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PubMed1998

Infection and reactive oxygen species.

In previous years the physiologic and pathophysiologic significance of reactive oxygen species (ROS) on sperm function has been recognized. The impact of ROS during the invasion, adhesion and multiplication of microorganisms in the male genital tract are largely unknown. However, it is known that the resulting activation of leukocytes leads to an increased generation of ROS. There is growing evidence that spermatozoa are protected from detrimental ROS effects by the powerful antioxidants in seminal plasma since disturbances of sperm function by ROS were demonstrated in the absence of seminal plasma, i.e., during epididymitis or after semen preparation. If seminal plasma is present, ROS generated by physiologic numbers of granulocytes (< 1 x 10(6) ml-1) apparently do not damage spermatozoa. Interestingly, ROS generated by leukocytes during male genital tract infections are critical for the techniques of semen preparation for assisted reproduction. These ROS impair sperm function if the protective effects of seminal plasma are not present. The relevance of ROS production by higher leukocyte numbers in human semen is presently unknown as is the relevance of ROS generated in the female reproductive tract.

Female↗

PubMed2005

Reactive oxygen species amplify glucose signalling in renal cells cultured under high glucose and in diabetic kidney.

Diabetic nephropathy is characterized by excessive accumulation of extracellular matrix (ECM) in the kidney. Reactive oxygen species (ROS) play a central role in the ECM synthesis and degradation in the glomeruli and tubulointerstitium leading to renal fibrosis. High glucose (HG) induces cellular ROS through protein kinase C (PKC)-dependent activation of NADPH oxidase and through mitochondrial metabolism. ROS thus generated activate signal transduction cascade (PKC, mitogen-activated protein kinases, and janus kinase/signal transducers and activators of transcription) and transcription factors (nuclear factor-kappaB, activated protein-1, and specificity protein-1), up-regulate transforming growth factor-beta1 (TGF-beta1), angiotensin II (Ang II), monocyte chemoattractant protein-1 (MCP-1), and plasminogen activator inhibitor-1 (PAI-1) gene and protein expression, and promote formation of advanced glycation end-products (AGE). PKC, TGF-beta1, Ang II, and AGE also induce cellular ROS and signal through ROS leading to enhanced ECM synthesis. NF-kappaB-MCP-1 pathway is activated by ROS and promotes monocyte recruitment and profibrotic process in the kidney. HG- and TGF-beta1-induced PAI-1 up-regulation is mediated by ROS and contribute to ECM accumulation via suppression of plasmin ativity. TGF-beta1-induced myofibroblast transformation of renal tubular epithelial cells (epithelial-mesenchymal transition) is also mediated by ROS and contribute to tubulointerstitial fibrosis. In summary, ROS transduce and amplify glucose signalling in renal cells under high glucose environment and play a critical role in excessive ECM deposition in the diabetic kidney. A better understanding of ROS production and removal will allow more effective therapeutic strategies in diabetic renal and other vascular complications.

Animals↗

PubMed2002

Generation of reactive oxygen and nitrogen species in contracting skeletal muscle: potential impact on aging.

Since the early 1980s biologists have recognized that skeletal muscle generates free radicals. Of particular interest are two closely related redox cascades--reactive oxygen species (ROS) and nitric oxide (NO) derivatives. The ROS cascade is initiated by superoxide anion radicals derived from the mitochondrial electron transport chain, the membrane-associated NAD(P)H oxidase complex, or other sources. NO is produced by two NO synthase isoforms constitutively expressed by muscle fibers. ROS and NO derivatives are produced continually and are detectable in both the cytosolic and extracellular compartments. Production increases during strenuous exercise. Both ROS and NO modulate contractile function. Under basal conditions, low levels of ROS enhance force production. Excessive ROS accumulation inhibits force, for example, during fatiguing exercise. NO inhibits skeletal muscle contraction, an effect that is partially mediated by cyclic GMP as a second messenger. With aging, redox modulation of muscle contraction may be altered by changes in the rates of ROS and NO production, the levels of endogenous antioxidants that buffer ROS and NO, and the sensitivities of regulatory proteins to ROS and NO action. The impact of aging on contractile regulation depends on the relative magnitude of these changes and their net effects on ROS and NO activities at the cellular level.

Aging↗

PubMed2005

Reactive oxygen species elicit apoptosis by concurrently disrupting topoisomerase II and DNA-dependent protein kinase.

Reactive oxygen species (ROS) are produced by all aerobic cells and have been implicated in the regulation of diverse cellular functions, including intracellular signaling, transcription activation, proliferation, and apoptosis. Salvicine, a novel diterpenoid quinone compound, demonstrates a broad spectrum of antitumor activities. Although salvicine is known to trap the DNA-topoisomerase II (Topo II) complex and induce DNA double-strand breaks (DSBs), its precise antitumor mechanisms remain to be clarified. In this study, we investigated whether salvicine altered the levels of ROS in breast cancer MCF-7 cells and whether these ROS contributed to the observed antitumoral activity. Our data revealed that salvicine stimulated intracellular ROS production and subsequently elicited notable DSBs. The addition of N-acetyl cysteine (NAC), an antioxidant, effectively attenuated the salvicine-induced ROS enhancement and subsequent DNA DSBs. Heat treatment reversed the accumulation of DNA DSBs, and the addition of NAC attenuated the Topo II-DNA cleavable complexes formation and the growth inhibition of salvicine-treated JN394top2-4 yeast cells, collectively indicating that Topo II is a target of the salvicine-induced ROS. On the other hand, when examining the impact of salvicine on DNA repair pathways, we unexpectedly observed that salvicine selectively down-regulated the catalytic subunit of DNA-dependent protein kinase (DNA-PK(CS)) protein levels and repressed DNA-PK kinase activity; both of these effects were attenuated by NAC pretreatment of MCF-7 cells. Finally and most importantly, NAC attenuated salvicine-induced apoptosis and cytotoxicity in MCF-7 cells. These results indicate that apart from its direct actions, salvicine generates ROS that modulate DNA damage and repair, contributing to the comprehensive biological consequences of salvicine treatment, such as DNA DSBs, apoptosis, and cytotoxicity in tumor cells. The finding of salvicine-induced ROS provides new evidence for the molecular mechanisms of this compound. Moreover, the effects of salvicine-induced ROS on Topo II and DNA-PK give new insights into the diverse biological activities of ROS.

Acetylcysteine↗

PubMed2003

Influence of induced reactive oxygen species in p53-mediated cell fate decisions.

The p53 tumor suppressor gene can induce either apoptosis or a permanent growth arrest (also termed senescence) phenotype in response to cellular stresses. We show that the increase in intracellular reactive oxygen species (ROS) associated with the magnitude of p53 protein expression correlated with the induction of either senescence or apoptosis in both normal and cancer cells. ROS inhibitors ameliorated both p53-dependent cell fates, implicating ROS accumulation as an effector in each case. The absence of Bax or PUMA strongly inhibited both p53-induced apoptosis and ROS increase, indicating an important role these p53 targets affecting mitochondrial function genes in p53-mediated ROS accumulation. Moreover, physiological p53 levels in combination with an exogenous ROS source were able to convert a p53 senescence response into apoptosis. All of these findings establish a critical role of ROS accumulation and mitochondrial function in p53-dependent cell fates and show that other ROS inducers can collaborate with p53 to influence these fate decisions. Thus, our studies imply that therapeutic agents that generate ROS are more likely to be toxic for normal cells than p53-negative tumor cells and provide a rationale for identifying therapeutic agents that do not complement p53 in ROS generation to ameliorate the cytotoxic side effects in normal cells.

Apoptosis↗

PubMed2006

O2 delivery and redox state are determinants of compartment-specific reactive O2 species in myocardial reperfusion.

Reperfusion of the ischemic myocardium leads to a burst of reactive O(2) species (ROS), which is a primary determinant of postischemic myocardial dysfunction. We tested the hypothesis that early O(2) delivery and the cellular redox state modulate the initial myocardial ROS production at reperfusion. Isolated buffer-perfused rat hearts were loaded with the fluorophores dihydrofluorescein or Amplex red to detect intracellular and extracellular ROS formation using surface fluorometry at the left ventricular wall. Hearts were made globally ischemic for 20 min and then reperfused with either 95% or 20% O(2)-saturated perfusate. The same protocol was repeated in hearts loaded with dihydrofluorescein and perfused with either 20 or 5 mM glucose-buffered solution to determine relative changes in NADH and FAD. Myocardial O(2) delivery during the first 5 min of reperfusion was 84.7 +/- 4.2 ml O(2)/min with 20% O(2)-saturated buffer and 354.4 +/- 22.8 ml O(2)/min with 95% O(2) (n = 8/group, P < 0.001). The fluorescein signal (intracellular ROS) was significantly increased in hearts reperfused with 95% O(2) compared with 20% O(2). However, the resorufin signal (extracellular ROS) was significantly increased with 20% O(2) compared with 95% O(2) during reperfusion. Perfusion of hearts with 20 mM glucose reduced the (.)NADH during ischemia (P < 0.001) and the (.)ROS at reperfusion (P < 0.001) compared with 5.5 mM-perfused glucose hearts. In conclusion, initial O(2) delivery to the ischemic myocardium modulates a compartment-specific ROS response at reperfusion such that high O(2) delivery promotes intracellular ROS and low O(2) delivery promotes extracellular ROS. The redox state that develops during ischemia appears to be an important precursor for reperfusion ROS production.

Adaptation, Physiological↗

PubMed2005

NAD(P)H oxidase and uncoupled nitric oxide synthase are major sources of glomerular superoxide in rats with experimental diabetic nephropathy.

Increased production of reactive oxygen species (ROS) in diabetes may be a common pathway linking diverse pathogenic mechanisms of diabetic vascular complications, including nephropathy. Assessment of the oxidative stress production pathway is therefore important for the prediction and prevention of diabetic complications. However, ROS production mechanisms remain unclear in diabetic glomeruli. To identify the source and determine the mechanisms of ROS production in the diabetic kidney, diabetes was induced with streptozotocin in rats. After 6 wk, glomerular ROS production had increased in the streptozotocin rat kidney, as assessed by dihydroethidium-derived chemiluminescence. ROS production was increased by the addition of NADH or L-arginine and was partially reduced by the addition of diphenylene iodonium or N(G)-nitro-L-arginine methyl ester, identifying NAD(P)H oxidase and nitric oxide (NO) synthase (NOS) as ROS sources. The mRNA and protein expression of endothelial NOS (eNOS), as measured by real-time RT-PCR and Western blotting, increased significantly (mRNA level, 1.3-fold; protein level, 1.8-fold). However, the dimeric form of eNOS was decreased in diabetic glomeruli, as measured by low-temperature SDS-PAGE. Production of renal ROS and NO by uncoupled NOS was imaged by confocal laser microscopy after renal perfusion of 2',7'-dichlorofluorescein diacetate (a ROS marker) and diaminorhodamine-4M AM (a NO marker) with L-arginine. Accelerated ROS production and diminished bioavailable NO caused by NOS uncoupling were noted in the diabetic kidney. Administration of tetrahydrobiopterin (BH4), a cofactor for eNOS, reversed the decreased dimeric form of eNOS and glomerular NO production. Our results indicate that NAD(P)H oxidase and uncoupling of eNOS contribute to glomerular ROS production, mediated by the loss of BH4 availability. These mechanisms are potential key targets for therapeutic interventions.

Animals↗

PubMedDate not supplied

Mitochondria in exercise-induced oxidative stress.

In recent years it has been suggested that reactive oxygen species (ROS) are involved in the damage to muscle and other tissues induced by acute exercise. Despite the small availability of direct evidence for ROS production during exercise, there is an abundance of literature providing indirect support that oxidative stress occurs during exercise. The electron transport associated with the mitochondrial respiratory chain is considered the major process leading to ROS production at rest and during exercise. It is widely assumed that during exercise the increased electron flow through the mitochondrial electron transport chain leads to an increased rate of ROS production. On the other hand, results obtained by in vitro experiments indicate that mitochondrial ROS production is lower in state 3 (ADP-stimulated) than in state 4 (basal) respiration. It is possible, however, that factors, such as temperature, that are modified in vivo during intense physical activity induce changes (uncoupling associated with loss of cytochrome oxidase activity) leading to increased ROS production. The mitochondrial respiratory chain could also be a potential source of ROS in tissues, such as liver, kidney and nonworking muscles, that during exercise undergo partial ischemia because of reduced blood supply. Sufficient oxygen is available to interact with the increasingly reduced respiratory chain and enhance the ROS generation. At the cessation of exercise, blood flow to hypoxic tissues resumes leading to their reoxygenation. This mimics the ischemia-reperfusion phenomenon, which is known to cause excessive production of free radicals. Apart from a theoretical rise in ROS, there is little evidence that exercise-induced oxidative stress is due to its increased mitochondrial generation. On the other hand, if mitochondrial production of ROS supplies a remarkable contribution to exercise-induced oxidative stress, mitochondria should be a primary target of oxidative damage. Unfortunately, there are controversial reports concerning the exercise effects on structural and functional characteristics of mitochondria. However, the isolation of mitochondrial fractions by differential centrifugation has shown that the amount of damaged mitochondria, recovered in the lightest fraction, is remarkably increased by long-lasting exercise.

Animals↗

PubMed2003

Oxidative stress mediates tumor necrosis factor-alpha-induced mitochondrial DNA damage and dysfunction in cardiac myocytes.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) and angiotensin II (Ang II) are implicated in the development and further progression of heart failure, which might be, at least in part, mediated by the production of reactive oxygen species (ROS). However, the cause and consequences of this agonist-mediated ROS production in cardiac myocytes have not been well defined. Recently, we demonstrated that increased ROS production was associated with mitochondrial DNA (mtDNA) damage and dysfunction in failing hearts. We thus investigated whether the direct exposure of cardiac myocytes to TNF-alpha and Ang II in vitro could induce mtDNA damage via production of ROS. METHODS AND RESULTS: TNF-alpha increased ROS production within cultured neonatal rat ventricular myocytes after 1 hour, as assessed by 2',7'-dichlorofluorescin diacetate fluorescence microscopy. TNF-alpha also decreased mtDNA copy number by Southern blot analysis in association with complex III activity, which was prevented in the presence of the antioxidant alpha-tocopherol. A direct exposure of myocytes to H2O2 caused a similar decrease in mtDNA copy number. In contrast, Ang II did not affect mtDNA copy number, despite the similar increase in ROS production. TNF-alpha-mediated ROS production and a decrease in mtDNA copy number were inhibited by the sphingomyelinase inhibitor D609. Furthermore, N-acetylsphingosine (C2-ceramide), a synthetic cell-permeable ceramide analogue, increased myocyte ROS production, suggesting that TNF-alpha-mediated ROS production and subsequent mtDNA damage were mediated by the sphingomyelin-ceramide signaling pathway. CONCLUSIONS: The intimate link between TNF-alpha, ROS, and mtDNA damage might play an important role in myocardial remodeling and failure.

Angiotensin II↗

PubMed1998

Endothelial NADPH oxidase as the source of oxidants in lungs exposed to ischemia or high K+.

We have previously demonstrated the generation of reactive oxygen species (ROS) in cultured bovine pulmonary artery endothelial cells (BPAECs) and in isolated perfused rat lungs exposed to high K+ and during global lung ischemia. The present study evaluates the NADPH oxidase pathway as a source of ROS in these models. ROS production, detected by oxidation of the fluorophore, dichlorodihydrofluorescein, increased 2.5-fold in BPAECs and 6-fold in rat or mouse lungs exposed to high (24 mmol/L) K+. ROS generation was markedly inhibited by diphenyliodonium, a flavoprotein inhibitor, and by the synthetic peptide PR-39, an inhibitor of NADPH oxidase assembly, whereas allopurinol had no effect. With ischemia (1 hour), ROS generation by rat and mouse lungs increased 7-fold; PR-39 showed concentration-dependent inhibition of ROS production, with 50% inhibition at 3 micromol/L PR-39. ROS production in lungs exposed to high K+ or ischemia was essentially abolished in mice with a "knockout" of gp91(phox), a membrane-localized cytochrome component of NADPH oxidase; increased ROS production by these lungs after anoxia/reoxygenation was similar to control. PR-39 also inhibited ischemia and the high K+-mediated increase in lung thiobarbituric acid reactive substance. Western blotting of BPAECs and immunocytochemistry of BPAECs and rat and mouse lungs showed the presence of p47phox, a cytoplasmic component of NADPH oxidase and the putative target for PR-39 inhibition. In situ fluorescence imaging in the intact lung demonstrated that the increased dichlorofluorescein fluorescence in these models of ROS generation was localized primarily to the pulmonary endothelium. These studies demonstrate that ROS production in lungs exposed to ischemia or high K+ results from assembly and activation of a membrane-associated NAPDH oxidase of the pulmonary endothelium.

Animals↗

PubMed2004

Changes in retinal pigment epithelial gene expression induced by rod outer segment uptake.

PURPOSE: Rod outer segment (ROS) uptake, a crucial function of the retinal pigment epithelium (RPE), probably involve multiple proteins, yet only a small number have so far been identified. The goal of this study was to find additional genes involved in ROS uptake and degradation by identifying ROS-induced gene expression changes. METHODS: Human RPE-derived ARPE-19 cells were harvested 3 and 12 hours after addition of bovine ROS. Gene expression profiles were compared with control cultures by using a custom human retina cDNA microarray and were validated by quantitative real-time RT-PCR (QPCR). ROS binding and internalization were quantitated with a fluorescence assay. RESULTS: Alterations in the expression levels of multiple genes (especially ones involved in transcriptional regulation, signal transduction, or protein modification) were detected 3 hours after ROS challenge, whereas by 12 hours most had returned to baseline. QPCR results corroborated the microarray results for seven of the eight genes tested. Time-course QPCR experiments on an independent sample set demonstrated characteristic temporal expression changes for each gene. Protein levels of one of these, plasminogen activator inhibitor-1 (PAI-1), were tested and found to parallel the mRNA changes. In addition, exogenous PAI-1 inhibited ROS uptake by RPE cells in vitro, consistent with its putative function in integrin receptor regulation. CONCLUSIONS: ROS uptake is associated with regulation of multiple RPE genes in a gene-specific temporal pattern. These genes are candidates for involvement in ROS uptake and degradation, particularly PAI-1, for which the study provided evidence suggesting that it may participate in the negative feedback of ROS uptake.

Animals↗

PubMed2006

Chronic ethanol feeding increases activation of NADPH oxidase by lipopolysaccharide in rat Kupffer cells: role of increased reactive oxygen in LPS-stimulated ERK1/2 activation and TNF-alpha production.

Reactive oxygen species (ROS) contribute to the development of chronic ethanol-induced liver injury. Although ROS modulate the activity of many signal transduction pathways, the molecular targets of ROS during ethanol exposure are not well understood. Here, we investigated whether specific ROS-sensitive signal transduction pathways contribute to increased tumor necrosis factor alpha (TNF-alpha) production by Kupffer cells after chronic ethanol feeding to rats. Lipopolysaccharide (LPS) rapidly increased ROS production, measured by dihydrorhodamine fluorescence, in Kupffer cells from ethanol- and pair-fed rats, and ROS production was 2.5-fold greater in ethanol-fed compared with pair-fed. Pretreatment with diphenyleneiodonium (DPI), which inhibits reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, normalized ROS production in Kupffer cells from ethanol-fed rats. LPS rapidly increased Rac1-guanosinetriphosphatase (GTPase) activity and p67(phox) translocation to the plasma membrane in Kupffer cells from pair-fed rats. After ethanol feeding, Rac1-GTPase activity was already increased over pair-fed at baseline and remained elevated over pair-fed after LPS stimulation. Further, LPS-stimulated p67(phox) translocation to the plasma membrane was enhanced after chronic ethanol feeding. LPS-stimulated extracellular signal-regulated kinase (ERK)1/2 and p38 phosphorylation, two signaling pathways regulated by ROS, were increased twofold in Kupffer cells from ethanol-fed rats compared with pair-fed controls. However, only LPS-stimulated ERK1/2 phosphorylation was inhibited by DPI, which also reduced LPS-stimulated TNF-alpha production in Kupffer cells from pair- and ethanol-fed rats. These results demonstrate that chronic ethanol feeding increases LPS-stimulated NADPH oxidase-dependent production of ROS in Kupffer cells. Further, ERK1/2 is an important target of NADPH oxidase-derived ROS in Kupffer cells, contributing to enhanced LPS-stimulated TNF-alpha production by Kupffer cells after chronic ethanol feeding.

Animals↗

PubMed2005

Reactive oxygen species as mediators of cardiac injury and protection: the relevance to anesthesia practice.

Reactive oxygen species (ROS) are central to cardiac ischemic and reperfusion injury. They contribute to myocardial stunning, infarction and apoptosis, and possibly to the genesis of arrhythmias. Multiple laboratory studies and clinical trials have evaluated the use of scavengers of ROS to protect the heart from the effects of ischemia and reperfusion. Generally, studies in animal models have shown such effects. Clinical trials have also shown protective effects of scavengers, but whether this protection confers meaningful clinical benefits is uncertain. Several IV anesthetic drugs act as ROS scavengers. In contrast, volatile anesthetics have recently been demonstrated to generate ROS in the heart, most likely because of inhibitory effects on cardiac mitochondria. ROS are involved in the signaling cascade for cardioprotection induced by brief exposure to a volatile anesthetic (termed "anesthetic preconditioning"). ROS, therefore, although injurious in large quantities, can have a paradoxical protective effect within the heart. In this review we provide background information on ROS formation and elimination relevant to anesthetic and adjuvant drugs with particular reference to the heart. The sources of ROS, the means by which they induce cardiac injury or activate protective signaling pathways, the results of clinical studies evaluating ROS scavengers, and the effects of anesthetic drugs on ROS are each discussed.

Anesthesia↗

PubMed1998

Gap junctional intercellular communication contributes to the contraction of rat osteoblast populated collagen lattices.

The contraction of native collagen lattices by resident mesenchymal cells mimics the organization of collagen during development and repair. Lattice contraction is cell density dependent, suggesting that cell-to-cell communications may contribute to the process. This possibility was investigated by comparing lattice contraction by four rat osteoblastic cell lines: ROS 17/2.8 cells (ROS); ROS transfected with an antisense cDNA sequence of the gap junctional protein connexin 43 (RCx16); ROS transfected with connexin 45 cDNA, a connexin not normally expressed in ROS cells (ROS/Cx45); and ROS transfected with cDNA encoding carboxy-terminal truncated Cx45 (ROS/Cx45tr). The cell coupling indices, which reflect gap junctional communication, were quantitated by the fluorescent dye scrape loading. ROS cells were well coupled (index 3.0), ROS/Cx45tr were better coupled (index 4.2), ROS/Cx45 were poorly coupled (index 1.7), and RCx16 showed no coupling (index 1.1). As determined by immunoblotting, the level of connexin 43 protein was increased in both ROS/Cx45tr and ROS/Cx45 cell lines compared with ROS cells, while the level in RCx16 cells was reduced. ROS populated collagen lattices (PCLs) contracted significantly more at day 5 (177 mm2 to 67 mm2) than ROS/Cx45tr (84 mm2), ROS/Cx45 (108 mm2), or RCx16 (114 mm2). Myosin ATPase activity, which is required for lattice contraction, was equivalent in all four cell lines, indicating that it was not responsible for inhibiting PCL contraction. ROS cells in collagen appeared elongated compared with the other cell lines which were more rounded. These experiments suggest gap junctional communication contributes to PCL contraction by resident osteoblasts.

Animals↗

PubMed2007

Role for mitochondrial reactive oxygen species in brain lipid sensing: redox regulation of food intake.

The ability for the brain to sense peripheral fuel availability is mainly accomplished within the hypothalamus, which detects ongoing systemic nutrients and adjusts food intake and peripheral metabolism as needed. Here, we hypothesized that mitochondrial reactive oxygen species (ROS) could trigger sensing of nutrients within the hypothalamus. For this purpose, we induced acute hypertriglyceridemia in rats and examined the function of mitochondria in the hypothalamus. Hypertriglyceridemia led to a rapid increase in the mitochondrial respiration in the ventral hypothalamus together with a transient production of ROS. Cerebral inhibition of fatty acids-CoA mitochondrial uptake prevented the hypertriglyceridemia-stimulated ROS production, indicating that ROS derived from mitochondrial metabolism. The hypertriglyceridemia-stimulated ROS production was associated with change in the intracellular redox state without any noxious cytotoxic effects, suggesting that ROS function acutely as signaling molecules. Moreover, cerebral inhibition of hypertriglyceridemia-stimulated ROS production fully abolished the satiety related to the hypertriglyceridemia, suggesting that hypothalamic ROS production was required to restrain food intake during hypertriglyceridemia. Finally, we found that fasting disrupted the hypertriglyceridemia-stimulated ROS production, indicating that the redox mechanism of brain nutrient sensing could be modulated under physiological conditions. Altogether, these findings support the role of mitochondrial ROS as molecular actors implied in brain nutrient sensing.

Animals↗

PubMed2006

Determination of intracellular reactive oxygen species and high mitochondrial membrane potential in Percoll-treated viable boar sperm using fluorescence-activated flow cytometry.

The use of frozen semen in the swine industry is limited by problems with viability and fertility compared with liquid semen. Part of the reduction in sperm motility and fertility associated with cryopreservation may be due to oxidative damage from excessive or inappropriate formation of reactive oxygen species (ROS). Chemiluminescence measurements of ROS are not possible in live cells and are problematic because of poor specificity. An alternative approach, flow cytometry, was developed to identify viable boar sperm containing ROS utilizing the dyes hydroethidine and 2', 7'-dichlorodihydrofluorescein diacetate as oxidizable substrates and impermeant DNA dyes to exclude dead sperm. The percentage of sperm with high mitochondrial transmembrane potential was determined by flow cytometry using the mitochondrial probe 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide with propidium iodide staining to exclude nonviable cells. Sperm were incubated with and without ROS generators and free radical scavengers. Basal ROS formation was low (less than 4%) and did not differ (P = 0.26) between viable fresh and frozen-thawed boar sperm. In addition, fresh and frozen-thawed viable sperm were equally susceptible (P = 0.20) to intracellular formation of ROS produced by xanthine/xanthine oxidase (94.4 and 87.9% of sperm, respectively). Menadione increased (P < 0.05) ROS formation, decreased (P < 0.05) JC-1-aggregate fluorescence intensity, and decreased (P < 0.05) motion variables by 25 to 60%. The mechanism of inhibition of motility by ROS formation may be related to a decrease in mitochondrial charge potential below a critical threshold. Catalase and superoxide dismutase treatment in the presence of xanthine/xanthine oxidase indicated that hydrogen peroxide was the primary intracellular ROS measured. Further, catalase, but not superoxide dismutase, was capable of attenuating ROS-induced inhibition of motility. Whereas basal intracellular hydrogen peroxide formation was low in viable fresh and frozen-thawed boar sperm, both were quite susceptible to external sources of hydrogen peroxide.

Animals↗

PubMed1990

Characterization of a galactosyltransferase in purified bovine rod outer segments.

Purified bovine rod outer segments (ROS) were used to study the transfer of labeled galactose from UDP-[3H]galactose to endogenous ROS glycoproteins, exogenous glycoproteins and N-acetylglucosamine (GlcNAc). The ROS reaction was also compared with that of the retinal microsomal fraction and milk galactosyltransferase. The results indicate that the ROS reaction was enhanced by exposure to light. Illumination, however, had no effect on the transfer of labeled galactose to either endogenous microsomal glycoproteins by retinal microsomal galactosyltransferase or the transfer of the sugar to ROS glycoproteins by milk galactosyltransferase. Manganese was most effective, followed by cobalt, as cofactor for the ROS enzyme. Calcium and magnesium produced about 60% of the activity observed with manganese. The ROS enzyme transferred minimal amounts of labeled galactose to asialo-agalactotransferrin or ovalbumin but readily transferred the sugar to GlcNAc. The latter reaction had an optimum pH of 6.3 and was linear for at least 90 min. It reached a maximum at about 30 mM GlcNAc and was inhibited by higher concentrations of the aminosugar and by low concentrations of alpha-lactalbumin. On the other hand, the transfer of galactose to ROS glycoproteins was not affected by low concentrations of alpha-lactalbumin. Our data suggest that the ROS galactosyltransferase may have a certain specificity towards its acceptor in the ROS. Its activation by light may indicate a role in the light-activated processes of the photoreceptor cell.

Acetylglucosamine↗

PubMed1996

A simple flow cytometric technique to quantify rod outer segment phagocytosis in cultured retinal pigment epithelial cells.

PURPOSE: The primary aim of this study was to develop and characterize a simple flow cytometric method of quantifying rod outer segment (ROS) phagocytosis in cultured retinal pigment epithelial (RPE) cells. A secondary aim was to compare the kinetics of ROS phagocytosis in an immortal human RPE cell line with untransformed human RPE cells. METHODS: Flow cytometry was performed on RPE cells that had been challenged with fluorescein isothiocyanate-labeled ROS (FITC-ROS) and phagocytosis was calculated by subtracting background cellular autofluorescence. RESULTS: Non-specific uptake of fluorescent label was negligible and RPE cells phagocytosed FITC-ROS and unlabeled ROS with equal efficacy. The kinetics of FITC-ROS phagocytosis in the D407 RPE cell line differed from early passage untransformed human RPE cultures. FITC-ROS phagocytosis proceeded at a fairly linear rate for the first 12 h in the 3 human cell cultures studied, but was rapid for the first 3 h before slowing in the D407 cells. Within all cell populations, there was a heterogeneity of phagocytic activity which varied with time. CONCLUSIONS: This automated technique for measuring phagocytosis is rapid, simple, highly accurate, avoids radiation hazards, and permits study of heterogeneity within cell populations. The biochemistry, physiology and pathophysiology of the interactions between retinal pigment epithelial (RPE) cells and photoreceptors continue to be areas of considerable research interest (1, 2, 3). Vital to such work is the ability to accurately quantify rod outer segment (ROS) phagocytosis by RPE cells. Current in vitro techniques of measuring ROS phagocytosis use either automated or manual methods to count phagosomes. While manual counting techniques offer the advantage of visual quality control, they are highly labor intensive, there is a practical limitation to the number of phagosomes that can be counted, and measurements suffer from relatively large standard errors (3). Automated methods include scintillation counting and flow cytometry. Problems with radiolabels include radiation hazards, nonspecific radiolabel uptake, and limited visual control (3). Flow cytometry, on the other hand, circumvents nearly all of these problems and may prove to be the optimal phagocytosis assay.

Adult↗