Modification of cysteine residues by N-ethylmaleimide inhibits annexin II tetramer mediated liposome aggregation

A role of cysteine residues in annexin II tetramer (AIIt)'s function was investigated using the sulfhydryl reagent N-ethylmaleimide (NEM). Incubation of AIIt with NEM resulted in a dose-dependent inhibition of AIIt-mediated liposome aggregation and loss of sulfhydryl groups of AIIt. The concentration effecting 50% inhibition was 0.18 mM. The inhibition was observed in all Ca2+ concentrations tested (1-1000 microM). NEM had no effects on liposome aggregation mediated by other annexins (I, III, and IV), indicating that the inhibitory effect caused by NEM modification is specific to AIIt. The NEM-treated AIIt still can bind to liposomes. However, once AIIt was bound to membrane, the cysteine residues were protected from NEM modification. Our results suggest that cysteine residues are critical for AIIt-mediated liposome aggregation.     

Fusion of lamellar body with plasma membrane is driven by the dual action of annexin II tetramer and arachidonic acid

Annexin II has been implicated in membrane fusion during the exocytosis of lamellar bodies from alveolar epithelial type II cells. Most previous studies were based on the fusion assays by using model membranes. In the present study, we investigated annexin II-mediated membrane fusion by using isolated lamellar bodies and plasma membrane as determined by the relief of octadecyl rhodamine B (R18) self-quenching. Immunodepletion of annexin II from type II cell cytosol reduced its fusion activity. Purified annexin II tetramer (AIIt) induced the fusion of lamellar bodies with the plasma membrane in a dose-dependent manner. This fusion is Ca2+-dependent and is highly specific to AIIt because other annexins (I and II monomer, III, IV, V, and VI) were unable to induce the fusion. Modification of the different functional residues of AIIt by N-ethylmaleimide, nitric oxide, or peroxynitrite abolished AIIt-mediated fusion. Arachidonic acid enhanced AIIt-mediated fusion and reduced its Ca2+ requirement to an intracellularly achievable level. This effect is due to membrane-bound arachidonic acid, not free arachidonic acid. Other fatty acids including linolenic acid, palmitoleic acid, myristoleic acid, stearic acid, palmitic acid, and myristic acid had little effect. AIIt-mediated fusion was suppressed by the removal of arachidonic acid from lamellar body and plasma membrane using bovine serum albumin. The addition of arachidonic acid back to the arachidonic acid-depleted membranes restored its fusion activity. Our results suggest that the fusion between lamellar bodies with the plasma membrane is driven by the synergistic action of AIIt and arachidonic acid.     

Fucoidan-dependent conformational changes in annexin II tetramer

Fucoidan, a sulfated fucopolysaccharide, mimics the fucosylated glycans of glycoproteins and has therefore been used as a probe for investigating the role of membrane polysaccharides in cell-cell adhesion. In the present report we have characterized the interaction of fucoidan with the Ca(2+)- and phospholipid-binding protein annexin II tetramer (AIIt). AIIt bound to fucoidan with an apparent K(d) of 1.24 +/- 0.69 nM (mean +/- SD, n = 3) with a stoichiometry of 0.010 +/- 0.001 mol of fucoidan/mol of AIIt (mean +/- SD, n = 3). The binding of fucoidan to AIIt was Ca(2+)-independent. Furthermore, in the presence but not the absence of Ca(2+), the binding of fucoidan to AIIt caused a decrease in the alpha-helical content from 32% to 7%. A peptide corresponding to a region of the p36 subunit of AIIt, F(306)-S(313), which contains a Cardin-Weintraub consensus sequence for heparin binding, was shown to undergo a conformational change upon fucoidan binding. This suggests that heparin and fucoidan bound to this region of AIIt. The binding of fucoidan but not heparin by AIIt also inhibited the ability of AIIt to bind to and aggregate phospholipid liposomes. These results suggest that the binding of AIIt to the carbohydrate conjugates of certain membrane glycoproteins may have profound effects on the structure and biological activity of AIIt.     

Interactions between cell surface protein disulphide isomerase and S-nitrosoglutathione during nitric oxide delivery.

In this study, we investigated the role of protein disulphide isomerase (PDI) in rapid metabolism of S-nitrosoglutathione (GSNO) and S-nitrosoalbumin (albSNO) and in NO delivery from these compounds into cells. Incubation of GSNO or albSNO (1 microM) with the megakaryocyte cell line MEG-01 resulted in a cell-mediated removal of each compound which was inhibited by blocking cell surface thiols with 5,5'-dithiobis 2-nitrobenzoic acid (DTNB) (100 microM) or inhibiting PDI with bacitracin (5mM). GSNO, but not albSNO, rapidly inhibited platelet aggregation and stimulated cyclic GMP (cGMP) accumulation (used as a measure of intracellular NO entry). cGMP accumulation in response to GSNO (1 microM) was inhibited by MEG-01 treatment with bacitracin or DTNB, suggesting a role for PDI and surface thiols in NO delivery. PDI activity was present in MEG-01 conditioned medium, and was inhibited by high concentrations of GSNO (500 microM). A number of cell surface thiol-containing proteins were labelled using the impermeable thiol specific probe 3-(N-maleimido-propionyl) biocytin (MPB). Pretreatment of cells with GSNO resulted in a loss of thiol reactivity on some but not all proteins, suggesting selective cell surface thiol modification. Immunoprecipitation experiments showed that GSNO caused a concentration-dependent loss of thiol reactivity of PDI. Our data indicate that PDI is involved in both rapid metabolism of GSNO and intracellular NO delivery and that during this process PDI is itself altered by thiol modification. In contrast, the relevance of PDI-mediated albSNO metabolism to NO signalling is uncertain.     

Mechanism of superoxide dismutase/H(2)O(2)-mediated nitric oxide release from S-nitrosoglutathione--role of glutamate

S-Nitrosoglutathione (GSNO), a physiologically relevant nitric oxide ((*)NO) donor, exhibits antioxidant, anti-ischemic, and antiplatelet properties. The exact mechanism of (*)NO release from GSNO in biological systems has not been determined. Both copper ions and copper-containing enzymes have been shown to catalyze (*)NO release from GSNO. In this study we observed that copper-zinc superoxide dismutase (Cu,ZnSOD) in the presence of H(2)O(2) caused a rapid decomposition of GSNO, forming oxidized glutathione (GSSG) and (*)NO. The cupric ions (Cu(2+)) released from Cu,ZnSOD were bound to the glutamate moiety of GSNO, yielding a 2:1 (GSNO)(2)Cu(2+) complex. Strong chelators of cupric ions, such as histidine and diethylenetriaminepentaacetic acid, inhibited the formation of (GSNO)(2)Cu(2+) complex, GSSG, and (*)NO. GSSG alone inhibited Cu(2+)-induced decomposition of GSNO. This effect is attributed to complexation of copper by GSSG. We conclude that binding of copper to GSNO is obligatory for (*)NO release from GSNO; however, the rate of this reaction was considerably slowed due to binding of Cu(2+) by GSSG. The glutamate moiety in GSNO and GSSG controls copper-catalyzed (*)NO release from GSNO. Cu,ZnSOD and H(2)O(2) enhanced peroxidation of unsaturated lipid that was inhibited by GSNO. The antioxidant function of GSNO is related to the sequestering of copper by GSNO and its ability to slowly release (*)NO. Implications of these findings are discussed in relation to GSNO-induced cardioprotection and to neuropathological processes.     

Characterization of the Ca2+-binding sites of annexin II tetramer

Annexin II heterotetramer (AIIt) is a multifunctional Ca(2+)-binding protein composed of two 11-kDa subunits and two annexin II subunits. The annexin II subunit contains three type II and two type III Ca(2+)-binding sites which are thought to regulate the interaction of AIIt with anionic phospholipid, F-actin, and heparin. In the present study we utilized site-directed mutagenesis to create AIIt mutants with inactive type III (TM AIIt), type II (CM AIIt), and both type II and III Ca(2+)-binding sites (TCM AIIt). Surprisingly, we found that in the presence of Ca(2+), the TM, CM, and TCM AIIt bound phospholipid and F-actin with similar affinity to the wild type AIIt (WT AIIt). Furthermore, the TCM mutant, and to a lesser extent the TM and CM AIIt displayed dose-dependent Ca(2+)-independent phospholipid aggregation and binding. While the TM and CM AIIt demonstrated Ca(2+)-dependent binding to F-actin, the binding of the TCM AIIt was Ca(2+)-independent. These results suggest that the type II or type III Ca(2+)-binding sites do not directly participate in anionic phospholipid or F-actin binding. We therefore propose that in the absence of Ca(2+), the type II and type III Ca(2+)-binding sites of AIIt stabilize a conformation of AIIt that is unfavorable for binding phospholipid and F-actin. Ca(2+) binding to these sites, or the inactivation of these Ca(2+)-binding sites by site-directed mutagenesis, results in a conformational change that promotes binding to anionic phospholipid and F-actin. Since the TM, CM, and TCM AIIt require Ca(2+) for binding to heparin, we also propose that novel Ca(2+)-binding sites regulate this binding event.     

Nitration of annexin II tetramer.

Annexin II tetramer (AII(t)) is a member of the Ca(2+)- and phospholipid-binding protein family and is implicated in membrane fusion during surfactant secretion. It had previously been shown that high concentrations of nitric oxide (NO) inhibit surfactant secretion from lung type II cells. NO reacts with superoxide (O(2)(-)) to form peroxynitrite (ONOO(-)), a tyrosine nitrating agent, which is found in lungs under certain pathological conditions. It is therefore hypothesized that nitration of AII(t) by ONOO(-) may be a mechanism for the NO inhibition of regulated exocytosis. We therefore performed in vitro studies to test effects of ONOO(-) on AII(t). Western blot analysis using anti-nitrotyrosine antibodies showed a dose-dependent nitration of tyrosine residues in AII(t) treated with ONOO(-). Nitration occurred on the core domain of the p36 subunit, as well as on the p11 subunit. ONOO(-) also caused the formation of dimers between p36 and p11 subunits which were stable in the presence of heating, SDS, and beta-mercaptoethanol. AII(t)-mediated liposome aggregation was inhibited by ONOO(-) with an IC(50) of approximately 30 microM. The inhibition was abolished by urate (a scavenger of ONOO(-) and *OH), but not by mannitol (a scavenger of *OH) or superoxide dismutase (a scavenger of O(2)(-)) and appeared to be specific to AII(t), since ONOO(-) only slightly influenced annexin I-mediated liposome aggregation. The conformational change of AII(t) induced by Ca(2+) had no effect on the inhibition. Furthermore, ONOO(-) only partially inhibited the binding of AII(t) to membranes. Nitration of AII(t) also occurred in intact A549 cells, a lung epithelial cell line, treated with ONOO(-). The results of this study suggest that AII(t)-mediated liposome aggregation was inhibited by nitration of the protein.     

Structural characterization of a dimeric complex between the short cytoplasmic domain of CEACAM1 and the pseudo tetramer of S100A10-Annexin A2 using NMR and molecular dynamics

AIIt, a heterotetramer of S100A10 (P11) and Annexin A2, plays a key role in calcium dependent, membrane associations with a variety of proteins. We previously showed that AIIt interacts with the short cytoplasmic domain (12 amino acids) of CEACAM1 (CEACAM1-SF). Since the cytoplasmic domains of CEACAM1 help regulate the formation of cis- or trans-dimers at the cell membrane, we investigated the possible role of their association with AIIt in this process. Using NMR and molecular dynamics, we show that AIIt and its pseudoheterodimer interacts with two molecules of short cytoplasmic domain isoform peptides, and that interaction depends on the binding motif 454-Phe-Gly-Lys-Thr-457 where Phe-454 binds in a hydrophobic pocket of AIIt, the null mutation Phe454Ala reduces binding by 2.5 fold, and the pseudophosphorylation mutant Thr457Glu reduces binding by three fold. Since these two residues in CEACAM1-SF were also found to play a role in the binding of calmodulin and G-actin at the membrane, we hypothesize a sequential set of three interactions are responsible for regulation of cis- to trans-dimerization of CEACAM1. The hydrophobic binding pocket in AIIt corresponds to a previously identified binding pocket for a peptide found in SMARCA3 and AHNAK, suggesting a conserved functional motif in AIIt allowing multiple proteins to reversibly interact with integral membrane proteins in a calcium dependent manner.     

Antiproliferative effect of nitric oxide on rat glomerular mesangial cells via inhibition of mitogen-activated protein kinase.

The effect of nitric oxide (NO) donors and lipopolysaccharide (LPS) on the proliferation of rat glomerular mesangial cells was characterized. Exogenous application of a NO donor inhibited serum-induced proliferation in a time- and dose-dependent manner. S-Nitrosoglutathione (GSNO) also increased cGMP generation and arachidonic acid release, but it did not cause any measurable increase in the cytosolic Ca2+ concentration. Chelation of cytosolic Ca2+ or inhibition of mitogen-activated protein kinase (MAPK) kinase had an inhibitory effect on proliferation, but neither enhanced the antiproliferative effect of GSNO. In contrast, inhibition of guanylate cyclase or phospholipase A2 had no effect on proliferation, but partially reversed GSNO-induced antiproliferation by approximately 98 and 65%, respectively. GSNO did not cause cell death. Incubation of cells with LPS induced endogenous NO generation and had an antiproliferative effect. LPS-induced antiproliferation was reversed completely by inhibition of nitric oxide synthase and partially by inhibition of guanylate cyclase or phospholipase A2. GSNO or LPS inhibited serum-induced MAPK activation, and both effects were partially reversed by inhibition of guanylate cyclase or phospholipase A2. Inclusion of 8-bromo-cGMP or arachidonic acid in the growth medium resulted in a similar antiproliferative effect. In conclusion, in rat glomerular mesangial cells, MAPK inhibition and an antiproliferative effect could be induced by either an increase in the cellular concentration of NO or exposure of the cells to LPS. Part of the effect of NO was attributable to the increased cellular cGMP generation and arachidonic acid release.     

S-nitrosophytochelatins: investigation of the bioactivity of an oligopeptide nitric oxide delivery system

This study investigates the in vitro bioactivity of S-nitrosophytochelatins (SNOPCs), oligopeptide analogues of S-nitrosoglutathione (GSNO), and their mechanisms of nitric oxide (NO) delivery. SNOPCs were more potent than GSNO in inhibiting platelet aggregation and stimulating vasorelaxation. Their potency was related to the number of S-nitrosated moieties per mole compound. Transnitrosation reactions with cell membrane surface components were shown to be the primary mode of NO delivery to intracellular targets for SNOPCs, while delivery via γ-glutamyl transpeptidase was unique to GSNO. Due to rapid NO release, larger SNOPCs elicited a more transitory effect compared to smaller compounds. The duration of effect was influenced by compound molecular weight, NO release kinetics, ability to undergo transnitrosation, and incubation time with tissues. In summary, a new oligopeptide NO delivery system based on SNOPCs was shown to be biologically active and can be used to investigate the mechanisms of NO delivery to intracellular targets.     

The effect of nitric oxide on glucose metabolism

Nitric oxide (NO) is an important bioactive signaling molecule that mediates a variety of normal physiological functions, which, if altered, could contribute to the genesis of many pathological conditions, including diabetes. In this study, we examined the possible diabetogenicity of NO by noting differences in the cellular binding of insulin in dogs treated with the NO donor, S-nitrosoglutathione (GSNO) compared to captopril-treated controls. GSNO administration resulted in an abnormality in glucose metabolism which was attributed to decreased binding of insulin to its receptor on the cell membrane of mononuclear leucocytes, 11.60 +/- 0.60% in GSNO-treated dogs compared with 18.10 +/- 1.90% in captopril-treated control (p < 0.05). The decreased insulin binding was attributed to decreased insulin receptor sites per cell, 21.43 +/- 2.51 x 10(4) in GSNO-treated dogs compared with 26.60 +/- 1.57 x 10(4) in captopril-treated controls (p < 0.05). Average affinity analysis of the binding data demonstrated that this decrease in insulin binding was also due to a decrease in average affinity of the receptor on mononuclear leucocytes for insulin. This was evident by a decrease in empty and filled site affinities in GSNO-treated dogs compared with that of captopril-treated dogs (p < 0.05). It appears that GSNO is exerting its effect by decreasing the number of insulin receptor sites and/or decreasing the average receptor affinity. These results provide evidence for a novel role of NO as a modulator of insulin binding and the involvement of NO in the aetiology of diabetes mellitus.     

Nitric oxide inhibits ornithine decarboxylase by S-nitrosylation.

Ornithine decarboxylase (ODC) is the initial enzyme in the polyamine synthetic pathway, and polyamines are required for cell proliferation. We have shown previously that nitric oxide (NO) inhibits ODC activity in Caco-2 cells and in crude cell lysate preparations. In this study we examined the mechanism by which NO inhibits the activity of purified ODC. NO, in the form of S-nitrosocysteine (CysNO), S-nitrosoglutathione (GSNO), or 1, 1-diethyl-2-hydroxy-2-nitroso-hydrazine (DEA/NO), inhibited enzyme activity in a concentration-dependent manner. CysNO (1 microM) inhibited ODC activity by approximately 90% and 3 microM GSNO by more than 70%. DEA/NO was less potent, inhibiting enzyme activity by 70% at a concentration of 30 microM. Inhibition of enzyme activity by CysNO, GSNO, or DEA/NO was reversible by addition of dithiothreitol or glutathione. Cuprous ion (Cu (I)) also reversed the inhibitory effect of these NO donor agents. The data presented here support the hypothesis that NO inhibits ODC activity via S-nitrosylation of a critical cysteine residue(s) on ODC.     

Construction and evaluation of nitric oxide generating vascular graft material loaded with organoselenium catalyst via layer-by-layer self-assembly

A new biomimetic material for artificial blood vessel with in situ catalytic generation of nitric oxide(NO) was prepared in this study. Organoselenium immobilized polyethyleneimine as NO donor catalyst and sodium alginate were alternately loaded onto the surface of electrospun polycaprolactone matrix via electrostatic layer-by-layer self-assembly. This material revealed significant NO generation when contacting NO donor S-nitrosoglutathione(GSNO). Adhesion and spreading of smooth muscle cells were inhibited on this material in the presence of GSNO, while proliferation of endothelial cells was promoted. In vitro platelet adhesion and arteriovenous shunt experiments demonstrated good antithrombotic properties of this material, with inhibited platelet activation and aggregation, and prevention of acute thrombosis. This study may provide a new method of improving cellular function and antithrombotic property of vascular grafts.     

The effect of nitric oxide on glucose metabolism

Nitric oxide (NO) is an important bioactive signaling molecule that mediates a variety of normal physiological functions, which, if altered, could contribute to the genesis of many pathological conditions, including diabetes. In this study, we examined the possible diabetogenicity of NO by noting differences in the cellular binding of insulin in dogs treated with the NO donor, S-nitrosoglutathione (GSNO) compared to captopril-treated controls. GSNO administration resulted in an abnormality in glucose metabolism which was attributed to decreased binding of insulin to its receptor on the cell membrane of mononuclear leucocytes, 11.60 ± 0.60% in GSNO-treated dogs compared with 18.10 ± 1.90% in captopril-treated control (p < 0.05). The decreased insulin binding was attributed to decreased insulin receptor sites per cell, 21.43 ± 2.51 × 10⁴ in GSNO-treated dogs compared with 26.60 ± 1.57 × 10⁴ in captopril-treated controls (p < 0.05). Average affinity analysis of the binding data demonstrated that this decrease in insulin binding was also due to a decrease in average affinity of the receptor on mononuclear leucocytes for insulin. This was evident by a decrease in empty and filled site affinities in GSNO-treated dogs compared with that of captopril-treated dogs (p < 0.05). It appears that GSNO is exerting its effect by decreasing the number of insulin receptor sites and/or decreasing the average receptor affinity. These results provide evidence for a novel role of NO as a modulator of insulin binding and the involvement of NO in the aetiology of diabetes mellitus. (Mol Cell Biochem 263: 29–34, 2004)     

Regulation of the RON receptor tyrosine kinase expression in macrophages: blocking the RON gene transcription by endotoxin-induced nitric oxide

Previous studies have shown that activation of the RON receptor tyrosine kinase inhibits inducible NO production in murine peritoneal macrophages. The purpose of this study is to determine whether inflammatory mediators such as LPS, IFN-gamma, and TNF-alpha regulate RON expression. Western blot analysis showed that RON expression is reduced in peritoneal macrophages collected from mice injected with a low dose of LPS. The inhibition was seen as early as 8 h after LPS challenge. Experiments in vitro also demonstrated that the levels of the RON mRNA and protein are diminished in cultured peritoneal macrophages following LPS stimulation. TNF-alpha plus IFN-gamma abrogated macrophage RON expression, although individual cytokines had no significant effect. Because LPS and TNF-alpha plus IFN-gamma induce NO production, we reasoned that NO might be involved in the RON inhibition. Two NO donors, S-nitroglutathione (GSNO) and (+/-)-S-nitroso-N-acetylpenicillamine (SNAP), directly inhibited macrophage RON expression when added to the cell cultures. Blocking NO production by NO inhibitors like TGF-beta prevented the LPS-mediated inhibitory effect. In Raw264.7 cells transiently transfected with a report vector, GSNO or SNAP inhibited the luciferase activities driven by the RON gene promoter. Moreover, GSNO or SNAP inhibited the macrophage-stimulating protein-induced RON phosphorylation and macrophage migration. We concluded from these data that RON expression in macrophages is regulated during inflammation. LPS and TNF-alpha plus IFN-gamma are capable of down-regulating RON expression through induction of NO production. The inhibitory effect of NO is mediated by suppression of the RON gene promoter activities.     

Pulmonary vasoconstriction by serotonin is inhibited by S-nitrosoglutathione

Nitric oxide (NO) functions as an endothelium-derived relaxing factor by activating guanylate cyclase to increase cGMP levels. However, NO and related species may also regulate vascular tone by cGMP-independent mechanisms. We hypothesized that naturally occurring NO donors could decrease the pulmonary vascular response to serotonin (5-HT) in the intact lung through chemical interactions with 5-HT(2) receptors. In isolated rabbit lung preparations and isolated pulmonary artery (PA) rings, 50-250 microM S-nitrosoglutathione (GSNO) inhibited the response to 0.01-10 microM 5-HT. The vasoconstrictor response to 5-HT was mediated by 5-HT(2) receptors in the lung, since it could be blocked completely by the selective inhibitor ketanserin (10 microM). GSNO inhibited the response to 5-HT by 77% in intact lung and 82% in PA rings. In PA rings, inhibition by GSNO could be reversed by treatment with the thiol reductant dithiothreitol (10 mM). 3-Morpholinosydnonimine (100-500 microM), which releases NO and O simultaneously, also blocked the response to 5-HT. Its chemical effects, however, were distinct from those of GSNO, because 5-HT-mediated vasoconstriction was not restored in isolated rings by dithiothreitol. In the intact lung, neither NO donor altered the vascular response to endothelin, which activates the same second-messenger vasoconstrictor system as 5-HT. These findings, which did not depend on guanylate cyclase, are consistent with chemical modification by NO of the 5-HT(2) G protein-coupled receptor system to inhibit vasoconstriction, possibly by S-nitrosylation of the receptor or a related protein. This study demonstrates that GSNO can regulate vascular tone in the intact lung by a reversible mechanism involving inhibition of the response to 5-HT.     

Spermine as a modulator of membrane fusion: interactions with acidic phospholipids

The interaction of spermine with acidic phospholipids was investigated for its possible relevance to membrane fusion. Equilibrium dialysis was used to measure the binding of spermine and calcium to large unilamellar vesicles (liposomes) of phosphatidate (PA) or phosphatidylserine (PS). Spermine bound to isolated PA and PS liposomes with intrinsic association constants of approximately 2 and 0.2 M-1, respectively. Above the aggregation threshold of the liposomes, the binding of spermine increased dramatically, especially for PA. The increased binding upon aggregation of PA liposomes was interpreted as evidence for the formation of a new binding complex after aggregation. Spermine enhanced calcium binding to PA, while it inhibited calcium binding to PS, under the same conditions. This difference explained the small effect of spermine on the overall rate of calcium-induced fusion of PS liposomes as opposed to the large effect on PA liposomes. The rate increase could be modeled by a spermine-induced increase in the liposome aggregation rate. The preference for binding of spermine to PA over PS suggested a preference for accessible monoesterified phosphate groups by spermine. This preference was confirmed by the large effects of spermine on aggregation and overall fusion rates of liposomes containing phosphatidylinositol 4,5-diphosphate. The large spermine effects on these liposomes compared with phosphatidate- or phosphatidylinositol-containing liposomes suggested that spermine has a strong specific interaction with phosphatidylinositol 4,5-diphosphate. Clearly, phosphorylation of phosphatidylinositol can lead to a large change in the spermine sensitivity of membrane fusion.     

Determination of the Escherichia coli S-nitrosoglutathione response network using integrated biochemical and systems analysis

During infection or denitrification, bacteria encounter reactive nitrogen species. Although the molecular targets of and defensive response against nitric oxide (NO) in Escherichia coli are well studied, the response elements specific to S-nitrosothiols are less clear. Previously, we employed an integrated systems biology approach to unravel the E. coli NO-response network. Here we use a similar approach to confirm that S-nitrosoglutathione (GSNO) primarily impacts the metabolic and regulatory programs of E. coli in minimal medium by reaction with homocysteine and cysteine and subsequent disruption of the methionine biosynthesis pathway. Targeting of homocysteine and cysteine results in altered regulatory activity of MetJ, MetR, and CysB, activation of the stringent response and growth inhibition. Deletion of metJ or supplementation with methionine strongly attenuated the effect of GSNO on growth and gene expression. Furthermore, GSNO inhibited the ArcAB two-component system. Consistent with the underlying nitrosative and thiol-oxidative chemistry, growth inhibition and the majority of the regulatory perturbations were dependent upon GSNO internalization by the Dpp dipeptide transporter. Contrastingly, perturbation of NsrR appeared to be a result of the submicromolar levels of NO released from GSNO and did not require GSNO internalization.     

Modulation of mast cell activity by a peptide agonist of the thrombin receptor: role of nitric oxide.

The effect of a thrombin receptor agonist peptide (TRAP-6) on the release of nitric oxide (NO) and platelet activating factor (PAF) from resting and calcium-ionophore (A23187)-activated rat peritoneal mast cells (RPMC) was studied using a platelet aggregation bioassay. RPMC spontaneously released NO, which inhibited TRAP-6-, ADP-, and PAF-stimulated platelet aggregation. This effect of NO was abolished by the addition of an NO binding agent, oxyhemoglobin (oxyHb), to the platelet suspension. The RPMC-induced suppression of platelet aggregation was completely inhibited by the NO-synthase inhibitor L-NAME. TRAP-6 and its high affinity analog haTRAP stimulated the rapid release of NO from RPMC. The effect of TRAP-6 was inhibited by pretreatment of the RPMC with L-NAME or with the inhibitor of the constitutive NO-synthase isoform (cNOS) calmidazolium. TRAP-6 inhibited PAF release from A23187-activated RPMC via an NO-dependent mechanism. Platelet aggregation induced by PAF release from activated RPMC was also confirmed in experiments using the PAF receptor antagonist ginkgolide B. Thus, TRAP-6 is a rapidly acting modulator of mast cell reactivity; it stimulates NO release and inhibits PAF secretion.