vitro and in cells validated the proteomic results and further suggested a role for thioredoxin in the denitrosylation and activation of inducible nitric oxide synthase and the protein kinase MEK1. Our findings contribute to a better understanding of the macrophage S-nitrosoproteome and the role of thioredoxin-mediated denitrosylation in nitric oxide signaling. The approach described here may prove generally useful for the identification and exploration of nitroso-proteomes under various physiological and pathophysiological conditions.

Regulation of Histone Deacetylase 6 Activity via S-Nitrosylation.

Nitric oxide (NO) is a gaseous regulatory factor produced by NO synthases (NOS) and it plays several critical roles via S-nitrosylation of protein cysteine residues. Histone deacetylase (HDAC) functions in the maintenance/balance of chromatin acetylation and contributes to transcriptional supression. It has been reported that S-nitrosylation of HDAC2 is involved in the regulation of deacetylase activity. However, it remains unknown whether other subtypes of the HDAC family are S-nitrosylated. In the present study, we found that HDAC6 is a target of NO. A biotin-switch assay revealed that endogenous HDAC6 is S-nitrosylated by both NO donors and NO derived from the inducible type of NOS in cells treated with cytokines. NO led to suppressed deacetylase activity in vitro and increased acetylated α-tubulin, a major substrate for HDAC6, in A549 cells. These findings suggest that S-nitrosylation of HDAC6 plays a pivotal role in the regulation of protein acetylation.

Steroid-sensitive gene 1 is a novel cyclic GMP-dependent protein kinase I substrate in vascular smooth muscle cells.

NO, via its second messenger cGMP, activates protein kinase GI (PKGI) to induce vascular smooth muscle cell relaxation. The mechanisms by which PKGI kinase activity regulates cardiovascular function remain incompletely understood. Therefore, to identify novel protein kinase G substrates in vascular cells, a λ phage coronary artery smooth muscle cell library was constructed and screened for phosphorylation by PKGI. The screen identified steroid-sensitive gene 1 (SSG1), which harbors several predicted PKGI phosphorylation sites. We observed direct and cGMP-regulated interaction between PKGI and SSG1. In cultured vascular smooth muscle cells, both the NO donor S-nitrosocysteine and atrial natriuretic peptide induced SSG1 phosphorylation, and mutation of SSG1 at each of the two predicted PKGI phosphorylation sites completely abolished its basal phosphorylation by PKGI. We detected high SSG1 expression in cardiovascular tissues. Finally, we found that activation of PKGI with cGMP regulated SSG1 intracellular distribution.