Decrease In ATP Content Research Articles

A New Type of Peroxisomal Acyl-Coenzyme A Synthetase from Arabidopsis thaliana Has the Catalytic Capacity to Activate Biosynthetic Precursors of Jasmonic Acid

Arabidopsis thaliana contains a large number of genes that encode carboxylic acid-activating enzymes, including nine long-chain fatty acyl-CoA synthetases, four 4-coumarate:CoA ligases (4CL), and 25 4CL-like proteins of unknown biochemical function. Because of their high structural and sequence similarity with bona fide 4CLs and their highly hydrophobic putative substrate-binding pockets, the 4CL-like proteins At4g05160 and At5g63380 were selected for detailed analysis. Following heterologous expression, the purified proteins were subjected to a large scale screen to identify their preferred in vitro substrates. This study uncovered a significant activity of At4g05160 with medium-chain fatty acids, medium-chain fatty acids carrying a phenyl substitution, long-chain fatty acids, as well as the jasmonic acid precursors 12-oxo-phytodienoic acid and 3-oxo-2-(2'-pentenyl)-cyclopentane-1-hexanoic acid. The closest homolog of At4g05160, namely At5g63380, showed high activity with long-chain fatty acids and 12-oxo-phytodienoic acid, the latter representing the most efficiently converted substrate. By using fluorescent-tagged variants, we demonstrated that both 4CL-like proteins are targeted to leaf peroxisomes. Collectively, these data demonstrate that At4g05160 and At5g63380 have the capacity to contribute to jasmonic acid biosynthesis by initiating the beta-oxidative chain shortening of its precursors.

Effects of 6-hydroxydopamine on mitochondrial function and glutathione status in SH-SY5Y human neuroblastoma cells

SH-SY5Y human neuroblastoma cells were incubated with 6-hydroxydopamine (6-OHDA) for 4 and 24 h to examine the mechanism of cell death and to determine the time-dependent effects of 6-OHDA on cellular glutathione status. After 4 h, 6-OHDA significantly depleted cellular ATP and GSH concentrations with only slight increases in cell death. GSH:GSSG ratios and mitochondrial membrane potential (Δ ψ m) were significantly decreased during 4 h incubations with 6-OHDA. High concentrations of 6-OHDA (⩾100 μM) induced oxidative stress and mitochondrial dysfunction in SH-SY5Y cells within 4 h leading to cell death. In 24 h incubations, 25 and 50 μM 6-OHDA significantly decreased ATP concentrations; however, significant increases in cell death were only observed with 50 μM 6-OHDA. 6-OHDA induced a concentration-dependent increase in GSH and total glutathione concentrations after 24 h. After exposure to 50 μM 6-OHDA, GSH concentrations were increased up to 12-fold after 24 h with no change in the GSH:GSSG ratio. Gene analysis suggests that the increase in GSH concentration was due to increased expression of the GSH synthesis genes glutamate cysteine ligase modifier and catalytic subunits. Our results suggest that 6-OHDA induces oxidative stress in SH-SY5Y cells resulting in an adaptive increase in cellular GSH concentrations.

Effect of Growth Hormone and Estrogen Administration on Hepatocyte Alterations in Old Ovariectomized Female Wistar Rats

Aging could be due to the accumulation of oxidative damage. On the other hand, growth hormone (GH) and estrogen deficiency induce deleterious effects on different tissues, and hormonal replacement could counteract these effects. We have investigated whether GH and estrogen administration modify some parameters related to oxidative stress and inflammation in hepatocytes isolated from old ovariectomized female rats. Twenty-two month-old ovariectomized animals were divided into control rats, rats treated with GH, rats treated with estradiol, and rats treated with GH+estradiol. Two-month-old intact female rats were used as young reference group. Hepatocytes were isolated, cultured, and CO and NO release, ATP, cyclic-guanosyl monophosphate (cGMP), and lipid peroxide (LPO) content of cells, as well as phosphatidylcholine (PC)synthesis, were measured. Hepatocytes isolated from old ovariectomized rats showed a decrease in ATP content and PC synthesis compared to young rats. Age also induced an increase in LPO, NO, CO, and cGMP. Treating old rats with GH significantly increased ATP and reduced CO and cGMP levels. Estradiol administration improved all the parameters that were altered. Co-administration of GH and estrogens induced a more marked effect than estrogens alone only in cGMP content. In conclusion, administration of estrogens to old ovariectomized females seemed to prevent oxidative changes in hepatocytes, whereas the effect of GH is not so evident.

Glycolysis plays a major role for adenosine triphosphate supplementation in mouse sperm flagellar movement.

The mammalian sperm must be highly motile for a long time to fertilize a egg. It has been supposed that ATP required for sperm flagellar movement depends predominantly on mitochondrial respiration. We assessed the contribution of mitochondrial respiration to mouse sperm motility. Mouse sperm maintained vigorous motility with high beat frequency in an appropriate solution including a substrate such as glucose. The active sperm contained a large amount of ATP. When carbonyl cyanide m-chlorophenylhydrazone (CCCP) was applied to suppress the oxidative phosphorylation in mitochondria, the vigorous motility was maintained and the amount of ATP was kept at the equivalent level to that without CCCP. When pyruvate or lactate was provided instead of glucose, both sperm motility and the amount of ATP were high. However, they were drastically decreased when oxidative phosphorylation was suppressed by addition of CCCP. We also found that sperm motility could not be maintained in the presence of respiratory substrates when glycolysis was suppressed. 2-Deoxy-d-glucose (DOG) had no effect on mitochondrial respiration assessed by a fluorescent probe, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), but, it inhibited motility and decreased ATP content when pyruvate or lactate were provided as substrates. The present results suggest that glycolysis has an unexpectedly important role in providing the ATP required for sperm motility throughout the length of the sperm flagellum.

Combined hyperthermia and chlorophyll-based photodynamic therapy: tumour growth and metabolic microenvironment.

The effects of combined and simultaneously applied localised 43°C hyperthermia (HT) and an antivascular bacteriochlorophyll-serine-based photodynamic therapy (Bchl-ser-PDT) on tumour growth and several microenvironmental parameters were examined. Rats bearing DS-sarcomas were allocated to treatment groups: (i) sham-treatment (control), (ii) Bchl-ser-PDT (20 mg kg−1 i.v.), (iii) localised HT, (iv) Bchl-ser-PDT+HT. The light source used was an infrared-A irradiator, which, by use of appropriate filters, delivered the different ranges of wavelengths required. Following treatment, tumour volume was monitored. The greatest tumour growth inhibition was seen with Bchl-ser-PDT+HT, and subsequent experiments identified the pathophysiological basis for this effect. Red blood cell flux in tumour microvessels declined rapidly upon Bchl-ser-PDT+HT, reaching approximately 10% of initial values by the end of treatment. Similarly, tumour oxygenation worsened, reaching almost anoxic levels by the end of the treatment period. Assessment of metabolic parameters showed a pronounced increase in lactate levels and a decrease in ATP concentrations after combined treatment. The results presented suggest that vascular collapse and flow stasis resulting in a deterioration of tumour oxygenation and a switch from oxidative to glycolytic glucose turnover are key elements in the tumour eradication seen with this novel approach in which an antivascular PDT and HT are combined and simultaneously applied.

Non-steroidal anti-inflammatory drugs affect the methotrexate transport in IEC-6 cells

Methotrexate (MTX) is used not only for the cancer chemotherapy but also for the treatment of rheumatic disease, often together with non-steroidal anti-inflammatory drugs (NSAIDs). MTX is actively cotransported with H + in the small intestine, mediated by a reduced folate carrier (RFC). The coadministration of some NSAIDs with MTX to rats caused a decrease of MTX absorption through the small intestine. This may be due to the uncoupling effect of oxidative phosphorylation of the NSAIDs. The present study investigated whether flufenamic acid, diclofenac and indomethacin, NSAIDs, decreased ATP content of rat-derived intestinal epithelial cell line IEC-6 cells and affected the MTX transport in IEC-6 cells. The MTX uptake in IEC-6 cells was dependent on medium pH and maximum around pH 4.5-5.5. The MTX uptake was composed of a transport inhibited by 4, 4′-diisothiocyanostilbene-2, 2′-disulfonic acid (DIDS) and a non-saturable one. The DIDS-sensitive component in the MTX uptake showed a saturation kinetics (Michaelis–Menten constant (Km): 3.91 ± 0.52 μM, Maximum velocity (Vmax): 94.66 ± 6.56 pmol/mg protein/5 min). The cellular ATP content in IEC-6 cells decreased significantly at 30 min after the cells were started to incubate with the NSAIDs (250 μM flufenamic acid, 500 μM diclofenac and 500 μM indomethacin). The MTX uptake in IEC-6 cells in the presence of the NSAIDs decreased with the reduction of cellular ATP content and showed a good correlation with the ATP content (correlation coefficient: 0.982). Thus it seems likely that the ATP content in IEC-6 cells with the NSAIDs decreased due to the uncoupling effect of oxidative phosphorylation of the NSAIDs, resulting in the inhibition of the secondary active transport of MTX in IEC-6 cells. The present results also suggest that IEC-6 cells are useful to evaluate the drug interaction relating to this carrier system.

The effect of lead ions on the energy metabolism of human erythrocytes in vitro

The aim of this work was to evaluate the influence of chronic exposure to lead ions on the parameters of energetic status of human erythrocytes in vitro. Umbilical cord erythrocytes were incubated with lead acetate at final lead ion concentrations ranging from 10 to 200 μg/dl. ATP, ADP, AMP, adenosine, GTP, GDP, GMP, guanosine, IMP, inosine, hypoxanthine, NAD and NADP concentrations in erythrocytes were determined using HPLC. Scanning electron micrographs of erythrocytes were taken. The mean concentrations of ATP, GTP, NAD and NADP, and mean values of adenylate energy charge (AEC) and GEC in cells incubated at the presence of lead ions were significantly lower after 20 h of incubation. Concentrations of purine degradation products (Ado, Guo, Ino) and Hyp were significantly higher. It is suggested that lead ions affect the energy metabolism of erythrocytes. Morphological changes in erythrocytes correspond to the increase of lead ions in the incubation mixture and to the decrease of ATP concentration in erythrocytes. A decrease in NAD and ATP concentration in erythrocytes could be a sensitive indicator of energy process disturbance, useful in monitoring in case of chronic lead exposure.

The role of uncoupling protein 2 in the development of type 2 diabetes.

Uncoupling proteins (UCP) are carriers expressed in the mitochondrial inner membrane that uncouple oxygen consumption by the respiratory chain from ATP synthesis. UCP2 is a member of the multigenic UCP family that is expressed in a wide range of tissues and organs. Possible functions of UCP2 include control of ATP synthesis, regulation of fatty acid metabolism and control of reactive oxygen species production. UCP2 expression in tissues involved in lipid and energy metabolism and mapping of the gene to a region linked to obesity and hyperinsulinemia prompted studies on the involvement of UCP2 in metabolic disorders, and especially in type 2 diabetes. In human adipose tissue and skeletal muscle, UCP2 expression is increased during fasting. The carrier was shown to be under the control of fatty acids and thyroid hormones in vivo. An upregulation has been observed in the liver during high-fat feeding and obesity. However, data in UCP2 gene knockout mice do not support a role for UCP2 in steatohepatitis. The most compelling metabolic role of UCP2 comes from studies in pancreatic beta cells. Overexpression in isolated pancreatic islets results in decreased ATP content and blunted glucose-stimulated insulin secretion. UCP2-deficient mice show an increased ATP level and an enhanced insulin secretion. Lack of UCP2 dramatically improves insulin secretion and decreases hyperglycemia in leptin-deficient mice. The role of UCP2 in the control of insulin secretion constitutes, to date, the most pertinent path to investigate in a therapeutic perspective.

Allosteric modulation of the mouse Kir6.2 channel by intracellular H+ and ATP.

The ATP-sensitive K+ (K(ATP)) channels are regulated by intracellular H+ in addition to ATP, ADP, and phospholipids. Here we show evidence for the interaction of H+ with ATP in regulating a cloned K(ATP) channel, i.e. Kir6.2 expressed with and without the SUR1 subunit. Channel sensitivity to ATP decreases at acidic pH, while the pH sensitivity also drops in the presence of ATP. These effects are more evident in the presence of the SUR1 subunit. In the Kir6.2 + SUR1, the pH sensitivity is reduced by about 0.4 pH units with 100 microM ATP and 0.6 pH units with 1 mM ATP, while a decrease in pH from 7.4 to 6.8 lowers the ATP sensitivity by about fourfold. The Kir6.2 + SUR1 currents are strongly activated at pH 5.9-6.5 even in the presence of 1 mM ATP. The modulations appear to take place at His175 and Lys185 that are involved in proton and ATP sensing, respectively. Mutation of His175 completely eliminates the pH effect on the ATP sensitivity. Similarly, the K185E mutant-channel loses the ATP-dependent modulation of the pH sensitivity. Thus, allosteric modulations of the cloned K(ATP) channel by ATP and H+ are demonstrated. Such a regulation allows protons to activate directly the K(ATP) channels and release channel inhibition by intracellular ATP; the pH effect is further enhanced with a decrease in ATP concentration as seen in several pathophysiological conditions.

β-Amyloid Fragment 25–35 Causes Mitochondrial Dysfunction in Primary Cortical Neurons

β-Amyloid deposition and compromised energy metabolism both occur in vulnerable brain regions in Alzheimer's disease. It is not known whether β-amyloid is the cause of impairment of energy metabolism, nor whether impaired energy metabolism is specific to neurons. Our results, using primary neuronal cultures, show that 24-h incubation with Aβ 25–35 caused a generalized decrease in the specific activity of mitochondrial enzymes per milligram of cellular protein, induced mitochondrial swelling, and decreased total mitochondrial number. Incubation with Aβ 25–35 decreased ATP concentration to 58% of control in neurons and 71% of control in astrocytes. Levels of reduced glutathione were also lowered by Aβ 25–35 in both neurons (from 5.1 to 2.9 nmol/mg protein) and astrocytes (from 25.2 to 14.9 nmol/mg protein). We conclude that 24-h treatment with extracellular Aβ 25–35 causes mitochondrial dysfunction in both astrocytes and neurons, the latter being more seriously affected. In astrocytes mitochondrial impairment was confined to complex I inhibition, whereas in neurons a generalized loss of mitochondria was seen.

Increased Nitric Oxide Production in Hepatocytes Is Involved in Liver Dysfunction Following Obstructive Jaundice

Background. Obstructive jaundice damages critical functions in the liver. However, the mechanisms involved in hepatic dysfunction are obscure. Nitric oxide is implicated in liver injury under various pathological conditions. We previously reported that proinflammatory cytokine interleukin-1β (IL-1β) stimulated the production of nitric oxide in hepatocytes, which was associated with mitochondrial dysfunction. Studies were performed to examine whether obstructive jaundice influences the production of nitric oxide in hepatocytes and alters hepatic energy metabolism.Material and methods. Hepatocytes were isolated and cultured from a rat model of obstructive jaundice or sham control. Nitric oxide production, ATP content, and ketone body ratio (acetoacetate/β-hydroxybutyrate; KBR) were compared between the two groups in the presence of IL-1β.Results. Hepatocytes obtained from obstructive jaundice rats markedly increased the levels of nitric oxide production stimulated by IL-1β compared with those from sham control. Western blot analysis revealed that the enhancement of nitric oxide production was a posttranslational event, since protein levels of inducible nitric oxide synthase (NOS) were unchanged between the two groups. IL-1β decreased cellular ATP content in obstructive jaundice but not in sham control. Further, the KBR, which is a marker of mitochondrial redox state, was lower in obstructive jaundice than in sham control. Addition of NG-monomethyl-l-arginine, an inhibitor of NOS, abolished the decreases in ATP content and KBR as well as the nitric oxide production.Conclusions. These results indicate that a priming of nitric oxide production following obstructive jaundice is associated with the alteration of hepatic energy metabolism in part through mitochondrial dysfunction. Regulation of nitric oxide production may be a useful therapy for preventing liver dysfunction in obstructive jaundice.

Association between mitochondrial dysfunction and severity and outcome of septic shock.

Sepsis-induced multiple organ failure is the major cause of mortality and morbidity in critically ill patients. However, the precise mechanisms by which this dysfunction is caused remain to be elucidated. We and others have shown raised tissue oxygen tensions in septic animals and human beings, suggesting reduced ability of the organs to use oxygen. Because ATP production by mitochondrial oxidative phosphorylation accounts for more than 90% of total oxygen consumption, we postulated that mitochondrial dysfunction results in organ failure, possibly due to nitric oxide, which is known to inhibit mitochondrial respiration in vitro and is produced in excess in sepsis. We did skeletal muscle biopsies on 28 critically ill septic patients within 24 h of admission to intensive care, and on nine control patients undergoing elective hip surgery. The biopsy samples were analysed for respiratory-chain activity (complexes I-IV), ATP concentration, reduced glutathione (an intracellular antioxidant) concentration, and nitrite/nitrate concentrations (a marker of nitric oxide production). Skeletal muscle ATP concentrations were significantly lower in the 12 patients with sepsis who subsequently died than in the 16 septic patients who survived (p=0.0003) and in controls (p=0.05). Complex I activity had a significant inverse correlation with norepinephrine requirements (a proxy for shock severity, p=0.0003) and nitrite/nitrate concentrations (p=0.0004), and a significant positive correlation with concentrations of reduced glutathione (p=0.006) and ATP (p=0.03). In septic patients, we found an association between nitric oxide overproduction, antioxidant depletion, mitochondrial dysfunction, and decreased ATP concentrations that relate to organ failure and eventual outcome. These data implicate bioenergetic failure as an important pathophysiological mechanism underlying multiorgan dysfunction.

Limitation to photosynthesis in water-stressed leaves: stomata vs. metabolism and the role of ATP.

Decreasing relative water content (RWC) of leaves progressively decreases stomatal conductance (gs), slowing CO2 assimilation (A) which eventually stops, after which CO2 is evolved. In some studies, photosynthetic potential (Apot), measured under saturating CO2, is unaffected by a small loss of RWC but becomes progressively more inhibited, and less stimulated by elevated CO2, below a threshold RWC (Type 1 response). In other studies, Apot and the stimulation of A by elevated CO2 decreases progressively as RWC falls (Type 2 response). Decreased Apot is caused by impaired metabolism. Consequently, as RWC declines, the relative limitation of A by g(s) decreases, and metabolic limitation increases. Causes of decreased Apot are considered. Limitation of ribulose bisphosphate (RuBP) synthesis is the likely cause of decreased Apot at low RWC, not inhibition or loss of photosynthetic carbon reduction cycle enzymes, including RuBP carboxylase/oxygenase (Rubisco). Limitation of RuBP synthesis is probably caused by inhibition of ATP synthesis, due to progressive inactivation or loss of Coupling Factor resulting from increasing ionic (Mg2+) concentration, not to reduced capacity for electron or proton transport, or inadequate trans-thylakoid proton gradient (ApH). Inhibition of Apot by accumulation of assimilates or inadequate inorganic phosphate is not considered significant. Decreased ATP content and imbalance with reductant status affect cell metabolism substantially: possible consequences are discussed with reference to accumulation of amino acids and alterations in protein complement under water stress.

Enhancement of Hyperthermia-induced Apoptosis by Local Anesthetics on Human Histiocytic Lymphoma U937 Cells

The combined effects of hyperthermia at 44 degrees C and local anesthetics on apoptosis in human histiocytic lymphoma U937 cells were investigated. When the cells were exposed to hyperthermia for l0 min marginal DNA fragmentation and nuclear fragmentation were observed. In the presence of amide-type local anesthetics further enhancement was found depending on concentration. The order of the concentration required for maximum induction was the reverse order of the lipophilicity (prilocaine > lidocaine > bupivacaine). Western blotting revealed that in hyperthermia there was initial release of Ca(2+) from the intracellular store site as indicated by increased expression of the type 1 inositol-1,4,5-trisphosphate receptor. However, the combination with lidocaine did not induce any further enhancement. Lidocaine enhanced the decrease in ATP content and the increase in intracellular Ca(2+) concentration in individual cells induced by hyperthermia. In addition, superoxide formation, decrease in the mitochondrial membrane potential, and activation of intracellular caspase-3 were found in the cells treated with hyperthermia and lidocaine. All of these were suppressed in part in the presence of the intracellular Ca(2+) ion chelator BAPTA-AM (bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl). The present results indicate that local anesthetics at optimal concentrations enhance hyperthermia-induced apoptosis via Ca(2+)- and mitochondria-dependent pathways. Initial release of Ca(2+) from intracellular store sites caused by hyperthermia and followed by the subsequent increase in the intracellular Ca(2+) concentration and the additional activation of the mitochondrial caspase-dependent pathway (partly regulated by intracellular Ca(2+) concentration) plays a crucial role in the enhancement of apoptosis induced by the combination of hyperthermia and lidocaine.

Mitochondrial respiratory chain as a new target for anti-ischemic molecules

Vascular diseases like thrombosis, myocardial infarction, cerebral ischemia or chronic venous insufficiency affect a high proportion of the population. They are all associated with more or less pronounced ischemic conditions. We have previously shown that some venotropic drugs display an anti-ischemic activity, i.e. they prevent the hypoxia-induced decrease in ATP content in cultured cells. The effect is due to the fact that these molecules maintain mitochondrial respiratory activity during hypoxia. Among them is bilobalide. Starting from the 3D structure of bilobalide, we designed new molecules presenting the same chemical features. They were synthesized and tested for their biological activity. As the parent compound, two of them, malonic acid dicyclopent-2-enyl ester (MRC2P119) and 2-oxo-3-oxa-bicyclo[3.1.0]hexane-1-carboxylic acid allyl ester (MRC2P57), were able to markedly increase the respiratory control ratio of isolated mitochondria. They are able to prevent the inhibition of complex I by amytal and of complex III by myxothiazol, but not the uncoupling of the respiration by carbonylcyanide m-chlorophenyl hydrazone ( m-CCP). Moreover, MRC2P119 and MRCP2P57 inhibit, in a dose-dependent way, the hypoxia-induced decrease in ATP content in endothelial cells as well as the subsequent activation of these cells as evidenced by an inhibition of the increase in neutrophil adherence to the endothelial cells induced by hypoxia. Finally, MRC2P119 prevent the hypoxia- and the hypoxia–reoxygenation-induced decrease in viability of SH-SY5Y neuroblastoma cells. In conclusion, we identified two new molecules, which display anti-ischemic properties when tested in vitro on endothelial and neuronal cell types. This anti-ischemic activity is probably due to a protection of complexes I and III of the mitochondrial respiratory chain.

Interactions of dietary fat and 2,5-anhydro-d-mannitol on energy metabolism in isolated rat hepatocytes

The fructose analog 2,5-anhydro-D-mannitol (2,5-AM) stimulates feeding in rats by reducing ATP content in the liver. These behavioral and metabolic effects occur with rats fed a high-carbohydrate/low-fat (HC/LF) diet, but they are prevented or attenuated when the animals eat high-fat/low-carbohydrate (HF/LC) food. To examine the metabolic bases for this effect of diet, we assessed the actions of 2,5-AM on ATP content, oxygen consumption, and substrate oxidation in isolated hepatocytes from rats fed one of the two diets. Compared with cells from rats fed the HC/LF diet ("HC/LF" cells), cells from rats fed the HF/LC diet ("HF/LC" cells) had similar ATP contents but lower oxygen consumption, decreased fructose, and increased palmitate oxidation. 2,5-AM did not decrease ATP content or oxygen consumption in HF/LC cells as much as it did in HC/LF hepatocytes, and it only affected fructose and palmitate oxidation in HC/LF cells. 31P-NMR spectroscopy indicated that differences in phosphate trapping accounted for differences in depletion of ATP by 2,5-AM. These results suggest that intake of the HF/LC diet prevents the eating response and attenuates the decline in liver ATP by shifting hepatocyte metabolism to favor fat over carbohydrate as an energy-yielding substrate.

Effect of tributyltin on adenylate content and enzyme activities of teleost sperm: a biochemical approach to study the mechanisms of toxicant reduced spermatozoa motility

The effects of tributyltin (TBT) on the energy metabolism and motility of fish spermatozoa were investigated in vitro in African catfish and common carp. A significant ( P<0.05) decrease of the duration and the intensity of motility was observed in catfish spermatozoa exposed to 0.27 μg/l TBT for 24 h. Exposure of catfish spermatozoa to 2.7–27 μg/l TBT caused an instant decrease in ATP content. In the presence of 27 μg/l TBT approximately 55% of the initial ATP concentration in catfish semen was lost after 60 min incubation while AMP concentrations increased and the total adenine nucleotide (TAN) pool remained unchanged. The reduction in sperm ATP levels could not be attributed to cell death since viability decreased only slightly over the period of exposure. In carp by contrast, none of the adenylates concentrations studied (ATP, ADP and AMP) were affected by TBT exposure at any experimental condition. However, carp sperm motility was significantly reduced by exposure to 2.7 μg/l TBT. Among the enzymes investigated only lactate dehydrogenase (LDH) in catfish sperm was significantly ( P<0.01) affected by 27 μg/l TBT treatment with a reduction in activity of approximately 75%. Compared with carp sperm before TBT exposure, that of catfish had lower adenylate contents and overall lower enzymatic activities; this explains its slower sperm velocity and shorter duration of movement as measured by computer assisted sperm analysis (CASA). The present in vitro study shows that catfish spermatozoa are more sensitive to TBT exposure (and probably to other toxicants) than those of carp.

Energy Metabolism of Erythrocytes in Lambs Chronically Exposed to Fluorine Compounds

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Different responses of astrocytes and neurons to nitric oxide: the role of glycolytically generated ATP in astrocyte protection.

It was recently proposed that in Jurkat cells, after inhibition of respiration by NO, glycolytically generated ATP plays a critical role in preventing the collapse of mitochondrial membrane potential (Deltapsi(m)) and thus apoptotic cell death. We have investigated this observation further in primary cultures of rat cortical neurons and astrocytes-cell types that differ greatly in their glycolytic capacity. Continuous and significant ( approximately 85%) inhibition of respiration by NO (1.4 microM at 175 microM O(2)) generated by [(z)-1-[2-aminoethyl]-N-[2-ammonioethyl]amino]diazen-1-ium-1,2 diolate (DETA-NO) initially (10 min) depleted ATP concentrations by approximately 25% in both cell types and increased the rate of glycolysis in astrocytes but not in neurons. Activation of glycolysis in astrocytes, as judged by lactate production, prevented further ATP depletion, whereas in neurons, which do not invoke this mechanism, there was a progressive decrease in ATP concentrations over the next 60 min. During this time, there was a persistent mitochondrial hyperpolarization and absence of apoptotic cell death in astrocytes, whereas in the neurons there was a progressive fall in Deltapsi(m) and increased apoptosis. After glucose deprivation or treatment with inhibitors of the F(1)F(0)-ATPase and adenine nucleotide translocase, astrocytes responded to NO with a fall in Deltapsi(m) and apoptotic cell death similar to the response in neurons. Finally, although treatment of astrocytes with NO partially prevented staurosporin-induced collapse in Deltapsi(m) and cell death, NO and staurosporin synergized in decreasing Deltapsi(m) and inducing apoptosis in neurons. These results demonstrate that although inhibition of cellular respiration by NO leads to neurotoxicity, it may also result in initial neuroprotection, depending on the glycolytic capacity of the particular cell.

Hypoxia-induced increase in intracellular calcium concentration in endothelial cells: role of the Na(+)-glucose cotransporter.

Hypoxia is a common denominator of many vascular disorders, especially those associated with ischemia. To study the effect of oxygen depletion on endothelium, we developed an in vitro model of hypoxia on human umbilical vein endothelial cells (HUVEC). Hypoxia strongly activates HUVEC, which then synthesize large amounts of prostaglandins and platelet-activating factor. The first step of this activation is a decrease in ATP content of the cells, followed by an increase in the cytosolic calcium concentration ([Ca(2+)](i)) which then activates the phospholipase A(2) (PLA(2)). The link between the decrease in ATP and the increase in [Ca(2+)](i) was not known and is investigated in this work. We first showed that the presence of extracellular Na(+) was necessary to observe the hypoxia-induced increase in [Ca(2+)](i) and the activation of PLA(2). This increase was not due to the release of Ca(2+) from intracellular stores, since thapsigargin did not inhibit this process. The Na(+)/Ca(2+) exchanger was involved since dichlorobenzamil inhibited the [Ca(2+)](i) and the PLA(2) activation. The glycolysis was activated, but the intracellular pH (pH(i)) in hypoxic cells did not differ from control cells. Finally, the hypoxia-induced increase in [Ca(2+)](i) and PLA(2) activation were inhibited by phlorizin, an inhibitor of the Na(+)-glucose cotransport. The proposed biochemical mechanism occurring under hypoxia is the following: glycolysis is first activated due to a requirement for ATP, leading to an influx of Na(+) through the activated Na(+)-glucose cotransport followed by the activation of the Na(+)/Ca(2+) exchanger, resulting in a net influx of Ca(2+).