Reduction of elevated proton leak rejuvenates mitochondria in the aged cardiomyocyte.

Aging-associated diseases, including cardiac dysfunction, are increasingly common in the population. However, the mechanisms of physiologic aging in general, and cardiac aging in particular, remain poorly understood. Age-related heart impairment is lacking a clinically effective treatment. Using the model of naturally aging mice and rats, we show direct evidence of increased proton leak in the aged heart mitochondria. Moreover, our data suggested ANT1 as the most likely site of mediating increased mitochondrial proton permeability in old cardiomyocytes. Most importantly, the tetra-peptide SS-31 prevents age-related excess proton entry, decreases the mitochondrial flash activity and mitochondrial permeability transition pore opening, rejuvenates mitochondrial function by direct association with ANT1 and the mitochondrial ATP synthasome, and leads to substantial reversal of diastolic dysfunction. Our results uncover the excessive proton leak as a novel mechanism of age-related cardiac dysfunction and elucidate how SS-31 can reverse this clinically important complication of cardiac aging.

ABT-737 sensitizes curcumin-induced anti-melanoma cell activity through facilitating mPTP death pathway.

In the current study, we studied the potential role of ABT-737, a novel Bcl-2 inhibitor, on curcumin-induced anti-melanoma cell activity in vitro. The associated mechanisms were also investigated. We demonstrated that ABT-737 significantly sensitized curcumin-induced activity against melanoma cells (WM-115 and B16 lines), resulting in substantial cell death and apoptosis with co-administration. At the molecular level, curcumin and ABT-737 synergistically induced mitochondrial permeability transition pore (mPTP) opening in melanoma cells, the latter was evidenced by mitochondrial membrane potential (MPP) reduction and mitochondrial complexation between cyclophilin-D (CyPD) and adenine nucleotide translocator 1 (ANT-1). Significantly, mPTP blockers, including cyclosporin A and sanglifehrin A, remarkably inhibited curcumin and ABT-737 co-administration-induced cytotoxicity against melanoma cells. Meanwhile, siRNA-mediated knockdown of CyPD or ANT-1, the two key components of mPTP, alleviated WM-116 cell death by the co-treatment. Collectively, we show that ABT-737 sensitizes curcumin-induced anti-melanoma cell activity probably through facilitating mPTP death pathway. ABT-737 could be further investigated as a potential curcumin adjuvant in melanoma and other cancer treatment.

Coenzyme A enhances activity of the mitochondrial adenine nucleotide translocator.

The adenine nucleotide translocator (ANT) accomplishes the exchange of ATP from the mitochondrial matrix with cytoplasmic ADP. While investigating the biochemical mechanism of retinoic acid (RA) on the ANT via retinoylation, we have found and subsequently demonstrated a positive influence of Coenzyme A (CoA) on the transport of ATP across the membranes of rat liver mitochondria. CoA enhances ANT activity in a dose-dependent manner modifying the V(max) (673.3+/-20.7 nmol ATP/mgprotein/min versus 155.0+/-1.9 nmol ATP/mgprotein/min), the IC(50) for the specific inhibitor carboxyatractyloside (CATR) (0.142+/-0.012 microM versus 0.198+/-0.011 microM) but not the K(m) (22.50+/-0.52 microM versus 22.19+/-0.98 microM). Data suggest a likely enzymatic involvement in the interaction between ANT and CoA. The effect of CoA is observed in mitochondria from several different tissues.

1,1-bis(3'-indolyl)-1-(p-substituted phenyl)methanes decrease mitochondrial membrane potential and induce apoptosis in endometrial and other cancer cell lines.

1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes, containing p-t-butyl (DIM-C-pPhtBu) and phenyl (DIM-C-pPhC(6)H(5)) substituents, are peroxisome proliferator-activated receptor gamma (PPARgamma) agonists; however, DIM-C-pPhtBu-induced growth inhibition and cell death in human HEC1A endometrial cancer cells is PPARgamma-independent. DIM-C-pPhtBu decreased mitochondrial membrane potential (MMP) and promoted the release of cytochrome c and caspase activation and nuclear uptake of endonuclease G leading to apoptosis of HEC1A cells. DIM-C-pPhtBu specifically targeted the mitochondrial permeability transition pore complex (PTPC) because the DIM-C-pPhtBu-induced pro-apoptotic responses were inhibited by atractyloside (Atra), a compound that specifically interacts with the inner mitochondrial membrane adenine nucleotide transport (ANT) proteins. At the dose of Atra used in this study (300 microM), this compound alone did not alter the PTPC but inhibited the mitochondriotoxic effects of DIM-C-pPhtBu. DIM-C-pPhtBu/DIM-C-pPhC(6)H(5) and Atra also differentially affected the ability of eosin-5-maleimide (EMA) to alkylate Cys160 in the ANT protein and Atra, but not DIM-C-pPhtBu, inhibited the exchange of ATP/ADP in isolated mitochondria suggesting that these pharmacophores act on different sites on the ANT protein. Results of this study show that the receptor-independent proapoptotic activity of DIM-C-pPhtBu and DIM-C-pPhC(6)H(5) were related to novel mitochondriotoxic activities involving inner mitochondrial ANT proteins.

Carboxyatractyloside effects on brown-fat mitochondria imply that the adenine nucleotide translocator isoforms ANT1 and ANT2 may be responsible for basal and fatty-acid-induced uncoupling respectively.

In brown-fat mitochondria, fatty acids induce thermogenic uncoupling through activation of UCP1 (uncoupling protein 1). However, even in brown-fat mitochondria from UCP1-/- mice, fatty-acid-induced uncoupling exists. In the present investigation, we used the inhibitor CAtr (carboxyatractyloside) to examine the involvement of the ANT (adenine nucleotide translocator) in the mediation of this UCP1-independent fatty-acid-induced uncoupling in brown-fat mitochondria. We found that the contribution of ANT to fatty-acid-induced uncoupling in UCP1-/- brown-fat mitochondria was minimal (whereas it was responsible for nearly half the fatty-acid-induced uncoupling in liver mitochondria). As compared with liver mitochondria, brown-fat mitochondria exhibit a relatively high (UCP1-independent) basal respiration ('proton leak'). Unexpectedly, a large fraction of this high basal respiration was sensitive to CAtr, whereas in liver mitochondria, basal respiration was CAtr-insensitive. Total ANT protein levels were similar in brown-fat mitochondria from wild-type mice and in liver mitochondria, but the level was increased in brown-fat mitochondria from UCP1-/- mice. However, in liver, only Ant2 mRNA was found, whereas in brown adipose tissue, Ant1 and Ant2 mRNA levels were equal. The data are therefore compatible with a tentative model in which the ANT2 isoform mediates fatty-acid-induced uncoupling, whereas the ANT1 isoform may mediate a significant part of the high basal proton leak in brown-fat mitochondria.

A new endogenous ATP analog (ApppI) inhibits the mitochondrial adenine nucleotide translocase (ANT) and is responsible for the apoptosis induced by nitrogen-containing bisphosphonates.

1. Bisphosphonates are currently the most important class of antiresorptive drugs used for the treatment of diseases with excess bone resorption. On the basis of their molecular mechanism of action, bisphosphonates can be divided into two pharmacological classes; nitrogen-containing (N-BPs) and non-nitrogen-containing bisphosphonates (non-N-BP). Both classes induce apoptosis but they evoke it differently; N-BPs by inhibiting the intracellular mevalonate pathway and protein isoprenylation, and non-N-BPs via cytotoxic ATP analog-type metabolites. N-BPs are not metabolized to ATP analogs, but we report here that these bisphosphonates can induce formation of a novel ATP analog (ApppI) as a consequence of the inhibition of the mevalonate pathway in cells. We also investigated whether ApppI is involved in the apoptosis induced by N-BPs. 2. Mass spectrometry and NMR were used to identify ApppI in N-BP treated osteoclasts, macrophages and glioma cells. The potency of different bisphosphonates to promote ApppI production was tested in J774 macrophages. The effects of ApppI on ADP/ATP translocase in isolated mitochondria and its capability to induce apoptosis in osteoclasts were also studied. 3. ApppI production correlated well with the capacity of N-BPs to inhibit mevalonate pathway. ApppI inhibited the mitochondrial ADP/ATP translocase and caused apoptosis in osteoclasts. 4. In conclusion, these findings provide the basis for a new mechanism of action for N-BPs. Some of these very potent bisphosphonates, such as zoledronic acid, represent a third class of bisphosphonates that can act both via the inhibition of the mevalonate pathway and by the blockade of mitochondrial ADP/ATP translocase, which is known to be involved in the induction of apoptosis.

Acute glutathione depletion restricts mitochondrial ATP export in cerebellar granule neurons.

Decreases in GSH pools detected during ischemia sensitize neurons to excitotoxic damage. Thermodynamic analysis predicts that partial GSH depletion will cause an oxidative shift in the thiol redox potential. To investigate the acute bioenergetic consequences, neurons were exposed to monochlorobimane (mBCl), which depletes GSH by forming a fluorescent conjugate. Neurons transfected with redox-sensitive green fluorescent protein showed a positive shift in thiol redox potential synchronous with the formation of the conjugate. Mitochondria within neurons treated with mBCl for 1 h failed to hyperpolarize upon addition of oligomycin to inhibit their ATP synthesis. A decreased ATP turnover was confirmed by monitoring neuronal oxygen consumption in parallel with mitochondrial membrane potential (Deltapsi(m)) and GSH-mBCl formation. mBCl progressively decreased cell respiration, with no effect on mitochondrial proton leak or maximal respiratory capacity, suggesting adequate glycolysis and a functional electron transport chain. This approach to "state 4" could be mimicked by the adenine nucleotide translocator inhibitor bongkrekic acid, which did not further decrease respiration when administered after mBCl. The cellular ATP/ADP ratio was decreased by mBCl, and consistent with mitochondrial ATP export failure, respiration could not respond to an increased cytoplasmic ATP demand by plasma membrane Na(+) cycling; instead, mitochondria depolarized. More prolonged mBCl exposure induced mitochondrial failure, with Deltapsi(m) collapse followed by cytoplasmic Ca(2+) deregulation. The initial bioenergetic consequence of neuronal GSH depletion in this model is thus an inhibition of ATP export, which precedes other forms of mitochondrial dysfunction.

Increased adenine nucleotide translocator 1 in reactive astrocytes facilitates glutamate transport.

A hallmark of central nervous system (CNS) pathology is reactive astrocyte production of the chronic glial scar that is inhibitory to neuronal regeneration. The reactive astrocyte response is complex; these cells also produce neurotrophic factors and are responsible for removal of extracellular glutamate, the excitatory neurotransmitter that rises to neurotoxic levels in injury and disease. To identify genes expressed by reactive astrocytes, we employed an in vivo model of the glial scar and differential display PCR and found an increase in the level of Ant1, a mitochondrial ATP/ADP exchanger that facilitates the flux of ATP out of the mitochondria. Ant1 expression in reactive astrocytes is regulated by transforming growth factor-beta1, a pluripotent CNS injury-induced cytokine. The significance of increased Ant1 is evident from the observation that glutamate uptake is significantly decreased in astrocytes from Ant1 null mutant mice while a specific Ant inhibitor reduces glutamate uptake in wild-type astrocytes. Thus, the astrocytic response to CNS injury includes an apparent increase in energy mobilization capacity by Ant1 that contributes to neuroprotective, energy-dependent glutamate uptake.