A key role for mitochondria in endothelial signaling by plasma cysteine/cystine redox potential

The redox potential of the plasma cysteine/cystine couple (E_hCySS) is oxidized in association with risk factors for cardiovascular disease (CVD), including age, smoking, type 2 diabetes, obesity, and alcohol abuse. Previous in vitro findings support a cause–effect relationship for extracellular E_hCySS in cell signaling pathways associated with CVD, including those controlling monocyte adhesion to endothelial cells. In this study, we provide evidence that mitochondria are a major source of reactive oxygen species (ROS) in the signaling response to a more oxidized extracellular E_hCySS. This increase in ROS was blocked by overexpression of mitochondrial thioredoxin-2 (Trx2) in endothelial cells from Trx2-transgenic mice, suggesting that mitochondrial thiol antioxidant status plays a key role in this redox signaling mechanism. Mass spectrometry-based redox proteomics showed that several classes of plasma membrane and cytoskeletal proteins involved in inflammation responded to this redox switch, including vascular cell adhesion molecule, integrins, actin, and several Ras family GTPases. Together, the data show that the proinflammatory effects of oxidized plasma E_hCySS are due to a mitochondrial signaling pathway that is mediated through redox control of downstream effector proteins.     

Reactive oxygen species,cellular redox systems,and apoptosis

Reactive oxygen species (ROS) are products of normal metabolism and xenobiotic exposure, and depending on their concentration, ROS can be beneficial or harmful to cells and tissues. At physiological low levels, ROS function as “redox messengers” in intracellular signaling and regulation, whereas excess ROS induce oxidative modification of cellular macromolecules, inhibit protein function, and promote cell death. Additionally, various redox systems, such as the glutathione, thioredoxin, and pyridine nucleotide redox couples, participate in cell signaling and modulation of cell function, including apoptotic cell death. Cell apoptosis is initiated by extracellular and intracellular signals via two main pathways, the death receptor- and the mitochondria-mediated pathways. Various pathologies can result from oxidative stress-induced apoptotic signaling that is consequent to ROS increases and/or antioxidant decreases, disruption of intracellular redox homeostasis, and irreversible oxidative modifications of lipid, protein, or DNA. In this review, we focus on several key aspects of ROS and redox mechanisms in apoptotic signaling and highlight the gaps in knowledge and potential avenues for further investigation. A full understanding of the redox control of apoptotic initiation and execution could underpin the development of therapeutic interventions targeted at oxidative stress-associated disorders.     

Thiol/disulfide redox states in signaling and sensing

Abstract

Rapid advances in redox systems biology are creating new opportunities to understand complexities of human disease and contributions of environmental exposures. New understanding of thiol–disulfide systems have occurred during the past decade as a consequence of the discoveries that thiol and disulfide systems are maintained in kinetically controlled steady states displaced from thermodynamic equilibrium, that a widely distributed family of NADPH oxidases produces oxidants that function in cell signaling and that a family of peroxiredoxins utilize thioredoxin as a reductant to complement the well-studied glutathione antioxidant system for peroxide elimination and redox regulation. This review focuses on thiol/disulfide redox state in biologic systems and the knowledge base available to support development of integrated redox systems biology models to better understand the function and dysfunction of thiol–disulfide redox systems. In particular, central principles have emerged concerning redox compartmentalization and utility of thiol/disulfide redox measures as indicators of physiologic function. Advances in redox proteomics show that, in addition to functioning in protein active sites and cell signaling, cysteine residues also serve as redox sensors to integrate biologic functions. These advances provide a framework for translation of redox systems biology concepts to practical use in understanding and treating human disease. Biological responses to cadmium, a widespread environmental agent, are used to illustrate the utility of these advances to the understanding of complex pleiotropic toxicities.     

Modulation of the matrix redox signaling by mitochondrial Ca~(2+)

Mitochondria sense,shape and integrate signals,and thus function as central players in cellular signal transduction. Ca2+ waves and redox reactions are two such intracellular signals modulated by mitochondria. Mitochondrial Ca2+ transport is of utmost physio-pathological relevance with a strong impact on metabolism and cell fate. Despite its importance,the molecular nature of the proteins involvedin mitochondrial Ca2+ transport has been revealed only recently. Mitochondrial Ca2+ promotes energy metabolism through the activation of matrix dehydrogenases and downstream stimulation of the respiratory chain. These changes also alter the mitochondrial NAD(P)H/NAD(P)+ ratio,but at the same time will increase reactive oxygen species(ROS) production. Reducing equivalents and ROS are having opposite effects on the mitochondrial redox state,which are hard to dissect. With the recent development of genetically encoded mitochondrial-targeted redoxsensitive sensors,real-time monitoring of matrix thiol redox dynamics has become possible. The discoveries of the molecular nature of mitochondrial transporters of Ca2+ combined with the utilization of the novel redox sensors is shedding light on the complex relation between mitochondrial Ca2+ and redox signals and their impact on cell function. In this review,we describe mitochondrial Ca2+ handling,focusing on a number of newly identified proteins involved in mitochondrial Ca2+ uptake and release. We further discuss our recent findings,revealing how mitochondrial Ca2+ influences the matrix redox state. As a result,mitochondrial Ca2+ is able to modulate the many mitochondrial redox-regulated processes linked to normal physiology and disease.     

Multicellular redox regulation: integrating organismal biology and redox chemistry

Early in the 20th century, Charles Manning Child attributed organismal gradients in metabolism to interactions among groups of cells. Metabolic gradients are now firmly grounded in redox chemistry, yet modern work on metabolic signaling has consistently focused on the cellular level. Multicellular redox regulation, however, may occur when redox state is determined by the behavior of a group of cells. For instance, typically an abundance of substrate will shift the redox state of mitochondria in the direction of reduction, leading to increased reactive oxygen species (ROS). These ROS, in turn, may modify the conformation and activity of proteins involved in signaling pathways, resulting in phenotypic changes. In contrast, if substrate triggers the contractions of a muscular structure comprising mitochondrion-rich cells, the resulting metabolic demand may shift the redox state in the direction of oxidation, with a corresponding decrease of ROS and different phenotypic effects. Indeed, colonial hydroids exemplify this process. Parallel examples may occur whenever mitochondria are concentrated in cells of structures that can respond to environmental perturbations with increased metabolic demand. In these circumstances, predicting the direction of metabolic signaling may require an understanding of events at the organismal level.     

Oxidant and redox signaling in vascular oxygen sensing mechanisms: basic concepts, current controversies, and potential importance of cytosolic NADPH

Vascular smooth muscle (VSM) derived from pulmonary arteries generally contract to hypoxia, whereas VSM from systemic arteries usually relax, indicating the presence of basic oxygen-sensing mechanisms in VSM that are adapted to the environment from which they are derived. This review considers how fundamental processes associated with the generation of reactive oxygen species (ROS) by oxidase enzymes, the metabolic control of cytosolic NADH, NADPH and glutathione redox systems, and mitochondrial function interact with signaling systems regulating vascular force in a manner that is potentially adapted to be involved in Po2 sensing. Evidence for opposing hypotheses of hypoxia, either decreasing or increasing mitochondrial ROS, is considered together with the Po2 dependence of ROS production by Nox oxidases as sensors potentially contributing to hypoxic pulmonary vasoconstriction. Processes through which ROS and NAD(P)H redox changes potentially control interactive signaling systems, including soluble guanylate cyclase, potassium channels, and intracellular calcium are discussed together with the data supporting their regulation by redox in responses to hypoxia. Evidence for hypothesized potential differences between systemic and pulmonary arteries originating from properties of mitochondrial ROS generation and the redox sensitivity of potassium channels is compared with a new hypothesis in which differences in the control of cytosolic NADPH redox by the pentose phosphate pathway results in increased NADPH and Nox oxidase-derived ROS in pulmonary arteries, whereas lower levels of glucose-6-phosphate dehydrogenase in coronary arteries may permit hypoxia to activate a vasodilator mechanism controlled by oxidation of cytosolic NADPH.     

Thiol redox proteomics: Characterization of thiol-based post-translational modifications

Redox post-translational modifications on cysteine thiols (redox PTMs) have profound effects on protein structure and function, thus enabling regulation of various biological processes. Redox proteomics approaches aim to characterize the landscape of redox PTMs at the systems level. These approaches facilitate studies of condition-specific, dynamic processes implicating redox PTMs and have furthered our understanding of redox signaling and regulation. Mass spectrometry (MS) is a powerful tool for such analyses which has been demonstrated by significant advances in redox proteomics during the last decade. A group of well-established approaches involves the initial blocking of free thiols followed by selective reduction of oxidized PTMs and subsequent enrichment for downstream detection. Alternatively, novel chemoselective probe-based approaches have been developed for various redox PTMs. Direct detection of redox PTMs without any enrichment has also been demonstrated given the sensitivity of contemporary MS instruments. This review discusses the general principles behind different analytical strategies and covers recent advances in redox proteomics. Several applications of redox proteomics are also highlighted to illustrate how large-scale redox proteomics data can lead to novel biological insights.     

Influence of vitamin C and vitamin E on redox signaling: Implications for exercise adaptations

The exogenous antioxidants vitamin C (ascorbate) and vitamin E (α-tocopherol) often blunt favorable cell signaling responses to exercise, suggesting that redox signaling contributes to exercise adaptations. Current theories posit that this antioxidant paradigm interferes with redox signaling by attenuating exercise-induced reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation. The well-documented in vitro antioxidant actions of ascorbate and α-tocopherol and characterization of the type and source of the ROS/RNS produced during exercise theoretically enable identification of redox-dependent mechanisms responsible for the blunting of favorable cell signaling responses to exercise. This review aimed to apply this reasoning to determine how the aforementioned antioxidants might attenuate exercise-induced ROS/RNS production. The principal outcomes of this analysis are (1) neither antioxidant is likely to attenuate nitric oxide signaling either directly (reaction with nitric oxide) or indirectly (reaction with derivatives, e.g., peroxynitrite); (2) neither antioxidant reacts appreciably with hydrogen peroxide, a key effector of redox signaling; (3) ascorbate but not α-tocopherol has the capacity to attenuate exercise-induced superoxide generation; and (4) alternate mechanisms, namely pro-oxidant side reactions and/or reduction of bioactive oxidized macromolecule adducts, are unlikely to interfere with exercise-induced redox signaling. Out of all the possibilities considered, ascorbate-mediated suppression of superoxide generation with attendant implications for hydrogen peroxide signaling is arguably the most cogent explanation for blunting of favorable cell signaling responses to exercise. However, this mechanism is dependent on ascorbate accumulating at sites rich in NADPH oxidases, principal contributors to contraction-mediated superoxide generation, and outcompeting nitric oxide and superoxide dismutase isoforms. The major conclusions of this review are: (1) direct evidence for interference of ascorbate and α-tocopherol with exercise-induced ROS/RNS production is lacking; (2) theoretical analysis reveals that both antioxidants are unlikely to have a major impact on exercise-induced redox signaling; and (3) it is worth considering alternate redox-independent mechanisms.     

Pyrroloquinoline quinone stimulates epithelial cell proliferation by activating epidermal growth factor receptor through redox cycling

Pyrroloquinoline quinone (PQQ), a redox cofactor for bacterial dehydrogenases, has been implicated to be an important nutrient in mammals functioning as a potent growth factor. However, the underlying molecular mechanisms have not been elucidated. The present study revealed that PQQ induces the activation (tyrosine autophosphorylation) of epidermal growth factor receptor (EGFR) and its downstream signaling in a ligand-independent manner, leading to increased cellular proliferation in an epithelial cell line A431. PQQ inhibited protein tyrosine phosphatase 1B (PTP1B), which negatively regulates the EGFR signaling by tyrosine dephosphorylation, to oxidatively modify the catalytic cysteine through its redox cycling activity to generate H(2)O(2). PQQ-inducible intracellular ROS production and EGFR activation were significantly suppressed by the pre-treatment with antioxidants. The intracellular redox state regulates the EGFR signaling through the redox-sensitive catalytic cysteine of PTP1B and modulates cell proliferation. Our data suggest that PQQ may stimulate epithelial cell proliferation by activating EGFR by oxidation and subsequent inactivation of PTP1B via its redox cycling. Our results provide novel insight into the mechanisms by which PQQ may function as a growth factor to contribute to mammalian growth.     

Primary antioxidant free radical scavenging and redox signaling pathways in higher plant cells

Antioxidants in plant cells mainly include glutathione, ascorbate, tocopherol, proline, betaine and others, which are also information-rich redox buffers and important redox signaling components that interact with cellular compartments. As an unfortunate consequence of aerobic life for higher plants, reactive oxygen species (ROS) are formed by partial reduction of molecular oxygen. The above enzymatic and non-enzymatic antioxidants in higher plant cells can protect their cells from oxidative damage by scavenging ROS. In addition to crucial roles in defense system and as enzyme cofactors, antioxidants influence higher plant growth and development by modifying processes from miotosis and cell elongation to senescence and death. Most importantly, they provide essential information on cellular redox state, and regulate gene expression associated with biotic and abiotic stress responses to optimize defense and survival. An overview of the literature is presented in terms of primary antioxidant free radical scavenging and redox signaling in plant cells. Special attention is given to ROS and ROS-anioxidant interaction as a metabolic interface for different types of signals derived from metabolisms and from the changing environment. This interaction regulates the appropriate induction of acclimation processes or execution of cell death programs, which are the two essential directions for higher plant cells.     

Higher plant antioxidants and redox signaling under environmental stresses

Main antioxidants in higher plants include glutathione, ascorbate, tocopherol, proline, betaine, and others, which are also information-rich redox buffers and important redox signaling components that interact with biomembrane-related compartments. As an evolutionary consequence of aerobic life for higher plants, reactive oxygen species (ROS) are formed by partial reduction of molecular oxygen. The above enzymatic and non-enzymatic antioxidants in higher plants can protect their cells from oxidative damage by scavenging ROS. In addition to crucial roles in defense system and as enzyme cofactors, antioxidants influence higher plant growth and development by modifying processes from mitosis and cell elongation to senescence and death. Most importantly, they provide essential information on cellular redox state, and regulate gene expression associated with biotic and abiotic stress responses to optimize defense and survival. An overview of the literature is presented in terms of main antioxidants and redox signaling in plant cells. Special attention is given to ROS and ROS-antioxidant interaction as a metabolic interface for different types of signals derived from metabolism and from the changing environment, which regulates the appropriate induction of acclimation processes or, execution of cell death programs, which are the two essential directions for higher plants.     

Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling

Reactive oxygen species (ROS) are generated during mitochondrial oxidative metabolism as well as in cellular response to xenobiotics, cytokines, and bacterial invasion. Oxidative stress refers to the imbalance due to excess ROS or oxidants over the capability of the cell to mount an effective antioxidant response. Oxidative stress results in macromolecular damage and is implicated in various disease states such as atherosclerosis, diabetes, cancer, neurodegeneration, and aging. Paradoxically, accumulating evidence indicates that ROS also serve as critical signaling molecules in cell proliferation and survival. While there is a large body of research demonstrating the general effect of oxidative stress on signaling pathways, less is known about the initial and direct regulation of signaling molecules by ROS, or what we term the "oxidative interface." Cellular ROS sensing and metabolism are tightly regulated by a variety of proteins involved in the redox (reduction/oxidation) mechanism. This review focuses on the molecular mechanisms through which ROS directly interact with critical signaling molecules to initiate signaling in a broad variety of cellular processes, such as proliferation and survival (MAP kinases, PI3 kinase, PTEN, and protein tyrosine phosphatases), ROS homeostasis and antioxidant gene regulation (thioredoxin, peroxiredoxin, Ref-1, and Nrf-2), mitochondrial oxidative stress, apoptosis, and aging (p66Shc), iron homeostasis through iron-sulfur cluster proteins (IRE-IRP), and ATM-regulated DNA damage response.     

Acid sphingomyelinase amplifies redox signaling in Pseudomonas aeruginosa-induced macrophage apoptosis

Recent studies indicate that distinct membrane microdomains, also named lipid rafts, and ceramide play an important role in infectious biology. Ceramide forms larger ceramide-enriched membrane platforms that are required for diverse signal transduction. In this study, we demonstrate that ceramide-enriched membrane platforms are critically involved in redox signaling that regulates alveolar macrophage apoptosis upon infection with Pseudomonas aeruginosa. In freshly isolated alveolar macrophages, P. aeruginosa infection results in rapid activation of acid sphingomyelinase (Asm), release of ceramide, and formation of ceramide-enriched membrane platforms, which are required for P. aeruginosa-induced activation of NADPH oxidase and production of reactive oxygen species (ROS). Inhibition of NADPH oxidase or removal of intracellular ROS reduced P. aeruginosa-induced activation of the Asm and formation of ceramide-enriched membrane platforms, suggesting that NADPH oxidase-derived ROS regulate Asm-initiated redox signaling in a positive feedback manner. Furthermore, stimulation of JNK and induction of apoptosis upon P. aeruginosa infections are dependent on NADPH oxidase-derived ROS. These findings indicate that ceramide-enriched membrane platforms are essential for amplification of Asm-mediated redox signaling, which mediates JNK activation and thereby apoptosis of alveolar macrophages upon P. aeruginosa infection.     

Detection of reactive oxygen species-sensitive thiol proteins by redox difference gel electrophoresis: implications for mitochondrial redox signaling

Reactive oxygen species (ROS) produced by the mitochondrial respiratory chain can be a redox signal, but whether they affect mitochondrial function is unclear. Here we show that low levels of ROS from the respiratory chain under physiological conditions reversibly modify the thiol redox state of mitochondrial proteins involved in fatty acid and carbohydrate metabolism. As these thiol modifications were specific and occurred without bulk thiol changes, we first had to develop a sensitive technique to identify the small number of proteins modified by endogenous ROS. In this technique, redox difference gel electrophoresis, control, and redox-challenged samples are labeled with different thiol-reactive fluorescent tags and then separated on the same two-dimensional gel, enabling the sensitive detection of thiol redox modifications by changes in the relative fluorescence of the two tags within a single protein spot, followed by protein identification by mass spectrometry. Thiol redox modification affected enzyme activity, suggesting that the reversible modification of enzyme activity by ROS from the respiratory chain may be an important and unexplored mode of mitochondrial redox signaling.     

A redox signaling mechanism for density-dependent inhibition of cell growth

Reactive oxygen species (ROS) have recently drawn significant attention as putative mitogenic mediators downstream of activated growth factor receptors and oncogenic Ras; however, the possibility that a redox-related mechanism also operates in the negative control of cell proliferation by inhibitory signals has not been investigated thus far. Here we show that the arrest of growth induced by cell confluence ("contact inhibition") is due, at least in part, to a decrease in the steady-state levels of intracellular ROS and the consequent impairment of mitogenic redox signaling. In confluent fibroblast cultures, the decrease in the concentration of oxygen species was associated with diminished activity of the small GTPase Rac-1, a signal transducer directly involved in the ligand-dependent generation of oxygen-derived molecules, and was effectively mimicked by exposure of sparse cultures to dithiothreitol (DTT) and inhibitors of enzymes (phospholipase A2 and lipoxygenase) acting in the arachidonic acid cascade downstream of growth factor receptors and Rac-1. Sparse fibroblasts treated with nontoxic amounts of DTT underwent growth arrest, whereas a low concentration of hydrogen peroxide significantly increased thymidine incorporation in confluent cultures, demonstrating a causal link between redox changes and growth control by cell density. Removal of oxygen species from sparse cultures was accompanied by a drastic decrease of protein tyrosine phosphorylation after epidermal growth factor stimulation, which, at a biochemical level, reproduced the signaling hallmarks of contact inhibition. Moreover, the cytosolic tyrosine phosphatase SHP-2 was identified as a putative target for redox signaling by cell density because the enzyme itself and the associated substrates appear markedly dephosphorylated in both confluent and reductant-treated cells after exposure to epidermal growth factor, and SHP-2 enzymatic activity is strongly activated by DTT in vitro. Taken together, these data support a model in which impaired generation of ROS and increased protein tyrosine phosphatase activity impede mitogenic signaling in contact-inhibited cells.     

Thiol‐based redox proteins in abscisic acid and methyl jasmonate signaling in Brassica napus guard cells

Reversibly oxidized cysteine sulfhydryl groups serve as redox sensors or targets of redox sensing that are important in various physiological processes. However, little is known about redox‐sensitive proteins in guard cells and how they function in stomatal signaling. In this study, Brassica napus guard‐cell proteins altered by redox in response to abscisic acid (ABA) or methyl jasmonate (MeJA) were identified by complementary proteomics approaches, saturation differential in‐gel electrophoresis and isotope‐coded affinity tagging. In total, 65 and 118 potential redox‐responsive proteins were identified in ABA‐ and MeJA‐treated guard cells, respectively. All the proteins contain at least one cysteine, and over half of them are predicted to form intra‐molecular disulfide bonds. Most of the proteins fall into the functional groups of ‘energy’, ‘stress and defense’ and ‘metabolism’. Based on the peptide sequences identified by mass spectrometry, 30 proteins were common to ABA‐ and MeJA‐treated samples. A total of 44 cysteines were mapped in the identified proteins, and their levels of redox sensitivity were quantified. Two of the proteins, a sucrose non‐fermenting 1‐related protein kinase and an isopropylmalate dehydrogenase, were confirmed to be redox‐regulated and involved in stomatal movement. This study creates an inventory of potential redox switches, and highlights a protein redox regulatory mechanism in ABA and MeJA signal transduction in guard cells.     

Glucohexaose-induced protein phosphatase 2C regulates cell redox status of cucumber seedling

Protein Phosphatase 2C (PP2C) is an important phosphatase-like protein in eukaryotic organisms that can negatively regulate protein kinase cascade abscisic acid (ABA) signal system through phosphorylation and carry out vital roles in various cell processes. The previous study indicated that the accumulation of reactive oxygen species (ROS) is a part of mechanism of glucohexaose-induced resistance in cucumber cotyledons, and CsPP2C80s might play a crucial role in processes related to ROS produce and signal transduction. To identify the mechanism of CsPP2C80s involved in glucohexaose and ABA signaling regulating cell redox status, the effects of glucohexaose and ROS inhibitor pretreatment on endogenous ABA content and ABA signaling genes expression levels of cucumber seedlings were analysed. These results suggest that cucumber CsPP2C80s are involved in ROS accumulation and ABA signal transduction pathway induced by glucohexaose, CsPP2C80s play a positive regulatory role in process of ABA combined with ABA receptors (PYLs) to activate SNF1-related protein kinases 2 (SnRK2s) and regulate NADPH oxidase to produce extracellular hydrogen peroxide (H₂O₂), providing unequivocal molecular evidence of PP2C-mediated ABA response mechanisms functioning in cell redox status induced by glucohexaose.     

Intraperoxisomal redox balance in mammalian cells: oxidative stress and interorganellar cross-talk

Reactive oxygen species (ROS) are at once unsought by-products of metabolism and critical regulators of multiple intracellular signaling cascades. In nonphotosynthetic eukaryotic cells, mitochondria are well-investigated major sites of ROS generation and related signal initiation. Peroxisomes are also capable of ROS generation, but their contribution to cellular oxidation-reduction (redox) balance and signaling events are far less well understood. In this study, we use a redox-sensitive variant of enhanced green fluorescent protein (roGFP2-PTS1) to monitor the state of the peroxisomal matrix in mammalian cells. We show that intraperoxisomal redox status is strongly influenced by environmental growth conditions. Furthermore, disturbances in peroxisomal redox balance, although not necessarily correlated with the age of the organelle, may trigger its degradation. We also demonstrate that the mitochondrial redox balance is perturbed in catalase-deficient cells and upon generation of excess ROS inside peroxisomes. Peroxisomes are found to resist oxidative stress generated elsewhere in the cell but are affected when the burden originates within the organelle. These results suggest a potential broader role for the peroxisome in cellular aging and the initiation of age-related degenerative disease.     

Mitochondria and the redox control of development in cnidarians

Mitochondria are the product of an ancient symbiosis between bacteria and host cells. While mitochondria function primarily in energy conversion, increasing amounts of evidence indicate that mitochondrial metabolic state can influence various emergent features of eukaryotic cells. Important intermediaries in such redox signaling include by-products of metabolism, particularly reactive oxygen species (ROS). This review uses cnidarians, a group of basally branching animals, to illustrate the many and varied effects of ROS on development. ROS from both mitochondria and algal symbionts are considered. Because some applications of ROS may lack specificity, the signaling demands of mitochondria and algae may to some extent conflict. An extensive algal symbiosis may thus be incompatible with a well-developed capacity for mitochondrial signaling.     

Multicellular redox regulation in an early-evolving animal treated with glutathione

Redox signaling has emerged as a unifying theme in many seemingly disparate disciplines. Such signaling has been widely studied in bacteria and eukaryotic organelles and is often mediated by reactive oxygen species (ROS). In this context, reduced glutathione (GSH) acts as an important intracellular antioxidant, diminishing ROS and potentially affecting redox signaling. Complementing this cell-level perspective, colonial hydroids can be a useful model for understanding organism-level redox signaling. These simple, early-evolving animals consist of feeding polyps connected by tubelike stolons. Colonies treated exogenously with GSH or reduced glutathione ethyl ester (GEE) were expected to show a morphological change to sheetlike growth typical of low levels of ROS. Contrary to expectations, diminished stolon branching and polyp initiation was observed. Such runnerlike growth is associated with higher levels of ROS, and surprisingly, such higher levels were found in GSH- and GEE-treated colonies. Further investigations show that GSH triggered a feeding response in hydroid polyps, increasing oxygen uptake but at the same time relaxing mitochondrion-rich contractile regions at the base of polyps. Diminished gastrovascular flow and increased emissions of mitochondrial ROS also correlated with the observed runnerlike growth. In contrast to cell-level, "bottom-up" views of redox signaling, here the phenotype may arise from a "top-down" interaction of mitochondrion-rich regions and organism-level physiology. Such multicellular redox regulation may commonly occur in other animals as well.