Mechanism of oxmetidine (SK&F 92994) cytotoxicity in isolated rat hepatocytes

Oxmetidine is an H2-receptor antagonist that has efficacy in the treatment of peptic ulcers. Isolated rat hepatocytes exposed to oxmetidine (0.5 mM) rapidly lost viability as estimated by increased leakage of lactate dehydrogenase, increased formation of plasma membrane surface blebs and decreased intracellular potassium concentration [K+]. Oxmetidine caused a reduction in hepatocyte reduced glutathione concentration that paralleled cell death; malondialdehyde formation was not observed. Hepatocyte respiration (O2 consumption) and intracellular ATP concentration were decreased markedly by oxmetidine in a concentration-related fashion. Oxmetidine (50 microM) blocked pyruvate/malate-supported state 3 (ADP-stimulated) respiration, caused a decrease in the ADP:0 ratio and a loss of respiratory control in isolated rat liver mitochondria. In contrast, oxmetidine did not block succinate-supported ADP-stimulated O2 consumption in isolated rat liver mitochondria. These data demonstrate that: 1) oxmetidine was cytotoxic to isolated rat hepatocytes in suspension and 2) the mechanism of oxmetidine-induced hepatocyte injury may be related to sustained inhibition of mitochondrial oxidative phosphorylation leading to decreased cellular ATP content and cell death. Although the exact site of action of oxmetidine within the mitochondrion has not been completely elucidated, it appears to reside in the inner mitochondrial membrane electron transport chain before ubiquinone oxidoreductase.     

Disposition of radioiodinated benzylguanidines in perfused rabbit lung: pharmacokinetics and effect of an organo-gold complexed antineoplastic agent

SK&F 104524 (bis-[1,2-bis(diphenylphosphino)-ethane]-gold(I)-lactate) ([Au(dppe)2]+) is a lipophilic phosphine-coordinated gold complex that has significant antineoplastic activity. However, this agent has significant hepato- and cardiotoxicity and preclinical preliminary experiments indicate potential pneumotoxic effects. Accordingly, we sought to evaluate the acute effects of [Au(dppe)2]+ on pulmonary removal of two radioiodinated benzylguanidines [metaiodobenzylguanidine (MIBG) and 3-iodo-4-amino-benzylguanidine (AIBG)]. Pulmonary removal of MIBG or AIBG was measured by application of indicator dilution techniques in rabbit lungs, perfused with Kreb's bicarbonate (3% albumin; 50 ml/min). A bolus containing either [125I]MIBG or--AIBG and [99mTc]sulfur colloid was injected into the pulmonary artery and effluent was diverted to an in-line nuclear detection system. In initial experiments we characterized some pharmacokinetic aspects of the disposition of these amines. Removal of MIBG [R(MIBG)] and R(AIBG) were 59 +/- 3% (n = 14) and 24 +/- 2% (n = 7), respectively. Addition of unlabeled norepinephrine, MIBG or AIBG (3-100 nmol) to the injection caused similar dose-dependent reductions in removal of either amine. Lowering the temperature (13 degrees C) or reducing the sodium concentration caused significant reductions in removal. After 15 min of perfusion with [Au(dppe)2]+ (50 micrograms/ml), perfusion pressure doubled and R(MIBG) and R(AIBG) were each decreased significantly. At 30 min, perfusion pressure returned to control values whereas there was still a 36 and 61% reduction in R(MIBG) and R(AIBG), respectively. These data confirm that removal of benzylguanidines involves a saturable, sodium- and temperature-dependent process similar to that responsible for the removal of norepinephrine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible mechanism of hepatocyte injury induced by diphenylamine and its structurally related nonsteroidal anti-inflammatory drugs

Diphenylamine is a common structure of nonsteroidal anti-inflammatory drugs (NSAIDs) to uncouple mitochondrial oxidative phosphorylation and to cause a decrease in hepatocellular ATP content and hepatocyte injury. The mechanism for acute cell injury induced by diphenylamine and its structurally related NSAIDs was investigated with rat liver mitochondria and freshly isolated hepatocytes, focusing on the relation to the uncoupling of oxidative phosphorylation. Incubation of mitochondria with diphenylamine as well as mefenamic acid and diclofenac caused pseudoenergetic mitochondrial swelling, indicating that these compounds induce mitochondrial membrane permeability transition. Diphenylamine also caused changes in safranine-binding spectra to mitochondria that was energized by succinate oxidation. This spectral shift indicates the loss of mitochondrial membrane potentials, which is known as one of the characteristics for uncouplers of oxidative phosphorylation, and also was caused by mefenamic acid and diclofenac. Incubation of hepatocytes with mefenamic acid, diclofenac, and diphenylamine diminished cellular ATP content, followed by leakage of lactose dehydrogenase from hepatocytes. Fructose, a low K(m) substrate for glycolysis, partially protected against the ATP depletion and hepatocyte injury induced by these compounds. Further addition of oligomycin, which blocks ATPase, pronounced the protection against cell injury. These results suggested that decreases in cellular ATP content, mainly caused by uncoupling of mitochondrial oxidative phosphorylation, were responsible for acute hepatocyte injury induced by diphenylamine and structurally related NSAIDs.     

Inhibition of mitochondrial electron transfer in rats by ethanethiol and methanethiol.

1. We have investigated the effects of ethanethiol, methanethiol and dimethyl sulphide on some metabolic processes of isolated rat hepatocytes, isolated mitochondria from liver and brain and ox-heart submitochondrial particles. 2. Ethanethiol, but not dimethyl sulphide, inhibited both gluconeogenesis and ureogenesis from various substrates in rat hepatocytes, depressed cellular ATP content and caused an increased reduction of the mitochondria. 3. Ethanethiol inhibited respiration in isolated rat-liver mitochondria with several substrates, both in the presence of ADP and phosphate or in the presence of an uncoupling agent. Ethanethiol also inhibited respiration in isolated rat-brain mitochondria. Dimethyl sulphide was much less effective in inhibiting mitochondrial respiration. 4. In ox-heart submitochondrial particles ethanethiol inhibited electron transfer between cytochrome c and oxygen. 5. Purified cytochrome c oxidase was inhibited by ethanethiol in a non-competitive manner. 6. Methanethiol inhibited cytochrome c oxidase and was an effective inhibitor of mitochondrial electron transfer, both in liver and brain. 7. The difference in inhibitory properties between ethanethiol, methanethiol and dimethyl sulphide observed in our experiments coincides with the difference in potency to elicit coma in rats. We suggest that inhibition of mitochondrial electron transfer by mercaptans may be relevant to the mechanism by which energy production in brain is depressed during hepatic coma.     

Mitochondrial function of isolated rat hepatocytes from normal and cirrhotic liver

Mitochondrial fractions were obtained from purely isolated hepatocytes of the normal and cirrhotic livers. Mitochondrial function of isolated hepatocytes was evaluated to compare between those from the normal and those from the cirrhotic livers in addition to the evaluation of the mitochondrial function of the normal and cirrhotic liver tissues. Respiratory control, ADP/O ratio and ATP synthesis were significantly lower in the cirrhotic liver tissue than those in the normal liver tissue. However, the mitochondrial function of isolated hepatocytes showed no difference between normal and cirrhotic groups. By electron microscopic examination, debris was more observed in the mitochondrial fractions from the normal and cirrhotic tissues compared to those from hepatocytes. However, no difference of the shape and size of the mitochondria was seen between the fraction from hepatocytes and that from the tissue in each group. These results suggest that as far as hepatocyte itself is concerned, the cirrhotic liver preserves the sufficient function as well as the normal liver.     

Hepatocyte mitochondrial metabolism is inhibited in neonatal rat endotoxaemia: effects of glutamine

Glutamine has beneficial effects on enterocytes and the immune system in sepsis, but its effects on hepatic metabolism remain unknown. The aim of the present study was to determine the effects of glutamine on hepatocyte energy metabolism under conditions of neonatal endotoxaemia. Suckling Wistar rats were injected intraperitoneally with 200 microg/kg lipopolysaccharide. Oxygen consumption was measured polarographically in hepatocytes respiring on either palmitate (0.5 mM) or palmitate plus glutamine (10 mM). Total hepatocyte oxygen consumption was similar in hepatocytes from control and endotoxic rats, but this was due to a decrease in intramitochondrial and an increase in extramitochondrial oxygen consumption in the cells from endotoxic animals. The addition of glutamine to hepatocytes from endotoxic rats restored intramitochondrial oxygen consumption to control levels. Although glutamine did not reverse the inhibition of the thermogenic proton leak observed in endotoxaemia, it significantly increased oxygen consumption due to mitochondrial ATP synthesis (P=0.03). Glutamine significantly increased the hepatocyte ATP/ADP ratio (P=0.02 compared with hepatocytes from endotoxic rats). Electron microscopy revealed morphological damage to the mitochondria of hepatocytes from endotoxic rats, and a return to a normal appearance with the addition of glutamine. We conclude that glutamine reverses the inhibition of mitochondrial metabolism that is observed in endotoxaemia. The effect is primarily at the level of ATP synthesis.     

Sulfonylureas inhibit metabolic flux through rat liver pyruvate carboxylase reaction

The effect of oral hypoglycemic sulfonylureas, tolbutamide and glyburide, on metabolic flux through the pyruvate carboxylase reaction was evaluated in liver mitochondria isolated from 24-hr fasted rats. Both these sulfonylureas inhibited the metabolic flux through the pyruvate carboxylase reaction in a concentration dependent manner. Half-maximal inhibition was achieved at tolbutamide and glyburide concentrations of 0.85 mM and 63.3 microM, respectively. Neither sulfonylurea altered the activity of pyruvate carboxylase or the Km of the enzyme for ATP and pyruvate. However, glyburide and tolbutamide decreased mitochondrial ATP content and elevated mitochondrial ADP and AMP levels. The decrease in mitochondrial ATP was greater with 400 microM glyburide compared with 2.0 mM tolbutamide. Glyburide also decreased mitochondrial acetyl-coenzyme A/CoASH ratio. Additionally, glyburide and tolbutamide stimulated pyruvate (5 mM) supported mitochondrial respiration in the absence of ADP. These data indicate that these sulfonylureas inhibit the metabolic flux through the pyruvate carboxylase reaction by decreasing mitochondrial ATP/ADP and acetyl-coenzyme A/CoASH ratios. Decreased mitochondrial nucleotide content and increased mitochondrial respiration caused by sulfonylureas suggest that these compounds may uncouple oxidative phosphorylation.     

The anti-inflammatory drug, nimesulide (4-nitro-2-phenoxymethane-sulfoanilide), uncouples mitochondria and induces mitochondrial permeability transition in human hepatoma cells: protection by albumin

Like other nonsteroidal anti-inflammatory drugs, nimesulide (4-nitro-2-phenoxymethane-sulfoanilide) triggers hepatitis in a few recipients. Although nimesulide has been shown to uncouple mitochondrial respiration and cause hepatocyte necrosis in the absence of albumin, mechanisms for cell death are incompletely understood, and comparisons with human concentrations are difficult because 99% of nimesulide is albumin-bound. We studied the effects of nimesulide, with or without a physiological concentration of albumin, in isolated rat liver mitochondria or microsomes and in human hepatoma cells. Nimesulide did not undergo monoelectronic nitro reduction in microsomes. In mitochondria incubated without albumin, nimesulide (50 microM) decreased the mitochondrial membrane potential (DeltaPsim), increased basal respiration, and potentiated the mitochondrial permeability transition (MPT) triggered by calcium preloading. In HUH-7 cells incubated for 24 h without albumin, nimesulide (1 mM) decreased the DeltaPsim and cell NADPH and increased the glutathione disulfide/reduced glutathione ratio and cell peroxides; nimesulide triggered MPT, ATP depletion, high cell calcium, and caused mostly necrosis, with rare apoptotic cells. Coincubation with either cyclosporin A (an MPT inhibitor) or the combination of fructose-1,6-diphosphate (a glycolysis substrate) and oligomycin (an ATPase inhibitor) prevented the decrease in DeltaPsim, ATP depletion, and cell death. A physiological concentration of albumin abolished the effects of nimesulide on isolated mitochondria or HUH-7 cells. In conclusion, the weak acid, nimesulide, uncouples mitochondria and triggers MPT and ATP depletion in isolated mitochondria or hepatoma cells incubated without albumin. However, in the presence of albumin, only a fraction of the drug enters cells or organelles, and uncoupling and toxicity are not observed.     

The critical role of mitochondrial energetic impairment in the toxicity of nimesulide to hepatocytes

We described the effects of nimesulide (N-[4-nitro-2-phenoxyphenyl]-methanesulfonamide) and its reduced metabolite in isolated rat hepatocytes. Nimesulide stimulated the succinate-supported state 4 respiration of mitochondria, indicating an uncoupling effect of the drug. Incubation of hepatocytes with nimesulide (0.1-1 mM) elicited a concentration- and time-dependent decrease in cell viability as assessed by lactate dehydrogenase leakage, a decrease of mitochondrial membrane potential as assessed by rhodamine 123 retention, and cell ATP depression. Nimesulide also decreased the levels of NAD(P)H and glutathione in hepatocytes, but the extent of the effects was less pronounced in relation to the energetic parameters; in addition, these effects did not imply the peroxidation of membrane lipids. The decrease in the viability of hepatocytes was prevented by fructose and, to a larger extent, by fructose plus oligomycin; it was stimulated by proadifen, a cytochrome P450 inhibitor. In contrast, the reduced metabolite of nimesulide did not present any of the effects observed for the parent drug. These results indicate that: 1) nimesulide causes injury to the isolated rat liver cells, 2) this effect is mainly mediated by impairment of ATP production by mitochondria due to uncoupling, and 3) on account of the activity of its nitro group, the parent drug by itself is the main factor responsible for its toxicity to the hepatocytes.     

Extraction of intracellular nucleosides and nucleotides with acetonitrile

Extraction of intracellular nucleosides and nucleotides with acetonitrile (ACN) and water was compared with extraction with 60 g/L perchloric acid (PCA), followed by neutralization with KOH, 1 mol/L. Freshly isolated rat bone-marrow and intestinal cells were incubated with radiolabeled 5-fluorouracil (FUra) and 5-fluorodeoxyuridine. The ribose and deoxyribose nucleosides and nucleotides of FUra, and ADP and ATP in the soluble extracts were separated by HPLC and measured by scintillation counting or ultraviolet absorbance. The insoluble precipitates were digested in 1 mol/L NaOH and analyzed for the radioactive macromolecule-bound nucleotides. Both extraction methods yielded the same total (i.e., soluble and insoluble) amount of radioactivity. However, the ACN method yielded significantly more FUra nucleosides and triphosphate nucleotides and ATP in the soluble fraction, and more proteins and macromolecule-bound nucleotides in the insoluble fraction. In the PCA method, the soluble fraction contained more monophosphate nucleotides and ADP than in the ACN method. The PCA extraction procedure promoted decomposition of ATP to ADP and interfered with the ion-pairing reversed-phase HPLC assay. The ACN extraction is faster (less than 5 min) than the PCA extraction (greater than 10 min). Moreover, the ACN in the soluble extract fraction can be removed by evaporation and thus does not interfere with the HPLC analysis. Thus the ACN method evidently is suitable for extraction of nucleosides and nucleotides.     

Purine nucleoside and nucleotide interactions on normal and subsensitive alpha adrenoreceptor responsiveness in guinea-pig vas deferens.

Adenosine and adenosine 5'-monophosphate (AMP) augment contractile responses to norepinephrine (NE) in isolated guinea-pig vas deferens. Dipyridamole slightly enhances, while theophylline antagonizes, adenosine effects on responses to NE. Adenosine triphosphate (ATP) and the nonhydrolyzable analog, adenosine 5'-(beta,gamma-imido)triphosphate (AppNp) depress responses to NE. Adenine and adenosine diphosphate (ADP) are ineffective in influencing alpha adrenoreceptor responsiveness. Repetitive stimulation of isolated vas deferens with maximal concentrations of NE markedly reduce the contractile response to test concentrations of 6 micron NE. Spontaneous resensitization of responses to NE to control levels occurs within 25 to 35 minutes after the end of desensitization treatment. Adenine nucleosides and nucleotides promote a more rapid rate of alpha adrenoreceptor resensitization, with a potency order: AMP greater than adenosine greater than ADP. Adenine and ATP did not influence the rate of alpha adrenoreceptor resensitization. The adenine nucleotides ADP, ATP and the analog AppNp elicit concentration-dependent contractions of guinea-pig vas deferens. Theophylline antagonizes this contractile activity to adenine nucleotides. AMP, adenosine and adenine are devoid of agonistic activity. In the presence of NE, however, AMP and adenosine produce contractile responses of isolated vas deferens strips, and the agonistic activity of ADP, ATP and AppNp is profoundly enhanced. Agonistic actions of purines in the presence of NE are antagonized by phentolamine much more effectively than by theophylline. The results suggest the existence of a purinergic receptor mediating excitatory responses of guinea-pig vas deferens. Furthermore, there appears to be mutual interaction between purinergic and alpha adrenoreceptor mechanisms. That adenyl derivatives are capable of augmenting subsensitive alpha adrenoreceptor responsiveness suggests that adenine nucleosides or nucleotides, released during sympathetic transmission, may be required for maintenance of normal alpha adrenoreceptor sensitivity.     

Mitochondrial dysfunction is an early manifestation of 1,1-dichloroethylene-induced hepatotoxicity in mice

Hepatotoxicity induced by 1,1-dichloroethylene (DCE) is mediated by cytochrome P450-dependent metabolism to reactive intermediates, including the epoxide. We have tested the hypothesis that mitochondria are a primary target of toxicity by investigating dose- and time-dependent effects of DCE on mitochondrial respiration. Hepatotoxicity, as assessed by serum alanine aminotransferase (ALT) activity, was evaluated. We have also determined the effectiveness of N-acetyl-L-cysteine (NAC) in protecting against respiratory perturbations and hepatotoxicity. Liver mitochondria were isolated 2 h after DCE (50, 75, 100, 125, and 150 mg/kg) treatment. Glutamate (complex I)- and succinate (complex II)-supported mitochondrial respiration was assessed by measurement of state 3 (ADP-stimulated) and state 4 (resting) rates of oxygen consumption. The corresponding respiratory control ratios (RCRs, state 3/state 4) and ADP:O ratios were then calculated. A DCE dose of 125 mg/kg significantly inhibited glutamate- and succinate-supported state 3 respiration, leading to a significant reduction in corresponding RCRs and ADP:O ratios. In time-dependent studies, state 3 respiration rates and RCRs for glutamate-supported respiration were significantly decreased as early as 20 min after DCE (125 mg/kg) treatment, whereas those for succinate-supported respiration were significantly decreased at 90 min. Additionally, ADP:O ratios for glutamate-supported respiration were significantly decreased starting at 60 min, and those for succinate-supported respiration at 90 min. Alterations in mitochondrial function preceded significant increases in ALT activity, which was first manifested at 2 h. Pretreatment with NAC (1200 mg/kg) abrogated DCE-induced GSH depletion and inhibited disturbances in mitochondrial respiration. Moreover, NAC protected against increased ALT activity, suggesting that the protective effect of NAC is due to increased GSH for conjugation reactions and/or its antioxidant property. These results showed that DCE-mediated mitochondrial dysfunction is an early event that preceded the onset of hepatotoxicity.     

Disruption of brain mitochondrial calcium sequestration by methylmercury.

In vitro effects of methylmercury (MeHg) on Ca2+ transport and respiratory control of mitochondria isolated from rat forebrain were examined to determine whether MeHg disrupted sequestration of Ca2+ by neuronal mitochondria. Uptake of 45Ca2+ by mitochondria and release of 45Ca2+ from preloaded mitochondria were measured in the presence and absence of ATP. Release of 45Ca2+ from preloaded mitochondria by MeHg was measured in the presence and absence of ruthenium red (RR), a putative inhibitor of the mitochondrial Ca2+ uptake uniporter. During incubation intervals ranging from 10 sec to 5 min, 10 microM MeHg reduced mitochondrial uptake of 45Ca2+ by about 50% and 100 microM MeHg completely prevented 45Ca2+ uptake. These effects of MeHg occurred in both the presence and absence of ATP. Exposure of mitochondria preloaded with 45Ca2+ to either 10 microM or 100 microM MeHg for 10 sec resulted in increased efflux of 45Ca2+ of 10% and 65%, respectively, in both the absence and presence of ATP. Loading mitochondria with 45Ca2+ in the presence of 20 microM RR reduced total uptake of 45Ca2+ and greatly attenuated MeHg-induced release of 45Ca2+ from mitochondria. RR did not inhibit the effects of MeHg on Ca2+ release by merely preventing the binding of MeHg to mitochondria because RR did not alter mitochondrial binding of methyl[203Hg]. The ratio of state 3 to state 4 respiration (respiratory control ratio) was measured as a means of assessing functional integrity of isolated mitochondria in the absence and presence of MeHg. Control ratios of from 3 to 5 were only marginally reduced by 2 microM MeHg but were greatly reduced by 10 and 20 microM MeHg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Toxicity of H2-receptor antagonists to isolated rat hepatocytes: structure-activity relationships

Oxmetidine is a potent and specific antagonist of the histamine H2-receptor. Oxmetidine is also cytotoxic to isolated rat hepatocytes through inhibition of mitochondrial oxidative phosphorylation. The purpose of this investigation was to test a variety of H2-receptor antagonists that are structural analogs of oxmetidine in an attempt to identify a critical structural component or a physicochemical property of the molecule which may be responsible for cytotoxicity. Six histamine receptor H2-antagonists were tested. The minimum drug concentrations that caused 100% cell death (leakage of intracellular lactate dehydrogenase and loss of intracellular potassium) ranged from 0.87 to 22.50 mM for the analogs tested. At toxic concentrations, two of the least potent analogs, SK&F 92909 and SK&F 9205A both caused a rapid decrease in hepatocyte O2 consumption and ATP content which occurred before any evidence of cell injury. The potency of these molecules as cytotoxicants to isolated hepatocytes did not correlate with their potency as histamine H2-receptor antagonists whereas there was a significant correlation between increasing potency and increasing octanol/water partition coefficients. These data suggest that lipid solubility may be a key factor in the cytotoxicity of this class of drugs to isolated rat hepatocytes.     

丹参对离体大鼠肝组织保护作用的实验研究

目的：探讨丹参对离体低温保存大鼠肝组织的保护作用及作用机理。方法：供肝切取采用模拟临床肝移植的标准取肝法，离体肝组织置于4℃保护液中。将72只SD大鼠随机分为3组：对照组（A组）32只（再根据不同保存时间分为1、2、3、6h4个亚组，每组各8只）、实验组（B组）32只（再根据不同保存时间分为1、2、3、6h4个亚组，每组各8只）和正常对照组8只（设为保存0h组）。肝脏保存1、2、3、6h后，测定肝细胞内三磷酸腺苷（ATP）等的含量，线粒体Ca^2＋含量及Ca^2＋-ATP酶活性。观察实验组、对照组肝细胞及线粒体形态学改变。结果：（1）实验组肝细胞ATP的含量、Ca^2＋-ATP酶活性、线粒体Ca^2＋含量与对照组比较，差异有显著性（P〈0．05）；（2）光电镜下见随保存时间延长，实验组细胞损伤较对照组轻微。结论：丹参能改善低温保存肝脏的能量代谢；减轻线粒体钙超载，减轻低温保存肝脏线粒体的损害，因此丹参能提高供肝保存质量，可用于供肝保存。     

Cellular mechanisms underlying the increase in cytosolic free calcium concentration induced by methylmercury in cerebrocortical synaptosomes from guinea pig

Neurotoxic mechanisms of methylmercury (Met-Hg) on presynaptic nerve terminals were studied using the synaptosomes from the cerebral cortex of guinea pig as a model. Cytosolic free calcium [Ca++)c was determined using intrasynaptically trapped fluorescence indicator, fura-2; the plasma membrane potential (delta Up) by measuring the diffusion potential of 86Rb+ and the mitochondrial membrane potential was monitored using the safranine method. Synaptosomal respiration, glycolysis and concentrations of ATP and ADP in the presence and absence of Met-Hg also were quantified. Met-Hg increased synaptosomal [Ca++]c by two distinctive mechanisms. Moderate elevation of [Ca++]c by 127 nM was observed at 30 microM Met-Hg, at which concentration synaptosomal respiration was inhibited completely, leading to partial depolarization of mitochondria. A 3-fold activation of anaerobic glycolysis upon inhibition of respiration was insufficient to sustain terminal energy levels. The delta Up did not depolarize significantly from the resting potential of--67 mV. Thus, the rise in [Ca++]c was due to the energy failure of the synaptosomes, which has been caused by Met-Hg. With 100 microM Met-Hg, [Ca++]c increased extensively by 882 nM. Upon addition of 100 microM Met-Hg the delta Up depolarized instantly dropping 36 mV within 1 min. Synaptosomes were severely energy-deprived, because anaerobic glycolysis was inhibited by 90% from the aerobic level and mitochondrial membrane potential dropped below the limit that could be detected by the safranine method. The proportion of fura-2 signal quenching by Mn++ also increased, indicating that the plasma membrane had become leaky. Thus, at high concentrations of Met-Hg, the rise in [Ca++]c was ascribed to increased ionic permeability of the plasma membrane. The contribution of presynaptic energy failure by Met-Hg is discussed as a possible biochemical mechanism underlying the neurotoxicity of organic mercury.     

Mechanism of hepatotoxicity in periportal regions of the liver lobule due to allyl alcohol: studies on thiols and energy status

The possible involvement of thiols and adenine nucleotides in the selective toxicity to periportal regions by allyl alcohol was evaluated in isolated perfused rat livers. Infusion of allyl alcohol (350 microM) for 20 min depleted hepatic glutathione content by 95% in both regions of the liver lobule yet damage was undetectable as indexed by release of lactate dehydrogenase or uptake of trypan blue. Perfusion for an additional 40 min in the absence of allyl alcohol resulted in lactate dehydrogenase release (2400 U/l) and uptake of trypan blue by 75% of hepatocytes in periportal regions of the liver lobule; however, dye was not taken up by cells in pericentral areas. Because thiol content was depleted in the undamaged pericentral area, it was concluded that thiol depletion alone cannot explain local toxicity to periportal regions by allyl alcohol. Perfusion with dithioerythritol (1.5 mM) prevented damage due to allyl alcohol totally. In contrast, addition of dithioerythritol 20 min after allyl alcohol did not prevent allyl alcohol-induced damage to periportal regions indicating that irreversible changes occur during the first 20 min which ultimately lead to damage. Fasting or pretreatment of rats with diethylmaleate (0.7 g/kg; 1 hr) to deplete glutathione decreased the T1/2 required for release of lactate dehydrogenase from 45 to 35 and 22 min, respectively. When methionine was infused into livers from diethylmaleate-treated rats, the T1/2 for release of lactate dehydrogenase by allyl alcohol was increased to 45 min. Infusion of allyl alcohol for 60 min also produced a significant decrease in ATP content and in the ATP/ADP ratio in periportal but not pericentral regions of the liver lobule.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations of hepatic ATP homeostasis and respiratory chain during development of non-alcoholic steatohepatitis in a rodent model

Background Mitochondrial dysfunction is considered a key player in non‐alcoholic steatohepatitis (NASH) but no data are available on the mitochondrial function and ATP homeostasis in the liver during NASH progression. In the present paper we evaluated the hepatic mitochondrial respiratory chain activity and ATP synthesis in a rodent model of NASH development. Materials and methods Male Wistar rats fed a High Fat/Methionine‐Choline Deficient (MCD) diet to induce NASH or a control diet (SHAM), and sacrificed after 3, 7 and 11 weeks. The oxidative phosphorylation, the F₀F₁ATPase (ATP synthase) and the ATP content were assessed in liver mitochondria. Results NASH mitochondria exhibited an increased rate of substrate oxidation at 3 weeks, which returned to below the normal level at 7 and 11 weeks, concomitantly with the coupling between the phosphorylation activity and the mitochondrial respiration (ADP/O). Uncoupling of NASH liver mitochondria did not allow the recovery of the maximal respiration rate at 7 and 11 weeks. The ATPase (ATP synthase) activity was similar in NASH and SHAM rats, but the mitochondrial ATP content was significantly lower in NASH livers. Conclusions The loss of hepatic ATP stores is not dependent on the F₀F₁‐ATPase but resides in the respiratory chain. Dysfunction of both Complex I and II of the mitochondrial respiratory chain during NASH development implies a mitochondrial adaptive mechanism occurring in the early stages of NASH.     

Plasma ectonucleotidases prevent desensitization of purinergic receptors in stored platelets: importance for platelet activity during thrombus formation

BACKGROUND: Platelets (PLTs) contain purinergic receptors for ATP (P2X1) and ADP (P2Y1 and P2Y12) that rapidly desensitize upon stimulation with these nucleotides. In vivo, this is antagonized by ectonucleotidases on the surface of endothelial cells and white blood cells (WBCs). The receptor desensitization of ATP- and ADP-induced responses of PLTs stored in plasma without WBCs was investigated. STUDY DESIGN AND METHODS: ATP- and ADP-induced PLT shape change (shear-induced) aggregation and Ca2+ signaling were measured in the presence or absence of plasma. Degradation of nucleotides in plasma was quantified by high-performance liquid chromatography. RESULTS: Washed PLTs became refractory for ATP and ADP in shape change, aggregation, and Ca2+ responses during a 90-minute incubation at 37 degrees C. The PLT responses mediated by P2X1, P2Y1, and P2Y12 receptors gradually reduced or disappeared. When plasma was present, however, the PLTs persistently showed high responses to ATP and ADP. Heat treatment of plasma abolished this effect. Also under conditions of flow and high shear, PLTs in plasma kept high P2X1 activity, mediating aggregate formation. In isolated plasma, not containing WBCs, nucleotides were degraded in the order of ADP/UDP>ATP/UTP. Degradation of ATP was partly inhibited by blocking the ecto-NTPDase CD39, whereas degradation of both ATP and ADP was inhibited by blocking ectopyrophosphatase/phosphodiesterase activity. Part of the nucleotide-degrading activities appeared to be membrane-bound. CONCLUSION: Ectonucleotidases in plasma preserve the functionality of P2X1 and P2Y receptors. Upon PLT storage, these plasma activities are essential to ensure adequate (shear-dependent) formation of aggregates and thrombi.     

Effect of Reye''s syndrome serum on isolated chinchilla liver mitochondria.

A general impairment of liver mitochondrial enzymes is central to Reye's syndrome (RS). The respiration of isolated liver mitochondria was measured after the addition of concentrated normal serum or RS serum derived from 12 patients. RS serum stimulates oxygen consumption in isolated rat liver mitochondria. This effect is due to the oxidation of uric acid by peroxisomes contaminating the preparation and a stimulation of mitochondrial respiration (1.05 +/- 0.14 nmol of O2/min X mg of protein; control 0.30 +/- 0.08 nmol O2/min X mg). The stimulation of respiration occurs in the presence of all respiratory substrates, is dependent on the amount of serum added, and represents an uncoupling of oxidative phosphorylation. RS serum reduces ATP formation by 15-76%. The uncoupling effect correlates with the amount of free fatty acid in the serum sample and resembles the effect induced by the addition of a dicarboxylic fatty acid. Dicarboxylic fatty acids, especially long-chain dicarboxylic acids, impair ATP formation. Dicarboxylic acids were found in the serum of all RS patients and comprised as much as 54% of the total serum free fatty acids. 90% of the serum dicarboxylic acids were of 16-18 carbon lengths. The amount of dicarboxylic acids in the RS serum corresponded directly with the reduction in ATP formation by the RS serum. This demonstrates that dicarboxylic acids occur in RS and may be important in the general impairment of mitochondrial function in RS and other disorders where they are present.