Protective stabilization of mitochondrial permeability transition and mitochondrial oxidation during mitochondrial Ca2+ stress by melatonin's cascade metabolites C3-OHM and AFMK in RBA1 astrocytes.

Cyclic 3-hydroxymelatonin (C3-OHM) and N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) are two major cascade metabolites of melatonin. We previously showed melatonin provides multiple levels of mitochondria-targeted protection beyond as a mitochondrial antioxidant during ionomycin-induced mitochondrial Ca2+ (mCa2+ ) stress in RBA1 astrocytes. Using noninvasive laser scanning fluorescence coupled time-lapse digital imaging microscopy, this study investigated whether C3-OHM and AFMK also provide mitochondrial levels of protection during ionomycin-induced mCa2+ stress in RBA1 astrocytes. Interestingly, precise temporal and spatial dynamic live mitochondrial images revealed that C3-OHM and AFMK prevented specifically mCa2+ -mediated mitochondrial reactive oxygen species (mROS) formation and hence mROS-mediated depolarization of mitochondrial membrane potential (△Ψm ) and permanent lethal opening of the MPT (p-MPT). The antioxidative effects of AFMK, however, were less potent than that of C3-OHM. Whether C3-OHM and AFMK targeted directly the MPT was investigated under a condition of "oxidation free-Ca2+ stress" using a classic antioxidant vitamin E to remove mCa2+ -mediated mROS stress and the potential antioxidative effects of C3-OHM and AFMK. Intriguingly, two compounds still effectively postponed "oxidation free-Ca2+ stress"-mediated depolarization of △Ψm and p-MPT. Measurements using a MPT pore-specific indicator Calcein further identified that C3-OHM and AFMK, rather than inhibiting, stabilized the MPT in its transient protective opening mode (t-MPT), a critical mechanism to reduce overloaded mROS and mCa2+ . These multiple layers of mitochondrial protection provided by C3-OHM and AFMK thus crucially allow melatonin to extend its metabolic cascades of mitochondrial protection during mROS- and mCa2+ -mediated MPT-associated apoptotic stresses and may provide therapeutic benefits against astrocyte-mediated neurodegeneration in the CNS.

RTA 408 Inhibits Interleukin-1ß-Induced MMP-9 Expression via Suppressing Protein Kinase-Dependent NF-κB and AP-1 Activation in Rat Brain Astrocytes.

Neuroinflammation is characterized by the elevated expression of various inflammatory proteins, including matrix metalloproteinases (MMPs), induced by various pro-inflammatory mediators, which play a critical role in neurodegenerative disorders. Interleukin-1ß (IL-1ß) has been shown to induce the upregulation of MMP-9 through nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)-reactive oxygen species (ROS)-dependent signaling pathways. N-(2-cyano-3,12-dioxo-28-noroleana-1,9(11)-dien-17-yl)-2-2-difluoropropanamide (RTA 408), a novel synthetic triterpenoid, has been shown to possess anti-oxidant and anti-inflammatory properties in various types of cells. Here, we evaluated the effects of RTA 408 on IL-1ß-induced inflammatory responses by suppressing MMP-9 expression in a rat brain astrocyte (RBA-1) line. IL-1ß-induced MMP-9 protein and mRNA expression, and promoter activity were attenuated by RTA 408. The increased level of ROS generation in RBA-1 cells exposed to IL-1ß was attenuated by RTA 408, as determined by using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and CellROX. In addition, the inhibitory effects of RTA 408 on MMP-9 expression resulted from the suppression of the IL-1ß-stimulated activation of Pyk2 (proline-rich tyrosine kinase), platelet-derived growth factor receptor ß (PDGFRß), Akt, ROS, and mitogen-activated protein kinases (MAPKs). Pretreatment with RTA 408 attenuated the IL-1ß-induced c-Jun phosphorylation, mRNA expression, and promoter activity. IL-1ß-stimulated nuclear factor-κB (NF-κB) p65 phosphorylation, translocation, and promoter activity were also attenuated by RTA 408. Furthermore, IL-1ß-induced glial fibrillary acidic protein (GFAP) protein and mRNA expression, and cell migration were attenuated by pretreatment with RTA 408. These results provide new insights into the mechanisms by which RTA 408 attenuates IL-1ß-mediated inflammatory responses and exerts beneficial effects for the management of brain diseases.

Melatonin as a mitochondria-targeted antioxidant: one of evolution's best ideas.

Melatonin is an ancient antioxidant. After its initial development in bacteria, it has been retained throughout evolution such that it may be or may have been present in every species that have existed. Even though it has been maintained throughout evolution during the diversification of species, melatonin's chemical structure has never changed; thus, the melatonin present in currently living humans is identical to that present in cyanobacteria that have existed on Earth for billions of years. Melatonin in the systemic circulation of mammals quickly disappears from the blood presumably due to its uptake by cells, particularly when they are under high oxidative stress conditions. The measurement of the subcellular distribution of melatonin has shown that the concentration of this indole in the mitochondria greatly exceeds that in the blood. Melatonin presumably enters mitochondria through oligopeptide transporters, PEPT1, and PEPT2. Thus, melatonin is specifically targeted to the mitochondria where it seems to function as an apex antioxidant. In addition to being taken up from the circulation, melatonin may be produced in the mitochondria as well. During evolution, mitochondria likely originated when melatonin-forming bacteria were engulfed as food by ancestral prokaryotes. Over time, engulfed bacteria evolved into mitochondria; this is known as the endosymbiotic theory of the origin of mitochondria. When they did so, the mitochondria retained the ability to synthesize melatonin. Thus, melatonin is not only taken up by mitochondria but these organelles, in addition to many other functions, also probably produce melatonin as well. Melatonin's high concentrations and multiple actions as an antioxidant provide potent antioxidant protection to these organelles which are exposed to abundant free radicals.

Melatonin Mitigates Mitochondrial Meltdown: Interactions with SIRT3.

Melatonin exhibits extraordinary diversity in terms of its functions and distribution. When discovered, it was thought to be uniquely of pineal gland origin. Subsequently, melatonin synthesis was identified in a variety of organs and recently it was shown to be produced in the mitochondria. Since mitochondria exist in every cell, with a few exceptions, it means that every vertebrate, invertebrate, and plant cell produces melatonin. The mitochondrial synthesis of melatonin is not photoperiod-dependent, but it may be inducible under conditions of stress. Mitochondria-produced melatonin is not released into the systemic circulation, but rather is used primarily in its cell of origin. Melatonin's functions in the mitochondria are highly diverse, not unlike those of sirtuin 3 (SIRT3). SIRT3 is an NAD+-dependent deacetylase which regulates, among many functions, the redox state of the mitochondria. Recent data proves that melatonin and SIRT3 post-translationally collaborate in regulating free radical generation and removal from mitochondria. Since melatonin and SIRT3 have cohabitated in the mitochondria for many eons, we predict that these molecules interact in many other ways to control mitochondrial physiology. It is predicted that these mutual functions will be intensely investigated in the next decade and importantly, we assume that the findings will have significant applications for preventing/delaying some age-related diseases and aging itself.

Long-term Aß exposure augments mCa2+-independent mROS-mediated depletion of cardiolipin for the shift of a lethal transient mitochondrial permeability transition to its permanent mode in NARP cybrids: a protective targeting of melatonin.

Mitochondrial dysfunction is a hallmark of amyloid ß-peptide (Aß)-induced neurodegeneration of Alzheimer's disease (AD). This study investigated whether mtDNA T8993G mutation-induced complex V inhibition, clinically associated with neurological muscle weakness, ataxia, and retinitis pigmentosa (NARP), is a potential risk factor for AD and the pathological link for long-term exposure of Aß-induced mitochondrial toxicity and apoptosis in NARP cybrids. Using noninvasive fluorescence probe-coupled laser scanning imaging microscopy and NARP cybrids harboring 98% mutant genes along with its parental 143B osteosarcoma cells, we demonstrated that Aß-augmented mitochondrial Ca(2+) (mCa(2+))-independent mitochondrial reactive oxygen species (mROS) formation for a cardiolipin (CL, a major mitochondrial protective phospholipid)-dependent lethal modulation of the mitochondrial permeability transition (MPT). Aß augmented not only the amount but also the propagation rate of mROS-induced mROS formation to significantly depolarize mitochondrial membrane potential (∆Ψ(m)) and reduce mCa(2+) stress. Aß-augmented mROS oxidized and depleted CL, thereby enhances mitochondrial fission and movement retardation, which promoted the NARP-augmented lethal transient-MPT (t-MPT) to switch to its irreversible mode of permanent-MPT (p-MPT). Interestingly, melatonin, a multiple mitochondrial protector, markedly reduced Aß-augmented mROS formation and therefore significantly reduced mROS-mediated depolarization of ∆Ψ(m), fission of mitochondria and retardation of mitochondrial movement to stabilize CL and hence the MPT. In the presence of melatonin, Aß-promoted p-MPT was reversed to a protective t-MPT, which preserved ∆Ψ(m) and lowered elevated mCa(2+) to sublethal levels for an enhanced mCa(2+)-dependent O(2) consumption. Thus, melatonin may potentially rescue AD patients associated with NARP symptoms.

Japanese encephalitis virus induces matrix metalloproteinase-9 expression via a ROS/c-Src/PDGFR/PI3K/Akt/MAPKs-dependent AP-1 pathway in rat brain astrocytes.

BACKGROUND: Japanese encephalitis virus (JEV) infection is a major cause of acute encephalopathy in children, which destroys central nervous system (CNS) cells, including astrocytes and neurons. Matrix metalloproteinase (MMP)-9 has been shown to degrade components of the basal lamina, leading to disruption of the blood-brain barrier (BBB) and to contribute to neuroinflammatory responses in many neurological diseases. However, the detailed mechanisms of JEV-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells) are largely unclear. METHODS: In this study, the effect of JEV on expression of MMP-9 was determined by gelatin zymography, western blot analysis, RT-PCR, and promoter assay. The involvement of AP-1 (c-Jun and c-Fos), c-Src, PDGFR, PI3K/Akt, and MAPKs in these responses were investigated by using the selective pharmacological inhibitors and transfection with siRNAs. RESULTS: Here, we demonstrate that JEV induces expression of pro-form MMP-9 via ROS/c-Src/PDGFR/PI3K/Akt/MAPKs-dependent, AP-1 activation in RBA-1 cells. JEV-induced MMP-9 expression and promoter activity were inhibited by pretreatment with inhibitors of AP-1 (tanshinone), c-Src (PP1), PDGFR (AG1296), and PI3K (LY294002), and by transfection with siRNAs of c-Jun, c-Fos, PDGFR, and Akt. Moreover, JEV-stimulated AP-1 activation was inhibited by pretreatment with the inhibitors of c-Src, PDGFR, PI3K, and MAPKs. CONCLUSION: From these results, we conclude that JEV activates the ROS/c-Src/PDGFR/PI3K/Akt/MAPKs pathway, which in turn triggers AP-1 activation and ultimately induces MMP-9 expression in RBA-1 cells. These findings concerning JEV-induced MMP-9 expression in RBA-1 cells imply that JEV might play an important role in CNS inflammation and diseases.

mtDNA T8993G mutation-induced mitochondrial complex V inhibition augments cardiolipin-dependent alterations in mitochondrial dynamics during oxidative, Ca(2+), and lipid insults in NARP cybrids: a potential therapeutic target for melatonin.

Mitochondrial dynamics including morphological fission and mitochondrial movement are essential to normal mitochondrial and cellular physiology. This study investigated how mtDNA T8993G (NARP)-induced inhibition of mitochondrial complex V altered mitochondrial dynamics in association with a protective mitochondrial phospholipid, cardiolipin (CL), as a potential therapeutic target. NARP cybrids harboring 98% of mtDNA T8993G genes and its parental osteosarcoma 143B cells were studied for comparison, and protection provided by melatonin, a potent mitochondrial protector, was explored. We demonstrate for the first time that NARP mutation significantly enhances apoptotic death as a result of three distinct lethal mitochondrial apoptotic insults including oxidative, Ca(2+), and lipid stress. In addition, NARP significantly augmented pathological depletion of CL. NARP-augmented depletion of CL results in enhanced retardation of mitochondrial movement and fission and later swelling of mitochondria during all insults. These results suggest that CL is a common and crucial pathological target for mitochondrial apoptotic insults. Furthermore, CL possibly plays a central role in regulating mitochondrial dynamics that are associated with NARP-augmented mitochondrial pathologies. Intriguingly, melatonin, by differentially preserving CL during various stresses (oxidation > Ca(2+) > lipid), rescues differentially CL-altered mitochondrial dynamics and cell death (oxidation > Ca(2+) > lipid). Thus, melatonin, in addition to being a mitochondrial antioxidant to antagonize mitochondrial oxidative stress, a mitochondrial permeability transition modulator to antagonize mitochondrial Ca(2+) stress, may stabilize directly CL to prevent its oxidization and/or depletion and, therefore, exerts great potential in rescuing CL-dependent mitochondrial dynamics-associated mitochondrial pathologies for treatment of NARP-induced pathologies and diseases.

Melatonin preserves the transient mitochondrial permeability transition for protection during mitochondrial Ca(2+) stress in astrocyte.

Cells have two modes of mitochondrial permeability transition (MPT) which produce virtually opposite pathophysiological outcomes of survival or death when responding to apoptotic insults. The transient-MPT (t-MPT) protects mitochondria, whereas the prolonged-MPT (p-MPT), once activated, triggers the 'point of no return' for apoptosis or necrosis. Our previous studies show that in addition to scavenging mitochondrial reactive oxygen species, melatonin targets mitochondrial Ca(2+) (mCa(2+))-mediated MPT for protection during mCa(2+)-mediated apoptosis in astrocytes. The precise mechanism for how melatonin modulates the MPT during mCa(2+) stress, however, remains unelucidated. With the application of fluorescence laser scanning imaging microscopy, this study demonstrated for the first time that melatonin does not inhibit the MPT pore, rather it crucially preserves the pore in its protective mode of t-MPT during mCa(2+) stress. Melatonin-preserved t-MPT importantly maintained mitochondrial membrane potential (ΔΨ(m)) which not only prevented depolarized ΔΨ(m)-induced p-MPT but also retained ΔΨ(m)-dependent ATP formation during disturbed Ca(2+) homeostasis. Additionally, the melatonin-preserved t-MPT allowed mitochondria to release the toxic overload of mCa(2+) to sublethal levels, which prevented mCa(2+)-mediated fission and mCa(2+)-dependent p-MPT and possibly also improved mCa(2+)-dependent ATP synthesis. Melatonin's effect in reducing the Ca(2+) load greatly diminished when the MPT was inhibited by cyclosporine A, suggesting its pore dependency as well as that a preserved t-MPT may be superior to a MPT inhibition in protecting mCa(2+)-mediated apoptosis. The unique modulation on the MPT provided by melatonin may have extraordinary therapeutic potential in the treatment of mCa(2+)-mediated astrocyte-associated neurodegenerative pathologies and diseases.

Visualization of melatonin's multiple mitochondrial levels of protection against mitochondrial Ca(2+)-mediated permeability transition and beyond in rat brain astrocytes.

Melatonin protects cells against various types of oxidative stress-induced apoptosis due primarily to its ability to effectively scavenge pathological and disease condition-augmented generation of mitochondrial reactive oxygen species (mROS). Once produced, mROS indiscriminately damage mitochondrial components and more importantly they crucially activate directly the mitochondrial permeability transition (MPT), one of the critical mechanisms for initiating post mitochondrial apoptotic signaling. Whether or not melatonin targets directly the MPT, however, remains inconclusive, particularly during oxidative stress. This study, thus, investigated this possibility of an 'oxidation free Ca(2+) stress' in the presence of vitamin E after ionomycin exposure as a sole Ca(2+)-mediated MPT in order to exclude melatonin's primary antioxidative effects as well as Ca(2+)-mediated oxidative stress. The studies were carried out using cultured rat brain astrocytes RBA-1. With the application of laser scanning multiple fluorescence imaging microscopy, we visualized for the first time multiple mitochondrial protective effects provided by melatonin during Ca(2+) stress. First, melatonin, due to its primary antioxidative actions, completely prevented mCa(2+)-induced mROS formation during ionomycin exposure. Secondly, when melatonin(')s antioxidative effects were prevented due to the addition of vitamin E, melatonin significantly prevented mCa(2+)-mediated MPT and apoptosis suggesting its direct targeting of the MPT. Surprisingly, in the presence of cyclosporin A, a MPT inhibitor, melatonin reduced further mCa(2+)-mediated apoptosis during ionomycin exposure also suggesting its targeting beyond the MPT. As astrocytes are actively involve in regulating synaptic transmission and neurovascular coupling in the CNS, these multiple mitochondrial layers of protection provided by melatonin against mCa(2+)-and/or mROS-mediated apoptosis in astrocytes may be crucial for future therapeutic prevention and treatment of astrocyte-mediated neurodegenerative diseases in the CNS.

BK-induced COX-2 expression via PKC-delta-dependent activation of p42/p44 MAPK and NF-kappaB in astrocytes.

Bradykinin (BK) is an inflammatory mediator, elevated levels in the region of several brain injury and inflammatory diseases. It has been shown to induce cyclooxygenase-2 (COX-2) expression implicating in inflammatory responses in various cell types. However, the signaling mechanisms underlying BK-induced COX-2 expression in astrocytes remain unclear. First, RT-PCR and Western blotting analysis showed that BK induced the expression of COX-2 mRNA and protein, which was inhibited by B(2) BK receptor antagonist Hoe140, suggesting the involvement of B(2) BK receptors. BK-induced COX-2 expression and translocation of PKC-delta from cytosol to membrane fraction were inhibited by rottlerin, suggesting that PKC-delta might be involved in these responses. This hypothesis was further supported by the transfection with a dominant negative plasmid of PKC-delta significantly blocked BK-induced COX-2 expression. BK-stimulated p42/p44 MAPK phosphorylation, COX-2 mRNA expression, and prostaglandin E(2) (PGE(2)) release were attenuated by PD98059, indicating the involvement of MEK/p42/p44 MAPK in this pathway. Accordingly, BK-stimulated phosphorylation of p42/p44 MAPK was attenuated by rottlerin, indicating that PKC-delta might be an upstream component of p42/p44 MAPK. Moreover, BK-induced COX-2 expression might be mediated through the translocation of NF-kappaB into nucleus which was blocked by helenalin, rottlerin and PD98059, implying the involvement of NF-kappaB. These results suggest that in RBA-1 cells, BK-induced COX-2 expression and PGE(2) release was sequentially mediated through PKC-delta-dependent activation of p42/p44 MAPK and NF-kappaB. Understanding the regulation of COX-2 expression and PGE(2) release induced by BK in astrocytes might provide a new therapeutic strategy of brain injury and inflammatory diseases.

Biogenic amines in the reduction of oxidative stress: melatonin and its metabolites.

N-acetyl-5-methoxytryptamine (melatonin) is an endogenous indoleamine produced by all vertebrate organisms. Its production in the pineal gland has been extensively investigated but other organs also synthesize this important amine. Melatonin's functions in organisms are diverse. The actions considered in the current review relate to its ability to function in the reduction of oxidative stress, i.e., molecular damage produced by reactive oxygen and reactive nitrogen species. Numerous publications have now shown that not only is melatonin itself an efficient scavenger of free radicals and related reactants, but so are its by-products cyclic 3-hydroxymelatonin, N1-acetyl-N2-formyl-5-methoxykynuramine, and others. These derivatives are produced sequentially when each functions in the capacity of a free radical scavenger. These successive reactions are referred to as the antioxidant cascade of melatonin. That melatonin has this function within cells has been observed in studies employing time lapse conventional, confocal and multiphoton fluorescent microscopy coupled with the use of appropriate mitochondrial-targeted fluorescent probes. The benefits of melatonin and its metabolites have been described in the brain where they are found to be protective in models of Parkinson's disease, Alzheimer's disease and spinal cord injury. The reader is reminded, however, that data not covered in this review has documented beneficial actions of these amines in every organ where they have been tested. The outlook for the use of melatonin in clinical trials looks encouraging given its low toxicity and high efficacy.

Visualizing common deletion of mitochondrial DNA-augmented mitochondrial reactive oxygen species generation and apoptosis upon oxidative stress.

Common deletion (CD) 4977 bp of mitochondrial DNA (mtDNA) disrupt specifically mitochondrial complex I, IV and V on the electron transport chain (ETC) and is closely associated with wide spectrums of clinical manifestations. To quantitatively investigate how CD-induced ETC defect alters mitochondrial reactive oxygen species (mROS) generation as well as down stream apoptotic signaling, we employed an established array of human CD cytoplasmic hybrids (cybrids) harboring 0%-80% of CD. Pathological effects of CD on the mitochondria were visualized at single cell level by the application of fluorescent probes coupled with conventional and multiphoton imaging microscopy. Intriguingly, we observed CD-augmented mROS generation omitted "threshold effect". CD-augmented mROS generation was associated with depolarized mitochondrial membrane potential (DeltaPsi(m)). Upon oxidative stress, the amount of CD-augmented mROS generation was greatly enhanced to cause pathological apoptotic deterioration including opening of the mitochondrial permeability transition, cytochrome c release, phosphatidylserine externalization and DNA fragmentation. In addition, heterogeneous mitochondrial dysfunctions were found in cybrids containing 80% of CD (D cybrids), i.e., low sensitive-D (LS-D, roughly 80%) and a super sensitive-D (SS-D, 20%). As compared to LS-D, SS-D had higher resting mROS level but slightly hyperpolarized DeltaPsi(m). Upon H2O2 treatment, much faster generation of mROS was observed which induced a faster depolarization of DeltaPsi(m) and later apoptotic deterioration in SS-D. We proposed a dose-dependent, feed-forward and self-accelerating vicious cycle of mROS production might be initiated in CD-induced ETC defect without threshold effect. As CD-augmented mROS generation is obligated to cause an enhanced pathological apoptosis, precise detection of CD-augmented mROS generation and their degree of heterogeneity in single cells may serve as sensitive pathological indexes for early diagnosis, prognosis and treatment of CD-associated diseases.

Evaluation of Anti-Inflammatory Effects of Helminthostachys zeylanica Extracts via Inhibiting Bradykinin-Induced MMP-9 Expression in Brain Astrocytes.

Phytochemicals present in vegetables, fruits, and herbs are believed to reduce the risk of several major diseases including cardiovascular or neurodegenerative disorders. The roots of the fern Helminthostachys zeylanica (L.) Hook. (Ophioglossaceae) have been used for centuries in the treatment of inflammation and as a folk medicine in several countries. The plant has been shown to possess an array of medicinal properties, including antioxidants and anti-inflammatory activities. Moreover, a rising level of matrix metalloproteinase-9 (MMP-9) has been found in blood fluid of these patients suffering from brain inflammatory diseases, which may be considered an inflammatory biomarker in several inflammatory diseases including the central nervous system (CNS) inflammation. Previously, we have demonstrated the signaling mechanisms of bradykinin (BK)-induced MMP-9 expression in brain astrocytes. Herein, we evaluate the effects of H. zeylanica extracts on BK-induced MMP-9 expression in brain astrocytes and its influencing mechanism. The results showed that H. zeylanica extracts, including E0, E1, and E2 significantly reduce MMP-9 induced by BK in brain astrocytes (RBA-1 cells). These H. zeylanica extracts can inhibit BK-stimulated phosphorylation of c-Src, Pyk2, and PKC(α/δ). Moreover, BK-stimulated NADPH oxidase (Nox)-derived reactive oxygen species (ROS) generation has also been attenuated by pretreatment with these extracts, suggesting that the H. zeylanica extracts have an antioxidative activity. We further demonstrated that the H. zeylanica extracts blocked activation of MAPKs (e.g., ERK1/2 and p38 MAPK) by BK. These data indicated that the H. zeylanica extracts may be has anti-inflammatory activity by reducing BK-induced ROS-dependent MMP-9 expression via these related pathways in brain astrocytes.

Enhanced generation of mitochondrial reactive oxygen species in cybrids containing 4977-bp mitochondrial DNA deletion.

The poor bioenergetic state in mitochondria containing mtDNA with the 4977-bp deletion has been well documented. However, information on mitochondrial reactive oxygen species (ROS) generation at rest or under intense oxidative stress in mitochondria lacking the 4977-bp mtDNA fragment inside intact living cells was insufficient. We used cybrids containing truncated mtDNA lacking the 4977-bp fragment and measured ROS levels inside cybrids by fluorescence probe, 2',7'-dichlorodihydrofluorescein (DCF), and confocal microscopy. Mitochondrial ROS at resting state was slightly higher in cybrids containing 4977-bp deletion mtDNA as compared to cybrids without mtDNA defects. For intense oxidative stress treatment, cybrids were treated with 5 mM H2O2 for 10 min. Consecutive DCF images were acquired after H2O2 had been washed away. Progressive increase of DCF signals, especially in the mitochondrial area, was observed in cybrids containing 4977-bp deletion mtDNA, even long after the brief, intense H2O2 treatment. This result suggests that a feed-forward, self-accelerating vicious cycle of mitochondrial ROS production could be initiated in cybrids containing 4977-bp deletion fragment mitochondria after brief, intense H2O2 treatment. This mechanism may play an important role in the pathophysiology of the disease process caused by mitochondria containing mtDNA with the 4977-bp deletion.

Mitochondrion-targeted photosensitizer enhances the photodynamic effect-induced mitochondrial dysfunction and apoptosis.

Recently, the mitochondrion has been considered as a novel pharmacological target for anticancer therapy due to its crucial role involved in arbitrating cell apoptosis. We have previously demonstrated that 488-nm laser irradiation induced a specific mitochondrial reactive oxygen species (mROS) formation and apoptotic death. In this study, we used a second generation of photosensitizers, the benzoporphyrin-derivative monoacid ring A (BPD-MA). We investigated specifically mechanisms at the mitochondrial level for BPD-MA coupled with 690-nm laser irradiation, the photodynamic effect (PDE) of BPD-MA, using conventional and laser scanning imaging microscopy in intact C6 glioma cells. We demonstrated BPD-MA localized mainly in the mitochondrial area. The phototoxicity induced by 1-10 J 690-nm laser irradiation was minor as compared to that induced by 488-nm laser irradiation. Unlike other mitochondrion-targeted photosensitizers, the dark toxicity induced by BPD-MA (0.05-5 mg/mL, effective doses used for the PDE) was relatively low. Nevertheless, the PDE of BPD-MA using 0.5 mg/mL coupled with 5J 690-nm irradiation induced profound and rapid (< 1 min) mitochondrial swelling, mROS formation, and severe plasma membrane blebbing as compared to that induced by 488-nm laser irradiation (< 10 min). Later, the PDE of BPD-MA resulted in positive propidium iodide cell-death stain and positive TUNEL apoptotic nuclear stain and DNA laddering. Finally, the PDT of BPD-MA also instantaneously promoted the mitochondrion to diminish its covalent binding with a mitochondrial marker, MitoTracker Green. We conclude that the PDT of BPD-MA targeted primarily and compellingly the mitochondrion to induce effective mitochondria-mediated apoptosis and thus may serve as a powerful photosensitizer for clinical cancer therapy.

Intracellular signalings underlying bradykinin-induced matrix metalloproteinase-9 expression in rat brain astrocyte-1.

Bradykinin (BK), an inflammatory mediator, has been shown to increase the expression of proteins such as matrix metalloproteinases (MMPs) on brain cells and contributes to the pathophysiology of inflammatory responses. However, the mechanisms regulating MMP-9 expression by BK in rat brain astrocytes-1 (RBA-1) remain unclear. Here we report that the mitogen-activated protein kinase (MAPK) and NF-kappaB pathways participate in the induction of MMP-9 expression induced by BK in RBA cells. Zymographic, Western blotting, and RT-PCR analyses showed that BK increased expression of MMP-9 mRNA and protein in a time- and concentration-dependent manner. BK-induced MMP-9 mRNA and protein expression was inhibited by MEK1/2 inhibitor PD98059, PI3-K inhibitor LY294002, and NF-kappaB inhibitor helenalin. In accordance with these findings, BK-induced phosphorylation of p42/p44 MAPK and Akt and activation of NF-kappaB was attenuated by prior treatment with PD98059, LY294002, and helenalin, respectively. The effects of BK on MMP-9 expression and p42/p44 MAPK and Akt phosphorylation were inhibited by B(2) receptor antagonist Hoe 140, indicating the involvement of B(2) receptors revealed by [(3)H]-BK binding assay. Furthermore, BK-stimulated translocation of NF-kappaB into the nucleus was revealed by Western blotting and immnofluorescence staining and blocked by Hoe140, PD98059, LY294002, and helenalin. Taken together, these results suggest that in RBA cells, activation of p42/p44 MAPK and Akt cascades mediated through NF-kappaB pathway are essential for BK-induced MMP-9 gene expression. This study may provide insights into the regulation of MMP-9 production in CNS, which may occur in vivo in pathological situations such as CNS inflammation and brain astrocytoma.

Bradykinin-stimulated p42/p44 MAPK activation associated with cell proliferation in corneal keratocytes.

Bradykinin (BK) is released into the tear-film in ocular allergic patients. BK has been shown to exert mitogenic effects on several cell types. However, the mechanisms underlying its action on corneal keratocytes (CKs) were largely unknown. This study was to investigate the mitogenic effect of BK on rabbit CKs linked to activation of p42/p44 mitogen-activated protein kinase (MAPK), assessed by [3H]thymidine incorporation and Western blotting analysis, respectively. BK stimulated [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner. Pretreatment with pertussis toxin attenuated the BK-induced responses. BK-stimulated responses were attenuated by inhibitors of selective B2 receptor (Hoe 140), phosphatidylinositol (PI)-PLC (U73122), an intracellular Ca2+chelator (BAPTA/AM), PKC (GF109203X), tyrosine kinase (genistein), and MEK1/2 (PD98059). BK also stimulated translocation of p42/p44 MAPK into nucleus and led to expression of c-fos and c-jun in CKs. These results demonstrate that in CKs, BK-stimulated phosphorylation of p42/p44 MAPK is mediated through the activation of BK B2 receptors and leads to cell proliferation.

Mitochondrial swelling and generation of reactive oxygen species induced by photoirradiation are heterogeneously distributed.

Abundant evidence has been gathered to show that overproduction of reactive oxygen species (ROS) can lead to the opening of the mitochondrial permeability transition pore (MPTP) and result in apoptosis in mammalian cells. The information regarding spatial and temporal regulation of intracellular ROS formation related to the MPTP opening, however, is relatively limited. In this study, we used a fluorescent probe, dihydro-2',7'-dichloroforescin (DCF), to detect intracellular ROS levels in different compartments of the cell in a time-resolved manner. The roles of mitochondrial ROS (mROS) in the MPTP opening and mitochondrial membrane potential drop were investigated by using H(2)DCFDA coloaded with a mitochondrial marker dye MitoTracker Red, and by a mitochondrial membrane potential dye tetramethyl rhodamine ethyl ester. We applied multiphoton laser scanning microscopy to avoid autooxidation and bleaching of DCF so that long-term visualization of intracellular ROS formation could be performed. Moreover, we noted that the resting mROS levels of different mitochondria were not homogeneous. After cells had been exposed to photoirradiation, the intracellular ROS gradually increased but the heterogeneity of mROS was maintained. Later, swelling was observed in mitochondria that contained higher levels of ROS, indicating the opening of the MPTP. In cells in which all the mitochondria swelled, they were translocated to the perinuclear area, which became the site of ROS production. At this stage, mROS reached the highest level concomitantly with a complete loss of mitochondrial membrane potential, indicating full opening of the MPTP. At the end, photoirradiation resulted in apoptotic cell death. In summary, we demonstrated by multiphoton laser scanning microscopy that photoirradiation induces heterogeneous intracellular ROS formation and mitochondrial permeability transition pore opening in single intact cells. These observations imply the existence of a microdomain in the regulation of mROS formation and subsequent opening of the MPTP.

Mitochondrial reactive oxygen species generation and calcium increase induced by visible light in astrocytes.

Mitochondria contain photosensitive chromophores that can be activated or inhibited by light in the visible range. Rather than utilizing light energy, however, mitochondrial electron transport oxidation-reduction reaction and energy coupling could be stimulated or damaged by visible light. Our previous work demonstrated that reactive oxygen species (ROS) were generated in cultured astrocytes after visible laser irradiation. With confocal fluorescence microscopy, we found that ROS were generated mostly from mitochondria. This mitochondrial ROS (mROS) formation plays a critical role in photoirradiation-induced phototoxicity and apoptosis. In this study, we measured changes of mitochondrial calcium level ([Ca(2+)](m)) in cultured astrocytes (RBA-1 cell line) irradiated with blue light and examined the association between mROS formation and [Ca(2+)](m) level changes. Changes of intracellular ROS and [Ca(2+)](m) were visualized using fluorescent probes 2',7'-dichlorodihydrofluorescein (DCF), and rhod-2. After exposure to visible light irradiation, RBA-1 astrocytes showed a rapid increase in ROS accumulation particularly in the mitochondrial area. Increase in [Ca(2+)](m) was also induced by photoirradiation. The levels of increase in DCF fluorescence intensity varied among different astrocytes. Some of the cells generated much higher levels of ROS than others. For those cells that had high ROS levels, mitochondrial Ca(2+) levels were also high. In cells that had mild ROS levels, mitochondrial Ca(2+) levels were only slightly increased. The rate of increase in DCF fluorescence seemed to be close to the rate of rhod-2 fluorescence increase. There is a positive and close correlation between mitochondrial ROS levels and mitochondrial Ca(2+) levels in astrocytes irradiated by visible light.

Hypoxic preconditioning-induced mitochondrial protection is not disrupted in a cell model of mtDNA T8993G mutation-induced F1F0-ATP synthase defect: the role of mitochondrial permeability transition.

Transient opening of the mitochondrial permeability transition pore plays a crucial role in hypoxic preconditioning-induced protection. Recently, the cyclophilin-D component of the mitochondrial permeability transition pore has been shown to interact with and regulate the F1F0-ATP synthase. However, the precise role of the F1F0-ATP synthase and the interaction between cyclophilin-D and F1F0-ATP synthase in the mitochondrial permeability transition pore and hypoxic preconditioning remain uncertain. Here we found that a 1-h hypoxic preconditioning delayed apoptosis and improved cell survival after stimulation with various apoptotic inducers including H2O2, ionomycin, and arachidonic acid in mitochondrial DNA T8993G mutation (NARP) osteosarcoma 143B cybrids, an F1F0-ATP synthase defect cell model. This hypoxic preconditioning protected NARP cybrid cells against focal laser irradiation-induced oxidative stress by suppressing reactive oxygen species formation and preventing the depletion of cardiolipin. Furthermore, the protective functions of transient opening of the mitochondrial permeability transition pore in both NARP cybrids and wild-type 143B cells can be augmented by hypoxic preconditioning. Disruption of the interaction between cyclophilin-D and F1F0-ATP synthase by cyclosporin A attenuated the mitochondrial protection induced by hypoxic preconditioning in both NARP cybrids and wild-type 143B cells. Our results demonstrate that the interaction between cyclophilin-D and F1F0-ATP synthase is important in the hypoxic preconditioning-induced cell protection. This finding improves our understanding of the mechanism of mitochondrial permeability transition pore opening in cells in response to hypoxic preconditioning, and will be helpful in further developing new pharmacological agents targeting hypoxia-reoxygenation injury and mitochondria-mediated cell death.