Inorganic polyphosphate's role in energy production and mitochondrial permeability transition pore opening in tick mitochondria.

Inorganic polyphosphate (polyP) is a biopolymer composed of phosphoanhydride-linked orthophosphate molecules. PolyP is engaged in a variety of cellular functions, including mitochondrial metabolism. Here, we examined the effects of polyP on electron transport chain enzymes and F1 Fo ATP synthase in tick embryos during embryonic development. The study found that polyPs containing medium and long chains (polyP15 and polyP65 ) enhanced the activity of complex I, complex II, complex III, and F1 Fo ATP synthase, while short polyP chains (polyP3 ) had no effect. The study also examined the activity of exopolyphosphatases (PPX) in various energy-demand situations. PPX activity was stimulated when ADP concentrations are high, characterizing a low-energy context. When complexes I-III and F1 Fo ATP synthase inhibitors were added in energized mitochondria, PPX activity decreased, whereas the mitochondrial uncoupler FCCP had no impact on PPX activity. Additionally, the study investigated the effect of polyP on mitochondrial swelling, finding that polyP causes mitochondrial swelling by increasing calcium effects on the mitochondrial permeability transition pore. The findings presented here to increase our understanding of the function of polyP in mitochondrial metabolism and its relationship to mitochondrial permeability transition pore opening in an arthropod model.

Mitochondrial responses to intracellular Ca2+ release following infrared stimulation.

Many studies of Ca2+ effects on mitochondrial respiration in intact cells have used electrical and/or chemical stimulation to elevate intracellular [Ca2+], and have reported increases in [NADH] and increased ADP/ATP ratios as dominant controllers of respiration. This study tested a different form of stimulation: brief temperature increases produced by pulses of infrared light (IR, 1,863 nm, 8-10°C for ∼5 s). Fluorescence imaging techniques applied to single PC-12 cells in low µM extracellular [Ca2+] revealed IR stimulation-induced increases in both cytosolic (fluo5F) and mitochondrial (rhod2) [Ca2+]. IR stimulation increased O2 consumption (porphyrin fluorescence), and produced an alkaline shift in mitochondrial matrix pH (Snarf1), indicating activation of the electron transport chain (ETC). The increase in O2 consumption persisted in oligomycin, and began during a decrease in NADH, suggesting that the initial increase in ETC activity was not driven by increased ATP synthase activity or an increased fuel supply to ETC complex I. Imaging with two potentiometric dyes [tetramethyl rhodamine methyl ester (TMRM) and R123] indicated a depolarizing shift in ΔΨm that persisted in high [K+] medium. High-resolution fluorescence imaging disclosed large, reversible mitochondrial depolarizations that were inhibited by cyclosporin A (CSA), consistent with the opening of transient mitochondrial permeability transition pores. IR stimulation also produced a Ca2+-dependent increase in superoxide production (MitoSox) that was not inhibited by CSA, indicating that the increase in superoxide did not require transition pore opening. Thus, the intracellular Ca2+ release that follows pulses of infrared light offers new insights into Ca2+-dependent processes controlling respiration and reactive oxygen species in intact cells.NEW & NOTEWORTHY Pulses of infrared light (IR) provide a novel method for rapidly transferring Ca2+ from the endoplasmic reticulum to mitochondria in intact cells. In PC12 cells the resulting ETC activation was not driven by increased ATP synthase activity or NADH. IR stimulation produced a Ca2+-dependent, reversible depolarization of ΔΨm that was partially blocked by cyclosporin A, and a Ca2+-dependent increase in superoxide that did not require transition pore opening.

Anastrozole-mediated modulation of mitochondrial activity by inhibition of mitochondrial permeability transition pore opening: an initial perspective.

The mitochondrial permeability transition pore (mtPTP) plays a vital role in altering the structure and function of mitochondria. Cyclophilin D (CypD) is a mitochondrial protein that regulates mtPTP function and a known drug target for therapeutic studies involving mitochondria. While the effect of aromatase inhibition on the mtPTP has been studied previously, the effect of anastrozole on the mtPTP has not been completely elucidated. The role of anastrozole in modulating the mtPTP was evaluated by docking, molecular dynamics and network-guided studies using human CypD data. The peripheral blood mononuclear cells (PBMCs) of patients with mitochondrial disorders and healthy controls were treated with anastrozole and evaluated for mitochondrial permeability transition pore (mtPTP) function and apoptosis using a flow cytometer. Spectrophotometry was employed for estimating total ATP levels. The anastrozole-CypD complex is more stable than cyclosporin A (CsA)-CypD. Anastrozole performed better than cyclosporine in inhibiting mtPTP. Additional effects included inducing mitochondrial membrane depolarization and a reduction in mitochondrial swelling and superoxide generation, intrinsic caspase-3 activity and cellular apoptosis, along with an increase in ATP levels. Anastrozole may serve as a potential therapeutic agent for mitochondrial disorders and ameliorate the clinical phenotype by regulating the activity of mtPTP. However, further studies are required to substantiate our preliminary findings.Communicated by Ramaswamy H. Sarma.

Modulation of mitochondrial permeability transition pore opening by Myricetin and prediction of its-drug-like potential using in silico approach.

Myricetin has been demonstrated to have multiple biological functions with promising research and development prospects. This study investigated the effect of myricetin on liver mitochondrial membrane permeability transition pores and its inhibitory potential on proteins that are important in the apoptotic process in silico. Mitochondrial swelling was assessed as changes in absorbance under succinate-energized conditions. Cytochrome c release, mitochondrial-lipid peroxidation, caspase 3 and 9 expressions, as well as calcium ATPase, were assessed. Pharmacokinetic properties of myricetin were predicted through the SwissADME server while the binding affinity of myricetin toward the proteins was computed using the AutodockVina Screening tool. The conformational stability of protein-ligand interactions was evaluated using molecular dynamics simulations analysis through the iMODS server. Myricetin inhibited the opening of the mitochondrial permeability transition pore and also reversed the increase in mitochondrial lipid peroxidation caused by calcium and other toxicants. Myricetin also caused a reduction in the expression of caspase 3 and 9 as well as calcium ATPase activity. The molecular docking results revealed that myricetin had a considerable binding affinity to the pocket site of caspase 3 and 9 as well as calcium ATPase. Myricetin showed a good drug-likeness based on the predicted pharmacokinetic properties as revealed by low CYP 450 inhibitory promiscuity and relatively low toxicity. It could therefore be suggested that myricetin could be useful in the management of diseases where too many apoptosis occur characterized by excessive tissue wastage such as neurodegenerative conditions and could as well play a role in protecting the physicochemical properties of membrane bilayers from free radical-induced severe cellular damage.

Glycolytic potential enhanced by blockade of pyruvate influx into mitochondria sensitizes prostate cancer to detection and radiotherapy.

OBJECTIVE: This study aimed to evaluate the effects of mitochondrial pyruvate carrier (MPC) blockade on the sensitivity of detection and radiotherapy of prostate cancer (PCa). METHODS: We investigated glycolysis reprogramming and MPC changes in patients with PCa by using metabolic profiling, RNA-Seq, and tissue microarrays. Transient blockade of pyruvate influx into mitochondria was observed in cellular studies to detect its different effects on prostate carcinoma cells and benign prostate cells. Xenograft mouse models were injected with an MPC inhibitor to evaluate the sensitivity of 18F-fluorodeoxyglucose positron emission tomography with computed tomography and radiotherapy of PCa. Furthermore, the molecular mechanism of this different effect of transient blockage towards benign prostate cells and prostate cancer cells was studied in vitro. RESULTS: MPC was elevated in PCa tissue compared with benign prostate tissue, but decreased during cancer progression. The transient blockade increased PCa cell proliferation while decreasing benign prostate cell proliferation, thus increasing the sensitivity of PCa cells to 18F-PET/CT (SUVavg, P = 0.016; SUVmax, P = 0.03) and radiotherapy (P < 0.01). This differential effect of MPC on PCa and benign prostate cells was dependent on regulation by a VDAC1-MPC-mitochondrial homeostasis-glycolysis pathway. CONCLUSIONS: Blockade of pyruvate influx into mitochondria increased glycolysis levels in PCa but not in non-carcinoma prostate tissue. This transient blockage sensitized PCa to both detection and radiotherapy, thus indicating that glycolytic potential is a novel mechanism underlying PCa progression. The change in the mitochondrial pyruvate influx caused by transient MPC blockade provides a critical target for PCa diagnosis and treatment.

Baicalein suppresses high glucose-induced inflammation and apoptosis in trophoblasts by targeting the miRNA-17-5p-Mfn1/2-NF-κB pathway.

INTRODUCTION: Controlling inflammation and apoptosis in trophoblasts is critical for treating gestational diabetes mellitus (GDM). Baicalein (Bai) exhibits anti-inflammatory and miRNA-related effects; however, its roles and mechanisms in GDM remain unknown. Therefore, we explored whether Bai inhibited inflammation and apoptosis in human trophoblasts (HTR8 cells) and analyzed its mechanisms. METHODS: HTR8 cells pretreated with Bai were subjected to the high-glucose (HG) stimulation before analyzing their viability, cytokine production and apoptosis, followed the expression profiles of small RNA sequencing data. The effects of miR-17-5p on the inflammation, mitochondrial fission, and apoptosis were investigated by ELISA, transmission electron microscopy and flow cytometry, respectively. Moreover, miR-17-5p, Mfn1/Mfn2 levels and mitochondrial morphology in human plasma and placental tissues from GDM-complicated and normal pregnant women were examined. RESULTS: Bai decreased the secretion of TNF-α, IL-1ß, IL-6 and apoptosis in HG-stimulated HTR8 cells, while miR-17-5p mediated the anti-inflammatory and anti-apoptotic effects of Bai. Mechanically, miR-17-5p targeted Mfn1/Mfn2 by affecting the mitochondrial fission and apoptosis via regulation of p-Drp1 (Ser 616) and p-NF-κB signaling. Moreover, overexpression of Mfn1/Mfn2 reversed miR-17-5p-elicited mitochondrial fission and inflammation in HG-stimulated HTR8 cells pretreated with Bai. Furhtermore, overexpression of Drp1 also reversed the anti-inflammatory effect of Mfn1/2 overexpression in HG-treated HTR8 cells via up-regulation of p65 phosphorylation. Finally, miR-17-5p was upregulated in human GDM plasma and placentas along with the reduced Mfn1/Mfn2. DISCUSSION: We are the first to demonstrate that bai exerts anti-inflammatory and anti-apoptotic effects on GDM, likely by targeting the miRNA-17-5p-Mfn1/2-NF-κB pathway.

Kigelia africana (Lam.) Benth leaf extract inhibits rat brain and liver mitochondrial membrane permeability transition pore opening.

The effect of Kigelia africana on mitochondrial membrane permeability transition has not been explored. In this study, the effect of a solvent fraction of Kigelia africana leaf extract on mitochondrial membrane permeability transition of rat brain and liver was evaluated. A methanol extract of K. africana leaves was fractionated into different solvents by vacuum liquid chromatography and following preliminary screening, the dichloromethane:ethylacetate (1:1) fraction was selected for further assays. Constituent phytochemicals in the fraction were revealed by thin-layer chromatography and further purification was carried out to characterize the compounds. Brain and liver mitochondria were isolated and used for mitochondrial membrane permeability transition and adenosine triphosphatase assays. Exogenous Ca2+ and Al3+ were used to trigger the mitochondrial membrane permeability transition opening. Physicochemical properties revealed by thin-layer chromatography showed that the isolated compounds were flavonoids. The extract inhibited mitochondrial membrane permeability transition opening in the presence and absence of triggering agents in brain and liver mitochondria. It also inhibited mitochondrial lipid peroxidation and adenosine triphosphatase activity. These results suggest that the extract can limit the rate of apoptosis via inhibition of mitochondrial membrane permeability transition which is pivotal to the mitochondrial apoptotic pathway and is an important therapeutic target in some pathological conditions.

Effects of Tl+ on the inner membrane thiol groups, respiration, and swelling in succinate-energized rat liver mitochondria were modified by thiol reagents.

The effects of both Tl+ and thiol reagents were studied on the content of the inner membrane free SH-groups, detected with Ellman reagent, and the inner membrane potential as well as swelling and respiration of succinate-energized rat liver mitochondria in medium containing TlNO3 and KNO3. These effects resulted in a rise in swelling and a decrease in the content, the potential, and mitochondrial respiration in 3 and 2,4-dinitrophenol-uncoupled states. A maximal effect was seen when phenylarsine oxide reacting with thiol groups recessed into the hydrophobic regions of the membrane. Compared with phenylarsine oxide, the effective concentrations of other reagents were approximately one order of magnitude higher in experiments with mersalyl and 4,4'-diisothiocyanostilbene-2,2'-disulfonate, and two orders of magnitude higher in experiments with tert-butyl hydroperoxide and diamide. The above effects of Tl+ and the thiol reagents became even more pronounced with calcium overload of mitochondria. However, the effects were suppressed by inhibitors of the mitochondrial permeability transition pore (cyclosporine A, ADP, and n-ethylmaleimide). These findings suggest that opening of the pore induced by Tl+ in the inner membrane can be dependent on the conformation state of the adenine nucleotide translocase, which depends on the activity of its thiol groups.

TIMM29 interacts with hepatitis B virus preS1 to modulate the HBV life cycle.

Hepatitis B virus (HBV), a major global health problem, can cause chronic hepatitis, liver cirrhosis, and hepatocellular carcinomas in chronically infected patients. However, before HBV infection can be adequately controlled, many mysteries about the HBV life cycle must be solved. In this study, TIMM29, an inner mitochondrial membrane protein, was identified as an interaction partner of the preS1 region of the HBV large S protein. The interaction was verified by both an immunoprecipitation with preS1 peptides and a GST-pulldown assay. Immunofluorescence studies also showed colocalization of preS1 and TIMM29. Moreover, it was determined that the preS1 bound with amino acids 92-189 of the TIMM29 protein. Infection of HBV in TIMM29-overexpressing NTCP/G2 cells resulted in a significant decrease of HBeAg and both extracellular particle-associated and core particle-associated HBV DNA without affecting cccDNA formation. Comparable results were obtained with TIMM29-overexpressing HB611 cells, which constitutively produce HBV. In contrast, knockout of TIMM29 in NTCP/G2 cells led to a higher production of HBV including HBeAg expression, as did knockout of TIMM29 in HB611. Collectively, these results suggested that TIMM29 interacts with the preS1 region of the HBV large S protein and modulates HBV amplification.

Targeted disruption of PDE3B, but not PDE3A, protects murine heart from ischemia/reperfusion injury.

Although inhibition of cyclic nucleotide phosphodiesterase type 3 (PDE3) has been reported to protect rodent heart against ischemia/reperfusion (I/R) injury, neither the specific PDE3 isoform involved nor the underlying mechanisms have been identified. Targeted disruption of PDE3 subfamily B (PDE3B), but not of PDE3 subfamily A (PDE3A), protected mouse heart from I/R injury in vivo and in vitro, with reduced infarct size and improved cardiac function. The cardioprotective effect in PDE3B(-/-) heart was reversed by blocking cAMP-dependent PKA and by paxilline, an inhibitor of mitochondrial calcium-activated K channels, the opening of which is potentiated by cAMP/PKA signaling. Compared with WT mitochondria, PDE3B(-/-) mitochondria were enriched in antiapoptotic Bcl-2, produced less reactive oxygen species, and more frequently contacted transverse tubules where PDE3B was localized with caveolin-3. Moreover, a PDE3B(-/-) mitochondrial fraction containing connexin-43 and caveolin-3 was more resistant to Ca(2+)-induced opening of the mitochondrial permeability transition pore. Proteomics analyses indicated that PDE3B(-/-) heart mitochondria fractions were enriched in buoyant ischemia-induced caveolin-3-enriched fractions (ICEFs) containing cardioprotective proteins. Accumulation of proteins into ICEFs was PKA dependent and was achieved by ischemic preconditioning or treatment of WT heart with the PDE3 inhibitor cilostamide. Taken together, these findings indicate that PDE3B deletion confers cardioprotective effects because of cAMP/PKA-induced preconditioning, which is associated with the accumulation of proteins with cardioprotective function in ICEFs. To our knowledge, our study is the first to define a role for PDE3B in cardioprotection against I/R injury and suggests PDE3B as a target for cardiovascular therapies.

Reno-protective role of flunarizine (mitochondrial permeability transition pore inactivator) against gentamicin induced nephrotoxicity in rats.

This study was aimed to evaluate the role of flunarizine on gentamicin (GEM) induced nephrotoxicity in rat. Administration of GEM (40 mg/kg, s.c. for 10 consecutive days) significantly increased blood urea nitrogen (BUN), N-acetyl ß-d-glucosaminidase (NAG), thiobarbituric acid reactive substances (TBARS) and total calcium whereas, decreased body weight, fractional excretion of sodium (FrNa), creatinine clearance (CrCl), reduced glutathione (GSH), mitochondrial cytochrome c oxidase (Cyt-C oxidase) and ATP levels resulting in nephrotoxicity. Further, flunarizine (100, 200 and 300 µmol/kg, p.o.) was administered to evaluate its renoprotective effect against GEM induced nephrotoxicity and the results were compared with cylcosporin A (CsA, 50 µmol/kg, p.o.). Flunarizine resulted in the attenuation of renal dysfunction and oxidative marker changes in rats subjected to GEM induced nephrotoxicity in a dose dependent manner. Medium and higher doses of flunarizine produced significant renal protective effect which was comparable to cyclosporin A. The results of this study clearly revealed that flunarizine protected the kidney against the nephrotoxic effect of GEM via mitochondrial permeability transition pore (MPTP) inactivation potential.