Inhibition of mitochondrial complex III induces differentiation in acute myeloid leukemia.

Although acute myeloid leukemia (AML) is a highly heterogeneous disease with diverse genetic subsets, one hallmark of AML blasts is myeloid differentiation blockade. Extensive evidence has indicated that differentiation induction therapy represents a promising treatment strategy. Here, we identified that the pharmacological inhibition of the mitochondrial electron transport chain (ETC) complex III by antimycin A inhibits proliferation and promotes cellular differentiation of AML cells. Mechanistically, we showed that the inhibition of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme in de novo pyrimidine biosynthesis, is involved in antimycin A-induced differentiation. The activity of antimycin A could be reversed by supplement of excessive amounts of exogenous uridine as well as orotic acid, the product of DHODH. Furthermore, we also found that complex III inhibition exerts a synergistic effect in differentiation induction combined with DHODH inhibitor brequinar as well as with the pyrimidine salvage pathway inhibitor dipyridamole. Collectively, our study uncovered the link between mitochondrial complex III and AML differentiation and may provide further insight into the potential application of mitochondrial complex III inhibitor as a mono or combination treatment in differentiation therapy of AML.

Inhibition of Human Cancer Cell Growth by Analogues of Antimycin A.

In a recent study, several new derivatives of antimycin A (AMA) were produced by means of a novel transacylation reaction, and these were shown to mediate selective toxicity toward cultured A549 human lung epithelial adenocarcinoma cells, as compared with WI-38 normal human lung fibroblasts. The purpose of our study was to investigate whether the analogues all expressed their cytotoxicity by the same mechanism. This was done by studying the effects of the compounds in several types of cell lines. In comparison with 2-O-methylantimycin, which acts at the locus of Bcl-2, none of the new derivatives exhibited a difference in cytotoxicity toward cells expressing different levels of Bcl-2. In cell lines that over- or underexpress estrogen or Her2 receptors, AMA analogue 2 exhibited Her2 receptor dependency at low concentration. Three compounds (1, 4, and 6) exhibited concentration-dependent increases in reactive oxygen species, with 6 being especially potent. Compounds 5 and 6 diminished mitochondrial membrane potential more potently than AMA, and 1 also displayed enhanced activity relative to 2-4. Interestingly, only 1 and AMA displayed strong inhibition of the respiratory chain, as measured by monitoring NADH (reduced nicotinamide adenine dinucleotide) oxidase. Because four of the analogues have positively charged substituents, two of these (4 and 6) were studied to see whether the observed effects were due to much higher level of accumulation within the mitochondria. Their presence in the mitochondria was not dramatically enhanced. Neither of the two presently characterized mechanisms of cell killing by AMA can fully account for the observed results.

Kitamycin C, a new antimycin-type antibiotic from Streptomyces antibioticus strain 200-09.

An actinomycete strain 200-09, isolated from a soil sample collected from the coast of Hawaii, USA, was identified as Streptomyces antibioticus on the basis of its morphological, physiological and biochemical characteristics as well as 16S rDNA analysis. A new antimycin-type antibiotic, kitamycin C (1), together with kitamycin A (2), kitamycin B (3), urauchmycin B (4), deisovaleryblastomycin (5) was isolated from a cultured broth of strain 200-09. The structure of the new compound was determined by spectroscopic data, including HR-ESI-MS and NMR. All the compounds exhibited antifungal activities against Candida albicans with MIC of about 25.0 µg mL-1.

Genetic Evidence for Cytochrome b Qi Site Inhibition by 4(1H)-Quinolone-3-Diarylethers and Antimycin in Toxoplasma gondii.

Toxoplasma gondii is an apicomplexan parasite that causes fatal and debilitating brain and eye disease. Endochinlike quinolones (ELQs) are preclinical compounds that are efficacious against apicomplexan-caused diseases, including toxoplasmosis, malaria, and babesiosis. Of the ELQs, ELQ-316 has demonstrated the greatest efficacy against acute and chronic experimental toxoplasmosis. Although genetic analyses in other organisms have highlighted the importance of the cytochrome bc1 complex Qi site for ELQ sensitivity, the mechanism of action of ELQs against T. gondii and the specific mechanism of ELQ-316 remain unknown. Here, we describe the selection and genetic characterization of T. gondii clones resistant to ELQ-316. A T. gondii strain selected under ELQ-316 drug pressure was found to possess a Thr222-Pro amino acid substitution that confers 49-fold resistance to ELQ-316 and 19-fold resistance to antimycin, a well-characterized Qi site inhibitor. These findings provide further evidence for ELQ Qi site inhibition in T. gondii and greater insight into the interactions of Qi site inhibitors with the apicomplexan cytochrome bc1 complex.

Comparative kinetics of Qi site inhibitors of cytochrome bc1 complex: picomolar antimycin and micromolar cyazofamid.

Antimycin and cyazofamid are specific inhibitors of the mitochondrial respiratory chain and bind to the Qi site of the cytochrome bc1 complex. With the aim to understand the detailed molecular inhibition mechanism of Qi inhibitors, we performed a comparative investigation of the inhibitory kinetics of them against the porcine bc1 complex. The results showed that antimycin is a slow tight-binding inhibitor of succinate-cytochrome c reductase (SCR) with Ki  = 0.033 ± 0.00027 nm and non-competitive inhibition with respect to cytochrome c. Cyazofamid is a classical inhibitor of SCR with Ki  = 12.90 ± 0.91 µm and a non-competitive inhibitor with respect to cytochrome c. Both of them show competitive inhibition with respect to substrate DBH2 . Further molecular docking and quantum mechanics calculations were performed. The results showed that antimycin underwent significant conformational change upon the binding. The energy barrier between the conformations in the crystal and in the binding pocket is ~13.63 kcal/mol. Antimycin formed an H-bond with Asp228 and two water-bridged H-bonds with Lys227 and His201, whereas cyazofamid formed only one H-bond with Asp228. The conformational change and the different hydrogen bonding network might account for why antimycin is a slow tight-binding inhibitor, whereas cyazofamid is a classic inhibitor.

Selenium supplementation protects trophoblast cells from mitochondrial oxidative stress.

INTRODUCTION: Oxidative stress plays an important role in the pathogenesis of preeclampsia, a placental disorder affecting approximately 7% of pregnancies. Trophoblast cells are susceptible to oxidative stress which causes increased cell death and placental turnover. In this study, inhibitors of the mitochondrial respiratory chain were utilised to induce oxidative stress and the effect that selenium supplementation had on trophoblast viability was investigated. METHODS: Trophoblast cells (BeWo, JEG-3 and Swan-71) were treated with Na Selenite (100 nM) or Selenomethionine (500 nM) to increase the biological activity of antioxidants Glutathione Peroxidase and Thioredoxin Reductase. The cells were then oxidatively stressed with the addition of increasing doses of Antimycin C and Rotenone and the Resazurin end point assay was used to assess cellular activity. RESULTS: There was a significant dose dependent decrease in the cellular activity in BeWo, JEG-3 and Swan-71 when treated for 4 h with increasing concentrations of Antimycin (40-320 µM) and Rotenone (100-800 nM). Prior incubation with Na Selenite and Selenomethionine was able to protect trophoblast cells from oxidative stress at Rotenone concentrations of 200 and 400 nM (P < 0.001) and Antimycin concentrations of 80-240 µM (P < 0.001). DISCUSSION: These data suggest that selenoproteins such as Glutathione Peroxidase and Thioredoxin Reductase have an important role in protecting trophoblast mitochondria from oxidative stress. CONCLUSIONS: This study emphasises the importance of maintaining an adequate selenium supply during pregnancy and especially in pregnancies complicated by conditions such as preeclampsia.

Honokiol isolated from Magnolia officinalis stimulates osteoblast function and inhibits the release of bone-resorbing mediators.

There has been a strong interest in searching for natural therapies for osteoporosis. Honokiol is a phenolic compound isolated from the bark of Magnolia officinalis, a plant widely used in traditional medicine. In the present study, the effects of honokiol on the function of osteoblastic MC3T3-E1 cells were studied. Honokiol caused a significant elevation of cell growth, alkaline phosphatase activity, collagen synthesis, mineralization, glutathione content, and osteoprotegerin release in the cells (P<0.05). Moreover, honokiol significantly (P<0.05) decreased the production of osteoclast differentiation inducing factors such as TNF-α, IL-6, and receptor activator of nuclear factor-kB ligand (RANKL) in the presence of antimycin A, which inhibits mitochondrial electron transport and has been used as a ROS generator. These results demonstrate that honokiol may have positive effects on skeletal structure.

Iron deficiency induces changes in riboflavin secretion and the mitochondrial electron transport chain in hairy roots of Hyoscyamus albus.

Hyoscyamus albus hairy roots secrete riboflavin under Fe-deficient conditions. To determine whether this secretion was linked to an enhancement of respiration, both riboflavin secretion and the reduction of 2,3,5-triphenyltetrazolium chloride (TTC), as a measure of respiration activity, were determined in hairy roots cultured under Fe-deficient and Fe-replete conditions, with or without aeration. Appreciable TTC-reducing activity was detected at the root tips, at the bases of lateral roots and in internal tissues, notably the vascular system. TTC-reducing activity increased under Fe deficiency and this increase occurred in concert with riboflavin secretion and was more apparent under aeration. Riboflavin secretion was not apparent under Fe-replete conditions. In order to examine which elements of the mitochondrial electron transport chain might be involved, the effects of the respiratory inhibitors, barbiturate, dicoumarol, malonic acid, antimycin, KCN and salicylhydroxamic acid (SHAM) were investigated. Under Fe-deficient conditions, malonic acid affected neither root growth, TTC-reducing activity nor riboflavin secretion, whereas barbiturate and SHAM inhibited only root growth and TTC-reducing activity, respectively, and the other compounds variously inhibited growth and TTC-reducing activity. Riboflavin secretion was decreased, in concert with TTC-reducing activity, by dicoumarol, antimycin and KCN, but not by SHAM. In Fe-replete roots, all inhibitors which reduced riboflavin secretion in Fe-deficient roots showed somewhat different effects: notably, antimycin and KCN did not significantly inhibit TTC-reducing activity and the inhibition by dicoumarol was much weaker in Fe-replete roots. Combined treatment with KCN and SHAM also revealed that Fe-deficient and Fe-replete roots reduced TTC in different ways. A decrease in the Fe content of mitochondria in Fe-deficient roots was confirmed. Overall, the results suggest that, under conditions of Fe deficiency in H. albus hairy roots, the alternative NAD(P)H dehydrogenases, complex III and complex IV, but not the alternative oxidase, are actively involved both in respiration and in riboflavin secretion.

Tiron, a ROS scavenger, protects human lung cancer Calu-6 cells against antimycin A-induced cell death.

Antimycin A (AMA) inhibits the mitochondrial electron transport between cytochromes b and c. However, the relationship between AMA and lung cancer cells is poorly understood. In this study, we investigated the involvement of reactive oxygen species (ROS) and glutathione (GSH) in AMA-treated lung cancer Calu-6 cell death. Treatment with AMA reduced cell viability in a dose-dependent manner for 72 h. The intracellular ROS levels were decreased in Calu-6 cells treated with low doses of AMA (10, 25 or 50 microM) at 72 h. However, the levels increased in cells treated with a high dose of 100 microM AMA. Levels of O2.- were significantly increased in AMA-treated cells at 72 h. The increases in ROS levels including O2.- in AMA-treated cells were observed within 10 min. Treatment with AMA reduced the intracellular GSH content. SOD activity was up-regulated in AMA-treated Calu-6 cells at 72 h. However, catalase activity was down-regulated by AMA. Treatment with tiron, a ROS scavenger, reduced the intracellular ROS levels, which were associated with a partial reduction of apoptosis. Treatment with exogenous SOD and catalase significantly inhibited loss of the mitochondrial transmembrane potential (DeltaPsim) in AMA-treated Calu-6 cells. In conclusion, our results suggest that the changes of intracellular ROS and GSH affect apoptosis in AMA-treated Calu-6 cells.

L-Proline uptake in Crithidia deanei is influenced by its endosymbiont bacterium.

Crithidia deanei, a monoxenic trypanosomatid, presents an endosymbiotic bacterium in its cytoplasm. Both the protozoan and the bacterium maintain intensive metabolic exchange, resulting in an interesting model to study the coevolution of metabolisms. The relevance of l-proline for the growth of C. deanei and its transport into these cells was studied. Both the endosymbiont-containing (wild) and the endosymbiont-free protozoa (aposymbiont or cured) strains, when grown in medium supplemented with l-proline, reached higher cell densities than those grown in unsupplemented media. We biochemically characterized the uptake of l-proline in both the wild (K(m)=0.153+/-0.022 mM, V(max)=0.239+/-0.011 nmol min(-1) per 4 x 10(7) cells) and the aposymbiont strains (K(m)=0.177+/-0.049 mM, V(max)=0.132+/-0.012 nmol min(-1) per 4 x 10(7) cells). These data suggest a single type of proline transporter whose activity is upregulated by the presence of the symbiotic bacterium. Proline transport was further characterized and was found to be insensitive to the extracellular concentration of Na+, but sensitive to K+ and pH. The abolition of proline uptake by respiratory chain inhibitors and valinomycin indicates that the proline transport in C. deanei is dependent on the plasma membrane K+ gradient.

Respiratory chain components involved in the glycerophosphate dehydrogenase-dependent ROS production by brown adipose tissue mitochondria.

Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H2DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.

Potent inhibitors of the Qi site of the mitochondrial respiration complex III.

A series of azole-fused salicylamides were prepared as analogues of antimycin and assayed for activity at complex III of the mitochondrial respiratory chain. The activity of these compounds approached that of antimycin in inhibitory potency and some showed growth reduction of Septoria nodorum in vitro. Compound 8a was shown to bind at the Qi site of complex III by red-shift titration of the bc1 complex.

Preclinical pharmacology of 2-methoxyantimycin A compounds as novel antitumor agents.

PURPOSE: The present study was designed to determine pharmacological and biochemical properties of 2-methoxyantimycin A analogs (OMe-A1, OMe-A2, OMe-A3, and OMe-A5), which are novel antitumor compounds, and provide a basis for future pharmaceutical development, preclinical evaluation, and clinical trials. METHODS: A high-performance liquid chromatography (HPLC) method was established and employed to assess the biostability of these analogs and to determine their pharmacokinetic properties in mice and rats. RESULTS: In vitro biostability of the 2-methoxyantimycin analogs was esterase-dependent, compound-dependent, and species-dependent. In the absence of esterase inhibitors, all of the analogs were relatively unstable. Stability was greater, however, in human and dog plasma than in rat and mouse plasma. In the presence of esterase inhibitors, OMe-A1 was stable at 37 degrees C for 60 min in mouse and rat plasma, moderately stable in human plasma, and unstable in dog plasma. OMe-A2 was generally stable in all types of plasma. OMe-A3 was stable in dog and rat plasma, but not in human or mouse plasma. OMe-A5 was stable in human and dog plasma, but not in mouse or rat plasma. Each of these analogs was highly bound to plasma proteins. Of S9 fractions from four species, human S9 was least efficient in metabolizing OMe-A3. Following an intravenous dose of OMe-A1 in mice, plasma levels decreased rapidly, with an initial half-life of 2.7 min and a terminal half life of 34 min. Following an intraperitoneal dose in mice, plasma levels decreased less rapidly with a terminal half-life of 215 min. Following an intravenous dose of OMe-A1 or OMe-A3 in rats, plasma levels decreased more rapidly with initial half-lives of about 1.0 min. At an equivalent dose, OMe-A3 had a faster clearance than OMe-A1. CONCLUSIONS: For 2-methoxyantimycin A analogs, species differences in biostability, metabolism, and pharmacokinetics may be pertinent in assessing their pharmacological and toxicological profiles and antitumor activity in humans.

Inhibition of mitochondrial bioenergetics: the effects on structure of mitochondria in the cell and on apoptosis.

The effects of specific inhibitors of respiratory chain, F(o)F(1)ATP synthase and uncouplers of oxidative phosphorylation on survival of carcinoma HeLa cells and on the structure of mitochondria in the cells were studied. The inhibitors of respiration (piericidin, antimycin, myxothiazol), the F(1)-component of ATP synthase (aurovertin) and uncouplers (DNP, FCCP) did not affect viability of HeLa cells, apoptosis induced by TNF or staurosporin and the anti-apoptotic action of Bcl-2. Apoptosis was induced by combined action of respiratory inhibitors and uncouplers indicating possible pro-apoptotic action of reactive oxygen species (ROS) generated by mitochondria. Short-term incubation of HeLa cells with the mitochondrial inhibitors and 2-deoxyglucose followed by 24-48 h recovery resulted in massive apoptosis. Apoptosis correlated to transient (3-4 h) and limited (60-70%) depletion of ATP. More prolonged or more complete transient ATP depletion induced pronounced necrosis. The inhibitors of respiration and uncouplers caused fragmentation of tubular mitochondria and formation of small round bodies followed by swelling. These transitions were not accompanied with release of cytochrome c into the cytosol and were fully reversible. The combined effect of respiratory inhibitors and uncouplers developed more rapidly indicating possible involvement of ROS generated by mitochondria. More prolonged (48-72 h) incubation with this combination of inhibitors caused clustering and degradation of mitochondria.

Effects of EGb 761 Ginkgo biloba extract on mitochondrial function and oxidative stress.

As major sources of reactive oxygen species (ROS), mitochondrial structures are exposed to high concentrations of ROS and may therefore be particularly susceptible to oxidative damage. Mitochondrial damage could play a pivotal role in the cell death decision. A decrease in mitochondrial energy charge and redox state, loss of transmembrane potential (depolarization), mitochondrial respiratory chain impairment, and release of substances such as calcium and cytochrome c all contribute to apoptosis. These mitochondrial abnormalities may constitute a part of the spectrum of chronic oxidative stress in Alzheimer's disease. Accumulation of amyloid beta (Abeta) in form of senile plaques is also thought to play a central role in the pathogenesis of Alzheimer's disease mediated by oxidative stress. In addition, increasing evidence shows that Abeta generates free radicals in vitro, which mediate the toxicity of this peptide. In our study, PC12 cells were used to examine the protective features of EGb 761(definition see editorial) on mitochondria stressed with hydrogen peroxide and antimycin, an inhibitor of complex III. In addition, we investigated the efficacy of EGb 761 in Abeta-induced MTT reduction in PC12 cells. Moreover, we examined the effects of EGb 761 on ROS levels and ROS-induced apoptosis in lymphocytes from aged mice after in vivo administration. Here, we will report that EGb 761 was able to protect mitochondria from the attack of hydrogen peroxide, antimycin and Abeta. Furthermore, EGb 761 reduced ROS levels and ROS-induced apoptosis in lymphocytes from aged mice treated orally with EGb 761 for 2 weeks. Our data further emphasize neuroprotective properties of EGb 761, such as protection against Abeta-toxicity, and antiapoptotic properties, which are probably due to its preventive effects on mitochondria.

Functional characterization of novel mutations in the human cytochrome b gene.

The great variability of the human mitochondrial DNA (mtDNA) sequence induces many difficulties in the search for its deleterious mutations. We illustrate these pitfalls by the analysis of the cytochrome b gene of 21 patients affected with a mitochondrial disease. Eighteen different sequence variations were found, five of which were new mutations. Extensive analysis of the cytochrome b gene of 146 controls found 20 supplementary mutations, thus further demonstrating the high variability of the cytochrome b sequence. We fully evaluated the functional relevance of 36 of these 38 mutations using indirect criteria such as the nature of the mutation, its frequency in controls, or the phylogenetic conservation of the mutated amino acid. When appropriate, the mtDNA haplotype, the heteroplasmic state of the mutation, its tissue distribution or its familial transmission were also assessed. The molecular consequences of the mutations, which appeared possibly deleterious in that first step of evaluation, were evaluated on the complex III enzymological properties and protein composition using specific antibodies that we have generated against four of its subunits. Two original deleterious mutations were found in the group of seven patients with overt complex III defect. Both mutations (G15150A (W135X) and T15197C (S151P)) were heteroplasmic and restricted to muscle. They had significant consequences on the complex III structure. In contrast, only two homoplasmic missense mutations with dubious clinical relevance were found in the patients without overt complex III defect.

Computerized analysis of cytochemical reactions for dehydrogenases and oxygraphic studies as methods to evaluate the function of the mitochondrial sheath in rat spermatozoa.

Cytochemical reactions for mitochondrial NADH-dependent dehydrogenases (diaphorase/NADH which is related to flavoprotein), NAD-dependent dehydrogenases (isocitrate, malate) and succinate dehydrogenase were carried out in rat spermatozoa. In addition to a morphological evaluation, the intensity of the reactions was assessed using a computer image analysing system (Quantimet 600 S). The intensity of the reactions was examined in sperm midpieces by measuring integrated optical density (IOD) and mean optical density (MOD). The activity of mitochondrial respiratory chain complexes was also analysed using the polarographic method. In the population of spermatozoa studied, all whole spermatozoa midpieces were completely filled with formazans, the product of the cytochemical reaction. These morphological findings corresponded to the values obtained for IOD and MOD for the given enzymes. In the oxygraphic studies, the spermatozoa demonstrated consumption of oxygen in the presence of substrates for I, II and IV complexes and their mitochondria revealed normal integrity and sensitivity to the substrates and inhibitors. However, the oxygraphic studies revealed differences between the sperm and somatic cells. These differences concerned the stimulation of pyruvate oxidation by malate, the lack of an effect of malonic acid on phenazine methosulphate (an acceptor of electrons) oxidation and the lack of an effect of cytochrome c on ascorbate oxidation. The cytochemical method, together with densitometric measurements, enables: (1) the reaction intensity to be determined objectively; (2) subtle and dramatic differences in reaction intensity to be revealed between spermatozoa that do not differ under morphological evaluation of the intensity; (3) possible defects within the mitochondrial sheath to be located and assessed in a large number of spermatozoa. This method can be used as a screening method alongside the routine morphological examination of spermatozoa. On the other hand, the oxygraphic method in the inner membrane of mitochondria can reveal functional changes which are related to the action of respiratory chain complexes and display characteristic features of mitochondria energy metabolism. The methods used are complementary and allow the complex evaluation of mitochondria in spermatozoa. Both methods can be used in experimental and clinical studies.

Determining and understanding the control of flux. An illustration in submitochondrial particles of how to validate schemes of metabolic control.

Two complementary methods were used to determine how the rate of respiration and that of ATP hydrolysis were controlled in rat liver submitochondrial particles. In the first, 'direct control analysis' method, respiration was titrated with malonate, antimycin or cyanide at 20, 30 and 37 degrees C, to determine the flux control exerted by succinate dehydrogenase, cytochrome bc1 complex and cytochrome c oxidase, respectively. Together, the three respiratory complexes only controlled the flux by about 50%, leaving the other 50% of flux control to the H+ leak. In the second, 'elasticity based' method, the elasticity coefficients of the respiratory chain or the H+-ATPase and the H+ leak towards the H+ gradient were determined. Then, the flux control coefficients were calculated using the connectivity and summation laws of metabolic control theory. The correspondence between the flux control coefficients determined in the two ways validated the two methods. This allowed us to use the second method to analyse what was the kinetic origin of the observed distribution of control. Control of ATP hydrolysis by the ATPase decreased with increasing ATPase activity; hence, the control exerted by the H+ leak increased with increasing ATPase activity, due to a diminishing elasticity towards the H+ gradient. Reverse electron transport was mainly controlled by the ATPase; the sum of flux control coefficients of succinate dehydrogenase, NADH-CoQ oxidoreductase, and H+-ATPase yielded a value greater than one, indicating that the H+ leak exerted a significant negative control on this pathway.

Graded ATP depletion can cause necrosis or apoptosis of cultured mouse proximal tubular cells.

The mechanisms of cell death induced by ATP depletion were studied in primary cultures of mouse proximal tubular (MPT) cells. Graded ATP depletion, ranging in severity from approximately 2 to 70% of control levels, was induced by incubating cells with either antimycin or 2-deoxyglucose, with varying concentrations of dextrose. We found that cells subjected to ATP depletion below approximately 15% of control died uniformly of necrosis. In contrast, cells subjected to ATP depletion between approximately 25 and 70% of control all died by apoptosis. The rapidity of cell death was proportional to the severity of reduction of cell ATP content and was independent of the mechanism of cell death. Renal growth factors, epidermal growth factor (EGF) and high-dose insulin, did not ameliorate apoptotic cell death induced by ATP depletion. We conclude that ATP depletion can cause either necrosis or apoptosis in MPT cells. Furthermore, we have identified a narrow range of ATP depletion (approximately 15 to 25% of control) representing a threshold that determines whether cells die by necrosis or apoptosis.

Requirement of histidine 217 for ubiquinone reductase activity (Qi site) in the cytochrome bc1 complex.

Folding models suggest that the highly conserved histidine 217 of the cytochrome b subunit from the cytochrome bc1 complex is close to the quinone reductase (Qi) site. This histidine (bH217) in the cytochrome b polypeptide of the photosynthetic bacterium Rhodobacter capsulatus has been replaced with three other residues, aspartate (D), arginine (R), and leucine (L). bH217D and bH217R are able to grow photoheterotrophically and contain active cytochrome bc1 complexes (60% of wild-type activity), whereas the bH217L mutant is photosynthetically incompetent and contains a cytochrome bc1 complex that has only 10% of the wild-type activity. Single-turnover flash-activated electron transfer experiments show that cytochrome bH is reduced via the Qo site with near native rates in the mutant strains but that electron transfer between cytochrome bH and quinone bound at the Qi site is greatly slowed. These results are consistent with redox midpoint potential (Em) measurements of the cytochrome b subunit hemes and the Qi site quinone. The Em values of cyt bL and bH are approximately the same in the mutants and wild type, although the mutant strains have a larger relative concentration of what may be the high-potential form of cytochrome bH, called cytochrome b150. However, the redox properties of the semiquinone at the Qi site are altered significantly. The Qi site semiquinone stability constant of bH217R is 10 times higher than in the wild type, while in the other two strains (bH217D and bH217L) the stability constant is much lower than in the wild type. Thus H217 appears to have major effects on the redox properties of the quinone bound at the Qi site. These data are incorporated into a suggestion that H217 forms part of the binding pocket of the Qi site in a manner reminiscent of the interaction between quinone bound at the Qb site and H190 of the L subunit of the bacterial photosynthetic reaction center.