KATP channel openers have opposite effects on mitochondrial respiration under different energetic conditions.

Mitochondrial (m) KATP channel opening has been implicated in triggering cardiac preconditioning. Its consequence on mitochondrial respiration, however, remains unclear. We investigated the effects of two different KATP channel openers and antagonists on mitochondrial respiration under two different energetic conditions. Oxygen consumption was measured for complex I (pyruvate/malate) or complex II (succinate with rotenone) substrates in mitochondria from fresh guinea pig hearts. One of two mKATP channel openers, pinacidil or diazoxide, was given before adenosine diphosphate in the absence or presence of an mKATP channel antagonist, glibenclamide or 5-hydroxydecanoate. Without ATP synthase inhibition, both mKATP channel openers differentially attenuated mitochondrial respiration. Neither mKATP channel antagonist abolished these effects. When ATP synthase was inhibited by oligomycin to decrease [ATP], both mKATP channel openers accelerated respiration for both substrate groups. This was abolished by mKATP channel blockade. Thus, under energetically more physiological conditions, the main effect of mKATP channel openers on mitochondrial respiration is differential inhibition independent of mKATP channel opening. In contrast, under energetically less physiological conditions, mKATP channel opening can be evidenced by accelerated respiration and blockade by antagonists. Therefore, the effects of mKATP channel openers on mitochondrial function likely depend on the experimental conditions and the cell's underlying energetic state.

Photobleaching of melanosomes from retinal pigment epithelium: I. Effects on protein oxidation.

Melanin in the long-lived melanosomes of the retinal pigment epithelium (RPE) may undergo photobleaching with aging, which appears to diminish the antioxidant function of melanin and could make photobleached melanosomes less efficient in protecting biomolecules from oxidative modification. Here we analyzed whether photobleaching of melanosomes affects their ability to modify the oxidation state of nearby protein. As conventional methods developed to study soluble antioxidants are not well suited for analysis of granules such as melanosomes, we developed a new analytic method to focus on particle surfaces that involves experimentally coating granules with the cytoskeletal protein beta-actin to serve as a reporter for local protein oxidation. Isolated porcine RPE melanosomes were photobleached with visible light to simulate aging, then photobleached melanosomes, untreated melanosomes and control particles (black latex beads) were actin coated and illuminated in a photosensitized cell free system. Protein was re-stripped from particles and analyzed for carbonylation by Western blotting. Quantitative densitometry showed no reproducible differences for protein associated with untreated melanosomes when compared with control particles. Melanin has both anti- and pro-oxidant functions when light irradiated, but neither of these functions predominated in the protein oxidation assay when untreated melanosomes were used. However, protein extracted from photobleached melanosomes showed markedly increased carbonylation, both of associated actin and of endogenous melanosomal protein(s), and the effect increased with extent of granule photobleaching. Photobleaching of RPE melanosomes therefore changes the oxidation state of protein endogenous to the organelle and reduces the ability of the granule to modify the oxidation of exogenous protein near the particle surface. The results support the growing body of evidence that photobleaching of RPE melanosomes, which is believed to occur with aging, changes the physicochemical properties of the organelle and reduces the likelihood that the granules perform an antioxidant function.

Clinical and experimental applications of NIR-LED photobiomodulation.

This review presents current research on the use of far-red to near-infrared (NIR) light treatment in various in vitro and in vivo models. Low-intensity light therapy, commonly referred to as "photobiomodulation," uses light in the far-red to near-infrared region of the spectrum (630-1000 nm) and modulates numerous cellular functions. Positive effects of NIR-light-emitting diode (LED) light treatment include acceleration of wound healing, improved recovery from ischemic injury of the heart, and attenuated degeneration of injured optic nerves by improving mitochondrial energy metabolism and production. Various in vitro and in vivo models of mitochondrial dysfunction were treated with a variety of wavelengths of NIR-LED light. These studies were performed to determine the effect of NIR-LED light treatment on physiologic and pathologic processes. NIRLED light treatment stimulates the photoacceptor cytochrome c oxidase, resulting in increased energy metabolism and production. NIR-LED light treatment accelerates wound healing in ischemic rat and murine diabetic wound healing models, attenuates the retinotoxic effects of methanol-derived formic acid in rat models, and attenuates the developmental toxicity of dioxin in chicken embryos. Furthermore, NIR-LED light treatment prevents the development of oral mucositis in pediatric bone marrow transplant patients. The experimental results demonstrate that NIR-LED light treatment stimulates mitochondrial oxidative metabolism in vitro, and accelerates cell and tissue repair in vivo. NIR-LED light represents a novel, noninvasive, therapeutic intervention for the treatment of numerous diseases linked to mitochondrial dysfunction.

Oxidative stress in ARPE-19 cultures: do melanosomes confer cytoprotection?

The pigment melanin has antioxidant properties that could theoretically reduce oxidative damage to the retinal pigment epithelium (RPE), perhaps protecting against retinal diseases with an oxidative stress component like age-related macular degeneration. To determine whether melanin confers cytoprotection on RPE cells, melanosomes or control particles were introduced by phagocytosis into the human cell line ARPE-19 and oxidative stress was induced chemically (H2O2 or tert-butyl hydroperoxide) or with visible light. Since the iron-binding capacity of melanin is important for its antioxidant function, experiments were performed to confirm that the melanosomes were not iron saturated. Cytotoxicity was assessed by measures of plasma or lysosomal membrane integrity, mitochondrial function, and cell-substrate reattachment. Oxidative stress protocols were critically evaluated to produce modest cytotoxicity, which might allow detection of a small cytoprotective effect as expected for melanosomes. Particle internalization alone had no effect on baseline metabolic activity or on major RPE antioxidants. Particles were tested in multiple oxidative stress experiments in which culture conditions known to affect stress-induced cytotoxicity, notably culture density, were varied. No testing condition or outcome measure revealed a consistent protective (or cytotoxic) effect of melanosomes, indicating that measures of lysosome stability or whole cell viability do not demonstrate an antioxidant role for RPE melanosomes. If the melanosome, an insoluble particle, performs a cytoprotective function within cells, its effects may be limited to the local environment of the organelle and undetectable by conventional methods.

Effects of photodegradation on the physical and antioxidant properties of melanosomes isolated from retinal pigment epithelium.

Melanosomes of the retinal pigment epithelium (RPE) are relatively long-lived organelles that are theoretically susceptible to changes induced by exposure to visible light. Here melanosomes were isolated from porcine RPE cells and subjected to high intensity visible light to determine the effects of illumination on melanosome structure and on the content and antioxidant properties of melanin. As compared to untreated melanosomes, illuminated granules showed morphologic changes consistent with photodegradation, which included variable reductions in electron density demonstrated by transmission electron microscopy (TEM), and particle fragmentation and surface disruption revealed by scanning electron microscopy (SEM) and atomic force microscopy. Illuminated melanosomes had lower melanin content, indicated by measures of absorbance and electron spin resonance (ESR) signal intensity, and reduced ability to bind iron, shown by chemical and ESR analyses. Compared to untreated melanosomes, ESR-spin trapping analyses further indicated that illuminated melanosomes show increased photogeneration of superoxide anion and reduced ability to inhibit the iron ion-catalyzed free radical decomposition of hydrogen peroxide. It appears therefore that visible light irradiation can disrupt the structure of RPE melanosomes and reduce the amount and antioxidant properties of melanin. Some of these changes occur in human RPE melanosomes with aging and the results obtained here suggest that visible light irradiation is at least partly responsible. The consequence of light-induced changes in RPE melanosomes may be a diminished capacity of melanin to help protect aged cells from oxidative damage, perhaps increasing the risk of diseases with an oxidative stress component such as age-related macular degeneration.

Photobiomodulation directly benefits primary neurons functionally inactivated by toxins: role of cytochrome c oxidase.

Far red and near infrared (NIR) light promotes wound healing, but the mechanism is poorly understood. Our previous studies using 670 nm light-emitting diode (LED) arrays suggest that cytochrome c oxidase, a photoacceptor in the NIR range, plays an important role in therapeutic photobiomodulation. If this is true, then an irreversible inhibitor of cytochrome c oxidase, potassium cyanide (KCN), should compete with LED and reduce its beneficial effects. This hypothesis was tested on primary cultured neurons. LED treatment partially restored enzyme activity blocked by 10-100 microm KCN. It significantly reduced neuronal cell death induced by 300 microm KCN from 83.6 to 43.5%. However, at 1-100 mm KCN, the protective effects of LED decreased, and neuronal deaths increased. LED significantly restored neuronal ATP content only at 10 microm KCN but not at higher concentrations of KCN tested. Pretreatment with LED enhanced efficacy of LED during exposure to 10 or 100 microm KCN but did not restore enzyme activity to control levels. In contrast, LED was able to completely reverse the detrimental effect of tetrodotoxin, which only indirectly down-regulated enzyme levels. Among the wavelengths tested (670, 728, 770, 830, and 880 nm), the most effective ones (830 nm, 670 nm) paralleled the NIR absorption spectrum of oxidized cytochrome c oxidase, whereas the least effective wavelength, 728 nm, did not. The results are consistent with our hypothesis that the mechanism of photobiomodulation involves the up-regulation of cytochrome c oxidase, leading to increased energy metabolism in neurons functionally inactivated by toxins.

Antioxidants and ocular cell type differences in cytoprotection from formic acid toxicity in vitro.

Retinal photoreceptors and retinal pigment epithelial (RPE) cells are among the cell types that are sensitive to poisoning with methanol and its toxic metabolite formic acid. When exposed to formic acid in vitro, cultured cell lines from photoreceptors (661W) and the RPE (ARPE-19) were previously shown to accumulate similar levels of formate, but cytotoxic effects are greater in 661W cells. Here catalase and glutathione were analyzed in the two retinal cell lines to determine whether differences in these antioxidant systems contributed to cell-type-specific differences in cytotoxicity. Cells were exposed to formic acid (pH 6.8) in the culture medium in the presence or absence of a catalase activity inhibitor, 3-amino-1,2,4-triazole (AT), or a glutathione synthesis inhibitor, buthionine L-sulfoximine (BSO). Catalase protein, catalase enzyme activity, glutathione, glutathione peroxidase activity, cellular ATP, and cytotoxicity were analyzed. Compared to ARPE-19, 661W cells show lower antioxidant levels: 50% less glutathione, glutathione peroxidase and catalase protein, and 90% less catalase enzyme activity. In both cell types, formic acid treatment produced decreases in glutathione and glutathione peroxidase, and glutathione synthesis inhibition with BSO produced greater ATP depletion and cytotoxicity than formic acid treatment alone. In contrast, formate exposure produced decreases in catalase protein and activity in 661W cells, but increases in activity in ARPE-19. Treatment with the catalase inhibitor AT increased the formate sensitivity only of the ARPE-19 cells. ARPE-19 cells, therefore, may be less susceptible to formate toxicity due to higher levels of antioxidants, especially catalase, which increases on formate treatment and which has a significant cytoprotective effect for the RPE cell line.

Attenuation of mitochondrial respiration by sevoflurane in isolated cardiac mitochondria is mediated in part by reactive oxygen species.

BACKGROUND: Anesthetic preconditioning protects against cardiac ischemia/reperfusion injury. Increases in reduced nicotinamide adenine dinucleotide and reactive oxygen species during sevoflurane exposure suggest attenuated mitochondrial electron transport as a trigger of anesthetic preconditioning. The authors investigated the effects of sevoflurane on respiration in isolated cardiac mitochondria. METHODS: Mitochondria were isolated from fresh guinea pig hearts, and mitochondrial oxygen consumption was measured in the presence of complex I (pyruvate) or complex II (succinate) substrates. The mitochondria were exposed to 0, 0.13, 0.39, 1.3, or 3.9 mM sevoflurane. State 3 respiration was determined after adenosine diphosphate addition. The reactive oxygen species scavengers manganese(III) tetrakis (4-benzoic acid) porphyrin chloride and N-tert-Butyl-a-(2-sulfophenyl)nitrone sodium (10 microM each), or the K(ATP) channel blockers glibenclamide (2 microM) or 5-hydroxydecanoate (300 microM), were given alone or before 1.3 mM sevoflurane. RESULTS: Sevoflurane attenuated respiration for both complex I and complex II substrates, depending on the dose. Glibenclamide and 5-hydroxydecanoate had no effect on this attenuation. Both scavengers, however, abolished the sevoflurane-induced attenuation for complex I substrates, but not for complex II substrates. CONCLUSION: The findings suggest that sevoflurane-induced attenuation of complex I is mediated by reactive oxygen species, whereas attenuation of other respiratory complexes is mediated by a different mechanism. The opening of mitochondrial K(ATP) channels by sevoflurane does not seem to be involved in this effect. Thus, reactive oxygen species formation may not only result from attenuated electron transport by sevoflurane, but it may also contribute to complex I attenuation, possibly leading to a positive feedback and amplification of sevoflurane-induced reactive oxygen species formation in triggering anesthetic preconditioning.

Formate, the toxic metabolite of methanol, in cultured ocular cells.

Methanol has neurotoxic actions on the human retina due to its metabolite, formic acid, which is a mitochondrial toxin. In methanol poisoned animals, morphologic changes were seen both in retinal photoreceptors and in cells of the underlying retinal pigment epithelium (RPE). Here the effects of formate exposure on the two retinal cell types were analyzed in more detail in vitro using photoreceptor (661W) and RPE (ARPE-19) cell lines. Cells were exposed for time courses from minutes to days to sodium formate at pH 7.4 or to formic acid at pH 6.8, to simulate the metabolic acidosis that accompanies methanol poisoning. Formate accumulation, cellular ATP, cytotoxicity (lactate dehydrogenase (LDH) release) and cell phenotype were analyzed. Formate accumulated with a similar biphasic pattern in both cell types, and to similar levels whether delivered as sodium formate or as formic acid. ATP changes with sodium formate treatment differed between cell types with only 661W cells showing a rapid (within minutes), transient ATP increase. The subsequent ATP decrease was earlier in 661W cells (6 h) than the ATP decrease in ARPE-19 cells (24 h), and although both cell types showed evidence of cytotoxicity, the effects were greater for 661W cells. Both cell types showed enhanced morphologic and biochemical changes with formic acid treatment including earlier and/or greater effects on ATP depletion and cytotoxicity; again effects were more pronounced in 661W cells. Formate therefore is toxic for both cell lines, with 661W cells exhibiting greater sensitivity. Medium pH also appears to play a significant role in formate toxicity in vitro.

Anesthetic preconditioning improves adenosine triphosphate synthesis and reduces reactive oxygen species formation in mitochondria after ischemia by a redox dependent mechanism.

BACKGROUND: Mitochondrial changes that characterize the heart after anesthetic preconditioning (APC) or the mechanisms by which mitochondrial triggering factors lead to protection are unknown. This study hypothesized that generation of reactive oxygen species (ROS) during APC is required to initiate the mitochondrial protective effects, and that APC leads to improved mitochondrial electron transport chain function and cardiac function during reperfusion. METHODS: Isolated guinea pig hearts were subject to 30 min ischemia and 120 min reperfusion. Prior to ischemia hearts were either untreated (I/R), or treated with sevoflurane (APC), in the presence or absence of the ROS scavenger tiron (TIR), or the superoxide dismutase mimetic MnTBAP (TBAP). Intracellular ROS were measured by spectrofluorometry using the fluorescent probe dihydroethidium (DHE). In another series of experiments, using the same protocol, hearts were reperfused for only 5 min and removed for measurement of adenosine triphosphate (ATP) synthesis by luciferin-luciferase luminometry and ROS generation by dichlorohydro-fluorescein (DCF) fluorescence in isolated mitochondria. RESULTS: The APC improved cardiac function and reduced infarction. Tiron or MnTBAP abrogated the protection afforded by APC. Mitochondrial ATP synthesis was decreased by 70 +/- 3% after IR alone, by only 7 +/- 3% after APC, by 69 +/- 2% after APC+TIR, and by 71 +/- 3% after APC + TBAP. Mitochondrial ROS formation (DCF) increased by 48 +/- 3% after IR alone, by 0 +/- 2% after APC, by 43 +/- 4% after APC + TIR, and by 46 +/- 3% after APC + TBAP. ROS generation (DHE) was increased in I/R group at 5 and 120 min reperfusion. This was attenuated by APC but this protective effect was abrogated in APC + TIR and APC + TBAP groups. CONCLUSIONS: The results indicate that ROS are central both in triggering and mediating APC, and that the mitochondrion is the target for these changes.