β-Amyloid-Induced Endothelial Necrosis and Inhibition of Nitric Oxide Production

Deposits of amyloid β-peptide (Aβ) in senile plaques and cerebral blood vessels is the prominent feature of Alzheimer's disease (AD), regardless of genetic predisposition. The cellular origin of cerebral deposits of Aβ or its precise role in the neurodegenerative process has not been established. Recently we demonstrated a novel action of β-amyloid on blood vessels—vasoactivity and endothelial damage through superoxide radicals. Since endothelial dysfunction is associated with vascular degenerative diseases, we examined the direct action of Aβ on endothelial cells in culture. Cells treated with Aβ displayed characteristics of necrotic cell death which was prevented by the free radical scavenging enzyme superoxide dismutase. Stimulation of endothelial nitric oxide (NO) production by the calcium ionophore, A23187, or bradykinin was inhibited by β-amyloid. We conclude that an imbalance of NO and oxygen radicals may mediate the Aβ-induced endothelial damage on endothelial cells in culture and may also contribute to a variety of pathophysiological conditions associated with aging: hypertension, cerebral ischemia, vasospasm, or stroke.     

Coordination of Nitric Oxide by Heme—Hemopexin

Hemopexin, which acts as an antioxidant by binding heme (K _d < 1 pM), is synthesized by hepatic parenchymal cells, by neurons of the central and peripheral nervous systems, and by human retinal ganglia. Two key regulatory molecules, nitric oxide (·NO) and carbon monoxide (CO), both bind to heme proteins and since ferroheme–hemopexin binds CO, the possible role of heme–hemopexin in binding ·NO was investigated. ·NO binds rapidly to hemopexin-bound ferroheme as shown by characteristic changes in the Soret and visible-region absorbance spectra. Circular dichroism spectra of ·NO–ferroheme-hemopexin in the Soret region exhibit an unusual bisignate feature with a zero crossover at the absorbance wavelength maximum, showing that exciton coupling is occurring. Notably, the ·NO complex of ferroheme–hemopexin is sufficiently avid and stable to allow hemopexin to bind this molecule in vivo and, thus, hemopexin may protect against NO-mediated toxicity especially in conditions of trauma and hemolysis.     

Nitric Oxide Synthase 2 Improves Proliferation and Glycolysis of Peripheral γδ T Cells

γδ T cells play critical roles in host defense against infections and cancer. Although advances have been made in identifying γδ TCR ligands, it remains essential to understand molecular mechanisms responsible for in vivo expansion of γδ T cells in periphery. Recent findings identified the expression of the inducible NO synthase (NOS2) in lymphoid cells and highlighted novel immunoregulatory functions of NOS2 in αβ T cell differentiation and B cell survival. In this context, we wondered whether NOS2 exerts an impact on γδ T cell properties. Here, we show that γδ T cells express NOS2 not only in vitro after TCR triggering, but also directly ex vivo. Nos2 deficient mice have fewer γδ T cells in peripheral lymph nodes (pLNs) than their wild-type counterparts, and these cells exhibit a reduced ability to produce IL-2. Using chemical NOS inhibitors and Nos2 deficient γδ T cells, we further evidence that the inactivation of endogenous NOS2 significantly reduced γδ T cell proliferation and glycolysis metabolism that can be restored in presence of exogenous IL-2. Collectively, we demonstrate the crucial role of endogenous NOS2 in promoting optimal IL-2 production, proliferation and glycolysis of γδ T cells that may contribute to their regulation at steady state.     

Enhancement of Nitric Oxide and Superoxide Generations by α-Tocopheryl Succinate and Its Apoptotic and Anticancer Effects

Tocopheryl succinate (TS), a succinyl ester of alpha-tocopherol (alpha-T), has been reported to have various biological activities. In this communication, we review the current findings about TS including our recent studies of its effects on nitric oxide (NO) and superoxide (O2-) generations implicated in cancer and atherosclerosis. First, we investigated the effect of TS on NO production in vascular smooth muscle cells (VSMC) under atherosclerosis-like conditions using lipopolysaccharide (LPS) and interferon-gamma (IFN). TS enhanced LPS/IFN-dependent NO production, but alpha-T itself did not. The enhancement by TS of NO production was inhibited by alpha-T but not by antioxidants such as ascorbic acid and 2[3]-t-butyl-4-hydroxyanisole (BHA). TS enhanced the amount of protein kinase Calpha (PKCalpha) in VSMC, and PKC inhibitors inhibited TS-enhanced NO production, suggesting that the enhancing effect of TS on NO production is caused by up-regulation of PKC. Second, we found that TS induced apoptosis in VSMC associated with increase in O2- generation via NADPH-dependent oxidase. We further observed that a mouse breast cancer cell line C127I was more susceptible for TS-induced apoptosis than a mouse breast normal cell line NmuMG, and that superoxide dismutase, alpha-T, and BHA inhibited TS-caused morphological cell damage in C127I. From these results, O2- itself and/or other reactive oxygen species are assumed to associate with TS-induced cell toxicity, and antioxidative defense systems are supposed to be lowered in cancer cells. Finally, we found that intravenous injection of TS vesicles completely inhibited the growth of melanoma cells B16-F1 inoculated on the back of hairless mice and enhanced their survival time.     

Does Restraining Nitric Oxide Biosynthesis Rescue from Toxins-Induced Parkinsonism and Sporadic Parkinson's Disease?

Nitric oxide (NO) is an important inorganic molecule of the biological system owing to diverse physiological implications. NO is synthesised from a semi-essential amino acid l-arginine. NO biosynthesis is catalysed by a family of enzymes referred to as nitric oxide synthases (NOSs). NO is accused in many acute and chronic illnesses, which include central nervous system disorders, inflammatory diseases, reproductive impairments, cancer and cardiovascular anomalies. Owing to very unstable nature, NO gets converted into nitrite, peroxynitrite and other reactive nitrogen species that could lead to nitrosative stress in the nigrostriatal system. Nitrosative stress is widely implicated in Parkinson's disease (PD), and its beneficial and harmful effects are demonstrated in in vitro, rodent and primate models of toxins-induced parkinsonism and in the blood, cerebrospinal fluid and nigrostriatal tissues of sporadic PD patients. The current article updates the roles of NO and NOSs in sporadic PD and toxins-induced parkinsonism in rodents along with the scrutiny of how inhibitors of NOSs could open a new line of approach to moderately rescue from PD pathogenesis based on the existing literature. The article also provides a perspective concerning the lack of ample admiration to such an approach and how to minimise the underlying lacunae.     

Lipopolysaccharide-induced Overproduction of Nitric Oxide and Overexpression of iNOS and Interleukin-1β Proteins in Zinc-deficient Rats

Zinc deficiency leads to decreased cellular immune responses. The overproduction of nitrogen species derived from inducible nitric oxide synthase (iNOS), its enzyme, and interleukine-1 beta (IL-1β), and inflammatory cytokine have been implicated in immune responses. The goal of this study was to investigate the effects of lipopolysaccharide (LPS)-induced changes in NO metabolites, iNOS, and IL-1β protein expression in the lungs of zinc-deficient rats. Male Sprague–Dawley rats (body weight, 100 g) were divided into two groups and were fed either a zinc-deficient diet (ZnD) or a zinc-containing diet (Cont). After 4 weeks on these diets, rats received a 10-mg/kg dose of LPS injected via the tail vein and were then maintained for an additional 72 h. To determine total NO concentrations in the blood, serum zinc concentration, iNOS protein expression, IL-1β, and iNOS immunohistochemistry, blood and lung samples were obtained at pre-LPS injection, 5, 24, and 72 h after injection. Total NO levels were significantly increased at 5, at 24, and at 72 h after LPS injection compared with pre-LPS injection level in ZnD group; significant changes in total NO levels was elevated at 5 h from at pre-LPS level but not significant changes from basal level at 24 and 72 h in the control group. Based on western blot analyses and immunohistochemistry, clear bands indicating iNOS and IL-1β protein expression and iNOS antibody-stained inflammatory cells were detected at 5 and 24 h in the ZnD group and 5 h in the Cont group, not observed at 24 and 72 h in the control group. These results suggest that zinc deficiency induces overexpression of iNOS and IL-1β proteins from inflammatory cells around the alveolar blood vessels, resulting in overproduction of total NO and persisted inflammatory response in the zinc-deficient rat lung. Taken together, overexpression of LPS-induced iNOS, overproduction of iNOS-derived NO, and overexpression of IL-1β may induce nitrosative and oxidative stresses in the lung, and these stresses may be involved low immunity of zinc deficiency states.     

��-Actin Association with Endothelial Nitric-oxide Synthase Modulates Nitric Oxide and Superoxide Generation from the Enzyme

Protein-protein interactions represent an important post-translational mechanism for endothelial nitric-oxide synthase (eNOS) regulation. We have previously reported that β-actin is associated with eNOS oxygenase domain and that association of eNOS with β-actin increases eNOS activity and nitric oxide (NO) production. In the present study, we found that β-actin-induced increase in NO production was accompanied by decrease in superoxide formation. A synthetic actin-binding sequence (ABS) peptide 326 with amino acid sequence corresponding to residues 326–333 of human eNOS, one of the putative ABSs, specifically bound to β-actin and prevented eNOS association with β-actin in vitro. Peptide 326 also prevented β-actin-induced decrease in superoxide formation and increase in NO and l-citrulline production. A modified peptide 326 replacing hydrophobic amino acids leucine and tryptophan with neutral alanine was unable to interfere with eNOS-β-actin binding and to prevent β-actin-induced changes in NO and superoxide formation. Site-directed mutagenesis of the actin-binding domain of eNOS replacing leucine and tryptophan with alanine yielded an eNOS mutant that exhibited reduced eNOS-β-actin association, decreased NO production, and increased superoxide formation in COS-7 cells. Disruption of eNOS-β-actin interaction in endothelial cells using ABS peptide 326 resulted in decreased NO production, increased superoxide formation, and decreased endothelial monolayer wound repair, which was prevented by PEG-SOD and NO donor NOC-18. Taken together, this novel finding indicates that β-actin binding to eNOS through residues 326–333 in the eNOS protein results in shifting the enzymatic activity from superoxide formation toward NO production. Modulation of NO and superoxide formation from eNOS by β-actin plays an important role in endothelial function.     

Cytokine and Nitric Oxide Levels in Patients with Sepsis – Temporal Evolvement and Relation to Platelet Mitochondrial Respiratory Function

Background

The levels of nitric oxide (NO) and various cytokines are known to be increased during sepsis. These signaling molecules could potentially act as regulators and underlie the enhancement of mitochondrial function described in the later phase of sepsis. Therefore, we investigated the correlation between observed changes in platelet mitochondrial respiration and a set of pro- and anti-inflammatory cytokines as well as NO plasma levels in patients with sepsis.

Methods and Results

Platelet mitochondrial respiration and levels of TNFα, MCP-1 (monocyte chemotactic protein-1), INFγ (interferon-γ), IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10 and IL-17 and NO were analyzed in 38 patients with severe sepsis or septic shock at three time points during one week following admission to the ICU. Citrate synthase, mitochondrial DNA and cytochrome c were measured as markers of cellular mitochondrial content. All mitochondrial respiratory states increased over the week analyzed (p<0.001). IL-8 levels correlated with maximal mitochondrial respiration on day 6–7 (p = 0.02, r² = 0.22) and was also higher in non-survivors compared to survivors on day 3–4 and day 6–7 (p = 0.03 respectively). Neither NO nor any of the other cytokines measured correlated with respiration or mortality. Cytochrome c levels were decreased at day 1–2 by 24±5% (p = 0.03) and returned towards values of the controls at the last two time points. Citrate synthase activity and mitochondrial DNA levels were similar to controls and remained constant throughout the week.

Conclusions

Out of ten analyzed cytokines and nitric oxide, IL-8 correlated with the observed increase in mitochondrial respiration. This suggests that cytokines as well as NO do not play a prominent role in the regulation of platelet mitochondrial respiration in sepsis. Further, the respiratory increase was not accompanied by an increase in markers of mitochondrial content, suggesting a possible role for post-translational enhancement of mitochondrial respiration rather than augmented mitochondrial mass.     

Peroxynitrite Mediates Nitric Oxide–Induced Blood–Brain Barrier Damage

Using the in vitro blood-brain barrier (BBB) model ECV304/C6, which consists of cocultures of human umbilical vein endothelial-like cells (ECV304) and rat glioma cells (C6), the role of peroxynitrite (OONO-) in nitric oxide (NO*)-mediated BBB disruption was evaluated. Endothelial cell cultures were exposed to NO* gas, in the presence or absence of the OONO- blocker FeTPPS. Separate exposure to NO* and OONO- resulted in endothelial cell cytotoxicity and a decline in barrier integrity. Unfortunately, FeTPPS induced significant detrimental effects on model BBB integrity at a concentration of 300 microM and above. At 250 microM (the highest concentration usable), FeTPPS displayed a trend toward prevention of NO* elicited perturbation of barrier integrity. Dichlorofluorescein diacetate is oxidized to fluorescent dichlorofluorescein by OONO- but only marginally by NO* or O2*-. We observed large and rapid increases in fluorescence in ECV304 preloaded cells following NO* exposure, which were blocked by FeTPPS. Furthermore, using an antinitrotyrosine antibody we detected the nitration of endothelial cell proteins following NO* exposure and conclude that NO*-mediated BBB dysfunction is predominantly elicited by OONO- and not NO*. Proposed mechanisms of NO*-mediated OONO- elicited barrier dysfunction and damage are discussed.     

Regulation of Nitric Oxide Production by δ-Opioid Receptors during Glaucomatous Injury

To determine the roles of nitric oxide in glaucomatous injury and its regulation by δ-opioid-receptor activation, animals were treated with: 1) a selective inducible nitric oxide synthase (iNOS) inhibitor (aminoguanidine; AG; 25 mg/kg, i.p.); 2) δ-opioid-receptor agonist (SNC-121; 1 mg/kg, i.p.); or 3) with both drugs simultaneously for 7 days, once daily. The loss in retinal ganglion cell (RGC) numbers and their function in glaucomatous eyes were significantly improved in the presence of AG or SNC-121; however, we did not see any significant additive or synergistic effects when animals were treated with both drugs simultaneously. The levels of nitrate-nitrite were significantly increased in the glaucomatous retina when compared with normal retina (normal retina 86±9 vs. glaucomatous retina 174±10 mM/mg protein), which was reduced significantly when animals were treated either with SNC-121 (121±7 mM/mg protein; P<0.05) or AG (128±10 mM/mg protein; P<0.05). Additionally, SNC-121-mediated reduction in nitrate-nitrite levels was not only blocked by naltrindole (a δ-opioid-receptor antagonist), but naltrindole treatment potentiated the nitrate-nitrite production in glaucomatous retina (235±4 mM/mg protein; P<0.001). As expected, naltrindole treatment also fully-blocked SNC-121-mediated retina neuroprotection. The nitrotyrosine level in the glaucomatous retina was also increased, which was significantly reduced in the SNC-121-treated animals. Additionally, the expression level of iNOS was clearly increased over the control levels in the glaucomatous retina and optic nerves, which was also reduced by SNC-121 treatment. In conclusion, our data support the notion that nitric oxide plays a detrimental role during glaucomatous injury and inhibition of nitric oxide production provided RGC neuroprotection. Furthermore, δ-opioid receptor activation regulates the production of nitric oxide via inhibiting the activity of iNOS in the retina and optic nerve.     

Steady Shear and Step Changes in Shear Stimulate Endothelium via Independent Mechanisms—Superposition of Transient and Sustained Nitric Oxide Production

We propose that fluid shear presents two distinct stimuli to endothelium—the rate of change of flow and flow itself, to which cells sense and respond via independent mechanochemical transduction pathways. We demonstrate that nitric oxide production occurs by two independent mechanisms; a G protein-dependent transient burst stimulated by rapid changes in flow, and a G protein-independent sustained production under steady or smoothly transitioned flow. The novel use of step, ramp, and impulse flow in this study to stimulate nitric oxide production allows the isolation of these individual production events. Impulse flow activates only the G protein-dependent transient burst, which ramp flow fails to stimulate yielding only the sustained response. Step flow, which contains both a rapid increase and a steady flow component, stimulates both pathways, with the response of the superposition of the transient burst and sustained production.     

Effects of Antioxidant and Nitric Oxide on Chemokine Production in TNF-α-stimulated Human Dermal Microvascular Endothelial Cells

Chemokines have been implicated convincingly in the driving of leukocyte emigration in different inflammatory reactions. Multiple signaling mechanisms are reported to be involved in intracellular activation of chemokine expression in vascular endothelial cells by various stimuli. Nevertheless, redox-regulated mechanisms of chemokine expression in human dermal microvascular endothelial cells (HDMEC) remain unclear. This study examined the effects of pyrrolidine dithiocarbamate (PDTC, 0.1?mM) and spermine NONOate (Sper-NO, 1?mM) on the secretion and gene expression of chemokines, interleukin (IL)-8, monocyte chemotactic protein (MCP)-1, regulated upon activation normal T cell expressed and secreted (RANTES), and eotaxin. This study also addresses PDTC and Sper-NO effects on activation of nuclear factor kappa B (NF-κB) induced by TNF-α (10?ng/ml). Treatment with TNF-α for 8?h significantly increased secretion of IL-8, MCP-1, and RANTES, but not of eotaxin, in cultured HDMEC. Up-regulation of these chemokines was suppressed significantly by pretreatment with PDTC or Sper-NO for 1?h, but not by 1?mM 8-bromo-cyclic GMP. The mRNA accumulation of IL-8, MCP-1, RANTES, and eotaxin, and activation of NF-κB were induced by TNF-α for 2?h; all were suppressed significantly by the above two pretreatments. These findings indicate that both secretion and mRNA accumulation of IL-8, MCP-1, and RANTES in HDMEC induced by TNF-α are inhibited significantly by pretreatment with PDTC or Sper-NO, possibly via blocking redox-regulated NF-κB activation. These results suggest that restoration of the redox balance using antioxidant agents or nitric oxide pathway modulators may offer new opportunities for therapeutic interventions in inflammatory skin diseases.     

Nitric Oxide and Protein Disulfide Isomerase Explain the Complexities of Unfolded Protein Response Following Intra-hippocampal Aβ Injection

Several pathways involved in regulation of intracellular protein integrity are known as the protein quality control (PQC) system. Molecular chaperones as the main players are engaged in various aspects of PQC system. According to the importance of these proteins in cell survival, in the present study, we traced endoplasmic reticulum-specific markers and chaperone-mediated autophagy (CMA)-associated factors as two main arms of PQC system in intra-hippocampal amyloid beta (Aβ)-injected rats during 10 days running. Data analysis from Western blot indicated that exposure to Aβ activates immunoglobulin heavy-chain-binding protein (Bip) which is the upstream regulator of unfolded protein responses (UPR). Activation of UPR system eventually led to induction of pro-apoptotic factors like CHOP, calpain, and caspase-12. Moreover, our data revealed that protein disulfide isomerase activity dramatically decreased after Aβ injection, which could be attributed to the increased levels of nitric oxide. Besides, Aβ injection induced levels of 2 members of heat shock proteins (Hsp) 70 and 90. Elevated levels of Hsps family members are accompanied by increased levels of lysosome-associated membrane protein type-2A (Lamp-2A) that are involved in CMA. Despite the reduction in CHOP, calpain, caspase-12, and Lamp-2A protein levels, the levels of molecular chaperones Bip, Hsps70, and 90 increased 10 days after Aβ injection in comparison to the control group. Based on our results, 10 days after Aβ injection, despite the activation of protective chaperones, markers associated with neurotoxicity were still elevated.     

Role and Interrelationship of Gα Protein,Hydrogen Peroxide,and Nitric Oxide in Ultraviolet B-Induced Stomatal Closure in Arabidopsis Leaves

Heterotrimeric G proteins have been shown to transmit ultraviolet B (UV-B) signals in mammalian cells, but whether they also transmit UV-B signals in plant cells is not clear. In this paper, we report that 0.5 W m⁻² UV-B induces stomatal closure in Arabidopsis (Arabidopsis thaliana) by eliciting a cascade of intracellular signaling events including Gα protein, hydrogen peroxide (H₂O₂), and nitric oxide (NO). UV-B triggered a significant increase in H₂O₂ or NO levels associated with stomatal closure in the wild type, but these effects were abolished in the single and double mutants of AtrbohD and AtrbohF or in the Nia1 mutants, respectively. Furthermore, we found that UV-B-mediated H₂O₂ and NO generation are regulated by GPA1, the Gα-subunit of heterotrimeric G proteins. UV-B-dependent H₂O₂ and NO accumulation were nullified in gpa1 knockout mutants but enhanced by overexpression of a constitutively active form of GPA1 (cGα). In addition, exogenously applied H₂O₂ or NO rescued the defect in UV-B-mediated stomatal closure in gpa1 mutants, whereas cGα AtrbohD/AtrbohF and cGα nia1 constructs exhibited a similar response to AtrbohD/AtrbohF and Nia1, respectively. Finally, we demonstrated that Gα activation of NO production depends on H₂O₂. The mutants of AtrbohD and AtrbohF had impaired NO generation in response to UV-B, but UV-B-induced H₂O₂ accumulation was not impaired in Nia1. Moreover, exogenously applied NO rescued the defect in UV-B-mediated stomatal closure in the mutants of AtrbohD and AtrbohF. These findings establish a signaling pathway leading to UV-B-induced stomatal closure that involves GPA1-dependent activation of H₂O₂ production and subsequent Nia1-dependent NO accumulation.Heterotrimeric G proteins, composed of α-, β-, and γ-subunits, are a key intracellular signaling molecule in both mammalian and plant systems. Classically, upon signal reception by a receptor coupled to the heterotrimer, the Gα-subunit separates from the Gβγ dimer, and either Gα or the Gβγ dimer can act as a functional unit and induce downstream signaling (Oldham and Hamm, 2008). In contrast to mammalian cells, where multiple α, β, and γ genes exist, there is only one prototypical Gα (GPA1), one Gβ (AGB1), and two known Gγ (AGG1 and AGG2) genes in Arabidopsis (Arabidopsis thaliana; Temple and Jones, 2007). Despite the comparative simplicity of players, G proteins have been shown to participate in multiple signaling pathways in Arabidopsis, including developmental processes, phytohormone responses, and responses to biotic and abiotic environmental signals such as pathogens, ozone, drought, and light (Assmann, 2005; Temple and Jones, 2007; Warpeha et al., 2007; Okamoto et al., 2009; Nilson and Assmann, 2010).Depletion of the stratospheric ozone layer results in increased levels of the sun’s UV-B radiation (280–315 nm) at the Earth’s surface. Although this influx of shortwave photons with high energy implies serious effects for all living organisms (Frohnmeyer and Staiger, 2003), UV-B is also a key environmental signal that initiates diverse responses in a range of organisms (Jansen and Bornman, 2012). Thus, understanding the mechanism of UV-B signal transduction in cells is very important. In recent years, significant progress has been made in identifying the molecular players and understanding the early mechanisms and functions of the UV-B perception and signaling pathway in plants. The perception of UV-B by UV RESISTANCE LOCUS8 (UVR8) followed by the interaction among UVR8, CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), and ELONGATED HYPOCOTYL5 (HY5) has emerged as a primary mechanism of the UV-B response that is crucial for UV-B acclimation and tolerance (Rizzini et al., 2011; Christie et al., 2012; Heijde and Ulm, 2012; Jansen and Bornman, 2012). However, few of the molecular players involved in UV-B signal transduction are currently known. In mammalian cells, heterotrimeric G proteins have been shown to mediate various UV-B-induced cellular responses, such as secretion of heparin-binding epidermal growth factor (HB-EGF), activation of mitogen-activated protein kinases, cyclooxygenase2 expression, and apoptosis in human keratinocytes (Seo et al., 2004, 2007; Seo and Juhnn, 2010), suggesting that G proteins are important molecular players in UV-B signal transduction. However, at present, whether G proteins participate in the responses of plant cells to UV-B is not known.Stomata embedded in the epidermis of terrestrial plants are important for CO₂ absorption and water transpiration and are possible points of entry for pathogens. Thus, the regulation of stomatal apertures is extremely important for the survival of plants. Phenotypic analyses of Arabidopsis mutants lacking the Gα- or Gβ-subunit show that these G proteins are involved in stomatal movement regulated by abscisic acid (ABA; Wang et al., 2001; Pandey and Assmann, 2004; Liu et al., 2007; Fan et al., 2008; Zhang et al., 2011), sphingosine-1-P (Coursol et al., 2003, 2005), phosphatidic acid (PA; Mishra et al., 2006), extracellular calmodulin (ExtCaM; Chen et al., 2004; Li et al., 2009), extracellular ATP (Hao et al., 2012), and the pathogen-associated molecular pattern flg22 (Zhang et al., 2008), suggesting that plant G proteins respond to various stimuli as key regulators of stomatal movement. On exposure to UV-B radiation, many plant species exhibit decreases in stomatal conductance and/or aperture under growth chamber, greenhouse, and field conditions (Musil and Wand, 1993; Nogués et al., 1999; Jansen and Noort, 2000). However, in some species, UV-B has been reported to induce either stomatal opening or stomatal closure, perhaps depending on the metabolic state of guard cells (Jansen and Noort, 2000). Furthermore, UV-B-inhibited photosynthesis is partially caused by stomatal limitation (He et al., 2004). Thus, understanding the mechanism of stomatal movement regulated by UV-B is extremely important for improving the resistance of plants to enhanced UV-B radiation, but, to date, it is poorly understood.Recently, compelling evidence emerged that hydrogen peroxide (H₂O₂) and nitric oxide (NO) function as signaling molecules in plants, mediating a range of responses to environmental stress including UV-B radiation (Neill et al., 2002; Qiao and Fan, 2008; Wilson et al., 2008). Increasing evidence also points to the role for H₂O₂ and NO as essential components in guard cell signaling. For example, both H₂O₂ and NO have been implicated in ABA-, salicylic acid (SA)-, ethylene-, ExtCaM-, and darkness-induced stomatal closure. Furthermore, several main cellular players in stomatal movement, such as mitogen-activated protein kinases, protein phosphatases, cytoskeleton, and ion channels, have already been identified as likely targets downstream of H₂O₂ or NO (Neill et al., 2008; Wang and Song, 2008; Huang et al., 2009; Li et al., 2009; Wilkins et al., 2011; Yemets et al., 2011). G protein signaling to the membrane-bound H₂O₂ synthetic enzyme, NADPH oxidase, has been implicated in the development of disease resistance and the apoptotic hypersensitive response in rice (Oryza sativa; Suharsono et al., 2002). Production of reactive oxygen species (ROS) in response to the air pollutant ozone is also impaired in a mutant lacking the Gα subunit (Joo et al., 2005). The heterotrimeric G proteins also participate in ROS metabolism in plant cells (Wei et al., 2008; Zhao et al., 2010). During stomatal movement, G proteins mediate H₂O₂ production induced by ABA (Zhang et al., 2011), ExtCaM (Chen et al., 2004; Li et al., 2009), and extracellular ATP (Hao et al., 2012) as well as NO production induced by ExtCaM in guard cells (Li et al., 2009). In addition, phospholipase Dα and its product PA, which interact with GPA1 during ABA inhibition of stomatal opening (Mishra et al., 2006), also promote ABA-induced ROS production (Zhang et al., 2009). These observations suggest that G proteins may be key regulators of H₂O₂ and NO production in plant cells, including guard cells. With regard to the stomatal movement regulated by UV-B radiation, our previous studies showed that H₂O₂ and NO generation are required for UV-B-induced stomatal closure (He et al., 2005, 2011a, 2011b). However, whether the UV-B-induced production of H₂O₂ and NO in guard cells is also regulated by G proteins remains unknown.In this study, we use Arabidopsis mutants (e.g. GPA1 null mutants gpa1-1 and gpa1-2; Nia1-2, Nia2-1, and Nia1-2/Nia2-5, which are defective in NO production; and AtrbohD, AtrbohF, and AtrbohD/AtrbohF, which are defective in producing H₂O₂) and pharmacological reagents to show that the G protein is involved in the regulation of UV-B-induced stomatal closure in Arabidopsis via sequential elucidation of H₂O₂ and NO, two key regulators of UV-B regulation of stomatal movements. Our results establish a linear signaling cascade in which the Gα protein transmits UV-B signals to elicit H₂O₂, which then elicits NO in guard cells to regulate UV-B-dependent stomatal closure.