RhoA inactivation by S-nitrosylation regulates vascular smooth muscle contractive signaling.

S-nitrosothiols derived from nitric oxide are known to regulate cell signaling through thiol modification. Since small G protein RhoA contains cysteine residues in the GTP-binding domain which is critical for its function, modification these thiols may alter RhoA activity and lead to changes in the downstream signaling such as myosin light chain phosphorylation. However, it is still unclear that if RhoA activity and its downstream signals might be modulated by S-nitrosothiols and if the two cysteine residues located in the GTP-binding domain are critical for the regulation. In this study we show that S-nitroso-L-cysteine (CSNO) blocked RhoA activation as determined by either GDP/GTP exchange, active RhoA binding to rhotekin or RhoA translocation. CSNO was shown to lead to RhoA nitrosylation and RhoA thiol oxidation status was found to be consistent with loss of its activity. Mutation of all 6 single cysteine residues to serine showed that purified recombinant C20S mutant and C26/20S mutant were resistant to CSNO, but interestingly, in the intact cells only the double C16/20S mutant was resistant to CSNO. Moreover, inhibition of RhoA activation led to Rho-kinase inhibition and inhibition of Rho pathway signaling by CSNO. In both smooth muscle cells and aortic tissue, the outcome was inhibition of agonist-stimulated MYPT1 phosphorylation and reduced levels of myosin light chain phosphorylation. These effects of CSNO on MYPT1 and myosin light chain phosphorylation appear to be cGMP-independent since they were unaffected by inhibition of guanylyl cyclase. In contrast to CSNO, spermine NONOate did not alter RhoA GDP/GTP exchange and the effects of this compound on myosin light chain phosphorylation were blocked by guanylyl cyclase inhibition. And importantly, in C16/20S overexpressed smooth muscle cells, MYPT1 phosphorylation was resistant to the inhibitory effect of CSNO. Together, these data suggest that S-nitrosothiols regulate myosin light chain phosphorylation by inhibiting RhoA/Rho-kinase signaling through modification of RhoA cysteine residues at 16 and 20 in its GTP-binding domain, which might be an important therapeutic target for diseases with imbalanced vascular resistance.

Direct methods for detection of protein S-nitrosylation.

S-nitrosylation of protein cysteine residues is known to be an important mechanism for nitric oxide signaling. However, the detection of protein S-nitrosylation is still challenging due to technical limitations of current methods. This chapter provides a brief review on recent developments of methods, which directly target S-nitroso moieties for detection. We also describe in detail the protocol of an organophosphine-based biotin labeling of protein S-nitroso moieties.

Reaction based fluorescent probes for hydrogen sulfide.

A reaction based fluorescence turn-on strategy for hydrogen sulfide (H(2)S) was developed. This strategy was based on a H(2)S-specific Michael addition-cyclization sequence. Other biological thiols such as cysteine and glutathione did not pursue the reaction and therefore did not turn on the fluorescence/consume the substrates. The probes showed good selectivity and sensitivity for hydrogen sulfide.

Identification of protein nitrosothiols using phosphine-mediated selective reduction.

Regulation of protein function by S-nitrosation of critical cysteines is known to be an important mechanism for nitric oxide signaling. Evidence for this comes from several different experimental approaches including the ascorbate-based biotin switch method. However technical problems with specificity and sensitivity of ascorbate reduction of S-nitrosothiols limit its usefulness and reliability. In the current study we report the use of triphenylphosphine ester derivatives to selectively reduce SNO bonds in proteins. After triphenylphosphine ester reduction, thiols were tagged with biotin or fluorescently labeled maleimide reagents. Importantly we demonstrate that these compounds are specific reductants of SNO in complex biological samples and do not reduce protein disulfides or protein thiols modified by hydrogen peroxide. Reduction proceeds efficiently in cell extracts and in whole fixed cells. Application of this approach allowed us to demonstrate S-nitrosation of specific cellular proteins, label S-nitrosoproteins in whole fixed cells (especially the nuclear compartment) and demonstrate S-nitrosoprotein formation in cells expressing inducible nitric oxide synthase.

Reductive ligation mediated one-step disulfide formation of S-nitrosothiols.

A one-step reductive ligation mediated disulfide formation of S-nitrosothiols was developed. This reaction involves the reaction of the S-nitroso group with phosphine-thioesters to form sulfenamide and thiolate intermediates, which then undergo a fast intermolecular disulfide formation to form stable conjugates. This reaction can be used to design new biosensors of S-nitrosated proteins.

Transport rather than diffusion-dependent route for nitric oxide gas activity in alveolar epithelium.

The pathway by which inhaled NO gas enters pulmonary alveolar epithelial cells has not been directly tested. Although the expected mechanism is diffusion, another route is the formation of S-nitroso-L-cysteine, which then enters the cell through the L-type amino acid transporter (LAT). To determine if NO gas also enters alveolar epithelium this way, we exposed alveolar epithelial-rat type I, type II, L2, R3/1, and human A549-cells to NO gas at the air liquid interface in the presence of L- and D-cysteine+/-LAT competitors. NO gas exposure concentration dependently increased intracellular NO and S-nitrosothiol levels in the presence of L- but not D-cysteine, which was inhibited by LAT competitors, and was inversely proportional to diffusion distance. The effect of L-cysteine on NO uptake was also concentration dependent. Without preincubation with L-cysteine, NO uptake was significantly reduced. We found similar effects using ethyl nitrite gas in place of NO. Exposure to either gas induced activation of soluble guanylyl cylase in a parallel manner, consistent with LAT dependence. We conclude that NO gas uptake by alveolar epithelium achieves NO-based signaling predominantly by forming extracellular S-nitroso-L-cysteine that is taken up through LAT, rather than by diffusion. Augmenting extracellular S-nitroso-L-cysteine formation may augment pharmacological actions of inhaled NO gas.

Pulmonary alveolar epithelial uptake of S-nitrosothiols is regulated by L-type amino acid transporter.

Nitric oxide (NO) effects are often mediated via S-nitrosothiol (SNO) formation; SNO uptake has recently been shown to be mediated in some cell types via system L-type amino acid transporters (LAT-1, 2). Inhaled NO therapy may exert some biological effects via SNO formation. We therefore sought to determine if pulmonary epithelial SNO uptake depended on LAT or peptide transporter 2 (PEPT2). Both LAT-1 and PEPT2 proteins were detected by immunoblot and immunocytochemistry in L2 cells and rat lung. We tested SNO uptake through the transporters by exposing rat alveolar epithelial cells (L2 and type II) to RSNOs: S-nitrosoglutathione, S-nitrosocysteinylglycine (SNO-Cys-Gly), S-nitrosocysteine (CSNO), and to NO donor diethylamine NONOate (DEA-NONOate). SNO was detected in cell lysates by ozone chemiluminescence. NO uptake was detected by fluorescence in alveolar epithelial cells loaded with 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) diacetate cultured in submersion and exposed to RSNOs and DEA NONOate. Addition of L-Cys but not D-Cys to RSNOs or DEA NONOate increased SNO and DAF-FM signal that was inhibited by coincubation with LAT competitors. Incubation of cells with PEPT2 substrate SNO-Cys-Gly showed no increase in SNO or DAF-FM signal unless incubated with L-Cys. This was unaffected by PEPT2 inhibition. We conclude that RSNOs (thionitrites, S-nitrosothiols) and NO enter alveolar epithelial cells predominantly by S-nitrosation of L-Cys, which is then imported through LAT.

Keap1 modification and nuclear accumulation in response to S-nitrosocysteine.

Keap1 is a key regulator of the Nrf2 transcription factor, which transactivates the antioxidant response element (ARE) and upregulates numerous proteins involved in antioxidant defense. Under basal conditions, Keap1 targets Nrf2 for ubiquitination and proteolytic degradation and as such is responsible for the rapid turnover of Nrf2. In response to oxidants and electrophiles, Nrf2 is stabilized and accumulates in the nucleus. The mechanism for this effect has been proposed to involve thiol-dependent modulation of Keap1 leading to loss of its ability to negatively regulate Nrf2. We have previously shown that nitric oxide and S-nitrosothiols cause nuclear accumulation of Nrf2 and upregulation of the ARE-regulated gene HO-1. Here we show that nitric oxide and S-nitrosocysteine (CSNO) cause time- and dose-dependent Keap1 thiol modification. These studies were carried out in HEK293 cells and in HEK293 cells overexpressing hemagglutinin-tagged Keap1. Furthermore we demonstrate that in response to CSNO Keap1 accumulates in the nucleus with a time course similar to that of Nrf2.

Functional characterization of two S-nitroso-L-cysteine transporters, which mediate movement of NO equivalents into vascular cells.

System L amino acid transporters have been shown to be responsible for cellular uptake of S-nitroso-L-cysteine (l-CSNO). In this study, we examined the characteristics of L-CSNO uptake in Xenopus laevis oocytes expressing system L transporters and found that uptake increased only when both 4F2 heavy chain (4F2HC) and either L-type amino acid transporter 1 (LAT1) or LAT2 light chain were coexpressed. The K(m) for transport was 57 +/- 8 microM for 4F2HC-LAT1 and 520 +/- 52 microM for 4F2HC-LAT2. Vascular endothelial and smooth muscle cells were shown to express transcripts for 4F2HC and for both LAT1 and LAT2. Transport of L-CSNO into red blood cells, endothelial cells, and smooth muscle cells was inhibited by 2-aminobicyclo(2.2.1)heptane-2-carboxylic acid (BCH) and by large neutral amino acids demonstrating functional system L transporters in each cell type. Uptake of L-CSNO led to accumulation of cellular S-nitrosothiols and inhibition of both growth factor-induced ERK phosphorylation and TNF-alpha-mediated IkappaB degradation. Similar effects were seen when cells were incubated simultaneously with S-nitrosoalbumin and L-cysteine but not with d-cysteine or with S-nitrosoalbumin alone. In each case, nitrosylation of proteins and cellular responses were blocked by BCH. Together, these data suggest that transmembrane movement of nitric oxide (NO) equivalents from the plasma albumin NO reservoir is mediated by cysteine, which serves as a carrier. The mechanism requires transnitrosylation from S-nitrosoalbumin to free cysteine and activity of system L transporters, thereby providing a unique pathway for cellular responses to S-nitrosoalbumin.

Redox regulation of PTEN by S-nitrosothiols.

PTEN (phosphatase with sequence homology to tensin) is a phosphatidylinositol 3,4,5-trisphosphate phosphatase that regulates many cellular processes. Activity of the enzyme is dependent on the redox state of the active site cysteine such that oxidation by H2O2 leads to inhibition. Because S-nitrosothiols are known to modify enzymes containing reactive cysteines, we hypothesized that S-nitrosothiols would oxidize PTEN and inhibit its phosphatase activity. In the present study, we show that S-nitrosocysteine (CSNO), S-nitrosoglutathione (GSNO), and S-nitroso-N-acetylpenicillamine (SNAP) reversibly oxidized recombinant PTEN. In addition, CSNO led to concentration- and time-dependent oxidation of endogenous cellular PTEN. However, in contrast, GSNO and SNAP were effective only when coincubated with cysteine, suggesting that these nitrosothiols must react with cysteine to form CSNO, which can be transferred across cell membranes. Oxidation of cellular PTEN resulted from thiol modification and led to reversible inhibition of phosphatase activity. Although oxidation of PTEN by H2O2 led to formation of an intramolecular disulfide, oxidation of PTEN by CSNO seemed to lead to formation of a mixed disulfide. Glutathionylation of cellular proteins by incubating cells with diamide or incubating cellular extracts with GSSG oxidized PTEN in a manner similar to that of CSNO. Overall, these data demonstrate for the first time that S-nitrosothiols oxidatively modify PTEN, leading to reversible inhibition of its phosphatase activity, and suggest that the oxidized species is a mixed disulfide.

Identification of stereoselective transporters for S-nitroso-L-cysteine: role of LAT1 and LAT2 in biological activity of S-nitrosothiols.

Many of the biological effects of nitric oxide are mediated by S-nitrosothiols. However, the mechanisms by which S-nitrosothiols transduce their activity across cell membranes are unclear. We show that the pathway responsible for the cellular effects of S-nitrosothiols is specific for S-nitrosocysteine (CSNO), is stereoselective, and requires direct uptake of intact L-CSNO. Transport is independent of extracellular sodium, competitively inhibited by leucine, and blocked by 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, a specific inhibitor of the system L amino acid transporter family. Other nitrosothiols such as S-nitrosoglutathione are not substrates for transport and require reaction with L-cysteine for activity. To show that system L family members mediate uptake, we expressed two members, LAT1 and LAT2, in Xenopus oocytes. Both LAT1 and LAT2, when co-expressed with 4F2 heavy chain, were found to efficiently transport L-CSNO. Mammalian cells were shown to express LAT1 and LAT2. A431 cells express both proteins, whereas T24 cells express only LAT1. Overexpression of LAT1 in T24 cells using recombinant adenoviruses led to increased uptake of L-CSNO, whereas knockdown using a specific small interfering RNA led to decreased uptake. These data definitively identify LAT1 and LAT2 as members of system L that mediate transmembrane movement of l-CSNO and suggest that system L family members are involved in the cellular activity of small molecular weight nitrosothiols.

Disulfiram inhibits activating transcription factor/cyclic AMP-responsive element binding protein and human melanoma growth in a metal-dependent manner in vitro, in mice and in a patient with metastatic disease.

The thiocarbamate alcoholism drug disulfiram blocks the P-glycoprotein extrusion pump, inhibits the transcription factor nuclear factor-kappaB, sensitizes tumors to chemotherapy, reduces angiogenesis, and inhibits tumor growth in mice. Thiocarbamates react with critical thiols and also complex metal ions. Using melanoma as the paradigm, we tested whether disulfiram might inhibit growth by forming mixed disulfides with critical thiols in a mechanism facilitated by metal ions. Disulfiram given to melanoma cells in combination with Cu2+ or Zn2+ decreased expression of cyclin A and reduced proliferation in vitro at lower concentrations than disulfiram alone. In electrophoretic mobility shift assays, disulfiram decreased transcription factor binding to the cyclic AMP-responsive element in a manner potentiated by Cu2+ ions and by the presence of glutathione, suggesting that thiocarbamates might disrupt transcription factor binding by inducing S-glutathionylation of the transcription factor DNA binding region. Disulfiram inhibited growth and angiogenesis in melanomas transplanted in severe combined immunodeficient mice, and these effects were potentiated by Zn2+ supplementation. The combination of oral zinc gluconate and disulfiram at currently approved doses for alcoholism also induced >50% reduction in hepatic metastases and produced clinical remission in a patient with stage IV metastatic ocular melanoma, who has continued on oral zinc gluconate and disulfiram therapy for 53 continuous months with negligible side effects. These findings present a novel strategy for treating metastatic melanoma by employing an old drug toward a new therapeutic use.

Lipopolysaccharide stimulates mitochondrial biogenesis via activation of nuclear respiratory factor-1.

Exposure to bacterial lipopolysaccharide (LPS) in vivo damages mitochondrial DNA (mtDNA) and interferes with mitochondrial transcription and oxidative phosphorylation (OXPHOS). Because this damage accompanies oxidative stress and is reversible, we postulated that LPS stimulates mtDNA replication and mitochondrial biogenesis via expression of factors responsive to reactive oxygen species, i.e. nuclear respiratory factor-1 (NRF-1) and mitochondrial transcription factor-A. In testing this hypothesis in rat liver, we found that LPS induces NRF-1 protein expression and activity accompanied by mRNA expression for mitochondrial transcription factor-A, mtDNA polymerase gamma, NRF-2, and single-stranded DNA-binding protein. These events restored the loss in mtDNA copy number and OXPHOS gene expression caused by LPS and increased hepatocyte mitotic index, nuclear cyclin D1 translocation, and phosphorylation of pro-survival kinase, Akt. Thus, NRF-1 was implicated in oxidant-mediated mitochondrial biogenesis to provide OXPHOS for proliferation. This implication was tested in novel mtDNA-deficient cells generated from rat hepatoma cells that overexpress NRF-1. Depletion of mtDNA (rhoo clones) diminished oxidant production and caused loss of NRF-1 expression and growth delay. NRF-1 expression and growth were restored by exogenous oxidant exposure indicating that oxidative stress stimulates biogenesis in part via NRF-1 activation and corresponding to recovery events after LPS-induced liver damage.

NOX5 NAD(P)H oxidase regulates growth and apoptosis in DU 145 prostate cancer cells.

Reactive oxygen species (ROS) appear to play an important role in regulating growth and survival of prostate cancer. However, the sources for ROS production in prostate cancer cells have not been determined. We report that ROS are generated by intact American Type Culture Collection DU 145 cells and by their membranes through a mechanism blocked by NAD(P)H oxidase inhibitors. ROS are critical for growth in these cells, because NAD(P)H oxidase inhibitors and antioxidants blocked proliferation. Components of the human phagocyte NAD(P)H oxidase, p22phox and gp91phox, as well as the Ca2+ concentration-responsive gp91phox homolog NOX5 were demonstrated in DU 145 cells by RT-PCR and sequencing. Although the protein product for p22phox was not detectable, both gp91phox and NOX5 were identified throughout the cell by immunostaining and confocal microscopy and NOX5 immunostaining was enhanced in a perinuclear location, corresponding to enhanced ROS production adjacent to the nuclear membrane imaged by 2',7'-dichlorofluorescin diacetate oxidation. The calcium ionophore ionomycin dramatically stimulated ferricytochrome c reduction in cell media, further supporting the importance of NOX5 for ROS production. Antisense oligonucleotides for NOX5 inhibited ROS production and cell proliferation in DU 145 cells. In contrast, antisense oligonucleotides to p22phox or gp91phox did not impair cell growth. Inhibition of ROS generation with antioxidants or NAD(P)H oxidase inhibitors increased apoptosis in cells. These results indicate that ROS generated by the newly described NOX5 oxidase are essential for prostate cancer growth, possibly by providing trophic intracellular oxidant tone that retards programmed cell death.

Regulation of protein tyrosine phosphatase 1B in intact cells by S-nitrosothiols.

Protein tyrosine phosphatases (PTPases) contain an active site cysteine which when oxidized leads to loss of phosphatase activity and accumulation of phosphoproteins. For example, oxidants produced following EGF stimulation inhibit PTP1B and enhance EGF receptor phosphorylation. Because NO-derived species also modify reactive thiols, we postulated that NO would reversibly inhibit PTP1B. In our studies we exposed A431 or Jurkat cells to NO donors and measured PTP1B activity or used 3-maleimidylpropionylbiocytin (MPB) to measure thiol redox status. Nitrosothiols led to a rapid inhibition of PTP1B through a mechanism that was greatly enhanced by addition of cysteine to the medium. Analysis of thiol oxidation status using immunoprecipitated PTP1B showed modification consistent with loss of activity. Both enzyme inhibition and modification were reversible in intact cells or after addition of DTT to cell lysates. While DTT reversed oxidation, ascorbate did not, suggesting that formation of a mixed disulfide (possibly glutathionylation) rather than S-nitrosylation accounts for PTP1B inhibition. Importantly, PTP1B inhibition by nitrosothiols led to EGF receptor phosphorylation even in the absence of exogenously added EGF. These findings suggest an important role for NO in modulating signaling pathways since inhibition of PTPases could potentially enhance or prolong activity of phosphoproteins.

An NAD(P)H oxidase regulates growth and transcription in melanoma cells.

Malignant melanoma cells spontaneously generate reactive oxygen species (ROS) that promote constitutive activation of the transcription factor nuclear factor-kappaB (NF-kappaB). Although antioxidants and inhibitors of NAD(P)H oxidases significantly reduce constitutive NF-kappaB activation and suppress cell proliferation (11), the nature of the enzyme responsible for ROS production in melanoma cells has not been determined. To address this issue, we now have characterized the source of ROS production in melanoma cells. We report that ROS are generated by isolated, cytosol-free melanoma plasma membranes, with inhibition by NAD(P)H oxidase inhibitors. The p22(phox), gp91(phox), and p67(phox) components of the human phagocyte NAD(P)H oxidase and the gp91(phox) homolog NOX4 were demonstrated in melanomas by RT-PCR and sequencing, and protein product for both p22(phox) and gp91(phox) was detected in cell membranes by immunoassay. Normal human epidermal melanocytes expressed only p22(phox) and NOX4. Melanoma proliferation was reduced by NAD(P)H oxidase inhibitors and by transfection of antisense but not sense oligonucleotides for p22(phox) and NOX4. Also, the flavoprotein inhibitor diphenylene iodonium inhibited constitutive DNA binding of nuclear protein to the NF-kappaB and cAMP-response element consensus oligonucleotides, without affecting DNA binding activity to activator protein-1 or OCT-1. This suggests that ROS generated in autocrine fashion by an NAD(P)H oxidase may play a role in signaling malignant melanoma growth.

Carbon monoxide promotes hypoxic pulmonary vascular remodeling.

CO is a biologically active gas that produces cellular effects by multiple mechanisms. Because cellular binding of CO by heme proteins is increased in hypoxia, we tested the hypothesis that CO interferes with hypoxic pulmonary vascular remodeling in vivo. Rats were exposed to inspired CO (50 parts/million) at sea level or 18,000 ft of altitude [hypobaric hypoxia (HH)], and changes in vessel morphometry and pulmonary pressure-flow relationships were compared with controls. Vascular cell single strand DNA (ssDNA) and proliferating cell nuclear antigen (PCNA) were assessed, and changes in gene and protein expression of smooth muscle alpha-actin (sm-alpha-actin), beta-actin, and heme oxygenase-1 (HO-1) were evaluated by Western analysis, RT-PCR, and immunohistochemistry. After 21 days of HH, vascular pressure at constant flow and vessel wall thickness increased and lumen diameter of small arteries decreased significantly. The presence of CO, however, further increased both pulmonary vascular resistance (PVR) and the number of small muscular vessels compared with HH alone. CO + HH also increased vascular PCNA and nuclear ssDNA expression compared with hypoxia, suggesting accelerated cell turnover. CO in hypoxia downregulated sm-alpha-actin and strongly upregulated beta-actin. CO also increased lung HO activity and HO-1 mRNA and protein expression in small pulmonary arteries during hypoxia. These data indicate an overall propensity of CO in HH to promote vascular remodeling and increase PVR in vivo.

Nitric oxide and differential effects of ATP on mitochondrial permeability transition.

The mitochondrial permeability transition pore (PTP) undergoes a calcium-dependent transition (MPT) that disrupts membrane potential and releases apoptogenic proteins. Because PTP opening is enhanced by oxidation of thiols at the so-called "S-site," we hypothesized that nitrogen monoxide (NO*) could enhance the open probability of the PTP, e.g., by S-nitrosylation or S-thiolation. At low NO donor concentrations (1 to 20 microM), PTP opening in succinate-energized liver mitochondria at nonlimiting calcium was delayed or unaffected, while it was accelerated by NO donors at 20 to 100 microM. At low donor concentrations, PTP opening was facilitated twofold by adenosine triphosphate (ATP), which normally delays PTP opening. Among NO donors, the oxatriazole GEA 3162, with an activation constant (Ka) of 1.9 microM at 500 microM ATP was more effective at enhancing pore transition than SIN-1 or SNAP. NO donor effects were superseded by diamide, which induces disulfide formation, but independent of SH-adduct formation by alkylation. NO-related changes in PTP function were accompanied by protein mixed disulfide formation, inhibited by dithiothreitol (DTT), and reversed by DTT after donor addition. PTP opening was stimulated in the presence of ATP by L-arginine-dependent NO production, i.e., mitochondrial NOS activity. ATP-facilitated pore opening was sensitive to atractyloside and depended on nucleotide interactions but not on hydrolysis, because specific nonhydrolyzable ATP analogs accelerated pore opening. These data indicate NO can influence pore transition by oxidation of thiols that produce conformational changes governing the ATP interaction at the adenine nucleotide transporter.