Tetramethylrhodamine Ethyl Ester Perchlorate Research Articles

Heterozygous variants in CTR9, which encodes a major component of the PAF1 complex, are associated with a neurodevelopmental disorder

CTR9 is a subunit of the PAF1 complex (PAF1C) that plays a crucial role in transcription regulation by binding CTR9 to RNA polymerase II. It is involved in transcription-coupled histone modification through promoting H3K4 and H3K36 methylation. We describe the clinical and molecular studies in 13 probands, harboring likely pathogenic CTR9 missense variants, collected through GeneMatcher. Exome sequencing was performed in all individuals. CTR9 variants were assessed through 3-dimensional modeling of the activated human transcription complex Pol II-DSIF-PAF-SPT6 and the PAF1/CTR9 complex. H3K4/H3K36 methylation analysis, mitophagy assessment based on tetramethylrhodamine ethyl ester perchlorate immunofluorescence, and RNA-sequencing in skin fibroblasts from 4 patients was performed. Common clinical findings were variable degrees of intellectual disability, hypotonia, joint hyperlaxity, speech delay, coordination problems, tremor, and autism spectrum disorder. Mild dysmorphism and cardiac anomalies were less frequent. For 11 CTR9 variants, de novo occurrence was shown. Three-dimensional modeling predicted a likely disruptive effect of the variants on local CTR9 structure and protein interaction. Additional studies in fibroblasts did not unveil the downstream functional consequences of the identified variants. We describe a neurodevelopmental disorder caused by (mainly) de novo variants in CTR9, likely affecting PAF1C function.

Chaperone-mediated autophagy controls the turnover of E3 ubiquitin ligase MARCHF5 and regulates mitochondrial dynamics

ABSTRACT As a highly dynamic organelle, mitochondria undergo constant fission and fusion to change their morphology and function, coping with various stress conditions. Loss of the balance between fission and fusion leads to impaired mitochondria function, which plays a critical role in the pathogenesis of Parkinson disease (PD). Yet the mechanisms behind mitochondria dynamics regulation remain to be fully illustrated. Chaperone-mediated autophagy (CMA) is a lysosome-dependent process that selectively degrades proteins to maintain cellular proteostasis. In this study, we demonstrated that MARCHF5, an E3 ubiquitin ligase required for mitochondria fission, is a CMA substrate. MARCHF5 interacted with key CMA regulators and was degraded by lysosomes. Severe oxidative stress compromised CMA activity and stabilized MARCHF5, which facilitated DNM1L translocation and led to excessive fission. Increase of CMA activity promoted MARCHF5 turnover, attenuated DNM1L translocation, and reduced mitochondria fragmentation, which alleviated mitochondrial dysfunction under oxidative stress. Furthermore, we showed that conditional expression of LAMP2A, the key CMA regulator, in dopaminergic (DA) neurons helped maintain mitochondria morphology and protected DA neuronal viability in a rodent PD model. Our work uncovers a critical role of CMA in maintaining proper mitochondria dynamics, and loss of this regulatory control may occur in PD and underlie its pathogenic process. Abbreviations: CMA: chaperone-mediated autophagy; DA: dopaminergic; DNM1L: dynamin 1 like; FCCP: carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; HSPA8: heat shock protein family A (Hsp70) member 8; LAMP2A: lysosomal associated membrane protein 2A; MARCHF5: membrane-associated ring-CH-type finger 5; MMP: mitochondria membrane potential; OCR: oxygen consumption rate; 6-OHDA: 6-hydroxydopamine; PD: Parkinson disease; SNc: substantia nigra pars compacta; TEM: transmission electron microscopy; TH: tyrosine hydroxylase; TMRE: tetramethylrhodamine ethyl ester perchlorate; WT: wild type.

Apelin-13 protects the heart against ischemia-reperfusion injury through the RISK-GSK-3β-mPTP pathway.

IntroductionApelin plays an important role in the protection against myocardial ischemia-reperfusion (I/R) injury, while the mechanism still remains unclear. In the current study, we aimed to evaluate the protective effect of apelin-13, and the main mechanism.Material and methodsThe in vivo I/R injury model (Sprague-Dawley rat) was established, then infarct size, expression levels of phospho-protein kinase B (p-Akt), phospho-extracellular signal-regulated kinase (p-ERK) and phospho-glycogen synthase kinase-3β (p-GSK-3β) were measured. The fluorescence intensity of tetramethylrhodamine ethyl ester perchlorate (TMRE) of the isolated myocardial cells was determined to evaluate the opening of the mitochondrial permeability transition pore (mPTP) caused by oxidant stress and hypoxia/reoxygenation.ResultsFor the established I/R injury model, apelin-13 and SB216763 (GSK-3β inhibitor) significantly reduced the infarct size (p < 0.05), which could be abolished by LY294002 (PI3K inhibitor), PD98059 (MEK inhibitor) and atractyloside (mPTP accelerator). The enhanced expression levels of p-Akt, p-ERK and p-GSK-3β caused by apelin-13 (p < 0.05) could be counteracted by LY294002 and PD98059. The reduced fluorescence intensity of TMRE in the H2O2/apelin-13 and H2O2/SB216763 treated groups was significantly lower (p < 0.05), indicating that apelin-13 and SB216763 could reduce the decline in mitochondrial membrane potential caused by oxidant stress, and the fluorescence intensity in the hypoxia/reoxygenation + apelin-13 group was significantly lower (p < 0.05), which suggested that apelin-13 could inhibit the mitochondrial membrane potential changes induced by hypoxia/reoxygenation.ConclusionsThe protective mechanism of apelin-13 might be that inactivation of GSK-3β could inhibit the opening of mPTP by activating PI3K/Akt and ERK1/2 involved in the reperfusion injury salvage kinase (RISK) pathway.

Novel effect of the inhibitor of mitochondrial cyclophilin D activation, N‐methyl‐4‐isoleucine cyclosporin, on renal calcium crystallization

To experimentally evaluate the clinical application of N-methyl-4-isoleucine cyclosporin, a novel selective inhibitor of cyclophilin D activation. In vitro, cultured renal tubular cells were exposed to calcium oxalate monohydrate crystals and treated with N-methyl-4-isoleucine cyclosporin. The mitochondrial membrane was stained with tetramethylrhodamine ethyl ester perchlorate and observed. In vivo, Sprague-Dawley rats were divided into four groups: a control group, an ethylene glycol group (administration of ethylene glycol to induce renal calcium crystallization), a N-methyl-4-isoleucine cyclosporin group (administration of N-methyl-4-isoleucine cyclosporin) and an ethylene glycol + N-methyl-4-isoleucine cyclosporin group (administration of ethylene glycol and N-methyl-4-isoleucine cyclosporin). Renal calcium crystallization was evaluated using Pizzolato staining. Oxidative stress was evaluated using superoxide dismutase and 8-hydroxy-deoxyguanosine. Mitochondria within renal tubular cells were observed by transmission electron microscopy. Cell apoptosis was evaluated using cleaved caspase-3. In vitro, calcium oxalate monohydrate crystals induced depolarization of the mitochondrial membrane potential, which was remarkably prevented by N-methyl-4-isoleucine cyclosporin. In vivo, ethylene glycol administration induced renal calcium crystallization, oxidative stress, mitochondrial collapse and cell apoptosis in rats, which were significantly prevented by N-methyl-4-isoleucine cyclosporin. Herein we first report a new treatment agent determining renal calcium crystallization through cyclophilin D activation.

Mitochondrial Staining Allows Robust Elimination of Apoptotic and Damaged Cells during Cell Sorting

High-speed fluorescence-activated cell sorting is relevant for a plethora of applications, such as PCR-based techniques, microarrays, cloning, and propagation of selected cell populations. We suggest a simple cell-sorting technique to eliminate early and late apoptotic and necrotic cells, with good signal-to-noise ratio and a high-purity yield. The mitochondrial potential dye, TMRE (tetramethylrhodamine ethyl ester perchlorate), was used to separate viable and non-apoptotic cells from the cell sorting samples. TMRE staining is reversible and does not affect cell proliferation and viability. Sorted TMRE(+) cells contained a negligible percentage of apoptotic and damaged cells and had a higher proliferative potential as compared with their counterpart cells, sorted on the basis of staining with DNA viability dye. This novel sorting technique using TMRE does not interfere with subsequent functional assays and is a method of choice for the enrichment of functionally active, unbiased cell populations.

Abstract 2893: Curcumin loaded magnetic nanoparticles for breast cancer therapeutics and imaging applications

Abstract Background: Magnetic nanoparticles (MNPs) with theranostic features (diagnosis and treatment) have attracted significant attention and will greatly improve the scope of nanomedicine in cancer applications. The aim of this study is to develop a novel MNPs formulation composed of an iron oxide core coated with α-cyclodextrin and pluronic polymer (F68), to load an anti-cancer drug (curcumin, CUR) and prevent particle aggregation, respectively. The therapeutic efficacy and imaging capabilities of this novel formulation were evaluated in breast cancer cell line models. Methods: The physico-chemical analyses of curcumin loaded MNPs (MNP-CUR) were performed using dynamic light scattering (DLS), transmission electron microscopy (TEM) and Fourier transform infrared (FTIR) analyses. MNP-CUR internalization was evaluated after 6 hrs incubation with MDA-MB-231 cancer cells by Prussian blue stain and TEM. Anti-cancer effects of the MNP-CUR formulation was determined by a tetrazolium based dye (MTT) and colony formation assays using “triple negative” MDA-MB-231 cancer cells. Tetramethylrhodamine, ethyl ester, perchlorate (TMRE), red mitochondrial superoxide indicator (Mitosox), and propidium iodide stains were used to evaluate the loss of mitochondrial membrane potential, reactive oxygen species (ROS) generation, and apoptosis/dead cells, respectively, to determine effects MNP-CUR on these cellular features. The imaging capability of this MNP-CUR formulation was analyzed using a magnetic resonance imaging (MRI) system. The magnetic targeting feature of the MNP-CUR formulation was evaluated using fluorescence microscopy. Results: We prepared MNP-CUR with an average aggregative particles size of ∼ 150 nm (individual particle grain size, ∼ 9-11 nm). Prussian blue stain data represent a preferential uptake of MNP-CUR in MDA-MB-231 cells in a dose dependant manner. TEM analysis demonstrates that accumulation of MNP-CUR nanoparticles in cancer cells indicate that particles are not attached on the surface of cells but internalized within the cells. The MNP-CUR formulation showed strong anti-cancer effects in breast cancer cells including “triple negative” MDA-MB-231 cancer cells compared to free curcumin. This formulation also enhanced loss of mitochondrial membrane potential, generation of ROS and apoptosis compared to free curcumin treatment. Additionally, the MNP-CUR formulation exhibits superior T2 imaging characteristics compared to T1. A significant increase of the targeting feature was observed with MNP-CUR in breast cancer cells. Conclusion: These data suggest that our novel MNP-CUR formulation exhibits potent anti-cancer activity along with imaging and magnetic targeting capabilities. This approach can be extended to pre-clinical and clinical use and may have importance in cancer treatment and cancer imaging in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2893. doi:1538-7445.AM2012-2893

High Glucose Induces Mitochondrial Morphology and Metabolic Changes in Retinal Pericytes

Mitochondrial dysfunction is known to play a role in retinal vascular cell loss, a prominent lesion of diabetic retinopathy. High glucose (HG) has been reported to induce mitochondrial fragmentation and dysfunction in retinal endothelial cells, contributing to apoptosis. In this study, the effects of HG on mitochondrial morphology, membrane potential, and metabolic changes and whether they could contribute to HG-induced apoptosis in retinal pericytes were investigated. Bovine retinal pericytes (BRPs) were grown in normal or HG medium for 7 days. Both sets of cells were double stained with mitochondrial membrane potential-independent dye and tetramethylrhodamine-ethyl-ester-perchlorate (TMRE) and imaged by confocal microscopy. The images were analyzed for average mitochondria shape, by using form factor and aspect ratio values, and membrane potential changes, by using the ratio between the red and green dye. BRPs grown in normal or HG medium were analyzed for transient changes in oxygen consumption and extracellular acidification with a flux analyzer and apoptosis by TUNEL assay. BRPs grown in HG media exhibited significant fragmentation of mitochondria and increased membrane potential heterogeneity compared with the BRPs grown in normal medium. Concomitantly, BRPs grown in HG showed reduced steady state and maximum oxygen consumption and reduced extracellular acidification. Number of TUNEL-positive pericytes was increased in HG condition as well. In HG condition, mitochondria of retinal pericytes display significant fragmentation, metabolic dysfunction, and reduced extracellular acidification. The detrimental effects of HG on mitochondrial function and cellular metabolism could play a role in the accelerated apoptosis associated with the retinal pericytes in diabetic retinopathy.

ABC transporters affect the detection of intracellular oxidants by fluorescent probes

Intracellular production of reactive oxygen species (ROS) plays an important role in the control of cell physiology. For the assessment of intracellular ROS production, a plethora of fluorescent probes is commonly used. Interestingly, chemical structures of these probes imply they could be substrates of plasma membrane efflux pumps, called ABC transporters. This study tested whether the determination of intracellular ROS production and mitochondrial membrane potential by selected fluorescent probes is modulated by the expression and activity of ABC transporters. The sub-clones of the HL-60 cell line over-expressing MDR1, MRP1 and BCRP transporters were employed. ROS production measured by luminol- and L-012-enhaced chemiluminescence and cytochrome c reduction assay showed similar levels of ROS production in all the employed cell lines. It was proved that dihydrorhodamine 123, dihexiloxocarbocyanine iodide, hydroethidine, tetrachloro-tetraethylbenzimidazolocarbo-cyanine iodide and tetramethylrhodamine ethyl ester perchlorate are substrates for MDR1; dichlorodihydrofluoresceine, hydroethidine and tetramethylrhodamine ethyl ester perchlorate are substrates for MRP1; dichlorodihydrofluoresceine, dihydrorhodamine 123, hydroethidine and tetrachloro-tetraethylbenzimidazolocarbo-cyanine iodide are substrates for BCRP. Thus, the determination of intracellular ROS and mitochondrial potential by the selected probes is significantly altered by ABC transporter activities. The activity of these transporters must be considered when employing fluorescent probes for the assessment of ROS production or mitochondrial membrane potential.

Apoptosis in esophagus and pancreas carcinoma cells induced by circulating microparticles is related to phosphatidyl serine and microparticle-associated caspases

Circulating microparticles (MPs) are recently discussed as "biologically active", participating in the pathology of diseases rather than being a marker of damaging processes. It was the purpose of the present study to investigate the effects of MPs, as isolated from the blood of healthy volunteers, on the induction of apoptosis and necrosis in cultured KYSE-270 esophageal and ASPC1 pancreas carcinoma cells. MPs were obtained from the blood of 20 healthy volunteers (11 women; mean age 33.3 years). Viability, apoptosis, and necrosis were determined by flow cytometry using Annexin V/propidium iodide and tetramethylrhodamine ethyl ester perchlorate (TMRE)/propidium iodide for staining. Incubation of KYSE and ASPC1 carcinoma cells with MPs (1-20.000/μl) for 48 h reduced significantly viability of the cells, and induced apoptosis, but not necrosis. This apoptotic effect was significant at a concentration of ≥1.000 MPs/μl in both cell types. Pre-treatment of MPs with either the global caspase inhibitor ZVAD-FMK or Annexin V which blocks phosphatidyl serine in the outer membrane of MPs with high affinity, almost abolished MP-induced apoptosis. A specific enzyme assay as well Western blot analysis confirmed the presence (activity, protein) of the apoptotic enzyme caspase-3 in MPs. Incubation of carcinoma cells with MPs (20.000/μl) resulted in an increase in caspase-3 protein in carcinoma cells; this increase could be prevented by pre-treatment of MPs with Annexin V. It is suggested that MPs induce concentration-dependent apoptosis in KSYE esophageal and ASPC1 pancreas carcinoma cells in vitro by transferring caspases into target cells. This process probably requires a target cell-MP interaction, and membrane-bound anionic phosphatidyl serine may be involved.

Induction of early apoptosis and reactive oxygen species (ROS) production by Tanzanian basidiomycete (&lt;i&gt;Cantharellus miomboensis&lt;/i&gt;)

Cantharellus miomboensis is a new basidiomycete fungus recently found in Miombo woodlands in Tanzania. In this study, crude extract was prepared from fruiting bodies of C. miomboensis and was in vitro screened for its cytotoxicity using Tetrazolium salt (3-(4,5-dimethlthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) in human cell lines namely; Hepatocellular carcinoma (HepG2), Human non-small cell lung carcinoma (H157) and Human colon adenocarcinoma (HT.29). Thereafter, pro-apoptotic effects were determined using biochemical changes in apoptotic cells. These included externalization of phospholipid phosphatidylserine (PS) using APO Percentage dye by flow cytometry and depolarization of mitochondrial membrane potential using Tetramethyl rhodamine ethyl ester perchlorate (TMRE) assay. The test extract was found to induce dose dependent PS externalization on human cell lines when treated with various concentrations (1 - 5 mg/ml) and completely depolarized the mitochondrial membrane potential after 6 hours on HepG2 cell line. When the extract was examined for ROS production using 2’,7’- dichlorofluorescin diacetate (DCFH-DA) staining, there was no ROS generation found in HepG2 cells. It is therefore concluded that C. miomboensis extract is able to induce apoptosis in HepG2 cells and PS externalization and loss of mitochondrial membrane potential in HepG2 cells appear to be independent of ROS production. © 2010 International Formulae Group. All rights reserved.Keywords: Miombo woodlands, apoptosis, basidiomycete, reactive oxygen species, carcinoma.

Respiration in Adipocytes is Inhibited by Reactive Oxygen Species

It is a desirable goal to stimulate fuel oxidation in adipocytes and shift the balance toward less fuel storage and more burning. To understand this regulatory process, respiration was measured in primary rat adipocytes, mitochondria, and fat-fed mice. Maximum O(2) consumption, in vitro, was determined with a chemical uncoupler of oxidative phosphorylation (carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)). The adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio was measured by luminescence. Mitochondria were localized by confocal microscopy with MitoTracker Green and their membrane potential (Delta psi(M)) measured using tetramethylrhodamine ethyl ester perchlorate (TMRE). The effect of N-acetylcysteine (NAC) on respiration and body composition in vivo was assessed in mice. Addition of FCCP collapsed Delta psi(M) and decreased the ATP/ADP ratio. However, we demonstrated the same rate of adipocyte O(2) consumption in the absence or presence of fuels and FCCP. Respiration was only stimulated when reactive oxygen species (ROS) were scavenged by pyruvate or NAC: other fuels or fuel combinations had little effect. Importantly, the ROS scavenging role of pyruvate was not affected by rotenone, an inhibitor of mitochondrial complex I. In addition, mice that consumed NAC exhibited increased O(2) consumption and decreased body fat in vivo. These studies suggest for the first time that adipocyte O(2) consumption may be inhibited by ROS, because pyruvate and NAC stimulated respiration. ROS inhibition of O(2) consumption may explain the difficulty to identify effective strategies to increase fat burning in adipocytes. Stimulating fuel oxidation in adipocytes by decreasing ROS may provide a novel means to shift the balance from fuel storage to fuel burning.

A Single Luminally Continuous Sarcoplasmic Reticulum with Apparently Separate Ca2+ Stores in Smooth Muscle

Whether or not the sarcoplasmic reticulum (SR) is a continuous, interconnected network surrounding a single lumen or comprises multiple, separate Ca2+ pools was investigated in voltage-clamped single smooth muscle cells using local photolysis of caged compounds and Ca2+ imaging. The entire SR could be depleted or refilled from one small site via either inositol 1,4,5-trisphosphate receptors (IP3R) or ryanodine receptors (RyR) suggesting the SR is luminally continuous and that Ca2+ may diffuse freely throughout. Notwithstanding, regulation of the opening of RyR and IP3R, by the [Ca2+] within the SR, may create several apparent SR elements with various receptor arrangements. IP3R and RyR may appear to exist entirely on a single store, and there may seem to be additional SR elements that express either only RyR or only IP3R. The various SR receptor arrangements and apparently separate Ca2+ storage elements exist in a single luminally continuous SR entity.

Intersectin-1s Regulates the Mitochondrial Apoptotic Pathway in Endothelial Cells

Intersectins (ITSNs) are multidomain adaptor proteins implicated in endocytosis, regulation of actin polymerization, and Ras/MAPK signaling. We have previously shown that ITSN-1s is required for caveolae fission and internalization in endothelial cells (ECs). In the present study, using small interfering RNA to knock down ITSN-1s protein expression, we demonstrate a novel role of ITSN-1s as a key antiapoptotic protein. Knockdown of ITSN-1s in ECs activated the mitochondrial pathway of apoptosis as determined by genomic DNA fragmentation, extensive mitochondrial fission, activation of the proapoptotic proteins BAK and BAX, and cytochrome c efflux from mitochondria. ITSN-1 knockdown acts as a proapoptotic signal that causes mitochondrial outer membrane permeabilization, dissipation of the mitochondrial membrane potential, and generation of reactive oxygen species. These effects were secondary to decreased activation of Erk1/2 and its direct activator MEK. Bcl-X(L) overexpression prevented BAX activation and the apoptotic ECs death induced by suppression of ITSN-1s. Our findings demonstrate a novel role of ITSN-1s as a negative regulator of the mitochondrial pathway-dependent apoptosis secondary to activation of the Erk1/2 survival signaling pathway.

Identification of a Novel Mitochondrial Complex Containing Mitofusin 2 and Stomatin-like Protein 2

A reverse genetics approach was utilized to discover new proteins that interact with the mitochondrial fusion mediator mitofusin 2 (Mfn2) and that may participate in mitochondrial fusion. In particular, in vivo formaldehyde cross-linking of whole HeLa cells and immunoprecipitation with purified Mfn2 antibodies of SDS cell lysates were used to detect an approximately 42-kDa protein. This protein was identified by liquid chromatography and tandem mass spectrometry as stomatin-like protein 2 (Stoml2), previously described as a peripheral plasma membrane protein of unknown function associated with the cytoskeleton of erythrocytes (Wang, Y., and Morrow, J. S. (2000) J. Biol. Chem. 275, 8062-8071). Immunoblot analysis with anti-Stoml2 antibodies showed that Stoml2 could be immunoprecipitated specifically with Mfn2 antibody either from formaldehyde-cross-linked and SDS-lysed cells or from cells lysed with digitonin. Subsequent immunocytochemistry and cell fractionation experiments fully supported the conclusion that Stoml2 is indeed a mitochondrial protein. Furthermore, demonstration of mitochondrial membrane potential-dependent import of Stoml2 accompanied by proteolytic processing, together with the results of sublocalization experiments, suggested that Stoml2 is associated with the inner mitochondrial membrane and faces the intermembrane space. Notably, formaldehyde cross-linking revealed a "ladder" of high molecular weight protein species, indicating the presence of high molecular weight Stoml2-Mfn2 hetero-oligomers. Knockdown of Stoml2 by the short interfering RNA approach showed a reduction of the mitochondrial membrane potential, without, however, any obvious changes in mitochondrial morphology.

Thrombin stimulates calcium‐dependent production of mitochondrial ROS in pulmonary microvascular endothelial cells

Vascular cells respond to various stimuli via enhanced production of reactive oxygen species (ROS). In endothelial cells, proteins that produce ROS include the flavoenzyme NADPH oxidase of cell membranes and the mitochondrial electron transport chain. Thrombin, an enzyme linked to atherosclerosis and restenosis injury, can trigger cellular oxidative stress, although the mechanism for its effects in endothelial cells is unclear. To determine the mechanism by which thrombin evokes endothelial cell signaling, murine pulmonary microvascular endothelial cells (MPMVECs) were simultaneously loaded with the calcium indicator dye Fluo-4/AM and the mitochondrial membrane potential indicator tetramethylrhodamine, ethyl ester, perchlorate (TMRE) and visualized by confocal microscopy. Thrombin stimulated a large calcium release from the intracellular stores that preceded mitochondrial membrane potential loss. Subsequently, mitochondrial superoxide production was enhanced as evidenced by an increase in fluorescence of the mitochondrial superoxide-sensitive fluorophore, MitoSOX Red. Thrombin treatment also increased nuclear HE fluorescence, an indicator of cellular superoxide production, and was unaffected in the prescence of the NADPH oxidase inhibitor Apocynin and the anion channel blocker DIDS. In contrast, Apocynin and DIDS ablated the HE fluorescence increase observed in response to Angiotensin II, a known stimulator of NADPH oxidase. Thus, thrombin stimulates ROS production in endothelial cells primarily via mitochondria, and may play an important role in the vascular response in areas adjacent to injury. [HL-60290 & HL-07748]

Mannheimia haemolytica leukotoxin induces apoptosis of bovine lymphoblastoid cells (BL-3) via a caspase-9-dependent mitochondrial pathway.

Mannheimia haemolytica is a key pathogen in the bovine respiratory disease complex. It produces a leukotoxin (LKT) that is an important virulence factor, causing cell death in bovine leukocytes. The LKT binds to the beta(2) integrin CD11a/CD18, which usually activates signaling pathways that facilitate cell survival. In this study, we investigated mechanisms by which LKT induces death in bovine lymphoblastoid cells (BL-3). Incubation of BL-3 cells with a low concentration of LKT results in the activation of caspase-3 and caspase-9 but not caspase-8. Similarly, the proapoptotic proteins Bax and BAD were significantly elevated, while the antiapoptotic proteins Bcl-2, Bcl(XL) and Akt-1 were downregulated. Following exposure to LKT, we also observed a reduction in mitochondrial cytochrome c and corresponding elevation of cytosolic cytochrome c, suggesting translocation from the mitochondrial compartment to the cytosol. Consistent with this observation, tetramethylrhodamine ethyl ester perchlorate staining revealed that mitochondrial membrane potential was significantly reduced. These data suggest that LKT induces apoptosis of BL-3 cells via a caspase-9-dependent mitochondrial pathway. Furthermore, scanning electron micrographs of mitochondria from LKT-treated BL-3 cells revealed lesions in the outer mitochondrial membrane, which are larger than previous reports of the permeability transition pore through which cytochrome c is usually released.

Adenoviral melanoma differentiation-associated gene 7 induces apoptosis in lung cancer cells through mitochondrial permeability transition-independent cytochrome c release

Objective: Melanoma differentiation-associated gene 7 is a novel tumor suppressor gene that induces apoptosis in lung cancer cells when delivered by adenoviral gene transfer as Ad-mda7. The mechanisms of action are not well defined but may involve release of cytochrome c from the mitochondria with subsequent caspase activation. Methods: The lung cancer cell lines A549 and H1299 were transduced with Ad-mda7, adenovirus containing the gene for p53 (Ad-p53), and control adenoviral luciferase vectors. Staurosporine was used as a positive control to induce cytochrome c release through mitochondrial permeability transition-dependent pores, whereas cyclosporine (INN: ciclosporin) was used to specifically inhibit these mitochondrial permeability transition-dependent pores. Apoptosis was evaluated with fluorescence-activated cell sorting analysis of subdiploid populations and mitochondrial membrane potential changes with tetramethylrhodamine ethylester perchlorate. Results: Melanoma differentiation-associated gene 7, transduced by Ad-mda7 into H1299 and A549 lung cancer cells, resulted in sharp increases in cytosolic cytochrome c levels followed by induction of apoptosis and cellular death. The release of cytochrome c from the mitochondria occurred without changes in the mitochondrial membrane potential. Unlike staurosporine treatment, transduction with Ad-p53 and Ad-mda7 caused releases of cytochrome c and apoptosis that were not blocked by cyclosporine, suggesting a mitochondrial permeability transition pore-independent pathway. Conclusions: Ad-mda7 induces apoptosis in lung cancer cells through mitochondrial cytochrome c release in a process that is not dependent on mitochondrial membrane potential changes and occurs through mitochondrial permeability transition-independent pores. This unique mechanism of action may allow treatment of patients with lung cancer resistant to mitochondrial permeability transition-dependent cell death processes.

Diazoxide opens the mitochondrial permeability transition pore and alters Ca2+ transients in rat ventricular myocytes.

The mitochondrial K(ATP) channel (mitoK(ATP)) has been implicated as an end effector or trigger of ischemic preconditioning (IP). Although a mitoK(ATP) opener, diazoxide, mimics IP, mechanisms for the cardioprotective action remain unclear. We measured Ca2+ transients (CaTs) and mitochondrial inner membrane potential (Deltapsi(m)) with confocal microscopy and the fluorescent probes fluo-4 and tetramethylrhodamine ethyl ester perchlorate in rat ventricular myocytes. Diazoxide increased the amplitudes and diastolic levels of CaTs dose dependently. The effects of diazoxide on CaTs were inhibited by the mitoK(ATP) antagonist sodium 5-hydroxydecanoic acid (100 micromol/L), whereas application of diazoxide caused little change in Deltapsi(m). After sarcoplasmic reticulum function was disabled with ryanodine and thapsigargin, the effects of diazoxide on CaTs were still observed. The opening of the mitochondrial permeability transition pore was monitored with fluorescent calcein. Diazoxide accelerated the leakage of calcein from mitochondrial matrix (16% of control; P<0.05), and this effect was inhibited by cyclosporin A (2 micromol/L). Cyclosporin A also abolished the effects of diazoxide on CaTs. Diazoxide oxidized flavoprotein fluorescence reversibly, and this effect was partially blunted by cyclosporin A (by 24%; P<0.05). We conclude that in rat ventricular myocytes, diazoxide modulates the opening of the mitochondrial permeability transition pore, resulting in an increase in CaTs independent of the changes in Deltapsi(m). The action of diazoxide on the mitochondrial permeability transition pore also affects the mitochondrial redox state.