Suppressors of superoxide production from mitochondrial complex III.

Mitochondrial electron transport drives ATP synthesis but also generates reactive oxygen species, which are both cellular signals and damaging oxidants. Superoxide production by respiratory complex III is implicated in diverse signaling events and pathologies, but its role remains controversial. Using high-throughput screening, we identified compounds that selectively eliminate superoxide production by complex III without altering oxidative phosphorylation; they modulate retrograde signaling including cellular responses to hypoxic and oxidative stress.

Effect of zinc oxide nanoparticles on the function of MC3T3-E1 osteoblastic cells.

Zinc oxide nanoparticles (ZnO NPs) can be ingested directly when used in food, food packaging, drug delivery, and cosmetics. This study evaluated the cellular effects of ZnO NPs (50 and 100 nm diameter particle sizes) on the function of osteoblastic MC3T3-E1 cells. ZnO NPs showed cytotoxicity at concentrations of above 50 µg/ml, and there was no significant effect of the size on the cytotoxicity of ZnO NPs. Within the testing concentrations of 0.01~1 µg/ml, which did not cause a marked drop in cell viability, ZnO NPs (0.1 µg/ml) caused a significant elevation of alkaline phosphatase activity, collagen synthesis, mineralization, and osteocalcin content in the cells (P < 0.05). Moreover, pretreatment with ZnO NPs (0.01~1 µg/ml) significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, and ATP loss. Measurement of reactive oxygen species (ROS) indicated decrease in ROS level upon exposure to ZnO nanoparticles (0.01 µg/ml). Hence, our study indicated that ZnO nanoparticles can have protective effects on osteoblasts at low concentrations where there are little or no observable cytotoxic effects.

Deoxyactein Isolated from Cimicifuga racemosa protects osteoblastic MC3T3-E1 cells against antimycin A-induced cytotoxicity.

Deoxyactein is one of the major constituents isolated from Cimicifuga racemosa. In the present study, we investigated the protective effects of deoxyactein on antimycin A (mitochondrial electron transport inhibitor)-induced toxicity in osteoblastic MC3T3-E1 cells. Exposure of MC3T3-E1 cells to antimycin A caused significant cell viability loss, as well as mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, intracellular calcium ([Ca(2+) ]i ) elevation and oxidative stress. Pretreatment with deoxyactein prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, [Ca(2+) ]i elevation and oxidative stress. Moreover, deoxyactein increased the activation of PI3K (phosphoinositide 3-kinase), Akt (protein kinase B) and CREB (cAMP-response element-binding protein) inhibited by antimycin A. All these data indicate that deoxyactein may reduce or prevent osteoblasts degeneration in osteoporosis or other degenerative disorders.

Estrogen protection against mitochondrial toxin-induced cell death in hippocampal neurons: antagonism by progesterone.

Previously we demonstrated that mitochondrial dysfunction plays a critical role in the pathogenesis of Alzheimer's disease. Further, we have shown that the neuroprotective effects of 17ß-estradiol (E2) are dependent upon mitochondrial function. In the current study, we sought to identify mitochondrial sites of E2 action that mediate neuroprotection by assessing the efficacy of E2 to protect neurons against inhibitors of mitochondrial respiration which target specific complexes within the respiratory chain. Subsequently, the impact of progesterone (P4) on E2-induced prevention against mitochondrial toxins was investigated. Mitochondrial inhibitors, rotenone, 3-NPA, antimycin, KCN, and oligomycin, exhibited concentration dependent toxicity in primary hippocampal neurons. The concentration inducing 30% cell death (LD30) was selected for analyses assessing the neuroprotective efficacy of ovarian hormones (E2 and P4). Pretreatment of hippocampal neurons with E2 significantly protected against 3-NPA (7.5mM) and antimycin (125 µM) induced cell death and was moderately neuroprotective against rotenone (3 µM). E2 was ineffective against KCN and oligomycin-induced cell death. Pretreatment with P4 was without effect against these mitochondrial inhibitors. Co-administration of P4 with E2 abolished E2 induced neuroprotection against 3-NPA and antimycin. Additional metabolic analyses indicated that E2 and P4 separately increased mitochondrial respiratory capacity whereas the co-administration of E2 and P4 resulted in diminished mitochondrial respiration. These findings indicate that E2 protects against mitochondrial toxins that target complexes I, II and III whereas P4 was without effect. The data also predict that continuous combined co-administration of estrogen and progesterone common to many hormone therapy regimens is unlikely to prevent the deficits in mitochondrial function.

Antimycin A-induced mitochondrial apoptotic cascade is mitigated by phenolic constituents of Phyllanthus amarus aqueous extract in Hep3B cells.

Antimycin A (AMA) treatment of cells blocks mitochondrial electron transport chain, and leads to elevated ROS generation, thereby causing damage to mtDNA, proteins and lipids, along with mitochondrial membrane depolarization, release of pro-apoptotic proteins into the cytoplasm, and induction of apoptosis. Prevention of such oxidative cellular damage by the aqueous extract of Phyllanthus amarus has been investigated in this study. The extract demonstrated significant potential in mitigating H(2)O(2)-induced membrane damage along with considerable recession in AMA-governed mitochondrial protein and lipid degradation in Hep3B cells. 8-OHdG analysis of mtDNA damage revealed substantial protective potential of the extract against mtDNA damage. SQ-PCR of selected mtDNA sequences confirmed the potential of the extract to alleviate levels of mtDNA damage. FACS analysis with JC-1 fluorescent dye established significant escalation of mitochondrial membrane potential by the extract in AMA-treated cells. Extract treatment resulted in a distinct decline in the degrees of AMA-induced release of cytochrome c and AIF into the cytoplasm along with consequent pacification of apoptosis. All protective efficiencies of the extract reported in this study were found to hold strong and significant (P<0.05) positive correlation to its total phenolic contents, thereby proving that polyphenolic constituents of P. amarus aqueous extract mitigate oxidative stress-induced cellular degeneration and aging.

Stress protein flux during recovery from simulated ischemia: induced heat shock protein 70 confers cytoprotection by suppressing JNK activation and inhibiting apoptotic cell death.

Multiple stress proteins are recruited in response to stress in living cells. There are limited reports in the literature analyzing multiple stress protein shifts and their functional consequences on stress response. Using two-dimensional electrophoresis we have analyzed shifts in stress protein profiles in response to energy deprivation as a model of ischemic injury to kidneys. A group of chaperones and stress-induced mitogen activated protein (MAP) kinases were analyzed. In addition to examining stress protein induction and phosphorylation we have also examined the mechanism of cytoprotection by heat shock protein 70 (Hsp70). Our results show that, of the different stress proteins examined, only binding protein (BiP) and Hsp70 were significantly induced upon energy deprivation. Other stress proteins, including Hsp27, calnexin, Hsp90 and ERp57 showed alterations in their phosphorylation profiles. Three different MAP kinases, namely p38, extracellular signal regulated kisase and c-jun N-terminal kinase (JNK) were activated in response to energy deprivation. While JNK activation was linked to apoptosis, activated-p38 was involved in phosphorylation of Hsp27. Study of inhibitors of Hsp70 induction or pre-induction of Hsp70 indicated that induced Hsp70 was involved in the suppression of JNK activation thereby inhibiting apoptotic cell death. Our results provide important insights into the flux in stress protein profiles in response to simulated ischemia and highlight the antiapoptotic, cytoprotective mechanism of Hsp70 action.

Clusterin protects renal tubular epithelial cells from gentamicin-mediated cytotoxicity.

Clusterin is a heterodimeric secreted glycoprotein that is upregulated after acute renal injury. In aminoglycoside nephrotoxicity, clusterin is induced in the tubular epithelium and increased levels are found in the urine. In this study, we developed an in vitro model of gentamicin-induced cytotoxicity in renal proximal tubule cells and tested whether clusterin protected these cells from injury. LLC-PK(1) cells were incubated with varying concentrations of gentamicin in serum-free media, and cytotoxicity was quantified by lactate dehydrogenase release and confirmed by vital dye exclusion. A dose-dependent increase in cytotoxicity occurred with gentamicin concentrations up to 27 mg/ml. Clusterin decreased cytotoxicity in a dose- and time-dependent manner at 6, 12, and 24 h, whereas albumin, used as a control protein, had no effect. In contrast to the aminoglycoside model, when cells were injured by depletion of ATP, clusterin had only a minimally protective effect. LLC-PK(1) cells did not express megalin, a receptor that can mediate the uptake of both clusterin and aminoglycosides into proximal tubule cells. Uptake of gentamicin into LLC-PK(1) cells was observed despite the absence of megalin. In conclusion, clusterin specifically protects against gentamicin-induced renal tubular cell injury by a megalin-independent mechanism.

Cytoprotective effect of ulinastatin on LLC-PK1 cells treated with antimycin A, gentamicin, and cisplatin.

The cytoprotective effect of ulinastatin was studied in LLC-PK1 cells treated with antimycin A, gentamicin, or cisplatin. All of the three agents induced a concentration-dependent increase in the release of lactate dehydrogenase and a decrease in the amount of remaining protein. In the cell injury models treated with 1.5 microM antimycin A, 10 mM gentamicin, and 0.3 mM cisplatin, ulinastatin tended to show a cytoprotective effect at a concentration of 3,000 U/ml and provided a significant protective effect at 10,000 U/ml. LLC-PK1 cells treated with 0.3 mM cisplatin, bovine serum albumin, and alpha1-acid glycoprotein at a concentration of 3.54 mg/ml, which is a comparable protein concentration to that of 10,000 U/ml ulinastatin, showed no protective effect but rather enhanced cell injury. These results suggest that ulinastatin exerts a direct protective effect on LLC-PK1 cells against various renal toxicities.

Protection of hepatocytes against death due to mitochondrial failure: effect of di-Calciphor on antimycin A-induced toxicity.

Di-Calciphor is a synthetic derivative of prostaglandin B1 that protects against cerebral and cardiac ischemia apparently by preserving mitochondrial function. To determine whether di-Calciphor specifically protects against mitochondrial failure, we studied its effects on mitochondrial functions in hepatocytes treated with the specific mitochondrial poison, antimycin A. The results show that 1 microM di-Calciphor protects against cell death at concentrations of antimycin A that inhibited mitochondrial respiration and caused cellular ATP depletion. Di-Calciphor did not protect against loss of ATP but did protect against the loss of mitochondrial delta psi and delta pH. In addition, di-Calciphor protected against antimycin A-induced loading of phosphate into mitochondria and an associated mitochondrial swelling. Thus, these results show that di-Calciphor protects against a specific mitochondrial poison and support the interpretation that di-Calciphor is a mitochondrial protective agent. In addition, the results suggest that the protection of the mitochondria involves preservation of mitochondrial ionic and osmotic stability and does not involve improved ATP supply.

The neurotoxicants strychnine and bicuculline protect renal proximal tubules from mitochondrial inhibitor-induced cell death.

Glycine-induced cytoprotection of renal proximal tubules exposed to chemical- or hypoxic/anoxic-induced cell death is shared by a few amino acid agonists of the neuronal strychnine-sensitive glycine receptor. The goal of this study was to determine if antagonists of the strychnine-sensitive glycine receptor attenuated the cytoprotective effects of glycine. Strychnine did not antagonize the cytoprotective effects of glycine in proximal tubules exposed to antimycin A. In contrast, strychnine was cytoprotective, was equipotent as glycine (EC50 = 0.4 mM), and the combination of strychnine and glycine was additive. Likewise, bicuculline and norharmane were cytoprotective but 20-50% less potent than glycine. These results suggest that glycine and strychnine act as a common site to produce proximal tubule cytoprotection, but this site does not share the same potency and agonist/antagonist properties as the neuronal strychnine-sensitive glycine receptor.

Alteration of chemoreflex phrenic responses by oligomycin in the rabbit.

The effects of carotid chemoreceptor stimulation by intracarotid injections of sodium cyanide (NaCN, 30 micrograms), antimycin A (AMC, 10 micrograms) and dopamine (DA, 10 micrograms) on phrenic nerve activity were studied before and after oligomycin (200 micrograms) in the rabbit. The excitatory responses to NaCN and AMC were abolished after intracarotid administration of oligomycin, whereas the DA-induced phrenic depression was only slightly diminished. In addition, the effects of hypoxia on phrenic nerve activity were also studied before and after oligomycin (200 micrograms) in some animals with denervated one carotid sinus nerve. The hypoxia-induced phrenic excitation was greatly reduced after intracarotid administration of oligomycin. These results indicate that the chemoreflex phrenic responses induced by NaCN, AMC and hypoxia are probably related to the phosphate potential in the carotid body.

[Effect of menadione and vicasol on mitochondrial energy during inhibition of initiation sites of the respiration chain]. / Deistvie menadiona i vikasola na énergetiku mitokhondrii pri ingibirovanii nachal'nykh uchastkov dykhatel'noi tsepi.

Menadione and vicasol completely restore the respiration rate of rat liver mitochondria after its inhibition by rotenone. Under the same conditions these compounds stimulate oxygen consumption by rabbit heart mitochondria up to 40% of the maximal uncoupled respiration rate in the presence of 5 mM glutamate and up to 30% of the maximal uncoupled respiration rate in a lymphocyte suspension containing glucose. Cyanide and dicumarol, specific inhibitors of DT-diaforase, completely suppress the stimulating effect of menadione and vicasol in isolated mitochondria and by 50% in lymphocyte suspensions. The DiS-C3-(5) fluorescence in lymphocyte suspensions suggests that the menadione and vicasol-induced respiration is capable of supporting the mitochondrial transmembrane potential in lymphocytes. Thus, in different tissues menadione and vicasol can restore oxygen consumption in mitochondria, in which the first and second energy coupling sites are inhibited.

Pyruvate blocks expression of sensitivity to antimycin A and chloramphenicol.

Selectivity in Chinese hamster cells with antimycin A and chloramphenicol depends on a metabolic balance which can be modulated by varying the level of exogenous pyruvate. The effects of both inhibitors are most clearly seen in pyruvate-free nutrients. Addition of 1 mM pyruvate in plating assays shifts dose-response curves for antimycin A or chloramphenicol to higher concentration levels and reduces the differential in response between sensitive and resistant cells. In mass populations, growth inhibition by antimycin A is reduced by adding pyruvate, and growth curves for sensitive and resistant cells tend to converge. These observations show that responses to antimitochondrial drugs can be conditioned by extrinsic factors and indicate the need for further definition of selective systems.

On the mechanism of the cyanide-insensitive alternative pathway of respiration in fungi and higher plants and the nature of the alternative terminal oxidase.

In connection with investigations on the mechanism of action of the systemic fungicide carboxin a new hypothesis was developed on the mechanism of the cyanide-insensitive pathway of respiration, which is widely distributed in fungi und higher plants, and on the nature of the alterative terminal oxidase. Based on own experimental results which are in concordance with the properties of the alternative pathway described in the literature, it is assumed that the nonheme-iron-sulfurprotein (FeSPp) of the succinodehydrogenase, which is located on the main route of the cytochrome mediated respiration, is the alternative terminal oxidase itself. This property seems to be based on the ability for autoxidation in some organisms where the Fe-atoms are oxygenized and reach the maximal coordination number of 6. By this mechanism it is explainable that carboxin which also attacks the normal electron flow at the FeSPp of the succinodehydrogenase is able to inhibit simultaneously under certain circumstances the cyanide-insensitive respiration organisms sensitive to carboxin.

Observations on cell volume, ultrastructure, mitochondrial conformation and vital-dye uptake in Ehrlich ascites tumor cells. Effects of inhibiting energy production and function of the plasma membrane.

The ultrastructure, volume, vital-dye uptake, ATP levels and amounts of intracelular water have been studied in Ehrlich ascites cells treated with two classes of inhibitors. Cell membrane injury with 10(-3) M parachloromercuribenzene sulfonate (PCMBS) resulted in a rapid increase in mean cell volume to 3 times the mean zero time value. Inhibition of respiration plus glycolysis with 10(-4) M iodoacetate (IAA) plus 10(-4) M antimycin resulted first in a slight decrease in volume and then later a slow increase. When glucose was present in the medium, this slow increase was very slight. Intracellular water determination followed closely the volume changes. Cell populations with decreased mean volumes showed dilatations of the ER and swelling of mitochondria, indicating that expansion of some intracellular compartments can occur at the expense of others. Vital-dye uptake in metabolic injury correlated to some extent with the presence of substrate, indicating that an effect of glucose on membrane permeability might be independent of its effects on ATP levels. Mitochondrial contractions appeared with PCMBS as early as 5 minutes, with or without glucose present. With glucose present, treatment with IAA and antimycin also resulted in similar contractions as well as ring-form contractions even at 5 minutes. Without glucose, IAA plus antimycin mainly produced the usual mitochondrial contractions and these developed much later in the course of the incubation, shortly before high-amplitude swelling. The relationship of these mitochondrial conformational changes to ADP/ATP ratios and ADP levels is discussed.