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PubMed1997

In vivo skeletal muscle mitochondrial function in Leber's hereditary optic neuropathy assessed by 31P magnetic resonance spectroscopy.

We used 31P magnetic resonance spectroscopy (31P-MRS) to assess in vivo skeletal muscle mitochondrial function in 10 Leber's hereditary optic neuropathy patients/carriers with a mitochondrial DNA (mtDNA) mutation at one of three nucleotide positions, 11,778, 14,484, and 3,460. We studied one affected patient for each mutation and two unaffected carriers with the 11,778 or 3,460 mutation and three carriers with 14,484. All subjects were homoplasmic except the two 3,460 carriers, who showed 80% and 15% of mutated mtDNA. 31P-MRS at rest disclosed some abnormalities in all subjects. In particular, the phosphorylation potential was below the normal range in all cases. During recovery from exercise, the maximum rate of mitochondrial ATP production (Vmax) was reduced to 27% of normal in the 11,778 mutation and to 53% in the 14,484 mutation patient/carrier groups. Mitochondrial Vmax was within the normal range in all subjects with the 3,460 mutation but correlated inversely with the percentage of mutated mtDNA. This in vivo study shows that the 11,778 mutation causes a mitochondrial impairment more severe than the 14,484 and that the 3,460 mutation results in only a mild depression of muscle mitochondrial function.

Adult↗

PubMed1982

Effect of oxygen radicals on heart mitochondrial function in alpha-tocopherol deficient rabbits.

The mitochondria extracted from hearts of alpha-tocopherol deficient rabbits showed a decreased mitochondrial function and an increased formation of oxygen radicals associated with a decreased superoxide dismutase activity. The exposure of the alpha-tocopherol deficient mitochondria to exogeneous oxygen radicals caused a marked decrease in the mitochondrial function with respect to control mitochondria, which was partially reversed by the addition in vitro of alpha-tocopherol.

Animals↗

PubMed1977

Liver and skeletal muscle mitochondrial function following burn injury.

The possibility of altered mitochondrial function consequent to burn injury was investigated. Mitochondria isolated from liver or skeletal muscle of burn-injured rats (20% tbs) were compared at 3 days postburn to shams and normal controls. Mitochondrial yields were the same for all groups. ADP;O ratios were in the theoretical ranges expected and did not differ among burn, sham, and normal animals. Respiratory control ratios (RCR's) were decreased in liver mitochondria, averaging 71.7% of normal for burned animals compared to 95.8% for the sham group. The loss of respiratory control in liver mitochondria implies inefficient use of substrate chemical energy and could contribute to postburn hypermetabolism. The different response of muscle mitochondria as compared to liver suggests that alterations may be organ specific.

Adenosine Diphosphate↗

PubMed2002

Effect of the new matrix metalloproteinase inhibitor RO-28-2653 on mitochondrial function.

Matrix metalloproteinases (MMPs) have recently become interesting as potential anticancer drugs. RO-28-2653 is a promising compound because of its antimetastatic and antiangiogenic activities. Due to the structural similarity of RO-28-2653 to mitochondriotoxic agents, speculation has arisen that this substance might impair mitochondrial function. We, therefore, investigated the effects of RO-28-2653 on mitochondrial enzymes and on the functional properties of isolated mitochondria and skinned muscle fibers from rat hearts. Results were compared to the action of amytal and 2,4-dinitrophenol (2,4-DNP), both of which are well documented mitochondriotoxic compounds. In contrast to 2,4-DNP, RO-28-2653 did not uncouple oxidative phosphorylation, although higher concentrations of the compound did impair mitochondrial function. Using malate/pyruvate as substrate, 50 microM of RO-28-2653 inhibited mitochondrial respiration in isolated mitochondria and skinned fibers by 23 and 11%, respectively while 2mM of amytal elicited almost complete inhibition of the mitochondrial respiration. RO-28-2653 (50 micro) inhibited succinate-dependent respiration in both systems by 43 and 24%, respectively while 2mM of amytal caused 41 and 23% inhibition, respectively. There was no change in the ADP/O ratios. RO-28-2653 (50 microM) did not significantly alter the activity of the respiratory chain complexes or succinate dehydrogenase, although citrate synthase (CS) was inhibited by upto 71%. This inhibition was non-competitive at a K(i) of 25+/-5 microM. Inhibitory effects in the presence of hydrophobic substances, such as BSA and Triton X-100, were significantly lower in both test systems. In conclusion, high concentrations of RO-28-2653 impair mitochondrial function, although compared to amytal and 2,4-DNP, this is rather low. The resultant impairment is less pronounced in the more complex skinned muscle fiber system, and is dependent on hydrophobic interactions.

Animals↗

PubMed1982

Mitochondrial bioenergetics during the initiation of mercuric chloride-induced renal injury. I. Direct effects of in vitro mercuric chloride on renal mitochondrial function.

Increasing data suggest that mitochondrial dysfunction may be an important early component of nephrotoxin-induced changes in renal cell function and viability. This study was designed to obtain more detailed information about the effects on several basic bioenergetic parameters of the direct interaction of Hg2+ with renal cortical mitochondria in vitro as a necessary prelude to studies of mitochondrial functional changes after treatment with mercuric chloride in vivo. Beginning at a threshold level of 2 nmol of Hg2+/mg of mitochondrial protein, Hg2+ induced marked stimulation of State 4 respiration, mild inhibition of State 3 respiration, and 2,4-dinitrophenol uncoupled respiration, a striking increase in atractyloside-insensitive ADP uptake and stimulation of both basal- and Mg2+-activated oligomycin-sensitive mitochondrial ATPase activity. These effects of Hg2+ could be prevented and reversed by the sulfhydryl reagent dithioerythritol and by albumin but were not affected by Mg2+. Detailed studies on the addition of HgCl2 to the preparation at different stages of the mitochondrial isolation procedure demonstrated that the presence of other proteins decreased mitochondrial Hg2+ binding, that the Hg2+ was not readily washed off the mitochondria by nonprotein-containing solutions, and that prolonged exposure of mitochondria to Hg2+ during the isolation procedure did not markedly alter its functional effects or their reversibility as assessed on the final mitochondrial preparation. These data provide an important basis for critically assessing the changes in function of mitochondria isolated after in vivo treatment with mercuric chloride.

Adenosine Diphosphate↗

PubMed2001

Alzheimer-associated neuronal thread protein-induced apoptosis and impaired mitochondrial function in human central nervous system-derived neuronal cells.

In Alzheimer Disease (AD), dementia is due to cell loss and impaired synaptic function. The cell loss is mediated by increased apoptosis, predisposition to apoptosis, and impaired mitochondrial function. Previous studies demonstrated that the AD7c-NTP neuronal thread protein gene is over-expressed in AD beginning early in the course of disease, and that in AD, AD7c-NTP protein accumulation in neurons co-localizes with phospho-tau-immunoreactivity. To determine the potential contribution of AD7c-NTP over-expression to cell loss in AD, we utilized an inducible mammalian expression system to regulate AD7c-NTP gene expression in human CNS-derived neuronal cells by stimulation with isopropyl-1-beta-D-thiogalactopyranoside (IPTG). IPTG induction of AD7c-NTP gene expression resulted in increased cell death mediated by apoptosis, impaired mitochondrial function, and increased cellular levels of the p53 and CD95 pro-apoptosis gene products as occur in AD. In addition, over-expression of AD7c-NTP was associated with increased levels of phospho-tau, but not amyloid-beta immunoreactivity. These results suggest that AD7c-NTP over-expression may have a direct role in mediating some of the important cell death cascades associated with AD neurodegeneration, and further establish a link between AD7c-NTP overexpression and the accumulation of phospho-tau in preapoptotic CNS neuronal cells.

Alzheimer Disease↗

PubMed2004

Sperm mobility: phenotype in roosters (Gallus domesticus) determined by mitochondrial function.

Previously, inheritance of sperm mobility entailed a maternal additive genetic effect, and sperm ATP content was correlated (r = 0.80) with phenotype. The present study was conducted to determine if mitochondrial function was critical to phenotypic expression. Whereas phenotype was independent of mitochondrial helix length, phenotype was correlated with sperm oxygen consumption (r = 0.83) using random-bred roosters. Aberrant mitochondria characterized immobile sperm, as evidenced by transmission-electron microscopy. Such mitochondria were swollen and contained disorganized cristae. Additional experiments were performed with roosters from lines selected for low or high sperm mobility. A threefold difference in sperm oxygen consumption was observed between lines. Single nucleotide polymorphisms were observed in mitochondrial DNA by sequencing replicate mitochondrial genomes from each line. An A-to-G substitution in the gene encoding tRNA(Arg) was inherited consistently, as evidenced by restriction fragment length polymorphism analysis using two male and two female progeny per family group and 14 family groups per line. Motile concentration in semen from low-line males was half that observed in semen from high-line males, as evidenced by computer-assisted sperm motion analysis. Likewise, 47% of sperm from low-line males contained aberrant mitochondria, compared to 4% for high-line males. In summary, sperm mobility phenotype was dependent on mitochondrial function, which in turn was altered by genetic selection. Fowl deferent duct fluid contains a high concentration of glutamate. We propose that variation in sperm mobility phenotype stems from the extent to which glutamate induces excessive mitochondrial Ca2+ uptake before ejaculation.

Animals↗

PubMed1995

Assessment of mitochondrial function and control in normal and diseased states.

Mitochondrial function in muscle in vivo can be quantitatively evaluated using 31-phosphorus nuclear magnetic resonance. In resting muscle, the concentrations of ions (e.g. H+, Na+) and two of the major bioenergetic components (inorganic phosphate and creatine) are determined by regulated transcellular transport processes. During recovery after exercise the kinetics and control of mitochondrial ATP synthesis can be established. During exercise the relative contributions to ATP synthesis of phosphocreatine (using creatine kinase), anaerobic glycogenolysis and oxidative phosphorylation are dissected and have been shown to change with time. The consequences of mitochondrial lesions and dysfunctions on these processes have been summarised.

Adenosine Triphosphate↗

PubMed1999

Mitochondrial function in human skeletal muscle is not impaired by high intensity exercise.

The hypothesis that high-intensity (HI) intermittent exercise impairs mitochondrial function was investigated with different microtechniques in human muscle samples. Ten male students performed three bouts of cycling at 130% of peak O2 consumption (V.O2,peak). Muscle biopsies were taken from the vastus lateralis muscle at rest, at fatigue and after 110 min recovery. Mitochondrial function was measured both in isolated mitochondria and in muscle fibre bundles made permeable with saponin (skinned fibres). In isolated mitochondria there was no change in maximal respiration, rate of adenosine 5'-triphosphate (ATP) production (measured with bioluminescence) and respiratory control index after exercise or after recovery. The ATP production per consumed oxygen (P/O ratio) also remained unchanged at fatigue but decreased by 4% (P<0.05) after recovery. In skinned fibres, maximal adenosine 5'-diphosphate (ADP)-stimulated respiration increased by 23% from rest to exhaustion (P<0.05) and remained elevated after recovery, whereas the respiratory rates in the absence of ADP and at 0.1 mM ADP (submaximal respiration) were unchanged. The ratio between respiration at 0.1 and 1 mM ADP (ADP sensitivity index) decreased at fatigue (P<0.05) but after the recovery period was not significantly different from that at rest. It is concluded that mitochondrial oxidative potential is maintained or improved during exhaustive HI exercise. The finding that the sensitivity of mitochondrial respiration to ADP is reversibly decreased after strenuous exercise may indicate that the control of mitochondrial respiration is altered.

Adenosine Diphosphate↗

PubMed1986

Mitochondrial function and intracellular calcium in anoxic cardiac myocytes.

Mitochondrial dysfunction has been implicated as the cause of irreversible injury in the ischemic heart. To circumvent artifacts associated with organelle isolation, mitochondrial function was studied in intact isolated, Ca2+-tolerant rat ventricular myocytes. After 30 min of anaerobic incubation, myocyte viability decreased from 76 +/- 1 to 33 +/- 4%. Basal O2 consumption rates (nanoatoms . mg cell protein-1 . min-1) were 17.1 +/- 1.3 in aerobic cells and 51.0 +/- 9.8 in anoxic cells. Carbonylcyanide-p-trifluoromethoxyphenyl hydrazone (FCCP)-stimulated rates were 65.5 +/- 9.2 and 84.5 +/- 15.3 in aerobic and anoxic cells, respectively. Respiratory control ratio was lower in anoxic cells: 2.3 +/- 0.3 versus 4.2 +/- 0.4 in aerobic cells. These data suggest that early anoxic mitochondrial injury is due to increased permeability of the inner membrane. Addition of pyruvate, malate, and FCCP to cells made permeable by digitonin resulted in similar maximal O2 consumption rates: 276.5 +/- 31.8 in aerobic and 299.3 +/- 31.9 in anoxic cells, suggesting the electron transport chain is intact in anoxic cells. For purposes of investigating whether anoxic mitochondrial dysfunction is secondary to cellular or mitochondrial Ca2+ overload, total cell Ca2+, cytosolic free Ca2+ levels (measured by null-point titration), and mitochondrial Ca2+ contents (measured as FCCP-releasable Ca2+) were measured. There were no differences in these three parameters between aerobic and anoxic cells, suggesting that mitochondrial dysfunction and irreversible hypercontraction of isolated cardiac myocytes exposed to 30 min of anoxia are not related to Ca2+ overload.

Animals↗

PubMed1996

Favourable, significant effect of the dose-dependent treatment with RU 38486 (RU) on the alterations of the hepatic mitochondrial function of diabetic rats.

In the present work, the effect "in vivo' of increasing doses of RU 38486 upon the hepatic mitochondrial function of diabetic rats has been studied. At the same time, the action of adrenalectomy and corticosterone restitution on this function were comparatively demonstrated. The parameters measured were oxygen consumption with the substrates: 3-hydroxybutyrate (HB), succinate (Suc) and malate-glutamate (Mal-glut) in intact liver mitochondria and the activities of 3-hydroxybutyrate dehydrogenase (HBD) and cytochrome c oxidase (Cyt.c oxid.) enzymes in broken liver mitochondria. The groups of animals studied were normal controls (N) and the following groups of diabetic rats: rats without any treatment (D), adrenalectomized rats (D+ADX), rats that were adrenalectomized and treated with corticosterone (D+ADX+C) and four groups treated with increasing oral doses of RU (in mg/kg body wt.), that is, 12.5 (D+RU1), 25.0 (D+RU2), 37.5 (D+RU3) and 50.0 (D+RU4). The results showed a tendency of increasing values of mitochondrial oxygen consumption in diabetic animals treated with RU. The favourable effect of increasing doses of RU on O2 consumption of diabetic rat liver mitochondria with each of the substrates showed a significant association as indicated by the values obtained for the correlation coefficients r (0.95, 0.97 and 0.99 according to the substrate HB, Succ or Mal-glut, respectively). Likewise, the correlation between the treatment with increasing doses of RU and the recovery of enzyme activities showed a significant dose-effect association with r 0.94 for HBD and r = 0.95 for Cyt.c oxid. Adrenalectomy showed a similar effect to treatment with the maximum dose of RU while corticosterone restitution gave measured values similar to those of the D group. In conclusion, the favourable, significant variation of the hepatic mitochondrial function of diabetic rats was demonstrated by the dose-dependent treatment with RU as seen by the correlation statistical study performed. At the same time, the pernicious effect that glucocorticoids exert upon such function in experimental diabetes was confirmed.

3-Hydroxybutyric Acid↗

PubMed1992

Mitochondrial function in type I cells isolated from rabbit arterial chemoreceptors.

1. In this, and the accompanying paper (Duchen & Biscoe, 1992), we test the hypothesis that the oxygen sensitivity of mitochondrial electron transport forms a basis for transduction in the carotid body, the primary peripheral arterial oxygen sensor. We here describe for isolated type I cells the changes in autofluorescence of mitochondrial NAD(P)H that accompany changes in PO2. 2. NAD(P)H autofluorescence (excitation, 340-360 nm; emission peak, 450 nm) increased with anoxia, reflecting a rise in the NAD(P)H/NAD(P) ratio. Graded increases in autofluorescence were seen in response to graded decreases in PO2, suggesting that mitochondrial function is progressively altered below a PO2 of about 60 mmHg. 3. A mitochondrial origin for the NAD(P)H autofluorescence was suggested by the mutual exclusion of the responses to anoxia and cyanide. 4. Oxidized flavoproteins fluoresce when excited at 450 nm with an emission peak at 550 nm. The small signals obtained under these conditions increased with uncoupler and showed a graded decrease with falling PO2 reflecting a rise in the FADH/FAD ratio. 5. Hypoxia raises [Ca2+]i. The hypoxia-induced changes in mitochondrial function were not secondary to this rise. A brief K(+)-induced depolarization leads to a transient increase in [Ca2+]i. At the same time there is a rapid decrease in NAD(P)H autofluorescence followed by an increase that far outlasts the rise in [Ca2+]i. This delayed increase in autofluorescence was smaller than was the increase with anoxia, even though K(+)-induced depolarization raised [Ca2+]i more than does anoxia. In Ca(2+)-free solutions the depolarization-induced changes were abolished, while those associated with hypoxia were maintained. 6. The changes of autofluorescence with K(+)-induced depolarization appear to reflect (i) oxidation of NAD(P)H by stimulation of respiration following mitochondrial Ca2+ uptake and (ii) reduction of NAD(P) by the Ca(2+)-dependent activation of mitochondrial dehydrogenases. This activation could last several minutes following only 100 ms depolarization, while the changes accompanying hypoxia closely followed the time course of the change in PO2. 7. In similarly isolated rat or mouse chromaffin cells and mouse dorsal root ganglion neurons under identical conditions, no measurable change in autofluorescence or in [Ca2+]i was seen until the PO2 fell below about 5 mmHg. 8. Carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) increases O2 consumption, oxidizing mitochondrial NADH and hence decreasing autofluorescence, (delta FFCCP). Blockade of electron transport by anoxia or CN- decreases O2 consumption, increasing mitochondrial NADH/NAD and autofluorescence (delta FCN). The fractional change in autofluorescence with FCCP, delta FFCCP/delta FFCCP+FCN), is thus a measure of resting O2 consumption.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

The effect of a chalcogenapyrylium dye with and without photolysis on mitochondrial function in normal and tumor cells.

A chalcogenapyrylium dye 8b, which is under investigation for the photodynamic therapy of malignant gliomas (brain tumors), was evaluated for inhibition of mitochondrial function both before and after exposure to laser light of 800 nm. Neoplastic and normal cells forced to use mitochondrial substrates were killed by the light-activation of intracellular 8b as well as exposure to classic mitochondrial inhibitors, rotenone and sodium azide. Correspondingly, cells in glucose-rich media showed little decrease in viability due to the photolysis of intracellular 8b or the presence of mitochondrial toxins. The toxicity of 8b without light activation was found to be the same regardless of the cell's energy source. Measurement of cellular ATP generated during treatment also showed the photolysis of intracellular 8b to be more inhibitory towards mitochondrial function than the unactivated parent compound. We conclude that the chalcogenapyrylium dyes localize to the mitochondrion and that photoactivation results in mitochondrial injury.

Adenosine Triphosphate↗

PubMed2005

Valsartan improves mitochondrial function in hearts submitted to acute ischemia.

The effect of valsartan, an angiotensin II-type I receptor blocker, on the mitochondrial function, was studied using an ex vivo animal model (hearts from Wistar rats), perfused in a Langendorff system and then submitted to global acute ischemia. Parameters evaluated were: membrane electrical potential (DeltaPsi, using a tetraphenylphosphonium-TPP+-electrode), oxygen consumption by the respiratory chain (Clark-type O2 electrode), phosphorylation lag phase (time necessary to phosphorylate a fixed amount of ADP) and ATP/ADP ratio (adenine nucleotides quantified by high-pressure liquid chromatography-HPLC). Valsartan acts preferentially in the phosphorylation, increasing ATP/ADP ratios (succinate: 1.6+/-0.4 versus 0.5+/-0.1--P<0.05; ascorbate/N,N,N',N'-tetramethyl-P-phenylenodiamine-TMPD: 1.1+/-0.2 versus 0.4+/-0.1--p<0.05 versus ischemia in the absence of valsartan) and decreasing lag phase (glutamate/malate: 50.0+/-9.6 s versus 127.2+/-19.03 s-84.6+/-16.2% versus 215.3+/-32.2%; P=0.01; succinate: 111.8+/-33.1 s versus 275.73+/-45.99 s-168.2+/-49.8% versus 414.9+/-69.2%; P=0.02 or ascorbate/TMPD: 11.0+/-3.9 s versus 62.4+/-11.63 s-34.9+/-12.4% versus 198.1+/-36.9%; P=0.001 versus ischemia in the absence of valsartan). This enables a higher energy production in hearts submitted to acute ischemia, for which having energy becomes critical to preserve mitochondrial function. These mechanisms allow us to better understand valsartan cytoprotection in ischemic cardiomyopathy.

Acute Disease↗

PubMed2005

Modulation of brain mitochondrial function by deprenyl.

The present study shows that deprenyl, a known inhibitor of monoamine oxidase B (MAO B), may generate changes in mitochondrial function. Brain submitochondrial membranes (SMP), synaptosomes and cytosolic fractions were incubated with different deprenyl concentrations and nitric oxide synthase (NOS) activity was measured. The effect of deprenyl on oxygen consumption, calcium-induced permeability transition and hydrogen peroxide (H(2)O(2)) production rates was studied in intact mitochondria. Respiratory complexes and monoamine oxidase activities were also measured in submitochondrial membranes. Incubation of brain submitochondrial membranes with deprenyl 10, 25 and 50 microM inhibited nitric oxide synthase activity in a concentration-dependent manner. The same effect was observed in cytosolic fractions and synaptosomes. Monoamine oxidase activity was inhibited at lower deprenyl concentrations (from 0.5 microM). Cytochrome oxidase (complex IV) activity was found 42% increased in the presence of 25 microM deprenyl in a condition of maximal nitric oxide synthase activity. Incubation of brain mitochondria with deprenyl 25 microM produced a 60% increase in oxygen uptake in state 3, but no significant changes were observed in state 4. Pre-incubation of brain mitochondria with deprenyl 0.5 and 1 microM inhibited calcium-induced mitochondrial permeability transition and decreased hydrogen peroxide production rates. Our results suggest that in vitro effects of deprenyl on mitochondrial function can occur through two different mechanisms, involving nitric oxide synthase inhibition and decreased hydrogen peroxide production.

Animals↗

PubMed1984

Verapamil pretreatment preserves mitochondrial function and tissue magnesium in the ischemic kidney.

These studies were designed to test the efficacy and possible mechanisms of the prevention of mitochondrial functional deterioration in renal ischemia by the slow-channel calcium blocker verapamil. Renal ischemia was induced in guinea pigs by a unilateral ligation of the renal artery for 30 or 60 min. In the pretreated animals verapamil was given twice a day over a 5-d period prior to the induction of ischemia. Sham-operated animals were used as normal controls. After 30 and 60 minutes, the kidneys were removed and used for mitochondrial isolation and analyses, total tissue Ca2+ and Mg2+ determinations, or for electron microscopy. Verapamil pretreatment completely blocked the decrease of mitochondrial Ca2+ uptake rate induced by 30 or 60 min of ischemia. The pretreatment delayed by 30 min the ischemic decrease of state 3 respiratory activity. Total tissue Ca2+ concentration was not altered by ischemia or verapamil pretreatment. Total tissue Mg2+ concentration, however, was significantly reduced in the ischemic kidney at 60 min. This reduction was prevented completely by verapamil pretreatment. These data suggest that the mitochondrial functional deterioration induced by 30 min of ischemia is a primary cellular insult secondarily leading to loss of tissue Mg2+. The point of irreversibility in the ischemic cell injury might be initiated by lowered tissue Mg2+/Ca2+ ratios.

Animals↗

PubMed2000

Assessment of equine sperm mitochondrial function using JC-1.

The fluorescent carbocyanine dye, JC-1, labels mitochondria with high membrane potential orange and mitochondria with low membrane potential green. Evaluation of mitochondrial membrane potential with JC-1 has been used in a variety of cell types, including bull spermatozoa; however, JC-1 staining has not yet been reported for equine spermatozoa. The aim of this study was to apply JC-1 staining and assessment by flow cytometry or a fluorescence microplate reader for evaluation of mitochondrial function of equine spermatozoa. Six ejaculates from four stallions were collected and centrifuged through a Percoll gradient (PERC). Spermatozoa were resuspended to 25 x 10(6) cells/mL, samples were split, and one sample was repeatedly flash frozen (FF) in LN2 and thawed. The following gradients of PERC:FF were prepared: 100:0 (100), 75:25(75), 50:50 (50), 25:75 (25) and 0:100 (0). Samples were stained with 2.0 microM JC-1 and assessed for staining by flow cytometry and by a fluorescence microplate reader. A total of 10,000 gated events was analyzed per sample with flow cytometry. The mean percentage of cells staining orange for the 100, 75, 50, 25 and 0 treatments was 92.5, 72.8, 53.4, 27.3 and 7.3, respectively. The expected percentage of spermatozoa forming JC-1 aggregates was correlated with the actual percentage of orange labeled sperm cells determined by flow cytometry (r2=0.98). Conversely, JC-1 monomer formation was negatively correlated with expected mitochondrial membrane potential (r2=-0.98). The blank corrected orange fluorescence, assessed by microplate assay, was significantly (P<0.0001) correlated with the expected (r2=0.49) and with the flow cytometric (r2=0.50) determination of percentage of spermatozoa with mitochondria of high membrane potential. Total orange and orange:green fluorescence was also correlated with mitochondrial function. These results indicate that JC-1 staining can accurately detect changes in mitochondrial membrane potential of equine spermatozoa. The relative fluorescence of JC-1 labeling patterns of equine spermatozoa can be accurately and objectively determined by flow cytometry and by a fluorescence microplate reader assay.

Animals↗

PubMed2005

Pyruvate modulates hepatic mitochondrial functions and reduces apoptosis indicators during hemorrhagic shock in rats.

BACKGROUND: Dysfunctional mitochondria have been widely accepted as one of the key targets and a mediator of secondary cell injury and organ failure during hemorrhagic shock (HS). The liver is known to be the first organ to display the signs of injury during HS. This report describes experiments to determine whether modulation of hepatic mitochondrial dysfunctions by pharmacologic agents could prevent liver injury in rats subjected to HS. METHODS: In this study, Sprague-Dawley rats were either treated as controls or subjected to computer-controlled arterial hemorrhage (40 mmHg) for 60 min followed by resuscitation with hypertonic saline, hypertonic beta-hydroxybutyrate, or hypertonic sodium pyruvate for the next 60 min before death. During the course of the experiment, animals were continuously monitored for hemodynamic and metabolic parameters. At the end of the experiment, the liver was excised and examined for oxidative injury, mitochondrial functions, expression of nitric oxide synthase, and indicators of apoptosis. RESULTS: In comparison to hypertonic saline and hypertonic beta-hydroxybutyrate, pyruvate significantly protected the liver from oxidative injury, prevented the up-regulation of nitric oxide synthase, inhibited pyruvate dehydrogenase deactivation, and improved cellular energy charge and mitochondrial functions. In addition, pyruvate also reduced cleavage of poly-adenosine diphosphate ribose polymerase by preventing leakage of mitochondrial cytochrome c in the liver of HS animals. CONCLUSIONS: These data suggest that modulation of mitochondrial metabolic functions is likely to be one of the important mechanisms by which pyruvate exerts its protective effects on the liver during HS and resuscitation in rats.

Animals↗