Bioenerg Biomembr Research Articles

MON-LB086 Single-Cell Transcriptional Profiling of Bone Cells Reveals Diversity of Osteoblasts

Recent studies using mouse genetics have revealed that, beyond ambulation, protection and ion homeostasis, bone plays an unexpected broader role in whole-organism physiology as an endocrine organ. The osteoblast-derived hormone osteocalcin (Ocn) is necessary for glucose homeostasis, insulin secretion, male fertility, brain development, cognition and adaptation to exercise (1,2). However, it remains unknown whether the diversity of bone’s functions is built upon diversity in osteoblast subtypes. Here, we perform single-cell RNA-seq to explore the diverse physiological functions of osteoblasts. While all osteoblasts expressed equally high levels of classically osteogenic genes like Runx2, and Type I Collagen, our analysis identified four transcriptionally distinct osteoblast populations. Strikingly, one osteoblast population expresses Ocn as well as several other genes, including vitamin D receptor Vdr and Wnt signaling modulator Dkk1, all of which have been implicated in osteoblasts as negative regulators of bone mass control (3,4,5). In contrast, other osteoblast populations are enriched for bone mineralization genes, voltage-gated ion channels, postsynaptic markers, and neurotrophic factors, while the Ocn high population is not. These molecular signatures further establish the neuroendocrine function of osteoblasts, which is further supported by the role of Ocn in the response to acute stressors, immunohistochemistry of postsynaptic markers in bone, and patch clamp studies showing that osteoblasts can fire action potentials (6). The distinct molecular signatures identified suggest a specialization of osteoblast populations that correspond to specific physiological roles such as hormone secretion, bone synthesis, mineralization and neuronal interaction. Reference: (1) Karsenty et al. 2012. Nature 481, 314. (2) Obri et al. 2018. Nat Rev Endocrinol 14, 174. (3) Ducy et al. 1996. Nature 382, 448 (4) Yamamoto et al. 2013. Endocrinology 154, 1008 (5) McDonald et al. 2018. Calcif Tissue Int. 102, 105 (6) Pangalos et al. 2011. J Bioenerg Biomembr 43, 311. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

A Synthetic Peptide from the N-Terminal of Hexokinase I Prevents the Interaction Between VDAC1 and SOD1 G93A Mutant Recovering the Viability of an ALS Cell Model

Superoxide Dismutase 1 mutants associate with 20-25% of familial Amyotrophic Lateral Sclerosis (ALS) cases, producing toxic aggregates on mitochondria, notably in spinal cord [1,2]. The Voltage Dependent Anion Channel isoform 1 (VDAC1), the main pore in the outer mitochondrial membrane [3] is a docking site for SOD1 G93A mutant in ALS mice and the physiological receptor of Hexokinase I (HK1) [4], which is poorly expressed in mouse spinal cord [5, www.brain-map.org]. Our results demonstrate that HK1 competes with SOD1 G93A for binding VDAC1, suggesting that in ALS spinal cord available HK1-binding sites could be used by SOD1 mutants for docking mitochondria, producing thus organelle dysfunction [6]. We tested this model by studying the action of a HK1-N-end based peptide (NHK1). This NHK1 peptide specifically interacts with VDAC1, inhibits the SOD1 G93A binding to mitochondria and restores the viability of ALS model NSC34 cells [6]. Altogether, our results suggest that NHK1 peptide could be developed as a therapeutic tool in ALS, predicting an effective role also in other proteinopathies.1. Magri A et al, Biochim Biophys Acta (2016) 1857:789-982. Vande Velde C et al, PNAS (2008) 105:4022-73. De Pinto V et al, Biochim Biophys Acta (1989) 987:1-74. Tomasello MF et al (2013) PlosOne 8, e815225. Pastorino JG & Hoek JB J Bioenerg Biomembr (2008) 40:171-826. Magri A et al, SciRep. 2016, in press.

Deletion of a unique loop in the mycobacterial F-ATP synthase γ subunit sheds light on its inhibitory role in ATP hydrolysis-driven H(+) pumping.

The F1 FO -ATP synthase is one of the enzymes that is essential to meet the energy requirement of both the proliferating aerobic and hypoxic dormant stages of the life cycle of mycobacteria. Most F-ATP synthases consume ATP in the α3 :β3 headpiece to drive the γ subunit, which couples ATP cleavage with proton pumping in the c ring of FO via the bottom of the γ subunit. ATPase-driven H(+) pumping is latent in mycobacteria. The presence of a unique 14 amino acid residue loop of the mycobacterial γ subunit has been described and aligned in close vicinity to the c-ring loop Priya R et al. (2013) J Bioenerg Biomembr 45, 121-129 Here, we used inverted membrane vesicles (IMVs) of fast-growing Mycobacterium smegmatis and a variety of covalent and non-covalent inhibitors to characterize the ATP hydrolysis activity of the F-ATP synthase inside IMVs. These vesicles formed a platform to investigate the function of the unique mycobaterial γ loop by deleting the respective loop-encoding sequence (γ166-179 ) in the genome of M. smegmatis. ATP hydrolysis-driven H(+) pumping was observed in IMVs containing the Δγ166-179 mutant protein but not for IMVs containing the wild-type F-ATP synthase. In addition, when compared to the wild-type enzyme, IMVs containing the Δγ166-179 mutant protein showed increased ATP cleavage and lower levels of ATP synthesis, demonstrating that the loop affects ATPase activity, ATPase-driven H(+) pumping and ATP synthesis. These results further indicate that the loop may affect coupling of ATP hydrolysis and synthesis in a different mode.

Early changes in membrane potential of Saccharomyces cerevisiae induced by varying extracellular K+, Na+ or H+ concentrations

Recently we introduced a fluorescent probe technique that makes possible to convert changes of equilibrium fluorescence spectra of 3,3'-dipropylthiadicarbocyanine, diS-C3(3), measured in yeast cell suspensions under defined conditions into underlying membrane potential differences, scaled in millivolts (Plasek et al. in J Bioenerg Biomembr 44: 559-569, 2012). The results presented in this paper disclose measurements of real early changes of plasma membrane potential induced by the increase of extracellular K(+), Na(+) and H(+) concentration in S. cerevisiae with and without added glucose as energy source. Whereas the wild type and the ∆tok1 mutant cells exhibited similar depolarization curves, mutant cells lacking the two Trk1,2 potassium transporters revealed a significantly decreased membrane depolarization by K(+), particularly at lower extracellular potassium concentration [K(+)]out. In the absence of external energy source plasma membrane depolarization by K(+) was almost linear. In the presence of glucose the depolarization curves exhibited an exponential character with increasing [K(+)]out. The plasma membrane depolarization by Na(+) was independent from the presence of Trk1,2 transporters. Contrary to K(+), Na(+) depolarized the plasma membrane stronger in the presence of glucose than in its absence. The pH induced depolarization exhibited a fairly linear relationship between the membrane potential and the pHo of cell suspensions, both in the wild type and the Δtrk1,2 mutant strains, when cells were energized by glucose. In the absence of glucose the depolarization curves showed a biphasic character with enhanced depolarization at lower pHo values.

Monocarboxylate Transporters Play an Important Role in Aerobicglycolysis in Multiple Myeloma

Abstract 1821 IntroductionIt has been reported that cancer cells utilize glycolysis pathway (non-oxidative breakdown of glucose) even in the presence of adequate oxygen to provide cancer cells with energy, called the Warburg effect (aerobic glycolysis) that ultimately leads to produce lactate. We reported in the last ASH meeting that aerobic glycolysis is up-regulated in multiple myeloma (MM) cells in patients with high serum LDH levels and aerobic glycolysis itself could serve as a novel therapeutic target in MM patients. Here we report an importance of lactate transporter for the growth and survival of MM cells. Lactate, produced from pyruvate by lactate dehydrogenase A (LDHA), is known as an important energy source for solid tumor cells and is associated with tumor angiogenesis and chemo-resistance (Pinheiro, C., et al. J Bioenerg Biomembr. 44:127–139, 2012). On the other hand, LDHB converts lactate to pyruvate, thus negatively regulating lactate production. It is known that lactate is pumped out through monocarboxylate trasnporter, MCT4, while MCT1 mainly imports lactate to inside of cells. However, roles of MCT1 and MCT4 in MM cells remain to be elucidated. We here investigated the roles of these two molecules in the growth and survival of MM cells. CD147, a purported chaperone protein for MCT1, was also examined. MethodsSix MM cell lines, RPMI8226, U266, KMS12BM, KMS12PE, KHM11, and KMM1 were employed. Six genes associated with glycolysis, i.e., LDHA, LDHB, MCT1-4, were examined using real time PCR analysis. Expressions of MCT1 and MCT4 were analyzed with western blotting. Expression of CD147 was investigated by flow cytometry. Lactate production into culture supernatants of MM cell lines were analyzed by using a lactate analyzer. An inhibitor of MCT1, a-cyano-4 hydroxycinnamic acid (CHC), was utilized to analyze cytotoxic effects on MM cells. AnnexinV/PI stained cells was analyzed by flow cytometry to quantify cytotoxicity. MCT1-expression was inhibited by using siRNA. Dichroloacetate (DCA), an inhibitor of PDK1, was utilized for inhibiting glycolysis. ResultsAccumulation of lactate was found in the supernatants of MM cell lines as cell density increased. Transporters of lactate, MCT1, MCT4 and CD147, were found in most MM cell lines at various levels, suggesting that transportation of lactate occurs through membrane of MM cells. To examine the role of lactate as a growth promotion factor, lactate was exogenously supplemented to KMS-12-PE cells. Interestingly, expressions of MCT1 and LDHB genes increased by the addition of lactate while those of MCT4 and LDHA only moderately changed (Fig. 1), suggesting that lactate was imported to cells through MCT1, then converted to pyrvate by LDHB. These results raised a possibility that lactate is utilized by MM cells as a growth factor. To examine the possibility, CHC, an inhibitor of MCT1, was supplemented to MM cell cultures. Interestingly, CHC induced apoptosis in MM cells in a dose dependent manner (Fig. 2). Moreover, inhibition of MCT1 gene by siRNA showed significant induction of apoptosis (Fig. 3), strongly suggesting that MCT1 plays a crucial role for survival of MM cells. Finally, we found a significant increase in the apoptosis of MM cells when CHC and DCA were simultaneously added in the culture (Fig.4), suggesting that MCT1 functions independently from glycolysis per se and that CHC and DCA act additively in starving lactate within MM cells. ConclusionOur results suggest that lactate is actively transported through monocarboxylate transporters. Given the results that exogenous lactate production increased MCT1 and LDHB expression, lactate should play a role as a regulator of lactate transportation and glycolysis as well as an important energy source. Because we found significant amount of lactate was produced from stromal cells obtained from MM patients, lactate may be supplied not only from MM cells themselves but also from micro-environment. Our finding that inhibition of MCT1 leads to cell death suggests that MCT1 could be a potential novel target molecule in MM therapy that could be stratified in combination with glycolysis inhibitor. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

D-Amino acid oxidase-induced oxidative stress, 3-bromopyruvate and citrate inhibit angiogenesis, exhibiting potent anticancer effects

Angiogenesis is critical for cancer growth and metastasis. Steps of angiogenesis are energy consuming, while vascular endothelial cells are highly glycolytic. Glioblastoma multiforme (GBM) is a highly vascular tumor and this enhances its aggressiveness. D-amino acid oxidase (DAO) is a promising therapeutic protein that induces oxidative stress upon acting on its substrates. Oxidative stress-energy depletion (OSED) therapy was recently reported (El Sayed et al., Cancer Gene Ther, 19, 1-18, 2012). OSED combines DAO-induced oxidative stress with energy depletion caused by glycolytic inhibitors such as 3-bromopyruvate (3BP), a hexokinase II inhibitor that depleted ATP in cancer cells and induced production of hydrogen peroxide. 3BP disturbs the Warburg effect and antagonizes effects of lactate and pyruvate (El Sayed et al., J Bioenerg Biomembr, 44, 61-79, 2012). Citrate is a natural organic acid capable of inhibiting glycolysis by targeting phosphofructokinase. Here, we report that DAO, 3BP and citrate significantly inhibited angiogenesis, decreased the number of vascular branching points and shortened the length of vascular tubules. OSED delayed the growth of C6/DAO glioma cells. 3BP combined with citrate delayed the growth of C6 glioma cells and decreased significantly the number and size of C6 glioma colonies in soft agar. Human GBM cells (U373MG) were resistant to chemotherapy e.g. cisplatin and cytosine arabinoside, while 3BP was effective in decreasing the viability and disturbing the morphology of U373MG cells.

Mitochondria from Dipodascus (Endomyces) magnusii and Yarrowia lipolytica yeasts did not undergo a Ca2+-dependent permeability transition even under anaerobic conditions

In this study we used tightly-coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts. The two yeast strains are good alternatives to Saccharomyces cerevisiae, being aerobes containing well structured mitochondria (thus ensuring less structural limitation to observe their appreciable swelling) and fully competent respiratory chain with three invariantly functioning energy conservation points, including Complex I, that can be involved in induction of the canonical Ca²⁺/P(i)-dependent mitochondrial permeability transition (mPTP pore) with an increased open probability when electron flux increases(Fontaine et al. J Biol Chem 273: 25734–25740, 1998; Bernardi et al. FEBS J 273:2077–2099, 2006). High amplitude swelling and collapse of the membrane potential were used as parameters for demonstrating pore opening. Previously (Kovaleva et al. J Bioenerg Biomembr 41:239–249, 2009; Kovaleva et al. Biochemistry (Moscow) 75: 297–303, 2010) we have shown that mitochondria from Y.lipolytica and D. magnusii were very resistant to the Ca²⁺overload combined with varying concentrations of P(i),palmitic acid, SH-reagents, carboxyatractyloside (an inhibitor of ADP/ATP translocator), as well as depletion of intramitochondrial adenine nucleotide pools, deenergization of mitochondria, and shifting to acidic pH values in the presence of high [P(i)]. Here we subjected yeast mitochondria to other conditions known to induce an mPTP in animal and plant mitochondria, namely to Ca²⁺ overload under hypoxic conditions (anaerobiosis). We were unable to observe Ca²⁺-induced high permeability of the inner membrane of D. magnusii and Y. lipolytica yeast mitochondria under anaerobic conditions, thus suggesting that an mPTP-like pore, if it ever occurs in yeast mitochondria, is not coupled with the Ca²⁺ uptake. The results provide the first demonstration of ATP-dependent energization of yeast mitochondria under conditions of anaerobiosis.

Structural elements of the C-terminal domain of subunit E (E133-222) from the Saccharomyces cerevisiae V1VO ATPase determined by solution NMR spectroscopy

Subunit E of the vacuolar ATPase (V-ATPase) contains an N-terminal extended α helix (Rishikesan et al. J Bioenerg Biomembr 43:187-193, 2011) and a globular C-terminal part that is predicted to consist of a mixture of α-helices and β-sheets (Grüber et al. Biochem Biophys Res Comm 298:383-391, 2002). Here we describe the production, purification and 2D structure of the C-terminal segment E₁₃₃₋₂₂₂ of subunit E from Saccharamyces cerevisiae V-ATPase in solution based on the secondary structure calculation from NMR spectroscopy studies. E₁₃₃₋₂₂₂ consists of four β-strands, formed by the amino acids from K136-V139, E170-V173, G186-V189, D195-E198 and two α-helices, composed of the residues from R144-A164 and T202-I218. The sheets and helices are arranged as β1:α1:β2:β3:β4:α2, which are connected by flexible loop regions. These new structural details of subunit E are discussed in the light of the structural arrangements of this subunit inside the V₁- and V₁V₀ ATPase.

A role for anions in ATP synthesis and its molecular mechanistic interpretation

ATP, the 'universal biological energy currency', is synthesized by utilizing energy either from oxidation of fuels or from light, via the process of oxidative and photo-phosphorylation respectively. The process is mediated by the enzyme F(1)F(0)-ATP synthase, using the free energy of ion gradients in the final energy catalyzing step, i.e., the synthesis of ATP from ADP and inorganic phosphate (P(i)). The details of the molecular mechanism of ATP synthesis are among the most important fundamental issues in biology and hence need to be properly understood. In this work, a role for anions in making ATP has been found. New experimental data has been reported on the inhibition of ATP synthesis at nanomolar concentrations by the potent, specific anion channel blockers 4,4'-diisothiocyanostilbene-2, 2'-disulphonic acid (DIDS) and tributyltin chloride (TBTCl). Based on these inhibition studies, attention has been drawn to anion translocation (in addition to proton translocation) as a requirement for ATP synthesis. The type of inhibition has been quantified and an overall kinetic scheme for mixed inhibition that explains the data has been evolved. The experimental data and the type of inhibition found have been interpreted in the light of the torsional mechanism of energy transduction and ATP synthesis (Nath J Bioenerg Biomembr 42:293-300, 2010a; J Bioenerg Biomembr 42:301-309, 2010b). This detailed and unified mechanism resolves long-standing problems and inconsistencies in the first theories (Slater Nature 172:975-978, 1953; Williams J Theor Biol 1:1-17, 1961; Mitchell Nature 191:144-148, 1961; Mitchell Biol Rev 41:445-502, 1966), makes several novel predictions that are experimentally verifiable (Nath Biophys J 90:8-21, 2006a; Process Biochem 41:2218-2235, 2006b), and provides us with a new and fruitful paradigm in bioenergetics. The interpretation presented here provides intelligent answers to the unexplained existing results in the literature. It is shown that mechanistic interpretation of the experimental data requires substantial addition to available conceptual foundations such that present concepts, theories, and mechanisms must be revised.

The Therapeutic Potential of Pyruvate

Pyruvate, the anionic form of a simple α-keto acid, plays a key role in intermediary metabolism, being the end-product of glycolysis and the starting substrate for the tricarboxylic acid (TCA) cycle. Additionally, pyruvate reacts rapidly and non-enzymatically with the reactive oxygen species (ROS), hydrogen peroxide (H2O2). Scavenging of H2O2 by endogenously generated pyruvate is probably a key cellular defense against oxidative stress, particularly in proliferating cells [ 1 Brand K. Aerobic glycolysis by proliferating cells: Protection against oxidative stress at the expense of energy yield. J Bioenerg Biomembr. 1997; 29: 355 Crossref PubMed Scopus (126) Google Scholar , 2 Brand K.A. Hermfisse U. Aerobic glycolysis by proliferating cells: A protective strategy against reactive oxygen species. FASEB J. 1997; 11: 388 Crossref PubMed Scopus (333) Google Scholar ].

Apoptosis: it's BAK to VDAC

VDAC's role in Bak‐dependent mitochondrial outer membrane permeabilization (MOMP) is controversial. Gordon Shore makes sense of it all in light of an EMBO reports paper that shows that VDAC mediates mitochondrial recruitment of Bak, thereby controlling tBid‐induced MOMP.

Physiological aspects of the mitochondrial cyclosporin A-insensitive palmitate/Ca2+-induced pore: tissue specificity, age profile and dependence on the animal’s adaptation to hypoxia

Earlier we found that being added to rat liver mitochondria, palmitic acid (Pal) plus Ca(2+) opened a cyclosporin A-insensitive pore, which remained open for a short time. Apparently, this pore is involved in the Pal-induced apoptosis and may also take part in the mitochondrial Ca(2+) recycling as a Ca(2+) efflux system (Belosludtsev et al. J Bioenerg Biomembr 38:113-120, 2006; Mironova et al. J. Bioenerg. Biomembr. 39:167-174, 2007). In this paper, we continue studying physiological and regulatory aspects of the pore. The following observations have been made. (1) Cardiolipin has been found to facilitate the Ca(2+)-induced formation of pores in the Pal-containing liposomal membranes. (2) The opening of Pal/Ca(2+)-induced pore is accompanied by the release of apoptosis-induced factor (AIF) from mitochondria. (3) The rate of Pal/Ca(2+)-induced swelling of rat liver mitochondria increases substantially with the age of animals. (4) Although the Pal/Ca(2+)-induced pore opens both in the liver and heart mitochondria, the latter require higher Pal concentrations for the pore to open. (5) The pore opening depends on the resistance of animals to hypoxia: in the highly resistant to hypoxia rats, the mitochondrial Pal/Ca(2+)-induced pore opens easier than in the low resistant animals, this being opposite for the classical, cyclosporin A-sensitive MPT pore. The adaptation of the low resistant rats to oxygen deficiency increases the sensitivity of their mitochondria to PalCaP inductors. The paper also discusses a possible role of the mitochondrial Pal/Ca(2+)-induced pore in the protection of tissues against hypoxia.

Microbial toxin’s effect on mitochondrial survival by increasing K+ uptake

We studied the effects of toxins, which inhibited the motility of boar spermatozoa, on rat liver mitochondria. The toxins studied were originally from bacteria isolated from moisture-damaged buildings where inhabitants exhibited symptoms, or from food causing poisoning. Some strains of Bacillus cereus and Streptomyces griseus produced potassium ionophoric peptides cereulide and valinomycin (Mikkola, et al., European Journal of Biochemistry 1999; 263: 112-117). Of interest is that channels were formed in black-lipid membranes (BLM) with a selectivity of K(+) > Na(+) at a concentration of 26 nM. Recently, bafilomycin A1--an inhibitor of V-H(+)ATPases--was found also to be a K(+)-specific ionophore active at nanomolar concentrations (Teplova, et al., J Bioenerg Biomembr 2007; 39: 321-329), while B. amyloliquefaciens produced amylosin, a cation channel-forming peptide with a higher selectivity for K(+) over Na(+) at around 200 nM concentrations (Mikkola, et al., Toxicon 2007; 49: 1158-1171). Of interest is that channels were formed in BLM with a selectivity of K(+) > Na(+) at a concentration of 26 nM. The ionophores and the channel-forming amylosin caused swelling of energized mitochondria due to uptake of K(+), loss of membrane potential, inhibition of maximal respiration rates due to loss of pyridine nucleotides, and inhibition of ATP synthesis. Various cell types may have different sensitivities to the effects of the ionophores. Thus, the mitochondrial membrane potential in neuronal cells was more sensitive to cereulide than in differentiated Paju cells (Teplova, et al., Acta Biochimica Polonica 2004; 51: 539-544). Swelling causes release of proapoptotic factors from mitochondria, which explains that undifferentiated neuronal cells were sensitive, while differentiated Paju cells were resistant, which probably is due to them having an increased expression of the antiapoptotic protein Bcl-2 and the neuroprotective stanniocalcin.

Induction of a non-specific permeability transition in mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts

In this study we used tightly-coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, possessing a respiratory chain with the usual three points of energy conservation. High-amplitude swelling and collapse of the membrane potential were used as parameters for demonstrating induction of the mitochondrial permeability transition due to opening of a pore (mPTP). Mitochondria from Y. lipolytica, lacking a natural mitochondrial Ca(2+) uptake pathway, and from D. magnusii, harboring a high-capacitive, regulated mitochondrial Ca(2+) transport system (Bazhenova et al. J Biol Chem 273:4372-4377, 1998a; Bazhenova et al. Biochim Biophys Acta 1371:96-100, 1998b; Deryabina and Zvyagilskaya Biochemistry (Moscow) 65:1352-1356, 2000; Deryabina et al. J Biol Chem 276:47801-47806, 2001) were very resistant to Ca(2+) overload. However, exposure of yeast mitochondria to 50-100 microM Ca(2+) in the presence of the Ca(2+) ionophore ETH129 induced collapse of the membrane potential, possibly due to activation of the fatty acid-dependent Ca(2+)/nH(+)-antiporter, with no classical mPTP induction. The absence of response in yeast mitochondria was not simply due to structural limitations, since large-amplitude swelling occurred in the presence of alamethicin, a hydrophobic, helical peptide, forming voltage-sensitive ion channels in lipid membranes. Ca(2+)- ETH129-induced activation of the Ca(2+)/H(+)-antiport system was inhibited and prevented by bovine serum albumin, and partially by inorganic phosphate and ATP. We subjected yeast mitochondria to other conditions known to induce the permeability transition in animal mitochondria, i.e., Ca(2+) overload (in the presence of ETH129) combined with palmitic acid (Mironova et al. J Bioenerg Biomembr 33:319-331, 2001; Sultan and Sokolove Arch Biochem Biophys 386:37-51, 2001), SH-reagents, carboxyatractyloside (an inhibitor of the ADP/ATP translocator), depletion of intramitochondrial adenine nucleotide pools, deenergization of mitochondria, and shifting to acidic pH values in the presence of high phosphate concentrations. None of the above-mentioned substances or conditions induced a mPTP-like pore. It is thus evident that the permeability transition in yeast mitochondria is not coupled with Ca(2+) uptake and is differently regulated compared to the mPTP of animal mitochondria.

The Relationship between the Aging- and Photo-Dependent T414G Mitochondrial DNA Mutation with Cellular Senescence and Reactive Oxygen Species Production in Cultured Skin Fibroblasts

Mutations in the mitochondrial genome (mtDNA) are thought to be one of the causes of age-dependent cellular decline through their detrimental effects on respiration or reactive oxygen species (ROS) production. However, for many mutations, this link has not been clearly established. This study aimed to further investigate the phenotypic importance of a T414G mutation within the control region of mtDNA, previously shown to accumulate in both chronologically and photoaged human skin. We demonstrate that during dermal skin fibroblast replication in vitro in five separate cultures obtained from elderly individuals, the T414G mutant load can either increase or decrease during progressive cell division, implying the absence of consistent selection against the mutation in this context. In support of this, by utilizing a cell-sorting approach, we demonstrate that the level of the T414G mutation does not directly correlate with increased or decreased mtDNA copy number, or markers of cellular ageing including lipofuscin accumulation or ROS production. By consequence, the mutation can be distributed with a bias towards either the proliferating or senescent cell populations depending on the cell line. In conclusion, we propose that this particular mutation may have little effect on ROS production and the onset of cellular senescence in cultured fibroblasts.

Causal Role of Apoptosis-Inducing Factor for Neuronal Cell Death Following Traumatic Brain Injury

Traumatic brain injury (TBI) consists of two phases: an immediate phase in which damage is caused as a direct result of the mechanical impact; and a late phase of altered biochemical events that results in delayed tissue damage and is therefore amenable to therapeutic treatment. Because the molecular mechanisms of delayed post-traumatic neuronal cell death are still poorly understood, we investigated whether apoptosis-inducing factor (AIF), a pro-apoptotic mitochondrial molecule and the key factor in the caspase-independent, cell death signaling pathway, plays a causal role in neuronal death following TBI. Using an in vitro model of neuronal stretch injury, we demonstrated that AIF translocated from mitochondria to the nucleus of neurons displaying axonal disruption, chromatin condensation, and nuclear pyknosis in a caspase-independent manner, whereas astrocytes remained unaffected. Similar findings were observed following experimental TBI in mice, where AIF translocation to the nucleus coincided with delayed neuronal cell death in both cortical and hippocampal neurons. Down-regulation of AIF in vitro by siRNA significantly reduced stretch-induced neuronal cell death by 67%, a finding corroborated in vivo using AIF-deficient harlequin mutant mice, where secondary contusion expansion was significantly reduced by 44%. Hence, our current findings demonstrate that caspase-independent, AIF-mediated signaling pathways significantly contribute to post-traumatic neuronal cell death and may therefore represent novel therapeutic targets for the treatment of TBI.

G Proteins

See related article, pages 1191–1199 Signaling through the activation of G proteins represents the most widely used signaling pathway in mammalian biology.1,2 Classically, a transmembrane receptor comprising seven transmembrane domains (G protein-coupled receptor, GPCR) is activated by an extracellular stimulus and transduces this information to heterotrimeric G proteins through a conformational change of the receptor protein. Throughout evolution, a large variety of GPCRs has evolved to detect a wide spectrum of signals ranging from photons and odorants to endogenous neurotransmitters and hormones such as the catecholamines. Consequently, the majority of currently used drugs act on GPCRs. On receptor-mediated activation of the G protein α-subunit, the bound GDP is exchanged against GTP and both the GTP-bound α-subunit as well as the βγ-subunits may activate downstream targets (Figure). As a GTPase, the α-subunit then rapidly initiates its own inactivation through GTP-hydrolysis. This GTPase cycle of G protein activation and deactivation is subject to regulation by the RGS family of proteins (regulators of G protein signaling) that activate the GTPase function and thereby negatively regulate signaling of GPCRs.3,4 G protein dependent signaling in the cardiovascular system. Classically G proteins are activated through agonist occupied G protein-coupled receptors (GPCRs). In the inactive state, the G protein α-subunit is bound to GDP, with a rapid exchange against GTP on activation. Both the α-subunit and the βγ-subunits can then activate diverse downstream signaling cascades. In addition G protein dependent signaling can be regulated independent from GPCR-activation through activators of G protein signaling (AGS) and nucleoside diphosphate kinase (NDPK). Membrane-bound NDPK B transfers a high energy phosphate onto G β-subunits, which is then transferred onto GDP to form GTP. It thereby localizes GTP-supply to the vincinity of G protein α-subunits and mediates local, receptor-independent activation of G protein α-subunits. It remains to be determined, …

Mitochondrial Ca2+ cycle mediated by the palmitate-activated cyclosporin a-insensitive pore

Earlier we found that in isolated rat liver mitochondria the reversible opening of the mitochondrial cyclosporin A-insensitive pore induced by low concentrations of palmitic acid (Pal) plus Ca(2+) results in the brief loss of Deltapsi [Mironova et al., J Bioenerg Biomembr (2004), 36:171-178]. Now we report that Pal and Ca(2+), increased to 30 and 70 nmol/mg protein respectively, induce a stable and prolonged (10 min) partial depolarization of the mitochondrial membrane, the release of Ca(2+) and the swelling of mitochondria. Inhibitors of the Ca(2+) uniporter, ruthenium red and La(3+), as well as EGTA added in 10 min after the Pal/Ca(2+)-activated pore opening, prevent the release of Ca(2+) and repolarize the membrane to initial level. Similar effects can be observed in the absence of exogeneous Pal, upon mitochondria accumulating high [Sr(2+)], which leads to the activation of phospholipase A(2) and appearance of endogenous fatty acids. The paper proposes a new model of the mitochondrial Ca(2+) cycle, in which Ca(2+) uptake is mediated by the Ca(2+) uniporter and Ca(2+) efflux occurs via a short-living Pal/Ca(2+)-activated pore.

Effects of vitamin C infusion and vitamin E-coated membrane on hemodialysis-induced oxidative stress

Chronic hemodialysis (HD) patients manifest anemia and atherosclerosis with associated oxidative stress. We explored whether intravenous infusion of vitamin C (VC) and/or use of vitamin E (VE)-coated dialysis membrane could palliate HD-evoked oxidative stress. Eighty patients undergoing chronic HD were enrolled and randomly assigned into four groups: HD with intravenous VC (n=20), HD with VE-coated dialyzer (n=20), HD with both (n=20), and HD with neither (n=20). We evaluated oxidative stress in blood and plasma, erythrocyte methemoglobin/ferricyanide reductase (red blood cells (RBC)-MFR) activity, plasma methemoglobin, and pro-inflammatory cytokines in these patients. All patients showed marked increases (14-fold) in blood reactive oxygen species (ROS) after HD. The types of ROS were mostly hydrogen peroxide, and in lesser amounts, O2*- and HOCl. HD resulted in decreased plasma VC, total antioxidant status, and RBC-MFR activity and increased plasma and erythrocyte levels of phosphatidylcholine hydroperoxide (PCOOH) and methemoglobin. Intravenous VC significantly palliated HD-induced oxidative stress, plasma and RBC levels of PCOOH, and plasma methemoglobin levels and preserved RBC-MFR activity. The VE-coated dialyzer effectively prevented RBCs from oxidative stress, although it showed a partial effect on the reduction of total ROS activity in whole blood. In conclusion, intravenous VC plus a VE-coated dialyzer is effective in palliating HD-evoked oxidative stress, as indicated by hemolysis and lipid peroxidation, and by overexpression of proinflammation cytokines in HD patients. Using VE-coated dialyzer per se is, however, effective in reducing lipid peroxidation and oxidative damage to RBCs.

Letter Regarding Article by Argaud et al, “Postconditioning Inhibits Mitochondrial Permeability Transition”

To the Editor: A recent article in Circulation 1 proposes that prevention of myocardial damage by postconditioning involves the inhibition of mitochondrial permeability transition pore (mPTP) opening. We find the idea attractive and in keeping with studies implicating mPTP in postischemic damage2 but believe this study is too preliminary to prove this hypothesis. The authors present 2 main findings to support their claim. The first is that NIM811, an mPTP inhibitor, presents an effect similar to postconditioning. This finding indisputably shows that mPTP opening is involved in reperfusion injury. The results of NIM811 and postconditioning were both partial, however, and possible additive effects of both treatments were not determined. As a result, these experiments do not exclude that postconditioning can act on a pathway independent of mPTP opening. The second finding the authors present is an experiment with isolated mitochondria, which indicates that larger Ca2+ loads are required to induce Ca2+ release in …