Decrease In ATP Content Research Articles

DEGRADATION OF ATP AND GLYCOGEN IN COD (GADUS MORHUA) MUSCLE DURING FREEZING

Changes in ATP, IMP, lactate and glycogen contents in the muscle of cod were followed during freezing at temperatures of −20C and −45C. ATP degradation was accompanied by a corresponding increase in IMP content. Simultaneous measurement of temperature showed that at both freezing rates, the greatest decrease in ATP content was observed when the temperature reached -0.8C. Glycolysis occurred during freezing of cod as indicated by an increase in lactate content. The changes found in all measured metabolites were more pronounced when freezing was performed at a slow rate compared to a fast rate due to the thermal arrest time at about 0.8C.

ATP-dependent Affinity Change of Na+-binding Sites of V-ATPase

V-type Na(+)-ATPase of Enterococcus hirae binds about six (6 +/- 1) Na(+) ions/enzyme molecule with a high affinity (Murata, T., Igarashi, K., Kakinuma, Y., and Yamato, I. (2000) J. Biol. Chem. 275, 13415-13419). After the addition of 5 mm ATP, the binding capacity dropped to about 2 (1.8 +/- 0.3) Na(+) ions/enzyme molecule, returning to the initial value concomitant with the decrease of ATP hydrolysis rate. These findings suggest that the affinity of four of six Na(+)-binding sites of the enzyme changes (lowers) in enzyme reaction. The ATP analogs (adenosine 5'-O-(3-thiotriphosphate) or 5'-adenylylimido-diphosphate), ADP, or aluminum fluoride that is postulated to trap ATPases at their transition state did not inhibit the Na(+) binding capacity significantly. Therefore, the affinity decrease of Na(+)-binding sites was unlikely to be due to ATP binding alone or at the transition state of ATP hydrolysis. In the presence of 5 mm ATP, the ATPase showed strong negative cooperativity (n(H) = 0.16 +/- 0.03) for Na(+) stimulation of ATPase activity. The Hill coefficient (n(H)) increased to 1 in parallel to the decrease of ATP concentration in the reaction mixture. Thus, the ATP-dependent affinity change cooperatively occurs in continuous enzyme reaction.

Excessive Hexosamines Block the Neuroprotective Effect of Insulin and Induce Apoptosis in Retinal Neurons

In addition to microvascular abnormalities, neuronal apoptosis occurs early in diabetic retinopathy, but the mechanism is unknown. Insulin may act as a neurotrophic factor in the retina via the phosphoinositide 3-kinase/Akt pathway. Excessive glucose flux through the hexosamine biosynthetic pathway (HBP) is implicated in the development of insulin resistance in peripheral tissues and diabetic complications such as nephropathy. We tested whether increased glucose flux through the HBP perturbs insulin action and induces apoptosis in retinal neuronal cells. Exposure of R28 cells, a model of retinal neurons, to 20 mm glucose for 24 h attenuated the ability of 10 nm insulin to rescue them from serum deprivation-induced apoptosis and to phosphorylate Akt compared with 5 mm glucose. Glucosamine not only impaired the neuroprotective effect of insulin but also induced apoptosis in R28 cells in a dose-dependent fashion. UDP-N-acetylhexosamines (UDP-HexNAc), end products of the HBP, were increased approximately 2- and 15-fold after a 24-h incubation in 20 mm glucose and 1.5 mm glucosamine, respectively. Azaserine, a glutamine:fructose-6-phosphate amidotransferase inhibitor, reversed the effect of 20 mm glucose, but not that of 1.5 mm glucosamine, on attenuation of the ability of insulin to promote cell survival and phosphorylate Akt as well as accumulation of UDP-HexNAc. Glucosamine also impaired insulin receptor processing in a dose-dependent manner but did not decrease ATP content. By contrast, in L6 muscle cells, glucosamine impaired insulin receptor processing but did not induce apoptosis. These results suggest that the excessive glucose flux through the HBP may direct retinal neurons to undergo apoptosis in a bimodal fashion; i.e. via perturbation of the neuroprotective effect of insulin mediated by Akt and via induction of apoptosis possibly by altered glycosylation of proteins. The HBP may be involved in retinal neurodegeneration in diabetes.

Influence of ribose on adenine salvage after intense muscle contractions.

The influence of ribose supplementation on skeletal muscle adenine salvage rates during recovery from intense contractions and subsequent muscle performance was evaluated using an adult rat perfused hindquarter preparation. Three minutes of tetanic contractions (60 tetani/min) decreased ATP content in the calf muscles by approximately 50% and produced an equimolar increase in IMP. Effective recovery of muscle ATP 1 h after contractions was due to reamination of IMP via the purine nucleotide cycle and was complete in the red gastrocnemius but incomplete in the white gastrocnemius muscle section. Adenine salvage rates in recovering muscle averaged 45 +/- 4, 49 +/- 5, and 30 +/- 3 nmol. h(-1). g(-1) for plantaris, red gastrocnemius, and white gastrocnemius muscle, respectively, which were not different from values in corresponding nonstimulated muscle sections. Adenine salvage rates increased five- to sevenfold by perfusion with approximately 4 mM ribose (212 +/- 17, 192 +/- 9, and 215 +/- 14 nmol. h(-1). g(-1) in resting muscle sections, respectively). These high rates were sustained in recovering muscle, except for a small (approximately 20%) but significant (P < 0.001) decrease in the white gastrocnemius muscle. Ribose supplementation did not affect subsequent muscle force production after 60 min of recovery. These data indicate that adenine salvage rates were essentially unaltered during recovery from intense contractions.

Mitochondrial K(ATP) channel activation reduces anoxic injury by restoring mitochondrial membrane potential.

Mitochondrial membrane potential (DeltaPsi(m)) is severely compromised in the myocardium after ischemia-reperfusion and triggers apoptotic events leading to cell demise. This study tests the hypothesis that mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel activation prevents the collapse of DeltaPsi(m) in myocytes during anoxia-reoxygenation (A-R) and is responsible for cell protection via inhibition of apoptosis. After 3-h anoxia and 2-h reoxygenation, the cultured myocytes underwent extensive damage, as evidenced by decreased cell viability, compromised membrane permeability, increased apoptosis, and decreased ATP concentration. Mitochondria in A-R myocytes were swollen and fuzzy as shown after staining with Mito Tracker Orange CMTMRos and in an electron microscope and exhibited a collapsed DeltaPsi(m), as monitored by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Cytochrome c was released from mitochondria into the cytosol as demonstrated by cytochrome c immunostaining. Activation of mitoK(ATP) channel with diazoxide (100 micromol/l) resulted in a significant protection against mitochondrial damage, ATP depletion, cytochrome c loss, and stabilized DeltaPsi(m). This protection was blocked by 5-hydroxydecanoate (500 micromol/l), a mitoK(ATP) channel-selective inhibitor, but not by HMR-1098 (30 micromol/l), a putative sarcolemmal K(ATP) channel-selective inhibitor. Dissipation of DeltaPsi(m) also leads to opening of mitochondrial permeability transition pore, which was prevented by cyclosporin A. The data support the hypothesis that A-R disrupts DeltaPsi(m) and induces apoptosis, which are prevented by the activation of the mitoK(ATP) channel. This further emphasizes the therapeutic significance of mitoK(ATP) channel agonists in the prevention of ischemia-reperfusion cell injury.

The von Hippel-Lindau gene product inhibits renal cell apoptosis via Bcl-2-dependent pathways.

Previous studies have reported a protective role for the von Hippel-Lindau (VHL) gene products against pro-apoptotic cellular stresses, but the mechanisms remain unclear. In this study, we examined the role of VHL in renal cells subjected to chemical hypoxia, using four VHL-negative and two VHL-positive cell lines. VHL-negative renal carcinoma cells underwent apoptosis following chemical hypoxia (short-term glucose deprivation and antimycin treatment), as evidenced by morphologic changes and internucleosomal DNA cleavage. Reintroduction of VHL expression prevented this apoptosis. VHL-negative cells displayed a significant (greater than 5-fold) activation of caspase 9 and release of cytochrome c into the cytosol following chemical hypoxia. In contrast, VHL-positive cells showed minimal caspase 9 activation, and absence of cytochrome c release under the same conditions. Caspase 8 was only minimally activated in both VHL-negative and -positive cells. In addition, VHL-positive cells displayed a striking up-regulation of Bcl-2 expression (5-fold) following chemical hypoxia. Antisense oligonucleotides to Bcl-2 significantly down-regulated Bcl-2 protein expression in VHL-positive cells and rendered them sensitive to apoptosis. Overexpression of Bcl-2 in VHL-negative cells conferred resistance to apoptosis. Our results suggest that VHL protects renal cells from apoptosis via Bcl-2-dependent pathways.

Attenuation of liver normothermic ischemia-reperfusion injury by preservation of mitochondrial functions with S-15176, a potent trimetazidine derivative

We investigated the antiischemic properties of a new compound, S-15176, in an experimental model of rat liver subjected to 120-min normothermic ischemia followed by 30-min reperfusion. Rats were divided into groups, pretreated with different doses of S-15176 (1.25, 2.5, 5 and 10 mg/kg/day by intramuscular injection) or solvent alone, and subjected to the ischemia-reperfusion process. Another group served as the sham-operated controls. Ischemia-reperfusion induced huge alterations of hepatocyte functions, namely, a decrease in ATP content and bile flow, and membrane leakage of alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT). These effects were associated with alterations in mitochondrial functions characterized by (1) a decrease in ATP synthesis, (2) a decrease in NAD(P)H levels and mitochondrial membrane potential, and (3) an increase in mitochondrial swelling reflecting the generation of permeability transition. Pretreatment of rats with S-15176 alleviated these deleterious ischemia-reperfusion effects at both the cellular and mitochondrial levels in a dose-dependent manner. The protection of mitochondrial functions was almost complete at a dosage of 10 mg/kg/day. In addition, in vitro, S-15176 totally abolished the swelling of isolated mitochondria induced by a calcium overload with an ic 50 value of 10 μM. These data demonstrate that S-15176 protects mitochondria against the deleterious effects of ischemia-reperfusion and suggest that this protective effect could be related to the inhibition of the mitochondrial permeability transition.

Effects of Lewisite on cell membrane integrity and energy metabolism in human keratinocytes and SCL II cells

Lewisite is a highly toxic arsenic compound which can cause skin damage. In the present study effects of Lewisite on cell membrane integrity and energy metabolism as well as antidotal effects of dl-2,3-dimercaptopropanesulfonate (DMPS), and meso-2,3-dimercaptosuccinic acid (m-DMSA) were investigated in a keratinocyte derived cell line (SCL II) and primary human keratinocytes (HK). Cells were incubated in Lewisite (60 μM) containing medium for 5 min. During the following 6 h lactate dehydrogenase (LDH) activity in the supernatant, intracellular ATP content, tetrazolium reduction, glucose consumption and lactate formation were measured. Glucose consumption and lactate production were decreased in both cell lines after Lewisite exposure. In SCL II cells an increase of LDH activity in the supernatant, a decrease of ATP content, and an impaired ability to reduce tetrazolium was found 3 h after Lewisite exposure. In HK cultures tetrazolium reduction was significantly decreased already after 2 h, whereas LDH increase in the supernatant and ATP content decrease occurred only at 6 h after Lewisite exposure. When DMPS or m-DMSA was added directly after Lewisite exposure to SCL II cells, glucose consumption and lactate formation were restored and LDH leakage was prevented. SCL II cells might be more prone to membrane damage whereas in keratinocytes mitochondrial impairment seems to be the predominant effect of Lewisite.

Importance of glycolysis for the energetics of anoxia-tolerant and anoxia-intolerant teleost hepatocytes.

The importance of glycolysis, as an ATP-producing and substrate-providing pathway, was studied in anoxia-tolerant (goldfish) and anoxia-intolerant (trout) hepatocytes. Inhibition of glycolysis with iodoacetic acid (IAA) left aerobic ATP production largely unaffected in hepatocytes from both species but caused a significant decrease of ATP contents in the goldfish cells. Ouabain-sensitive oxygen consumption (osVo2), an estimate of mitochondrial ATP production coupled to ATP consumption by the Na(+) pump, was significantly reduced in IAA-treated goldfish hepatocytes, whereas it was unaltered in trout hepatocytes. Partial reduction of mitochondrial respiration, achieved by titration with cyanide (CN), strongly stimulated glycolytic flux but did not affect ATP contents of hepatocytes from both species. Under these conditions, osVo2 became undetectable. Rb(+)-uptake rates, providing a direct estimate of Na(+)-pump activity, were in good agreement with estimates derived from osVo2 in IAA-treated cells, showing a decrease in goldfish and no change in trout. However, they indicated persistent Na(+)-pump activity despite the lack of osVo2 in CN-treated cells. Overall, these data indicate that in goldfish hepatocytes Na(+)-pump activity is more dependent on glycolytic ATP production as compared to trout hepatocytes. Protein synthesis of goldfish hepatocytes was inhibited in IAA- and CN-treated cells, possibly reflecting the hierarchical organization of energy metabolism. In trout hepatocytes, protein synthesis could be sustained at control levels, given that energetic substrate provision was not limited.

Renal interstitial atp responses to changes in arterial pressure during alterations in tubuloglomerular feedback activity.

We recently demonstrated a direct relationship between autoregulation-related changes in renal vascular resistance (RVR) and renal interstitial ATP concentrations. To assess the possible role for extracellular ATP in the regulation of tubuloglomerular feedback (TGF)-mediated autoregulatory adjustments in RVR, renal interstitial ATP concentrations were measured with microdialysis probes in anesthetized dogs at different renal arterial pressures (RAPs) within the autoregulatory range during augmented and diminished activity of the TGF mechanism. Stepwise reductions in RAP from ambient pressure (129+/-3 mm Hg) to 102+/-2 mm Hg (step 1) and 75+/-1 mm Hg (step 2) resulted in significant decreases in ATP concentrations from 9.0+/-0.8 to 6.3+/-0.6 nmol/L in step 1 and to 4.2+/-0.5 nmol/L in step 2. Changes in RVR were highly correlated with changes in ATP concentrations (r=0.86, P<0.001, n=12). Acetazolamide (100 microgram. kg(-1). min(-1), n=6), which increases solute delivery to the macula densa, thus augmenting TGF activity, significantly decreased renal blood flow (RBF) by -16+/-2% and glomerular filtration rate (GFR) by -22+/-4% and increased ATP concentrations from 8.4+/-0.7 to 15.5+/-1.4 nmol/L. Although basal RBF and GFR levels were reduced by the acetazolamide infusion, autoregulation efficiency was maintained, and interstitial ATP concentrations were significantly decreased in response to reductions in RAP by -36+/-4% in step 1 and by -54+/-2% in step 2. The relationship between changes in RVR and interstitial ATP concentrations was preserved during acetazolamide treatment (r=0.80, P<0.01). Inhibition of the TGF mechanism by furosemide significantly increased RBF by 33+/-6% and GFR by 13+/-2% and decreased ATP concentrations from 8.9+/-1.4 to 5.0+/-0.8 nmol/L (n=6). Furosemide caused marked impairment of RBF and GFR autoregulatory efficiency (by -14+/-3% and -11+/-3% in step 1 and by -26+/-2% and -18+/-4% in step 2, respectively). In the furosemide-treated kidneys, interstitial ATP levels remained low and were not altered during reductions in RAP (4.7+/-0.7 nmol/L in step 1 and 4.7+/-0.8 nmol/L in step 2), and changes in RVR did not exhibit a correlation with changes in ATP concentrations (r=0.22, P=0.30). These data support the hypothesis that extracellular ATP contributes to autoregulatory adjustments in RVR that are mediated by changes in activity of the TGF mechanism.

UV-A irradiation induces a decrease in the mitochondrial respiratory activity of human NCTC 2544 keratinocytes

UV-A irradiation caused a dose-dependent decrease in cellular oxygen consumption (56%) and ATP content (65%) in human NCTC 2544 keratinocytes, one hour after treatment. This effect was partially reversed by maintaining the irradiated cells in normal culture conditions for 24h. Using malate/glutamate or succinate as substrates for mitochondrial electron transport, the oxygen uptake of digitoninpermeabilised cells was greatly inhibited following UV-A exposure. These results strongly suggest that UV-A irradiation affects the state 3 respiration of the mitochondria. However, under identical conditions, UV-A exposure did not reduce the mitochondrial transmembrane potential. The antioxidant, vitamin E inhibited UV-A-induced lipid peroxidation, but did not significantly prevent the UV-A-mediated changes in cellular respiration nor the decrease in ATP content, suggesting that these effects were not the result of UV-A dependent lipid peroxidation. UV-A irradiation also led to an increase in MnSOD gene expression 24 hours after treatment, indicating that the mitochondrial protection system was enhanced in response to UV-A treatment. These findings provide evidence that impairment of mitochondrial respiratory activity is one of the early results of UV-A irradiation for light doses much lower than the minimal erythemal dose.

Phosphatase inhibitors prevent HSP27 dephosphorylation, destruction of stress fibrils, and morphological changes in endothelial cells during ATP depletion.

Pretreatment with phosphatase inhibitors did not affect the decrease in ATP content in endothelial cells, but inhibited HSP27 dephosphorylation and redistribution, damages to actin cytoskeleton, and morphological changes in cells. Our results suggest that inhibition of stress-induced HSP27 dephosphorylation protects cells from ischemia-induced damages.

Glucose and the ATP paradox in yeast

A sustained decrease in the intracellular ATP concentration has been observed when extra glucose was added to yeast cells growing aerobically under glucose limitation. Because glucose degradation is the main source of ATP-derived free energy, this is a counter-intuitive phenomenon, which cannot be attributed to transient ATP consumption in the initial steps of glycolysis. We present a core model for aerobic growth in which glucose supplies carbon, as well as free energy, for biosynthesis. With Metabolic Control Analysis and numerical simulations, we demonstrate that the decrease in the ATP concentration can be reproduced if the biosynthetic route is more strongly activated by carbon substrates than is the catabolic (ATP-producing) route.

Glucose and the ATP paradox in yeast

A sustained decrease in the intracellular ATP concentration has been observed when extra glucose was added to yeast cells growing aerobically under glucose limitation. Because glucose degradation is the main source of ATP-derived free energy, this is a counter-intuitive phenomenon, which cannot be attributed to transient ATP consumption in the initial steps of glycolysis. We present a core model for aerobic growth in which glucose supplies carbon, as well as free energy, for biosynthesis. With Metabolic Control Analysis and numerical simulations, we demonstrate that the decrease in the ATP concentration can be reproduced if the biosynthetic route is more strongly activated by carbon substrates than is the catabolic (ATP-producing) route.

Renal Interstitial ATP Responses to Changes in Arterial Pressure During Alterations in Tubuloglomerular Feedback Activity

P90 We have recently demonstrated a direct relationship between autoregulatory related changes in renal vascular resistance (RVR) and changes in renal interstitial ATP levels. To examine further the possible role for extracellular ATP in the regulation of tubuloglomerular feedback (TGF)-mediated autoregulatory adjustments in RVR, experiments were performed in 8 anesthetized dogs to assess interstitial ATP concentrations in response to changes in renal arterial pressure (RAP) during augmented and diminished activity of the TGF mechanism. Using a microdialysis method, interstitial ATP levels were assessed at different levels of RAP before and during intra-arterial administration of acetazolamide (ACZ; 100 μg/kg/min, n=4) which increases solute delivery to the macula densa thus augmenting TGF activity, and after furosemide treatment (10 μg/kg/min, n=4) which inactivates the TGF mechanism. During the control period, stepwise reductions in RAP within the autoregulatory range from ambient pressure (126±4 mmHg) to 100±2 mmHg (step-1: S1) and 76±2 mmHg (step-2: S2) resulted in significant decreases in ATP concentrations from 8.9±0.7 to 6.3±0.6 nM (S1) and 4.0±0.5 nM (S2). ACZ decreased renal blood flow (RBF; -22±6%) and glomerular filtration rate (GFR; -28±4%), and increased RVR (22±2%) and ATP concentrations (7.9±0.9 to 14.3±1.8 nM). During ACZ infusion, the autoregulatory efficiency of RBF and GFR were maintained, and ATP levels were significantly decreased in responses to reductions in RAP (8.8±0.8 nM in S1 and 6.5±0.7 nM in S2). Inhibition of the TGF mechanism by furosemide increased RBF (32±9%) and GFR (17±2%), and decreased RVR (25±6%); ATP levels concentrations decreased from 8.7±1.1 to 5.4±0.9 nM. Furosemide caused marked impairment of RBF and GFR autoregulatory efficiency (by -17±3 and -16±6% in S1 and by -31±5 and -23±7% in S2, respectively). In addition, pressure-induced changes in ATP levels were completely prevented by furosemide (5.3±0.4 nM in S1 and 5.5±0.5 nM in S2). These data support the hypothesis that extracellular ATP contributes to the RVR adjustments that are elicited by changes in activity of the TGF mechanism.

Effect of venotropic drugs on the respiratory activity of isolated mitochondria and in endothelial cells.

Several drugs used in the treatment of chronic peripheral ischaemic and venous diseases, i.e. aescine, Cyclo 3, Ginkor Fort, hydroxyethylrutosides, naftidrofuryl, naphthoquinone and procyanidolic oligomers, were tested on the mitochondrial respiratory activity. The results show that all these drugs protected human endothelial cells against the hypoxia-induced decrease in ATP content. In addition, they all induced a concentration-dependent increase in respiratory control ratio (RCR) of liver mitochondria pre-incubated with the drugs for 60 min. The drugs were divided into two groups according to their effects. The first group (A), comprising aescine, Ginkor Fort, naftidrofuryl and naphthoquinone, increased RCR by decreasing state 4 respiration rate. The second group of drugs (B), comprising hydroxyethylrutosides, procyanidolic oligomers and Cyclo 3, increased RCR by increasing state 3 respiration rate. The drugs of group A were able to prevent the inhibition of complexes I and III respectively by amytal and antimycin A while the first two drugs of group B increased adenine nucleotide translocase activity. Cyclo 3 inhibited the carbonylcyanide m-chlorophenyl hydrazone (mCCP)-induced uncoupling of mitochondrial respiration. None of these seven drugs could protect complexes IV and V, respectively, from inhibition by cyanide and oligomycin. When tested on endothelial cells the drugs of group A, in contrast to group B, prevented the decrease in ATP content induced by amytal or antimycin A. The present results suggest that the protective effects on mitochondrial respiration activity by these venotropic drugs may explain their protective effect on the cellular ATP content in ischaemic conditions and some of their beneficial therapeutic effect in chronic vascular diseases.

Mechanism of Ca(2+) overload in endothelial cells exposed to simulated ischemia.

Several studies have shown that myocardial ischemia leads to functional failure of endothelial cells (EC) whereby disturbance of Ca(2+) homeostasis may play an important role. The mechanisms leading to Ca(2+) disbalance in ischemic EC are not fully understood. The aim of this study was to test effects of different components of simulated ischemia (glucose deprivation, anoxia, low extracellular pH (pH(o)) and lactate) on Ca(2+) homeostasis in EC. Cytosolic Ca(2+) (Ca(i)), cytosolic pH (pH(i)) and ATP content were measured in cultured rat coronary EC. In normoxic cells 60 min glucose deprivation at pH(o) 7.4 had no effect on pH(i). It only slightly increased Ca(i) and decreased ATP content. Reduction of pH(o) to 6.5 under these conditions led to marked cytosolic acidosis and Ca(i) overload, but had no effect on ATP content. Anoxia at pH(o) 6.5 had no additional effect on Ca(i) overload, but significantly reduced cellular ATP. Addition of 20 mmol/l lactate to anoxia at pH(o) 6.5 accelerated Ca(i) overload due to faster cytosolic acidification. Acidosis-induced Ca(i) overload was prevented by inhibition of Ca(2+) release channels of endoplasmic reticulum (ER) with 3 micromol/l ryanodine or by pre-emptying the ER with thapsigargin. Re-normalisation of pH(o) for 30 min led to recovery of pH(i), but not of Ca(i). The ischemic factors leading to cytosolic acidosis (low pH(o) and lactate) cause Ca(i) overload in endothelial cells, while anoxia and glucose deprivation play only a minor role. The ER is the main source for this Ca(i) rise. Ca(i) overload is not readily reversible.

Constitutive Regulation of Cardiac Fatty Acid Metabolism through Peroxisome Proliferator-activated Receptor α Associated with Age-dependent Cardiac Toxicity

The peroxisome proliferator-activated receptor alpha (PPARalpha) is a member of the nuclear receptor superfamily and mediates the biological effects of peroxisome proliferators. To determine the physiological role of PPARalpha in cardiac fatty acid metabolism, we examined the regulation of expression of cardiac fatty acid-metabolizing proteins using PPARalpha-null mice. The capacity for constitutive myocardial beta-oxidation of the medium and long chain fatty acids, octanoic acid and palmitic acid, was markedly reduced in the PPARalpha-null mice as compared with the wild-type mice, indicating that mitochondrial fatty acid catabolism is impaired in the absence of PPARalpha. In contrast, constitutive beta-oxidation of the very long chain fatty acid, lignoceric acid, did not differ between the mice, suggesting that the constitutive expression of enzymes involved in peroxisomal beta-oxidation is independent of PPARalpha(.) Indeed, PPARalpha-null mice had normal levels of the peroxisomal beta-oxidation enzymes except the D-type bifunctional protein. At least seven mitochondrial fatty acid-metabolizing enzymes were expressed at much lower levels in the PPARalpha-null mice, whereas other fatty acid-metabolizing enzymes were present at similar or slightly lower levels in the PPARalpha-null, as compared with wild-type mice. Additionally, lower constitutive mRNA expression levels of fatty acid transporters were found in the PPARalpha-null mice, suggesting a role for PPARalpha in fatty acid transport and catabolism. Indeed, in fatty acid metabolism experiments in vivo, myocardial uptake of iodophenyl 9-methylpentadecanoic acid and its conversion to 3-methylnonanoic acid were reduced in the PPARalpha-null mice. Interestingly, a decreased ATP concentration after exposure to stress, abnormal cristae of the mitochondria, abnormal caveolae, and fibrosis were observed only in the myocardium of the PPARalpha-null mice. These cardiac abnormalities appeared to proceed in an age-dependent manner. Taken together, the results presented here indicate that PPARalpha controls constitutive fatty acid oxidation, thus establishing a role for the receptor in cardiac fatty acid homeostasis. Furthermore, altered expression of fatty acid-metabolizing proteins seems to lead to myocardial damage and fibrosis, as inflammation and abnormal cell growth control can cause these conditions.

Effect of alpha-phenyl-N-tert-butyl nitrone (PBN) on fetal cerebral energy metabolism during intrauterine ischemia and reperfusion in rats.

The objective of the present study was to explore whether a free radical spin trap agent, alpha-phenyl-N-tert-butyl nitrone (PBN), influences bioenergetic failure induced in the 20-day-old fetal brain by 30 min of intrauterine ischemia in Wistar rats. Fetal brains were frozen in situ at the end of ischemia and after 1, 2, and 4 h of recirculation for analysis of ATP, ADP, AMP, and lactate. PBN or vehicle was given 1 h after recirculation. Tissue oxygen tension was evaluated in placental and fetal cerebral tissues throughout the whole periods of 30 min of ischemia and 4 h of recirculation. Ischemia was associated with a decrease in ATP concentration and an increase in lactate concentration (p < 0.001). Recirculation (1 and 2 h) led to a recovery of ATP concentration, but continued reflow (4 h) was associated with a secondary deterioration of high-energy phosphates (p < 0.01). Lactate concentration increased during this recovery period. This deterioration was prevented by PBN (p < 0.05). After 30 min of ischemia, tissue oxygen tension in placenta and fetal brain decreased to about 30% and 50% of control, respectively. However, recirculation brought about a recovery of oxygen delivery. The results indicate that although during the early time period after ischemia fetal cerebral energy metabolism is maintained by an acceleration of the anaerobic glycolytic rate, secondary deterioration of cellular bioenergetic state develops in the immature fetal brain. This deterioration may be due to mitochondrial dysfunction, which may be induced by oxygen-derived free radicals, and not by compromised microcirculation.

Inhibition of neutrophil superoxide production by uremic concentrations of guanidino compounds.

In uremia, diminished reactive oxygen intermediate production is an important consequence of impaired neutrophil function. The effects of guanidino compounds, which are known uremic toxins, on neutrophil reactive oxygen intermediate production in vitro were studied. Neutrophils from healthy volunteers were exposed for 3 h to individual guanidino compounds or mixed guanidino compounds (GCmix), at concentrations observed in uremic plasma. After removal of the guanidino compounds, the neutrophils were activated by adhesion, N-formylmethionylleucylphenylalanine, phorbol myristate acetate, or opsonized zymosan, and superoxide production was measured by monitoring lucigenin chemiluminescence. The direct effects of guanidino compounds on superoxide production in activated neutrophils were also measured. The energy status (ATP and creatine phosphate), antioxidant status (total glutathione), and glycolytic flux (lactate production) were measured. GCmix pretreatment decreased superoxide production in activated neutrophils (activated by N-formylmethionylleucylphenylalanine or zymosan) by 50% (P < 0.01), decreased ATP concentrations by 60% (P < 0.05), and inhibited glycolytic flux (lactate production) by 45% (P < 0.01) but did not alter glutathione concentrations. Simultaneous GCmix exposure and activation did not inhibit NADPH oxidase activity in cell lysates but inhibited superoxide formation in zymosan-activated intact neutrophils; this inhibition was reversed after removal of the guanidino compounds. Guanidinosuccinic acid, guanidinopropionic acid, and guanidinobutyric acid, when tested individually, were each as potent as GCmix. The inhibition of neutrophil superoxide generation by guanidino compounds results from decreased energy status. Micromolar concentrations of guanidino compounds significantly inhibit neutrophil metabolism, with serious implications for the functions of neutrophils in host defenses.