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Reactive oxygen species and nitric oxide signaling in bystander cells.

ABSTRACT: It is now well accepted that radiation induced bystander effects can occur in cells exposed to media from irradiated cells. The aim of this study was to follow the bystander cells in real time following addition of media from irradiated cells and to determine the effect of inhibiting these signals. A human keratinocyte cell line, HaCaT cells, was irradiated (0.005, 0.05 and 0.5 Gy) with ? irradiation, conditioned medium was harvested after one hour and added to recipient bystander cells. Reactive oxygen species, nitric oxide, Glutathione levels, caspase activation, cytotoxicity and cell viability was measured after the addition of irradiated cell conditioned media to bystander cells. Reactive oxygen species and nitric oxide levels in bystander cells treated with 0.5Gy ICCM were analysed in real time using time lapse fluorescence microscopy. The levels of reactive oxygen species were also measured in real time after the addition of extracellular signal-regulated kinase and c-Jun amino-terminal kinase pathway inhibitors. ROS and glutathione levels were observed to increase after the addition of irradiated cell conditioned media (0.005, 0.05 and 0.5 Gy ICCM). Caspase activation was found to increase 4 hours after irradiated cell conditioned media treatment (0.005, 0.05 and 0.5 Gy ICCM) and this increase was observed up to 8 hours and there after a reduction in caspase activation was observed. A decrease in cell viability was observed but no major change in cytotoxicity was found in HaCaT cells after treatment with irradiated cell conditioned media (0.005, 0.05 and 0.5 Gy ICCM). This study involved the identification of key signaling molecules such as reactive oxygen species, nitric oxide, glutathione and caspases generated in bystander cells. These results suggest a clear connection between reactive oxygen species and cell survival pathways with persistent production of reactive oxygen species and nitric oxide in bystander cells following exposure to irradiated cell conditioned media.

SUBMITTER: Jella KK

PROVIDER: S-EPMC5886541 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Reactive oxygen species and nitric oxide signaling in bystander cells.

Jella Kishore Kumar KK Moriarty Roisin R McClean Brendan B Byrne Hugh J HJ Lyng Fiona M FM

PloS one 20180405 4

It is now well accepted that radiation induced bystander effects can occur in cells exposed to media from irradiated cells. The aim of this study was to follow the bystander cells in real time following addition of media from irradiated cells and to determine the effect of inhibiting these signals. A human keratinocyte cell line, HaCaT cells, was irradiated (0.005, 0.05 and 0.5 Gy) with γ irradiation, conditioned medium was harvested after one hour and added to recipient bystander cells. Reactiv ...[more]

PMID: 29621312

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Project description:Vascular tone is controlled by the L-arginine/nitric oxide (NO) pathway, and NO bioavailability is strongly affected by hyperglycaemia-induced oxidative stress. Insulin leads to high expression and activity of human cationic amino acid transporter 1 (hCAT-1), NO synthesis and vasodilation; thus, a protective role of insulin on high D-glucose-alterations in endothelial function is likely. Vascular reactivity to U46619 (thromboxane A2 mimetic) and calcitonin gene related peptide (CGRP) was measured in KCl preconstricted human umbilical vein rings (wire myography) incubated in normal (5 mmol/L) or high (25 mmol/L) D-glucose. hCAT-1, endothelial NO synthase (eNOS), 42 and 44 kDa mitogen-activated protein kinases (p42/44mapk), protein kinase B/Akt (Akt) expression and activity were determined by western blotting and qRT-PCR, tetrahydrobiopterin (BH4) level was determined by HPLC, and L-arginine transport (0-1000 μmol/L) was measured in response to 5-25 mmol/L D-glucose (0-36 hours) in passage 2 human umbilical vein endothelial cells (HUVECs). Assays were in the absence or presence of insulin and/or apocynin (nicotinamide adenine dinucleotide phosphate-oxidase [NADPH oxidase] inhibitor), tempol or Mn(III)TMPyP (SOD mimetics). High D-glucose increased hCAT-1 expression and activity, which was biphasic (peaks: 6 and 24 hours of incubation). High D-glucose-increased maximal transport velocity was blocked by insulin and correlated with lower hCAT-1 expression and SLC7A1 gene promoter activity. High D-glucose-increased transport parallels higher reactive oxygen species (ROS) and superoxide anion (O2•-) generation, and increased U46619-contraction and reduced CGRP-dilation of vein rings. Insulin and apocynin attenuate ROS and O2•- generation, and restored vascular reactivity to U46619 and CGRP. Insulin, but not apocynin or tempol reversed high D-glucose-increased NO synthesis; however, tempol and Mn(III)TMPyP reversed the high D-glucose-reduced BH4 level. Insulin and tempol blocked the high D-glucose-increased p42/44mapk phosphorylation. Vascular dysfunction caused by high D-glucose is likely attenuated by insulin through the L-arginine/NO and O2•-/NADPH oxidase pathways. These findings are of interest for better understanding vascular dysfunction in states of foetal insulin resistance and hyperglycaemia.

Project description:The enteric pathogen Salmonella enterica can interact with parts of the plant immune system despite not being a phytopathogen. Previous transcriptomic profiling of S. enterica associating with tomato suggested that Salmonella was responding to oxidative and nitrosative stress in the plant niche. We aimed to investigate whether Salmonella was eliciting generation of reactive oxygen species (ROS) and nitric oxide (NO), two components of the microbe-associated molecular pattern (MAMP)-triggered immunity (MTI) of plants. We also sought to determine whether this interaction had any measurable effects on Salmonella colonization of plants. Biochemical, gene expression and on-plant challenge assays of tomato vegetative and fruit organs were conducted to assess the elicitation of ROS and NO in response to Salmonella Newport association. The counter bacterial response and the effect of NO and ROS on Salmonella colonization was also investigated. We detected H2O2 in leaves and fruit following challenge with live S. Newport (p < 0.05). Conversely, NO was detected on leaves but not on fruit in response to S. Newport (p < 0.05). We found no evidence of plant defense attenuation by live S. Newport. Bacterial gene expression of S. Newport associating with leaves and fruit were indicative of adaptation to biotic stress in the plant niche. The nitrosative stress response genes hmpA and yoaG were significantly up-regulated in S. Newport on leaves and fruit tissue compared to tissue scavenged of NO or ROS (p < 0.05). Chemical modulation of these molecules in the plant had a restrictive effect on bacterial populations. Significantly higher S. Newport titers were retrieved from H2O2 scavenged leaves and fruit surfaces compared to controls (p < 0.05). Similarly, S. Newport counts recovered from NO-scavenged leaves, but not fruit, were higher compared to control (p < 0.05), and significantly lower on leaves pre-elicited to produce endogenous NO. We present evidence of Salmonella elicitation of ROS and NO in tomato, which appear to have a restricting effect on the pathogen. Moreover, bacterial recognition of ROS and NO stress was detected. This work shows that tomato has mechanisms to restrict Salmonella populations and ROS and NO detoxification may play an important role in Salmonella adaptation to the plant niche.

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Reactive oxygen species and nitric oxide signaling in bystander cells.

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Reactive oxygen species and nitric oxide signaling in bystander cells.

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