Addition Of GSNO Research Articles

Nitric oxide improves the effect of 1-methylcyclopropene extending the tomato (Lycopersicum esculentum L.) fruit postharvest life

Tomato fruit ripening and postharvest display a complex net of metabolic pathways, which are controlled by different signaling molecules and hormones that coordinate this physiological process. The inhibition of the ethylene action with 1-methylcyclopropene (1-MCP) has proved to be an excellent tool for maintaining fruit quality during postharvest. Along with these findings, nitric oxide (NO) demonstrated different effects on several species in fruit postharvest. The aim of this work was to evaluate the joint effect of 1-MCP and a NO donor (S-nitrosoglutathione, GSNO) during tomato postharvest at 23 °C. Breaker tomatoes were harvested from a local grower and divided in the following treatments: untreated, 1 mM GSNO, 0.5 μL L−1 of 1-MCP, and the combination of 1 mM GSNO + 0.5 μL L−1 1-MCP. Fruit was stored at 23 °C and analyzed after 5 and 10 d. According to the results, the combination of 1-MCP and the NO donor delayed fruit softening at days 5 and 10, reduced the ethylene synthesis significantly at day 5 and elevated the respiration rate at 10 d, compared to the individual treatments of 1-MCP and GSNO alone. However, the solely application of 1-MCP, independently from the addition of GSNO, showed a strong effect in the rise of the total titratable acidity content, together with the citric acid content. 1-MCP also up-regulated the CAT, SOD and POD activities at 5 and 10 d after harvest. These findings may suggest that some fruit quality parameters in tomato postharvest are controlled by ethylene, while others need the coordination of nitric oxide as a signal molecule.

An exogenous source of nitric oxide modulates zinc nutritional status in wheat plants

The effect of addition of the nitric oxide donor S-nitrosoglutathione (GSNO) on the Zn nutritional status was evaluated in hydroponically-cultured wheat plants (Triticum aestivum cv. Chinese Spring). Addition of GSNO in Zn-deprived plants did not modify biomass accumulation but accelerated leaf senescence in a mode concomitant with accelerated decrease of Zn allocation to shoots. In well-supplied plants, Zn concentration in both roots and shoots declined due to long term exposure to GSNO. A further evaluation of net Zn uptake rate (ZnNUR) during the recovery of long-term Zn-deprivation unveiled that enhanced Zn-accumulation was partially blocked when GSNO was present in the uptake medium. This effect on uptake was mainly associated with a change of Zn translocation to shoots. Our results suggest a role for GSNO in the modulation of Zn uptake and in root-to-shoot translocation during the transition from deficient to sufficient levels of Zn-supply.

Potential problems and pitfalls with the use of S‐nitrosoglutathione and other S‐nitrosothiols in physiology‐oriented basic science

Potential problems and pitfalls with the use of <i>S</i>‐nitrosoglutathione and other <i>S</i>‐nitrosothiols in physiology‐oriented basic science

Mechanisms of Cystic Fibrosis Transmembrane Conductance Regulator Activation by S-Nitrosoglutathione

We investigated the mechanisms by which S-nitrosoglutathione (GSNO) alters cystic fibrosis transmembrane conductance regulator (CFTR) mediated chloride (Cl(-)) secretion across Calu-3 cells, an extensively used model of human airway gland serous cells. Confluent monolayers of Calu-3 cells, grown under an air-liquid interface, were mounted in Ussing chambers for the measurements of chloride short circuit current (I(sc)) and trans-epithelial resistance (R(t)). Addition of GSNO into the apical compartment of these chambers resulted in significant and sustained increase of I(sc) with an IC(50) of 3.2 +/- 1 mum (mean +/- 1 S.E.; n = 6). Addition of either glibenclamide or pre-treatment of Calu-3 cells with the soluble guanylate cyclase inhibitor 1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one totally prevented the GSNO-induced increase of I(sc). Conversely, BAY 41-2272, a sGC stimulator, increased I(sc) in a dose-response fashion. The GSNO increase of I(sc) was reversed by addition of two phosphatases (PP2A1, PP2A2) into the apical compartment of Ussing chambers containing Calu-3 monolayers. Oxy-myoglobin (oxy-Mb, 300 mum) added into the apical compartment of Ussing chambers either prior or after GSNO either completely prevented or immediately reversed the increase of I(sc). However, smaller concentrations of oxy-Mb (1-10 mum), sufficient to scavenge NO in the medium (as assessed by direct measurement of NO in the Ussing chamber using an ISO-NO meter) decreased I(sc) partially. Oxy-Mb did not reverse the increase of I(sc) following addition of GSNO and cysteine (50 mum). These findings indicate that GSNO stimulates Cl secretion via both cGMP-dependent and cGMP-independent mechanisms.

Consumption of S-nitrosoglutathione by polymorphonuclear leukocyte lysates

John F Hunt, Elizabeth Erwin, John W Vaughan, Benjamin M Gaston University of Virginia, Charlottesville, VA S-nitrosoglutathione (GSNO) is an endogenous bronchodilator depleted in the severe asthmatic airway. Eosinophils and neutrophils are found in elevated levels in bronchoalveolar lavage and biopsies of asthmatic lung, and exhaled nitric oxide (NO) levels are high. We hypothesized that polymorphonuclear leukocytes possess a catabolic enzyme for GSNO that releases NO. In this study, we isolated human peripheral blood eosinophils and neutrophils by density gradient and subsequent magnetic separation and subjected the cells to detergent lysis and homogenization in protease inhibitors in cold conditions. Homogenized cell lysates were suspended in 0.5% fetal bovine serum with 2 mM EDTA. Fifty micromolar GSNO was added to the cell homogenates which then were assayed over time for residual GSNO by gas-phase chemiluminescent detection after reduction to NO in Cu +1 -cysteine. Both eosinophil and neutrophil homogenates rapidly consumed GSNO compared to control (0.5% FBS + EDTA), with 33-50% comparative reduction in GSNO by 6 minutes, but limited difference from control in rate of consumption thereafter. In contrast, there were no differences from control in GSNO consumption by cell lysates after allowing them to degrade in a 37 degree water bath for 2 hours prior to addition of GSNO. These initial results suggest there is a GSNO consumptive pathway present in peripheral blood polymorphonuclear cell lysates. This pathway can be degraded with time, likely through proteolysis (if enzymatic) or elimination of NO-reactive species such as superoxide. If enzymatic, then consumption of a necessary cofactor could explain the cessation of effect after 6 minutes. This work supported by AAAAI Education and Research Trust Faculty Development Award, GlaxoSmithKline, Virginia Thoracic Society, NIH. JH is a Parker B. Francis Fellow in Pulmonary Medicine. 991 sisRelati°nshiPof Neuroblastoma°f TRK-A Statement to Clinical Stage and Progno-

Role of mitogen-activated protein kinases in S-nitrosoglutathione-induced macrophage apoptosis.

The inflammatory mediator nitric oxide (NO*) promotes apoptotic cell death based on morphological evidence, accumulation of the tumor suppressor p53, caspase-3 activation, and DNA fragmentation in RAW 264.7 macrophages. Since nitrosothiols may actually be the predominant form of biologically active NO* in vivo, we used S-nitrosoglutathione (GSNO) to study activation of extracellular signal-regulated protein kinases1/2 (ERK1/2), c-Jun N-terminal kinases/stress-activated protein kinases (JNK1/2), and p38 kinases. Moreover, we determined the role of mitogen-activated protein kinase signaling in the apoptotic transducing ability of GSNO. ERK1/2 became activated in response to GSNO after 4 h and remained active for the next 20 h. Blocking the ERK1/2 pathway by the mitogen-activated protein kinase kinase inhibitor PD 98059 enhanced GSNO-elicited apoptosis. p38 was activated as well, but inhibition of p38 with SB 203580 left apoptosis unaltered. Activation of JNK1/2 by GSNO showed maximal kinase activities between 2 and 8 h. Attenuating JNK1/2 by antisense-depletion eliminated the pro-apoptotic action of low GSNO concentrations (250 microM), whereas apoptosis proceeded independently of JNK1/2 at higher doses of the NO donor (500 microM). Decreased apoptosis by JNK1/2 depletion prevented p53 accumulation after the addition of GSNO, which positions JNK1/2 upstream of the p53 response at low agonist concentrations. In line, JNK1/2 activation proceeded unaltered in p53-antisense transfected macrophages. However, with higher GSNO concentrations apoptotic transducing pathways, including p53 accumulation, were JNK1/2 unrelated. The regulation of mitogen-activated protein kinases by GSNO may help to define cell protective and destructive actions of reactive nitrogen species.

Nitric Oxide Inhibited Peroxyl and Alkoxyl Radical Formation with Concomitant Protection against Oxidant Injury in Intestinal Epithelial Cells

A model compound of lipid peroxidation,tert-butyl hydroperoxide (tBOOH), was usedin vitroto investigate (i). the generation of tBOOH-derived peroxyl and alkoxyl radicals by rat intestinal epithelial cells or enterocytes and (ii) the role of nitric oxide (NO) on cell-generated free radical formation and cellular cytotoxicity. Peroxyl, alkoxyl, and methyl radicals were detected and characterized by direct and spin-trapping electron paramagnetic resonance spectroscopy in incubations containing tBOOH and hematin, enterocytes, or intestinal epithelial cell line-6 cells. The direct interactions of tBOOH-derived radicals and NO from nitrosoglutathione (GSNO), nitrosoacetyl penicillamine (SNAP), or 1-{b3-aminopropy-4-(3-aminopropylammonio)} butylamino-diazeniumdiolate (SpNONOate) were demonstrated as their levels were depleted in these incubations. SNAP, not GSNO or SpNONOate, was capable of trapping methyl radical produced during hematin-catalyzed decomposition of tBOOH. Cellular cytotoxicity expressed by percentage of dead cells and lactate dehydrogenase was increased with tBOOH treatment. Addition of GSNO, SNAP, or SpNONOate suppressed tBOOH-induced elevation of cell cytotoxicity. The NO donor precursor glutathione, acetylpenicillamine, or spermine did not have any effects on tBOOH-derived radical generation or cell cytotoxicity. These findings demonstrated free radical–free radical reactions between NO- and tBOOH-derived alkoxyl and peroxyl radicals generated by enterocytes. These reactions, at least in part, describe the protective role of NO from hydroperoxide-induced injury in intestinal epithelial cells.