Estrogen-responsive nitroso-proteome in uterine artery endothelial cells: role of endothelial nitric oxide synthase and estrogen receptor-β

Covalent adduction of a NO moiety to cysteines (S‐nitrosylation or SNO) is a major route for NO to directly regulate protein functions. In uterine artery endothelial cells (UAEC), estradiol‐17β (E2) rapidly stimulated protein SNO that maximized within 10–30 min post‐E2 exposure. E2‐bovine serum albumin stimulated protein SNO similarly. Stimulation of SNO by both was blocked by ICI 182, 780, implicating mechanisms linked to specific estrogen receptors (ERs) localized on the plasma membrane. E2‐induced protein SNO was attenuated by selective ERβ, but not ERα, antagonists. A specific ERβ but not ERα agonist was able to induce protein SNO. Overexpression of ERβ, but not ERα, significantly enhanced E2‐induced SNO. Overexpression of both ERs increased basal SNO, but did not further enhance E2‐stimulated SNO. E2‐induced SNO was inhibited by N‐nitro‐L ‐arginine‐methylester and specific endothelial NO synthase (eNOS) siRNA. Thus, estrogen‐induced SNO is mediated by endogenous NO via eNOS and mainly ERβ in UAEC. We further analyzed the nitroso‐proteomes by CyDye switch technique combined with two‐dimensional (2D) fluorescence difference gel electrophoresis. Numerous nitrosoprotein (spots) were visible on the 2D gel. Sixty spots were chosen and subjected to matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry. Among the 54 identified, nine were novel SNO‐proteins, 32 were increased, eight were decreased, and the rest were unchanged by E2. Tandom MS identified Cys139 as a specific site for SNO in GAPDH. Pathway analysis of basal and estrogen‐responsive nitroso‐proteomes suggested that SNO regulates diverse protein functions, directly implicating SNO as a novel mechanism for estrogen to regulate uterine endothelial function and thus uterine vasodilatation. J. Cell. Physiol. 227: 146–159, 2012. © 2011 Wiley Periodicals, Inc.     

Glycogen synthase kinase-3β is involved in C-reactive protein-induced endothelial cell activation

C-reactive protein (CRP) is a significant contributor to atherosclerosis and a powerful predictor of cardiovascular risk. The role of CRP in endothelial cell (EC) activation has been extensively investigated, but the underlying mechanisms have not been fully elucidated. The effect of glycogen synthase kinase-3β (GSK-3β) on CRP-induced EC activation was evaluated in this study. We observed that CRP decreased endothelial nitric oxide synthase (eNOS) activity during EC activation. CRP also activated GSK-3β by dephosphorylating its Ser9 level and reducing β-catenin protein expression in a time-dependent manner. We also found that the GSK-3β inhibitors TDZD-8 and SB415286 partially restored eNOS activity and suppressed the release of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 from ECs. These data provide new evidence for the involvement of GSK-3β in EC activation.     

β Common receptor integrates the erythropoietin signaling in activation of endothelial nitric oxide synthase

Erythropoietin (EPO), the key hormone for erythropoiesis, also increases nitric oxide (NO) bioavailability in endothelial cells (ECs), yet the definitive mechanisms are not fully understood. Increasing evidence has demonstrated that β common receptor (βCR) plays a crucial role in EPO-mediated non-hematopoietic effects. We investigated the role of βCR in EPO-induced endothelial NO synthase (eNOS) activation in bovine aortic ECs (BAECs) and the molecular mechanisms involved. Results of confocal microscopy and immunoprecipitation analyses revealed that βCR was colocalized and interacted with EPO receptor (EPOR) in ECs. Inhibition of βCR or EPOR by neutralizing antibodies or small interfering RNA abolished the EPO-induced NO production. Additionally, blockage of βCR abrogated the EPO-induced increase in the phosphorylation of eNOS, Akt, Src, or Janus kinase 2 (JAK2). Immunoprecipitation analysis revealed that treatment with EPO increased the interaction between βCR and eNOS, which was suppressed by inhibition of Src, JAK2, or Akt signaling with specific pharmacological inhibitors. Furthermore, EPO-induced EC proliferation, migration, and tube formation were blocked by pretreatment with βCR antibody and Src, JAK2, or PI3K/Akt inhibitors. Moreover, in vivo experiments showed that EPO increased the level of phosphorylated eNOS, Src, JAK2, and Akt, as well as βCR-eNOS association in aortas and promoted the angiogenesis in Matrigel plug, which was diminished by βCR or EPOR neutralizing antibodies. Our findings suggest that βCR may play an integrative role in the EPO signaling-mediated activation of eNOS in ECs.     

Can modulation of endothelial nitric oxide synthase explain the vasculoprotective actions of statins?

Statin-based drugs are the mainstay of therapy for coronary artery disease. Recent insights into the cellular mechanisms of this class of drugs suggest that modulation of nitric oxide synthesis contributes to their beneficial actions. These effects are independent of their lipid-lowering effects and result in enhanced nitric oxide release.     

β-amyloid decreases detectable endothelial nitric oxide synthase in human erythrocytes: a role for membrane acetylcholinesterase

Until few years ago, many studies of Alzheimer's disease investigated the effects of this syndrome in the central nervous system. Only recently, the detection of amyloid beta peptide (Aβ) in the blood has evidenced the necessity to extend studies on extraneuronal cells, particularly on erythrocytes. Aβ is also present in brain capillaries, where it interacts with the erythrocytes, inducing several metabolic and functional alterations. Recently, functionally active endothelial type nitric oxide synthase (eNOS) was discovered in human erythrocytes. The goal of the present study was to evidence the effect of Aβ on erythrocyte eNOS. We found that Aβ following to 24-h exposure causes a decrease in the immune staining of erythrocyte eNOS. Concurrently, Aβ alters erythrocyte cell morphology, decreases nitrites and nitrates levels, and affects membrane acetylcholinesterase activity. Propidium, an acetylcholinesterase inhibitor, was able to reverse the effects elicited by Aβ. These events could contribute to the vascular alterations associated with Alzheimer's disease disease. Copyright ? 2012 John Wiley & Sons, Ltd.     

Another activation switch for endothelial nitric oxide synthase: why does it have to be so complicated?

Regulation of the endothelial isoform of nitric oxide synthase (eNOS) appears to be much more complex in comparison to that of other NOS isoforms. A recent paper has expanded the regulation of the enzyme to the realm of sphingolipid signaling, specifically implicating that sphingosine 1-phosphate, endothelial differentiation gene (Edg) receptors and Akt kinase induce a signal transduction pathway via phosphorylation of a serine residue in eNOS. Bradykinin, a nonapeptide formed by enzymatic cleavage of a plasma protein precursor, activates eNOS by an independent pathway that does not require serine phosphorylation, suggesting a complex interplay of signals in the control of endothelial formation of nitric oxide.     

Erythropoietin prevents sepsis-related acute kidney injury in rats by inhibiting NF-κB and upregulating endothelial nitric oxide synthase

The pathophysiology of sepsis involves complex cytokine and inflammatory mediator networks, a mechanism to which NF-κB activation is central. Downregulation of endothelial nitric oxide synthase (eNOS) contributes to sepsis-induced endothelial dysfunction. Erythropoietin (EPO) has emerged as a major tissue-protective cytokine in the setting of stress. We investigated the role of EPO in sepsis-related acute kidney injury using a cecal ligation and puncture (CLP) model. Wistar rats were divided into three primary groups: control (sham-operated); CLP; and CLP+EPO. EPO (4,000 IU/kg body wt ip) was administered 24 and 1 h before CLP. Another group of rats received N-nitro-l-arginine methyl ester (l-NAME) simultaneously with EPO administration (CLP+EPO+l-NAME). A fifth group (CLP+EPOtreat) received EPO at 1 and 4 h after CLP. At 48 h postprocedure, CLP+EPO rats presented significantly higher inulin clearance than did CLP and CLP+EPO+l-NAME rats; hematocrit levels, mean arterial pressure, and metabolic balance remained unchanged in the CLP+EPO rats; and inulin clearance was significantly higher in CLP+EPOtreat rats than in CLP rats. At 48 h after CLP, creatinine clearance was significantly higher in the CLP+EPO rats than in the CLP rats. In renal tissue, pre-CLP EPO administration prevented the sepsis-induced increase in macrophage infiltration, as well as preserving eNOS expression, EPO receptor (EpoR) expression, IKK-α activation, NF-κB activation, and inflammatory cytokine levels, thereby increasing survival. We conclude that this protection, which appears to be dependent on EpoR activation and on eNOS expression, is attributable, in part, to inhibition of the inflammatory response via NF-κB downregulation.     

Glycogen synthase kinase 3β inhibition enhanced proliferation,migration and functional re-endothelialization of endothelial progenitor cells in hypercholesterolemia microenvironment

Hypercholesterolemia impairs the quantity and function of endothelial progenitor cell. We hypothesized that glycogen synthase kinase 3β activity is involved in regulating biological function of endothelial progenitor cells in hypercholesterolemia microenvironment. For study, endothelial progenitor cells derived from apolipoprotein E-deficient mice fed with high-fat diet were used. Glycogen synthase kinase 3β activity was interfered with glycogen synthase kinase 3β inhibitor lithium chloride or transduced with replication defective adenovirus vector expressing catalytically inactive glycogen synthase kinase 3β (GSK3β-KM). Functions of endothelial progenitor cells, proliferation, migration, secretion and network formation of endothelial progenitor cells were assessed in vitro. The expression of phospho-glycogen synthase kinase 3β, β-catenin and cyclinD1 in endothelial progenitor cells was detected by Western blot. The in vivo function re-endothelialization and vasodilation were also analyzed by artery injury model transplanted with glycogen synthase kinase 3β-inhibited endothelial progenitor cells. We demonstrated that while the proliferation, migration, network formation as well as VEGF and NO secretion were impaired in apolipoprotein E-deficient endothelial progenitor cells, glycogen synthase kinase 3β inhibition significantly improved all these functions. Apolipoprotein E-deficient endothelial progenitor cells showed decreased phospho-glycogen synthase kinase 3β, β-catenin and cyclinD1 expression, whereas these signals were enhanced by glycogen synthase kinase 3β inhibition and accompanied with β-catenin nuclear translocation. Our in vivo model showed that glycogen synthase kinase 3β inhibition remarkably increased re-endothelial and vasodilation. Taken together, our data suggest that inhibition of glycogen synthase kinase 3β is associated with endothelial progenitor cell biological functions both in vitro and in vivo. It might be an important interference target in hypercholesterolemia microenvironment.     

Nitric oxide (NO) traffic in endothelial NO synthase. Evidence for a new NO binding site dependent on tetrahydrobiopterin?

Nitric oxide (NO) traffic within the reduced ferrous-nitrosyl complex of endothelial nitric-oxide synthase (eNOS) has been studied by ultrafast time-resolved absorption spectroscopy. In the presence of tetrahydrobiopterin, the rate of NO rebinding to the heme upon photodissociation depends on the NO concentration. The time scale of this process, picoseconds to nanoseconds, precludes a diffusion from the solution toward the protein medium, and altogether the data point at a new NO binding site within the protein. Comparison of the kinetics of pterin-bound and -depleted eNOS points out that the existence of this new site depends on the presence of tetrahydrobiopterin. The new non-heme site may act as a "doorstep" to the heme pocket and control NO escape from eNOS.     

Purification and characterization of beta-cyanoalanine synthase and cysteine synthases from potato tubers: are beta-cyanoalanine synthase and mitochondrial cysteine synthase same enzyme?

beta-Cyanoalanine synthase (CAS; EC 4.4.1.9) and two kinds of cysteine synthases (CS; EC 4.2.99.8) have been purified from the particulate fraction of potato tubers. By DEAE Sephacel and Resource PHE chromatography, CAS activity was separated from two CS activities, designated as CS-1 and CS-2. The molecular masses of CAS, CS-1 and CS-2 were estimated to be 37, 39 and 34 kDa, respectively, by SDS-PAGE analysis. The purified CAS had CS activity, and both CS-1 and CS-2 had CAS activity. However, CAS and CSs had significant differences in kinetic characters. The antibody raised against purified CAS discriminated CAS from CSs, whereas the antibody raised against purified CS-2 recognized CS-1 and CS-2 but not CAS. The molecular mass and the partial amino acid sequence of CS-2 were similar to those of the cytosolic CS of potato, whereas the molecular mass of CS-1 was similar to that of the plastidic CS. The partial amino acid sequence of CAS was similar to those of CS isozymes, especially the mitochondrial CS isolated from spinach.     

Amyloid β induces adhesion of erythrocytes to endothelial cells and affects endothelial viability and functionality

It has been suggested that amyloid β-peptide (Aβ) might mediate the adhesion of erythrocytes to the endothelium which could disrupt the properties of endothelial cells. We provide evidence here that Aβ actually induced the binding of erythrocytes to endothelial cells and decreased endothelial viability, perhaps by the generation of oxidative and inflammatory stress. These changes are likely to contribute to the pathogenesis of Alzheimer's disease.     

Increased blood–brain barrier permeability and endothelial abnormalities induced by vascular endothelial growth factor

The early effects of intracerebrally infused vascular endothelial growth factor (VEGF) on the blood–brain barrier (BBB) to endogenous albumin were studied using a quantitative immunocytochemical procedure. In addition, transmission electron microscopy was used to observe morphological changes induced in brain vasculature. A solution of VEGF in saline (40 ng/10 μl) was infused into the parieto-occipital cortex of mice, which were killed 10 min, 30 min, and 24 h afterwards. Untreated mice and mice that received infusion of saline only were used as controls. For immunocytochemical evaluation, ultrathin sections of immersion-fixed brain samples embedded in Lowicryl K4M were exposed to anti-albumin antiserum followed by protein A-gold. Simultaneously, other brain samples embedded in Spurr resin were used for ultrastructural examination. Morphometric and statistical analysis indicated that as soon as 10 min after infusion of VEGF, 33% of vascular profiles were leaking albumin, and this value increased at 30 min to 92%. This effect of VEGF appears to be of rather short duration because after 24 h, only 27% of vascular profiles showed signs of leakage. The results of ultrastructural observations indicate that VEGF (30 min post-infusion) induces several changes in microvascular segments located in the area of intracerebral infusion of VEGF. These changes consist of the appearance of interendothelial gaps; fragmentation of the endothelium with formation of segmental, fenestrae-like narrowings; degenerative changes of the vascular basement membrane; and the appearance of fibrin gel in the vessel lumen. At 24 h post-infusion, solitary diaphragmed fenestrae appeared in attenuated segments of the endothelium in a few microvascular profiles. These changes, which are interpreted to be preparatory steps for angiogenesis, affect the structural integrity of the vascular segments, leading to extravasation of blood plasma proteins, including albumin. © 1998 Chapman and Hall     

Structural and functional characterization of α-isopropylmalate synthase and citramalate synthase, members of the LeuA dimer superfamily

The manipulation of modular regulatory domains from allosteric enzymes represents a possible mechanism to engineer allostery into non-allosteric systems. Currently, there is insufficient understanding of the structure/function relationships in modular regulatory domains to rationally implement this methodology. The LeuA dimer regulatory domain represents a well-conserved, novel fold responsible for the regulation of two enzymes involved in branched chain amino acid biosynthesis, α-isopropylmalate synthase and citramalate synthase. The LeuA dimer regulatory domain is responsible for the feedback inhibition of these enzymes by their respective downstream products. Both enzymes display multidomain architecture with a conserved N-terminal TIM barrel catalytic domain and a C-terminal (βββα)2 LeuA dimer domain joined by a flexible linker region. Due to the similarity of three-dimensional structure and catalytic mechanism combined with low sequence similarity, we propose these enzymes can be classified as members of the LeuA dimer superfamily. Despite their similarity, members of the LeuA dimer superfamily display diversity in their allosteric mechanisms. In this review, structural aspects of the LeuA dimer superfamily are discussed followed by three examples highlighting the diversity of allosteric mechanisms in the LeuA dimer superfamily.     

α-Isopropylmalate synthase from Alcaligenes eutrophus H 16

The -isopropylmalate synthase (EC 4.1.3.12) from Alcaligenes eutrophus H 16 was inhibited by l-leucine and -ketoisocaproate. The extent of inhibition was influenced by substrate- and inhibitor concentrations as well as by the pH. Intermediary plateaus, which always appeared in the inhibition curves, suggested cooperative effects. The maximal Hill coefficient was found to be two. At low concentrations of leucine the inhibition mechanism was of the competitive type with respect to substrate acetyl coenzyme A and of the noncompetitive type with respect to substrate -ketoisovalerate. The inhibition was specifically relieved by the addition of valine or isoleucine. The anomalous effect of temperature on enzyme activity was diminished by leucine. The Arrhenius energy of the reaction increased from about 11 kcal/mole in the absence of leucine to about 18 kcal/mole in the presence of leucine. The further addition of valine reversed this effect. The physiological relevance of the -ketoisocaproate-mediated inhibition is discussed.Abbreviations IPM -isopropylmalate - KIC -ketoisocaproate - KIV -ketoisovalerate - DTNB 5:5 Dithiobis-(2-nitrobenzoe acid)