一氧化氮信号通路与血管功能

一氧化氮-环磷鸟苷-依赖蛋白激酶信号通路是血管功能调制的关键机制之一,本文对这一机制近年来来研究进展,特别是相关信号分子二聚体化的氧化还原调节及生理意义,及可溶性鸟苷酸环化酶催化产生的环磷肌苷作为一个新的血管收缩信使分子作简要介绍。     

The Interaction Between the Mu Opioid Receptor and Filamin A

Our laboratory embarked on research to discover proteins the interaction of which with the mu opioid receptor (MOPr) is required for its function and regulation. We performed yeast two-hybrid screens, using the carboxy tail of the human MOPr as bait and a human brain library. This yielded a number of proteins that seemed to bind to the MOPr C-tail. The one we chose to study in detail was filamin A (FLNA). Evidence was obtained that there was indeed protein–protein binding between the C-tail of MOPr and FLNA. A human melanoma cell line (M2) lacking the gene for FLNA and a control cell line (A7) which differed from M2 only in having been transfected with the gene for FLNA and expressing the FLNA protein were made available to us. We transfected these cell lines with the gene for MOPr and used them in our studies. The absence of FLNA strongly reduced MOPr downregulation as well as desensitization of adenylyl cyclase inhibition and G protein activation. A recent finding, published here for the first time, is that FLNA is required for the activation by mu opioid agonists of the MAP kinase p38. Deletion studies indicated that the MOPr binding site on FLNA is in the 24th repeat, close to its C-terminal. It was further found that FLNA lacking the N-terminal actin binding domain is as capable as full length FLNA to restore cells to control status, suggesting that actin binding is not required. A surprising finding was that upregulation of MOPr by morphine and some agonist analogs occurs in M2 cells lacking FLNA, whereas normal receptor downregulation takes place in A7 cells.     

Pregnancy Augments G Protein Estrogen Receptor (GPER) Induced Vasodilation in Rat Uterine Arteries via the Nitric Oxide - cGMP Signaling Pathway

Background

The regulation of vascular tone in the uterine circulation is a key determinant of appropriate uteroplacental blood perfusion and successful pregnancy outcome. Estrogens, which increase in the maternal circulation throughout pregnancy, can exert acute vasodilatory actions. Recently a third estrogen receptor named GPER (G protein-coupled estrogen receptor) was identified and, although several studies have shown vasodilatory effects in several vascular beds, nothing is known about its role in the uterine vasculature.

Aim

The aim of this study was to determine the function of GPER in uterine arteries mainly during pregnancy. Uterine arteries were isolated from nonpregnant and pregnant rats.

Methods

Vessels were contracted with phenylephrine and then incubated with incremental doses (10⁻¹²–10⁻⁵ M) of the selective GPER agonist G1.

Results

G1 induced a dose-dependent vasodilation which was: 1) significantly increased in pregnancy, 2) endothelium-dependent, 3) primarily mediated by NO/cGMP pathway and 4) unaffected by BK_ca channel inhibition.

Conclusion

This is the first study to show the potential importance of GPER signaling in reducing uterine vascular tone during pregnancy. GPER may therefore play a previously unrecognized role in the regulation of uteroplacental blood flow and normal fetus growth.     

Nitric Oxide Signaling in Plant Responses to Abiotic Stresses

Nitric oxide (NO) plays important roles in diverse physiological processes In plants. NO can provoke both beneficial and harmful effects, which depend on the concentration and location of NO in plant cells. This review is focused on NO synthesis and the functions of NO in plant responses to abiotic environmental stresses. Abiotic stresses mostly induce NO production in plants. NO alleviates the harmfulness of reactive oxygen species, and reacts with other target molecules, and regulates the expression of stress responsive genes under various stress conditions.     

植物细胞一氧化氮信号转导研究进展

一氧化氮（nitric oxide,NO）作为重要的信号分子,调控植物的种子萌发、根形态建成和花器官发生等许多生长发育过程,并参与气孔运动的调节以及植物对多种非生物胁迫和病原体侵染的应答过程。已经知道,精氨酸依赖的NOS途径和亚硝酸盐依赖的NR途径是植物细胞NO产生的主要酶促合成途径。NO及其衍生物能够直接修饰底物蛋白的金属基团、半胱氨酸和酪氨酸残基,通过金属亚硝基化、巯基亚硝基化和Tyr．硝基化等化学修饰方式,调节靶蛋白的活性,并影响cGMP和Ca2＋信使系统等下游信号途径,调控相应的生理过程。最新的一些研究结果也显示,MAPK级联系统与NO信号转导途径之间存在复杂的交叉调控。此外,作为活跃的小分子信号,NO和活性氧相互依赖并相互影响,共同介导了植物的胁迫应答和激素响应过程。文章综述了植物NO信号转导研究领域中一些新的研究进展,对NO与活性氧信号途径间的交叉作用等也作了简要介绍。     

A Computationally Designed Water-Soluble Variant of a G-Protein-Coupled Receptor: The Human Mu Opioid Receptor

G-protein-coupled receptors (GPCRs) play essential roles in various physiological processes, and are widely targeted by pharmaceutical drugs. Despite their importance, studying GPCRs has been problematic due to difficulties in isolating large quantities of these membrane proteins in forms that retain their ligand binding capabilities. Creating water-soluble variants of GPCRs by mutating the exterior, transmembrane residues provides a potential method to overcome these difficulties. Here we present the first study involving the computational design, expression and characterization of water-soluble variant of a human GPCR, the human mu opioid receptor (MUR), which is involved in pain and addiction. An atomistic structure of the transmembrane domain was built using comparative (homology) modeling and known GPCR structures. This structure was highly similar to the subsequently determined structure of the murine receptor and was used to computationally design 53 mutations of exterior residues in the transmembrane region, yielding a variant intended to be soluble in aqueous media. The designed variant expressed in high yield in Escherichia coli and was water soluble. The variant shared structural and functionally related features with the native human MUR, including helical secondary structure and comparable affinity for the antagonist naltrexone (K _d  = 65 nM). The roles of cholesterol and disulfide bonds on the stability of the receptor variant were also investigated. This study exemplifies the potential of the computational approach to produce water-soluble variants of GPCRs amenable for structural and functionally related characterization in aqueous solution.     

Nitric Oxide Upregulates Proteasomal Protein Degradation in Neurons

Nitric oxide (NO) is involved in many neuronal functions such as neuromodulation and intracellular signaling. Recent studies have demonstrated that nitric oxide is involved in regulation of proteasomal protein degradation. However, its role in neuronal protein degradation still remains unclear. In our study, we investigated the influence of endogenous nitric oxide production in this process. We have shown that nitric oxide synthase blockade prevents decline of the Ub^G76V-GFP fluorescence (GFP-based proteasomal protein degradation reporter) in neuronal processes of the cultured hippocampal neurons. It suggests that nitric oxide may regulate ubiquitin-dependent proteasomal protein degradation in neurons. Also, we have confirmed that the NO synthesis blockade alone significantly impairs long-term potentiation, and demonstrated for the first time that simultaneous blockade of the NO and proteins synthesis leads to the long-term potentiation amplitude rescue to the control values. Obtained results suggest that nitric oxide is involved in the protein degradation in proteasomes in physiological conditions.     

Filamin A Mutant Lacking Actin-Binding Domain Restores Mu Opioid Receptor Regulation in Melanoma Cells

We have previously reported that the protein filamin A (FLA) binds to the carboxyl tail of the mu opioid receptor (MOPr). Using human melanoma cells, which do not express filamin A, we showed that receptor down-regulation, functional desensitization and trafficking are deficient in the absence of FLA (Onoprishvili et al. Mol Pharmacol 64:1092–1100, 2003). Since FLA has a binding domain for actin and is a member of the family of actin cytoskeleton proteins, it is usually assumed that FLA functions via the actin cytoskeleton. We decided to test this hypothesis by preparing cDNA coding for mutant FLA lacking the actin binding domain (FLA-ABD) and expressing FLA-ABD in the human melanoma cell line M2 (M2-ABD cell line). We report here that this mutant is capable of restoring almost as well as full length FLA the down-regulation of the human MOPr. It is similarly very effective in restoring functional desensitization of MOPr, as assessed by the decrease in G-protein activation after chronic exposure of M2-ABD cells to the mu agonist DAMGO. We also found that A7 cells, expressing wild type FLA, exhibit rapid activation of the MAP kinases, ERK 1 and 2, by DAMGO, as shown by a rise in the level of phospho-ERK 1 and 2. This is followed by rapid dephosphorylation (inactivation), which reaches basal level between 30 and 60 min after DAMGO treatment. M2 cells show normal activation of ERK 1 and 2 in the presence of DAMGO, but very slow inactivation. The rapid rate of MAPK inactivation is partially restored by FLA-ABD. We conclude that some functions of FLA do not act via the actin cytoskeleton. It is likely that other functions, not studied here, may require functional binding of the MOPr-FLA complex to actin. Special issue article in honor of Dr. Ji-Sheng Han.     

Mu Opioids Induce Biased Signaling at the Full-Length Seven Transmembrane C-Terminal Splice Variants of the mu Opioid Receptor Gene,Oprm1

Cellular and Molecular Neurobiology - The biased signaling has been extensively studied in the original mu opioid receptor (MOR-1), particularly through G protein and β-arrestin2 signaling...     

Nitric Oxide: A Unique Endogenous Signaling Molecule in Vascular Biology

The properties of nitric oxide as an endogenous cell signaling molecule in vascular biology are described.     

Aversive Stress Reduces Mu Opioid Receptor Expression in the Intercalated Nuclei of the Rat Amygdala

Cellular and Molecular Neurobiology - The amygdala plays an important role in the integration of responses to noxious and fearful stimuli. Sensory information from many systems is integrated in the...     

Expression and Purification of Functional Human Mu Opioid Receptor from E.coli

N-terminally his-tagged human mu opioid receptor, a G protein-coupled receptor was produced in E.coli employing synthetic codon-usage optimized constructs. The receptor was expressed in inclusion bodies and membrane-inserted in different E.coli strains. By optimizing the expression conditions the expression level for the membrane-integrated receptor was raised to 0.3–0.5 mg per liter of culture. Milligram quantities of receptor could be enriched by affinity chromatography from IPTG induced cultures grown at 18°C. By size exclusion chromatography the protein fraction with the fraction of alpha-helical secondary structure expected for a 7-TM receptor was isolated, by CD-spectroscopy an alpha-helical content of ca. 45% was found for protein solubilised in the detergent Fos-12. Receptor in Fos-12 micelles was shown to bind endomorphin-1 with a K_D of 61 nM. A final yield of 0.17 mg functional protein per liter of culture was obtained.     

Anti-Analgesic Effect of the Mu/Delta Opioid Receptor Heteromer Revealed by Ligand-Biased Antagonism

Delta (DOR) and mu opioid receptors (MOR) can complex as heteromers, conferring functional properties in agonist binding, signaling and trafficking that can differ markedly from their homomeric counterparts. Because of these differences, DOR/MOR heteromers may be a novel therapeutic target in the treatment of pain. However, there are currently no ligands selective for DOR/MOR heteromers, and, consequently, their role in nociception remains unknown. In this study, we used a pharmacological opioid cocktail that selectively activates and stabilizes the DOR/MOR heteromer at the cell surface by blocking its endocytosis to assess its role in antinociception. We found that mice treated chronically with this drug cocktail showed a significant right shift in the ED₅₀ for opioid-mediated analgesia, while mice treated with a drug that promotes degradation of the heteromer did not. Furthermore, promoting degradation of the DOR/MOR heteromer after the right shift in the ED₅₀ had occurred, or blocking signal transduction from the stabilized DOR/MOR heteromer, shifted the ED₅₀ for analgesia back to the left. Taken together, these data suggest an anti-analgesic role for the DOR/MOR heteromer in pain. In conclusion, antagonists selective for DOR/MOR heteromer could provide an avenue for alleviating reduced analgesic response during chronic pain treatment.     

A Kainate Receptor Linked to Nitric Oxide Synthesis from Arginine

In slices of young rat cerebellum, the glutamate analogue kainate induced a large accumulation of cyclic GMP, which was inhibited by non-N-methyl-D-aspartate antagonists. Quisqualate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate evoked only small cyclic GMP responses and inhibited the effect of kainate. When tested in cerebellar cell suspensions, glutamate was also a potent antagonist of the cyclic GMP response to kainate. Superoxide dismutase enhanced the response in the isolated cells, whereas haemoglobin and methylene blue were inhibitory. The response in slices was Ca2+ dependent, augmented by arginine, and inhibited by L-NG-monomethylarginine in a manner that could be reversed by additional arginine. It is concluded that stimulation of kainate receptors leads to activation of the enzyme that synthesizes nitric oxide from arginine and that activation of soluble guanylate cyclase by the released nitric oxide accounts for the cyclic GMP generation.     

Fibrinogen-Dependent Signaling in Microvascular Erythrocyte Function: Implications on Nitric Oxide Efflux

Experimental evidence has shown that plasma fibrinogen plays a key role as a major cardiovascular risk factor, acting directly to trigger erythrocyte aggregation in occlusive vascular disease. However, due to the complex and hitherto unclear interaction between fibrinogen and the erythrocyte membrane, no study has yet evaluated the effects of fibrinogen, under physiological range values, on the erythrocyte nitric oxide (NO) mobilization. Taking into consideration the potential NO-derived molecules, we have raised the hypothesis that fibrinogen, under physiological conditions, may act to influence blood flow via erythrocyte NO modulation. In this in vitro study whole-blood samples were harvested from healthy subjects, erythrocyte suspensions were incubated in the absence (control aliquots) and presence of different fibrinogen concentrations and levels of NO, nitrite, nitrate and S-nitroglutathione (GSNO) were determined. Our results showed, when compared with control aliquots, that the presence of fibrinogen modulates the NO mobilization in erythrocytes by (1) decreasing erythrocyte NO efflux levels (P < 0.001); (2) increasing levels of intraerythrocytic NO oxidative metabolites, namely, nitrite (P < 0.0001) and nitrate (P < 0.0001); and (3) enhancing the formation of GSNO (P < 0.001). In conclusion, this study provides new insights into an unknown mechanism by which fibrinogen modulates the erythrocyte capacity to supply NO, the effects of which on inflammation profiles (generally associated with blood hyperviscosity and hyperaggregation) still need to be elucidated. Also, increased erythrocyte GSNO levels may be associated with platelet NO metabolism, its activation status and hypotension, which may be extremely relevant in the clinical setting as biomarkers.     

Interaction Between Polyamine and Nitric Oxide Signaling in Adaptive Responses to Drought in Cucumber

The effect of polyamines (PAs) on nitric oxide (NO) generation was investigated in cucumber (Cucumis sativus cv. Dar) primary leaves using bio-imaging with an NO-selective fluorophor, DAF-2DA. Seedlings pretreated with PAs and subjected to water deficit showed early (after 5 h) and transient NO production. The amplitude of the response depended on the form of the applied polyamine. Spermine (1.0 mM) and spermidine (1.0 mM) induced higher NO-dependent fluorescence compared with putrescine (1.0 mM) and the control. The NO production was blocked by tungstate, an inhibitor of nitrate reductase, and partially by an inhibitor of nitric oxide synthase (NOS-like) enzymes. NO donor administration preceding drought had no effect on endogenous PA levels but was positively correlated with an alleviation of water deficit-induced membrane permeability and lipid peroxidation. Application of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a membrane-permeable NO scavenger, markedly reversed the NO donor effects. Similarly, pretreating seedlings with PAs resulted in lower ion leakage from the membrane and modified lipid peroxidation. The results indicate that NO may act downstream of PAs in cucumber seedlings under water stress.     

The Critical Role of Nitric Oxide Signaling,via Protein S-Guanylation and Nitrated Cyclic GMP,in the Antioxidant Adaptive Response

A nitrated guanine nucleotide, 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP), is formed via nitric oxide (NO) and causes protein S-guanylation. However, intracellular 8-nitro-cGMP levels and mechanisms of formation of 8-nitro-cGMP and S-guanylation are yet to be identified. In this study, we precisely quantified NO-dependent formation of 8-nitro-cGMP in C6 glioma cells via liquid chromatography-tandem mass spectrometry. Treatment of cells with S-nitroso-N-acetylpenicillamine led to a rapid, transient increase in cGMP, after which 8-nitro-cGMP increased linearly up to a peak value comparable with that of cGMP at 24 h and declined thereafter. Markedly high levels (>40 μm) of 8-nitro-cGMP were also evident in C6 cells that had been stimulated to express inducible NO synthase with excessive NO production. The amount of 8-nitro-cGMP generated was comparable with or much higher than that of cGMP, whose production profile slightly preceded 8-nitro-cGMP formation in the activated inducible NO synthase-expressing cells. These unexpectedly large amounts of 8-nitro-cGMP suggest that GTP (a substrate of cGMP biosynthesis), rather than cGMP per se, may undergo guanine nitration. Also, 8-nitro-cGMP caused S-guanylation of KEAP1 in cells, which led to Nrf2 activation and subsequent induction of antioxidant enzymes, including heme oxygenase-1; thus, 8-nitro-cGMP protected cells against cytotoxic effects of hydrogen peroxide. Proteomic analysis for endogenously modified KEAP1 with matrix-assisted laser desorption/ionization time-of-flight-tandem mass spectrometry revealed that 8-nitro-cGMP S-guanylated the Cys⁴³⁴ of KEAP1. The present report is therefore the first substantial corroboration of the biological significance of cellular 8-nitro-cGMP formation and potential roles of 8-nitro-cGMP in the Nrf2-dependent antioxidant response.