Effect of hypoxia and reoxygenation on metabolic pathways in rat hepatocytes.

BACKGROUND: The mechanisms whereby rat hepatocytes undergo irreversible injury due to a lack of oxygen have not been established. METHODS: Liver cells were used for reperfusion injury, and four compartmentalized pathways were evaluated during hypoxia (N2/CO2, 19:1) for 30 min followed by oxygen (O2/CO2, 19:1) for 30 min. RESULTS: Cell viability decreased during the hypoxic, but not during the reoxygenation, phase. Glycogenolysis, as measured by glucose release, was significantly increased during hypoxia as compared to controls in oxygen (205 +/- 15 vs. 155 +/- 10 nmol glucose/mg protein/h, respectively), and did not return to normal levels by reoxygenation. Gluconeogenesis was importantly decreased during hypoxia (102 +/- 10 vs. 8 +/- 2 nmol glucose/mg protein/h) with partial recovery during reoxygenation. Ureagenesis diminished in hypoxia, but recovered during reoxygenation. Additionally, 3-hydroxybutyrate formation was augmented by hypoxia, with some recovery when oxygen was present. CONCLUSIONS: These results suggest that compartmentalized pathways are protected from hypoxic injury in isolated hepatocytes, and also suggest it as a model to test the idea that enzymes of those pathways are organized into multienzyme complexes in vivo.

Extracellular ATP inhibits agonist-induced mobilization of internal calcium in human platelets.

Our previous studies have demonstrated that platelets possess ATP purinergic receptors in addition to the ADP, P2T, receptor. Occupancy of the P2 receptor by ATP inhibited agonist-induced platelet aggregation. This study demonstrated that the mechanism of inhibition may involve ATP inhibition of agonist-induced mobilization of internal calcium. Within the cardiovascular system, the ATP inhibition of calcium mobilization is unique to platelets. All other cell types in the cardiovascular system, where calcium mobilization is affected by extracellular ATP, responded with an increased mobilization as opposed to inhibition. The platelet inhibitory response to ATP was enhanced by the addition of an ATP generating system, creatine phosphate/phosphocreatine kinase. ATP and ATP analogues were found to inhibit calcium mobilization with a rank order of alpha beta-methylene ATP, beta gamma-methylene ATP approximately ATP > benzoyl ATP > 2 methylthio ATP which is a characteristic of P2x-like receptors. The inhibitory effect of ATP could be abrogated by prolonged treatment of platelets with the P2x desensitizing agent, alpha beta-methylene ATP. Also, UTP and CTP were approximately as effective inhibitors as ATP while GTP was not. ATP competition with ADP for the P2T receptor was excluded in studies with platelets derived from an aspirin-treated individual which were essentially insensitive to ADP. The agonist-induced calcium mobilization and inhibition by ATP occurred with the thromboxane A2 mimetic, U46619, collagen and thrombin; however, the kinetics of mobilization varied somewhat with the different agonists. The responses to extracellular ATP were independent of extracellular Ca2+, where 1 mM calcium or 0.3 mM EGTA was added to the reaction mixture. The inhibition of calcium mobilization coupled to inhibition of platelet aggregation by extracellular ATP may serve an important physiologic role. ATP, released from activated platelets at localized sites of vascular injury, may help to limit the size of the platelet plug-clot that, if left unregulated, could occlude the injured blood vessel.

Intramitochondrial localization of alanine aminotransferase in rat-liver mitochondria: comparison with glutaminase and aspartate aminotransferase.

The removal of the outer mitochondrial membrane and hence of constituents of the intermembrane space in rat-liver mitochondria using digitonin showed that phosphate-dependent glutaminase, alanine and aspartate aminotransferase were localized in the mitoplasts. Further fractionation of mitoplasts following their sonication resulted in 90% of glutaminase, 98% of alanine aminotransferase and 48% of aspartate aminotransferase being recovered in the soluble fraction while the remainder of each enzyme was recovered in the sonicated vesicles fraction. These results indicated that glutaminase and alanine aminotransferase were soluble matrix enzymes, the little of each enzyme recovered in the sonicated vesicles fraction being probably due to entrapment in the vesicles. Aspartate aminotransferase had dual localization, in the inner membrane and matrix with the high specific activity in sonicated vesicles confirming its association with the membrane. Activation experiments suggested that the membrane-bound enzyme was localized on the inner side of the inner mitochondrial membrane.

Effects of tri-Calciphor (trimer of 16, 16-dimethyl-15-dehydroprostaglandin B1) on glucose metabolism in liver cells.

Phosphorylase activity of isolated rat liver cells was increased about 2-fold on addition of tri-Calciphor (trimer of 16, 16-dimethyl-15-dehydroprostaglandin B1), epinephrine or the Ca2+ ionophore A23187, in all cases presumably due to an increase in cytosolic Ca2+. Extracellular Ca2+ was required with A23187, but not with either tri-Calciphor or epinephrine. Tri-Calciphor, however, did not stimulate a sustained release of glucose from hepatocytes as compared to the other Ca2+ mobilizing agents, even at concentrations 10-fold higher than that required to stimulate the phosphorylase activity. Tri-Calciphor did not alter the glucose release by epinephrine. It is concluded that tri-Calciphor can alter cytosolic Ca2+, but that its mechanism of action is more complex than that of a simple Ca2+ ionophore.

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

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

Tri-Calciphor (16,16-dimethyl-15-dehydroprostaglandin B1 trimer)-mediated mitochondrial Ca2+ movements: modulation by phosphate.

The trimeric derivative of 16,16-dimethyl-15-dehydroprostaglandin B1 (termed tri-Calciphor), which protects tissues against ischemic damage, induced Ca2+ efflux and swelling in mitochondria in the absence of phosphate, Mg2+ and ATP. When glutamate/malate rather than succinate was the substrate, higher tri-Calciphor concentrations were required for the ionophoretic activity. Ca2+ efflux and mitochondrial swelling induced by tri-Calciphor were completely inhibited by ATP, phosphate and Mg2+ added together, and partially inhibited with phosphate plus either ATP or Mg2+. Between 0 and 7 microM added Ca2+ and in the presence of phosphate, ATP and Mg2+, tri-Calciphor stimulated the uptake of Ca2+ by mitochondria and increased the efficiency of buffering of extramitochondrial Ca2+. Thus, depending on the assay conditions, two different effects involving Ca2+ movements and mitochondria are observed with tri-Calciphor.

Neuroprotective activity of dimer of 16,16'-dimethyl-15-dehydroprostaglandin B1 (di-Calciphor) in cerebral ischemia.

Post-ischemic treatment of di-Calciphor (16,16'-dimethyl-15- dehydroprostaglandin B1) significantly improves animal survival and prevents ischemia-induced neurodegeneration of vulnerable forebrain regions assessed with histochemical and biochemical techniques in gerbils. Neuronal degeneration seen by Cresyl violet staining and silver impregnation in the CA1 sector of the hippocampus and the dorso-lateral sector of the striatum was significantly reduced in animals treated with di-Calciphor. In addition, the early onset of selective degradation of calpain I substrates spectrin and microtubule-associated protein (MAP2) in these same vulnerable regions was prevented. The lack of adverse side effects may facilitate the potential therapeutic use of this drug in preventing neuronal damage caused by stroke.

MK-801 is neuroprotective but does not improve survival in severe forebrain ischemia.

The effects of MK-801 on postischemic recovery, survival and neuronal preservation in the cortex, hippocampus and striatum were studied in Mongolian gerbils. The drug was administered 30 min prior to 20 of min forebrain ischemia induced by bilateral ligation of the carotids. Neurological recovery and survival were monitored for 7 days. At the end of the monitoring period neuronal damage was analyzed in the brains of the survivors in both groups. Treatment with MK-801 did not improve either neurological recovery or end-point survival. However, significant (P < 0.01) neuronal protection was observed in the hippocampi and striata of the drug treated animals while cortical neurons were not significantly protected. These findings demonstrate that protection against ischemic neuronal damage can be observed without concomitant improvement in either postischemic neurological recovery or survival. Protection of selectively vulnerable brain regions, often used as the predictor of the therapeutic potential of an agent, does not appear to correlate well with postischemic survival in this animal model of ischemia.

Nuclear translocation of aflatoxin B1 - protein complex.

The in vitro binding of [3H]-AFB1 to various proteins was studied by equilibrium dialysis. At 23 +/- 1 degree C, [3H]-AFB1 binding activity (mmol/mol) decreased as follows: pyruvate kinase > albumin-NLS > albumin > carbonic anhydrase > RNase > histones. The nuclear translocation and activation of AFB1 and AFB-protein complexes was investigated using isolated rat liver nuclei in the presence of ATP and a NADPH regenerating system. Proteins containing NLS such as histones and albumin-NLS facilitated AFB1 translocation into the nucleus where activation and adduct formation took place.

Protective effect of the dimer of 16,16-diMePGB1 against KCN-induced mitochondrial failure in hepatocytes.

The dimer and trimer of 16,16-dimethyl-15-dehydroprostaglandin B1 (16,16-diMePGB1) previously have been shown to have protective effects on mitochondrial function. To examine the potential mechanisms involved in protection against mitochondrial failure, we have studied the effects of the dimer of 16,16-diMe-PGB1 (dicalciphor) on mitochondrial function in hepatocytes exposed to KCN. Addition of micromolar concentrations of dicalciphor provided substantial protection against KCN-induced toxicity in a concentration- and time-dependent manner. Dicalciphor, however, had no effect on total or mitochondrial ATP losses in KCN-treated cells. The dimer prevented the marked loss of mitochondrial membrane potential (delta psi) and delta pH that occurs as a result of KCN treatment and prevented KCN-induced loading of phosphate in mitochondria. Furthermore, the dimer of 16,16-diMePGB1 also prevented KCN-induced mitochondrial and cellular swelling. These results demonstrate that dicalciphor protects against KCN-induced damage and that this protection is associated with regulation of specific mitochondrial ion transport functions.

A novel treatment of global cerebral ischaemia with a glycine partial agonist.

Chronic treatment of gerbils with 1-aminocyclopropanecarboxylic acid (a high affinity, partial agonist at strychnine-insensitive glycine receptors) resulted in a 3-fold increase in survival, a significant improvement in neurological status, and an extensive protection of vulnerable brain regions following severe forebrain ischaemia. A bolus of 1-aminocyclopropanecarboxylic acid 30 min prior to ischaemia did not further improve outcome compared to gerbils receiving their last injection 24 h prior to ischaemia. These findings are consistent with the hypothesis that chronic treatment with a glycine partial agonist desensitizes the N-methyl-D-aspartate receptor complex. Pharmacological intervention at the strychnine-insensitive glycine receptor may be an effective means of ameliorating the consequences of neuronal degeneration caused by excitotoxic phenomena.

Treatment of severe brain ischemia with di- and tri-Calciphor (dimer and trimer of 16,16'-dimethyl prostaglandin B1).

Following 20 min occlusion of both carotid arteries, female gerbils were subjected to treatment with di- or tri-Calciphor (dimer or trimer of 16,16'-dimethyl prostaglandin B1). Dimer was injected i.p. at 5 and 10 mg/kg at 5 min and again at 24 h, 30 min and 24 h, 60 min and 24 h or 180 min and 24 h postischemia (N = 25/group). Trimer was given i.p. at 5, 10 or 15 mg/kg at 5 min and 24 h postischemia (N = 25/group.) The controls (N = 25) were injected with the vehicle. Neurological status and postischemic survival of the animals were monitored for 14 days postischemia. Survival of the treated gerbils was significantly improved following the treatment with either di- or tri-Calciphor administered at 10 mg/kg at 5 min and 24 h postischemia (36 vs. 68% di- and 64% tri-Calciphor, P less than 0.05), and with di-Calciphor at 5 mg/kg at 180 min and 24 h postischemia (64%). All other treatment regimens with either drug resulted in a numerical, statistically insignificant improvement. In addition, treatment with either drug reduced the intensity of postischemic neurological impairment. Treatment with di-Calciphor injected at 10 mg/kg at 5 min and 24 h post 20 min ischemia substantially reduced the period of postischemic locomotor hyperactivity. The drug had no impact on either body temperature or blood pressure. There is evidence that the effects of Calciphor may be mediated via calcium regulatory mechanisms. The results of the present study are discussed in the light of such possibility.

Protective effect of dicalciphor during mitochondrial failure.

Mammalian cells differ considerably in the duration of anoxia which they can tolerate despite the fact that dramatic bioenergetic changes occur rapidly. Previous studies indicate that the ability to tolerate anoxia is at least partly due to an endogenous signal transduction system that senses O2 deficiency and signal altered ion transport functions in the mitochondria. The responses included inhibition of ATP synthase, ADP/ATP exchange, inorganic phosphate uptake, mitochondrial swelling, and loss of the mitochondrial proton-motive force. An important distinction between KCN toxicity and anoxia is that KCN does not elicit these protective mechanisms. Thus, the ability of a compound to elicit these mechanisms in KCN-treated cells provides an assay for potential agonists of the endogenous protective mechanisms.

The in vivo disposition of aflatoxin B1 in rat liver.

The disposition of a non-toxic i.p. dose of [3H]-aflatoxin B1 (0.70 micrograms/kg) in the blood, plasma, and liver was studied in male Wistar rats. Uptake into the blood, plasma, and liver was biphasic; there was an initial rapid rise (0-2 hr) followed by a second phase (2-12 hr) of a gradual increase. Most of the radioactivity in the blood was bound noncovalently to albumin. Distribution of radioactivity in the subcellular fractions of liver showed that the microsomes exhibited the highest labeling which increased over the time course; labeling of the cytosol reached a maximum at 2 hr then decreased to a new steady state, whereas the mitochondria and nuclei reached a plateau. When the content of aflatoxin B1 in the nuclear subfractions was examined, greater than 92% of the total radioactivity was found in the deoxyribonucleoprotein fraction, and 84% of this was bound noncovalently. These results suggest that aflatoxin B1 is transported from the site of injection through the blood to the liver and its subcellular and subnuclear fractions primarily in a noncovalent form.

Isolated hepatocytes are capable of excreting aflatoxin metabolites.

The intra- and extra-cellular concentrations of AFB1, AFM1, AFP1, AFQ1 and their conjugates were quantitatively determined after 60 min of incubation with [3H]-AFB1 (1500 pmol/10(8) cells). Comparing the total concentrations of water-soluble conjugates, the eight fold greater amounts found in the medium (718 pmol) than in the cell (86 pmol) indicate that these detoxication products were excreted soon after they were formed. When the cells were perturbed with butylated hydroxyanisole (BHA), a noncompetitive inhibitor of the mixed-function oxidases, accumulations of intracellular AFB1 and extracellular AFB1 were observed. In a cell-free microsomal system, the AFB1 was metabolized at a slower rate than in intact cells. When the activation of AFB1 is blocked, the accumulation of intracellular AFB1 and decreased internalization of AFB1 suggest that AFB1 uptake, translocation of AFB1 from site of entry to site of actions, oxidation by cytochrome P-450-dependent monoxygenase, metabolic detoxication and conjugation reactions, and excretion of water soluble metabolites are linked.

Control of mitochondrial matrix calcium: studies using fluo-3 as a fluorescent calcium indicator.

Fluo-3, a fluorescent Ca2+ indicator, is sequestered by isolated rat liver mitochondria and is an effective probe for evaluating the concentration and kinetics of change of mitochondrial matrix ionized calcium ([Ca2+]m) under a variety of conditions. At the wavelengths employed, there is no significant interference by auto-fluorescence. There is an insignificant release of the indicator over four hours and the loading and presence of fluo-3 has no effect on respiratory rate or oxidative phosphorylation. The [Ca2+]m steady state can be altered by the assay conditions, i.e. the presence of extra-mitochondrial Ca2+, Mg2+ phosphate and respiratory inhibitors. The total matrix ionized calcium represents a small percent (less than 0.01%) of the total mitochondrial calcium.

2(3)-tert-butyl-4-hydroxyanisole inhibits oxidative metabolism of aflatoxin B1 in isolated rat hepatocytes.

Previous studies indicate that dietary administration of phenolic antioxidants, 2(3)-tert-butyl-4-hydroxyanisole (BHA) and 3,5-di-tert-butyl-4-hydroxytoluene, inhibits the carcinogenic effect of a number of chemical carcinogens including aflatoxin B1 (AFB1). Induction of hepatic enzymes, such as glutathione S-transferase, UDP-glucuronyltransferase, and epoxide hydrolase, has been shown to be responsible for the reduction of AFB1 cytotoxic and carcinogenic effects. The effect of BHA on AFB1 activation was examined in vitro utilizing isolated rat hepatocytes and liver microsomes. In hepatocytes, the total AFB1 content and bound form of AFB1 were 3.4 and 1.4 pmol/10(6) cells, respectively. In the cell-free microsomal activating system, 2.2 pmol were activated per mg of microsomal protein during 60 min of incubation. BHA (0.1-0.5 mM) inhibited AFB1 activation and binding in both systems in a dose-dependent manner; in hepatocytes, 90% inhibition was observed at 0.5 mM. Analyzing various AFB1 adducts, BHA (0.25 mM)-treated hepatocytes contained a significantly reduced amount of AFB1 macromolecular adducts. The antioxidant neither stimulated nor inhibited the cytosolic glutathione S-transferase and microsomal UDP-glucuronyltransferase activities. Analysis of various hydroxylated (aflatoxins M1 and Q1 (AFM1 and AFQ1] and demethylated (aflatoxin P1 (AFP1] metabolites of AFB1 in both the conjugated and unconjugated form indicated that there was a 30-50% reduction of unconjugated AFP1, AFQ1, and AFM1, whereas AFB1 was increased 3-fold. There was no significant change of conjugated metabolites. The effect of BHA on AFB1 activation in hepatocytes was compared with that of other cytochrome P-450 inhibitors; the ED50 values of SKF 525A, BHA, and metyrapone were 9 microM, 40 microM, and 280 microM, respectively. In the cell-free microsomal system, biotransformation of AFB1 to AFP1, AFM1, and AFQ1 was also inhibited. Kinetic analysis of p-nitroanisole O-demethylase activity of rat liver microsomes demonstrated that BHA inhibited noncompetitively with an apparent Ki of 90 microM. In the absence of enzyme induction, the phenolic antioxidant, BHA, blocks the oxidative biotransformation of AFB1 in isolated hepatocytes.

Studies of the cation binding properties of an oligomeric derivative of prostaglandin B1.

The ionophoretic activity of PGBx, an oligomeric mixture synthesized from 15-dehydro PGB1, with different cations was measured using arsenazo III-entrapped liposomes. The order of ionophoretic activity was Zn2+ greater than Co2+ greater than Mn2+ greater than Cu2+ greater than Ca2+ greater than Ba2+ greater than Sr2+ greater than Mg2+. The intrinsic fluorescence of PGBx was quenched by the binding of divalent cations as well as by La3+ and H+. Quenching by K+ and Na+ was minimal. The order of quenching strength of divalent cations was Zn2+ greater than Co2+ greater than Cu2+ = Mn2+ greater than Ca2+ greater than Ba2+ greater than Sr2+ greater than Mg2+. Binding affinities of these cations determined by a murexide indicator method were in good agreement with that determined by the fluorescence quenching reaction. The cation binding affinity of PGBx in aqueous solutions correlates with the ionophoretic activity in liposomes. The binding affinity for K+ was estimated from the inhibition by K+ of Ca2+ binding by PGBx. Although PGBx has a lower selectivity for divalent cation binding than the ionophore A23187, the characteristics of the binding affinity of these two compounds for various ions were similar. The pK of PGBx as determined by fluorescence quenching was 6.7. The molecular weight of the divalent cation binding unit was estimated to be about 680, with each PGBx molecule having three such binding sites. The binding of Ca2+ to such a site is one-to-one.

Modification of protein synthetic components by aflatoxin B1.

Molecular sites of perturbation by the hepatocarcinogen aflatoxin B1 (AFB1) in the protein synthesis initiation complex were assessed using isolated hepatocytes and a cell-free activating system containing microsomes and cytoplasmic ribonucleoprotein complexes (cRPC). Ribosomal proteins showed no detectable modification by the toxin in either system. With hepatocytes, initiation factors demonstrated only slight modification by AFB1. RNAs from both hepatocytes and the cell-free system with microsomes and cRPC were modified, with poly(A)-containing RNA exhibiting at least a 5-fold higher modification than poly(A)-lacking RNA. The poly(A)-lacking RNAs were modified in the order 28S rRNA greater than 18S rRNA greater than 5-6S rRNA greater than 4S tRNA. Guanine was the target base of AFB1, but only 10% of the AFB1-GMP adducts were on guanines located in a poly(G) region. These results suggest that guanine modification in RNAs may be responsible for the observed inhibition of translational initiation by AFB1 to a greater extent than modification of either ribosomal intrinsic or associated proteins.

Calcium ionophoretic activity of chemically synthesized oligomeric derivatives of prostaglandin B1.

Chemically synthesized dimers, trimers and tetramers of 15-dehydroprostaglandin B1 and 16,16'-dimethyl-15-dehydroprostaglandin B1 facilitate the release of Ca2+ from isolated rat liver mitochondria. The parent monomeric prostaglandins had no significant activity. The rate of release was stimulated by exogenous K+ or Na+, suggesting an antiport exchange of monovalent cations for intra-mitochondrial Ca2+. The activity depended upon the presence of ruthenium red, which prevented recycling of Ca2+; comparison of the activity with A23187 and carbonyl cyanide p-trifluoromethoxyphenylhydrazone indicated that the prostaglandin B1 oligomers were functioning as ionophores and the release of Ca2+ was not caused by an uncoupling of oxidative phosphorylation. The oligomers caused a major decrease in the membrane potential but only when the mitochondria were preloaded with exogenous Ca2+, and even then, the Ca2+ efflux was completed before the membrane potential decreased to less than 90 mV. The oligomeric molecules were able to form supramolecular aggregates in the presence of Ca2+ as detected by light scattering. They extracted Ca2+ into an organic phase, and translocated Ca2+ from one aqueous domain to another across an organic barrier; K+ and Na+ modulated these processes. The prostaglandin B1 derivatives also translocated Rb+ from one aqueous phase to another across an organic barrier when Ca2+ was translocated.