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.     

The Involvement of Intracellular Glutathione in Methyl Jasmonate Signaling in Arabidopsis Guard Cells

We examined the involvement of intracellular glutathione (GSH) in methyl jasmonate (MeJA) signaling. The chlorina1-1 (ch1-1) mutation decreased GSH in guard cells and narrowed the stomatal aperture. GSH monoethyl ester increased intracellular GSH, diminishing this phenotype. GSH did not affect MeJA-induced reactive oxygen species production or cytosolic Ca²⁺ oscillation, suggesting that GSH modulates MeJA signaling downstream of production and oscillation.     

Lrp4 Modulates Extracellular Integration of Cell Signaling Pathways in Development

The extent to which cell signaling is integrated outside the cell is not currently appreciated. We show that a member of the low-density receptor-related protein family, Lrp4 modulates and integrates Bmp and canonical Wnt signalling during tooth morphogenesis by binding the secreted Bmp antagonist protein Wise. Mouse mutants of Lrp4 and Wise exhibit identical tooth phenotypes that include supernumerary incisors and molars, and fused molars. We propose that the Lrp4/Wise interaction acts as an extracellular integrator of epithelial-mesenchymal cell signaling. Wise, secreted from mesenchyme cells binds to BMP''s and also to Lrp4 that is expressed on epithelial cells. This binding then results in the modulation of Wnt activity in the epithelial cells. Thus in this context Wise acts as an extracellular signaling molecule linking two signaling pathways. We further show that a downstream mediator of this integration is the Shh signaling pathway.     

The Roles of CATALASE2 in Abscisic Acid Signaling in Arabidopsis Guard Cells

We investigated the roles of catalase (CAT) in abscisic acid (ABA)-induced stomatal closure using a cat2 mutant and an inhibitor of CAT, 3-aminotriazole (AT). Constitutive reactive oxygen species (ROS) accumulation due to the CAT2 mutation and AT treatment did not affect stomatal aperture in the absence of ABA, whereas ABA-induced stomatal closure, ROS production, and [Ca²⁺]_cyt oscillation were enhanced.     

Cyclic GMP transporters

The biokinetics of guanosine 3',5'-cyclic monophosphate (cGMP) is characterized by three distinct processes: synthesis by guanylate cyclases (GCs), conversion of cGMP to GMP by cyclic nucleotide phosphodiesterases (PDEs) and the excretion of unchanged cGMP by transport proteins in the cell membrane. Efflux is observed in virtually all cell types including cells which originate from brain. Studies of intact cells, in which metabolic inhibitors and probenecid reduced extrusion of cGMP and wherein cGMP was extruded against concentration gradients, indicated the existence of ATP requiring organic anion transport system(s). Functional studies of inside-out vesicles have revealed cGMP transport systems wherein translocation is coupled to hydrolysis of ATP. The extrusion of cGMP is inhibited by a number of unrelated compounds and this indicates that cGMP is substrate for multispecific transporters. Recent transfection studies suggest that members of the MRP (multidrug resistance protein) family; MRP4, MRP5 and MRP8 translocate cGMP across the cell membrane. Many of the MRPs have been detected in brain. In addition tertiary active transport by the organic anion transporter family has also been identified. At least one member (OAT1) shows relative high affinity for cGMP and is also expressed in brain. The biological significance of cGMP transporters has to be clarified. Their role in cGMP biokinetics, being responsible for one of the cellular elimination pathways, is well established. However, there is growing evidence that extracellular cGMP has effects on cell physiology and pathophysiology by an auto- or paracrine mechanism.     

The TOR Pathway Modulates the Structure of Cell Walls in Arabidopsis

Plant cell growth is limited by the extension of cell walls, which requires both the synthesis and rearrangement of cell wall components in a controlled fashion. The target of rapamycin (TOR) pathway is a major regulator of cell growth in eukaryotes, and inhibition of this pathway by rapamycin reduces cell growth. Here, we show that in plants, the TOR pathway affects cell wall structures. LRR-extensin1 (LRX1) of Arabidopsis thaliana is an extracellular protein involved in cell wall formation in root hairs, and lrx1 mutants develop aberrant root hairs. rol5 (for repressor of lrx1) was identified as a suppressor of lrx1. The functionally similar ROL5 homolog in yeast, Ncs6p (needs Cla4 to survive 6), was previously found to affect TOR signaling. Inhibition of TOR signaling by rapamycin led to suppression of the lrx1 mutant phenotype and caused specific changes to galactan/rhamnogalacturonan-I and arabinogalactan protein components of cell walls that were similar to those observed in the rol5 mutant. The ROL5 protein accumulates in mitochondria, a target of the TOR pathway and major source of reactive oxygen species (ROS), and rol5 mutants show an altered response to ROS. This suggests that ROL5 might function as a mitochondrial component of the TOR pathway that influences the plant''s response to ROS.     

Targeting CB2-GPR55 Receptor Heteromers Modulates Cancer Cell Signaling

The G protein-coupled receptors CB₂ (CB₂R) and GPR55 are overexpressed in cancer cells and human tumors. Because a modulation of GPR55 activity by cannabinoids has been suggested, we analyzed whether this receptor participates in cannabinoid effects on cancer cells. Here we show that CB₂R and GPR55 form heteromers in cancer cells, that these structures possess unique signaling properties, and that modulation of these heteromers can modify the antitumoral activity of cannabinoids in vivo. These findings unveil the existence of previously unknown signaling platforms that help explain the complex behavior of cannabinoids and may constitute new targets for therapeutic intervention in oncology.     

Guard Cell Chloroplasts Are Essential for Blue Light-Dependent Stomatal Opening in Arabidopsis

Blue light (BL) induces stomatal opening through the activation of H⁺-ATPases with subsequent ion accumulation in guard cells. In most plant species, red light (RL) enhances BL-dependent stomatal opening. This RL effect is attributable to the chloroplasts of guard cell, the only cells in the epidermis possessing this organelle. To clarify the role of chloroplasts in stomatal regulation, we investigated the effects of RL on BL-dependent stomatal opening in isolated epidermis, guard cell protoplasts, and intact leaves of Arabidopsis thaliana. In isolated epidermal tissues and intact leaves, weak BL superimposed on RL enhanced stomatal opening while BL alone was less effective. In guard cell protoplasts, RL enhanced BL-dependent H⁺-pumping and DCMU, a photosynthetic electron transport inhibitor, eliminated this effect. RL enhanced phosphorylation levels of the H⁺-ATPase in response to BL, but this RL effect was not suppressed by DCMU. Furthermore, DCMU inhibited both RL-induced and BL-dependent stomatal opening in intact leaves. The photosynthetic rate in leaves correlated positively with BL-dependent stomatal opening in the presence of DCMU. We conclude that guard cell chloroplasts provide ATP and/or reducing equivalents that fuel BL-dependent stomatal opening, and that they indirectly monitor photosynthetic CO₂ fixation in mesophyll chloroplasts by absorbing PAR in the epidermis.     

Cyclic and Noncyclic Photophosphorylation in Isolated Guard Cell Chloroplasts from Vicia faba L

High rates of both cyclic and noncyclic photophosphorylation were measured in chloroplast lamellae isolated from purified guard cell protoplasts from Vicia faba L. Typical rates of light-dependent incorporation of ³²P into ATP were 100 and 190 micromoles ATP per milligram chlorophyll per hour for noncyclic (water to ferricyanide) and cyclic (phenazine methosulfate) photophosphorylation, respectively. These rates were 50 to 80% of those observed with mesophyll chloroplasts. Noncyclic photophosphorylation in guard cell chloroplasts was completely inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea supporting the notion that photophosphorylation is coupled to linear electron flow from photosystem II to photosystem I. Several lines of evidence indicated that contamination by mesophyll chloroplasts cannot account for the observed photophosphorylation rates.

A comparison of the photon fluence dependence of noncyclic photophosphorylation in mesophyll and guard cell chloroplasts showed significant differences between the two preparations, with half saturation at 0.04 and 0.08 millimole per square meter per second, respectively.

     

Atrial Natriuretic Hormones Raise the Level of Cyclic GMP in Neural Cell Lines

Atriopeptin III and related atrial natriuretic peptide hormones strongly elevate the level of cyclic GMP in three neural tumor cell lines. At peptide concentrations of 1 microM clear-cut plateaus of the dose-response curves are not yet reached. Atriopeptin III increases the intracellular concentration of cyclic GMP to a maximum in the course of 30-40 min. The effect of atriopeptin III on the cellular cyclic GMP level is independent of the concentration of extracellular Ca2+ and is not affected by the Ca2+ ionophore A23187. These results suggest (1) that atrial natriuretic hormones may play an important role in the nervous system, and (2) that cultured neural cells may be useful tools in the elucidation of the mechanisms of action of these hormones.     

Guard Cell Signaling Dynamics and Rapid Chip-based Cloning of Ion Accumulation Mutants

Guard cells have been developed as a model system for dissecting hormone,ion channel and calcium signal transduction in response to physiological stimuli. Abscisic acid causes elevations in the cytosolic Ca^2 concentration in guard cells. We identified reactive oxygen species（ROS) activation of Ca^2 -permeable channels     

Early ABA Signaling Events in Guard Cells

The plant hormone abscisic acid (ABA) regulates a wide variety of plant physiological and developmental processes, particularly responses to environmental stress, such as drought. In response to water deficiency, plants redistribute foliar ABA and/or upregulate ABA synthesis in roots, leading to roughly a 30-fold increase in ABA concentration in the apoplast of stomatal guard cells. The elevated ABA triggers a chain of events in guard cells, causing stomatal closure and thus preventing water loss. Although the molecular nature of ABA receptor(s) remains unknown, considerable progress in the identification and characterization of its downstream signaling elements has been made by using combined physiological, biochemical, biophysical, molecular, and genetic approaches. The measurable events associated with ABA-induced stomatal closure in guard cells include, sequentially, the production of reactive oxygen species (ROS), increases in cytosolic free Ca²⁺ levels ([Ca²⁺]_i), activation of anion channels, membrane potential depolarization, cytosolic alkalinization, inhibition of K⁺ influx channels, and promotion of K⁺ efflux channels. This review provides an overview of the cellular and molecular mechanisms underlying these ABA-evoked signaling events, with particular emphasis on how ABA triggers an “electronic circuitry” involving these ionic components.     

Cyclic AMP and cyclic GMP in hyperresponsiveness

Cyclic-AMP has been shown to cause a hyperresponse in blood pressure change in conjunction with norepinephrine in the anesthetized rat system. Recent experiments show that the antagonist to angiotensin II, Sar1-Thr8 angiotensin II, abolishes the hyperresponse produced by c-AMP. This is interpreted to mean that the added response caused by c-AMP is mediated through angiotensin II. When the antagonist is removed, the hyperresponse is observed to return. The experiments with cyclic-GMP indicate that the hyperresponse seen with c-AMP is not only absent, but a constant decrease in response to norepinephrine is observed as long as c-GMP is present. The decrease in blood pressure change in the presence of c-GMP suggests that the 10-5M c-GMP causes a relaxation of vascular smooth muscle. These two cyclic nucleotides seem to produce their effects by two completely different mechanisms.     

The Arabidopsis ATP-binding Cassette Protein AtMRP5/AtABCC5 Is a High Affinity Inositol Hexakisphosphate Transporter Involved in Guard Cell Signaling and Phytate Storage

Arabidopsis possesses a superfamily of ATP-binding cassette (ABC) transporters. Among these, the multidrug resistance-associated protein AtMRP5/AtABCC5 regulates stomatal aperture and controls plasma membrane anion channels of guard cells. Remarkably, despite the prominent role of AtMRP5 in conferring partial drought insensitivity upon Arabidopsis, we know little of the biochemical function of AtMRP5. Our phylogenetic analysis showed that AtMRP5 is closely related to maize MRP4, mutation of which confers a low inositol hexakisphosphate kernel phenotype. We now show that insertion mutants of AtMRP5 display a low inositol hexakisphosphate phenotype in seed tissue and that this phenotype is associated with alterations of mineral cation and phosphate status. By heterologous expression in yeast, we demonstrate that AtMRP5 encodes a specific and high affinity ATP-dependent inositol hexakisphosphate transporter that is sensitive to inhibitors of ABC transporters. Moreover, complementation of the mrp5-1 insertion mutants of Arabidopsis with the AtMRP5 cDNA driven from a guard cell-specific promoter restores the sensitivity of the mutant to abscisic acid-mediated inhibition of stomatal opening. Additionally, we show that mutation of residues of the Walker B motif prevents restoring the multiple phenotypes associated with mrp5-1. Our findings highlight a novel function of plant ABC transporters that may be relevant to other kingdoms. They also extend the signaling repertoire of this ubiquitous inositol polyphosphate signaling molecule.