Victoria Chagoya de Sánchez

Effects of adenosine administration on the function and membrane composition of liver mitochondria in carbon tetrachloride-induced cirrhosis

The effect of chronic carbon tetrachloride (CCl4) administration on liver mitochondria function and the protective action of adenosine on CCl4-induced damage were assessed in rats made cirrhotic by long-term exposure to the hepatotoxin (8 weeks). The CCl4 treatment decreased the ADP-stimulated oxygen consumption, respiratory control, and ADP/O values, mainly for substrates oxidation of site I, in isolated mitochondria. This impaired mitochondrial capacity for substrate oxidation and ATP synthesis was accompanied by an important diminution (approximately 30 mV) of membrane electrical potential. Disturbances of the mitochondrial membrane, induced by CCl4 treatment, were also evidenced as increased mitochondria swelling and altered oscillatory states of mitochondrial volume, both energy-linked processes. The deleterious effects of CCl4 on mitochondrial function were also reflected as a deficient activity of the malate-aspartate shuttle that correlated with abnormal distribution of cholesterol and phospholipids in membranes obtained from submitochondrial particles. Adenosine treatment of CCl4-poisoned rats partially prevented the alterations in mitochondria membrane composition and prevented, almost completely, the impairment of mitochondria function induced by CCl4. Although the nature of the protective action of adenosine on CCl4-induced mitochondria injury remains to be elucidated, such action at this level might play an important role in the partial prevention of liver damage induced by the CCl4.

In vivo modification of the energy charge in the liver cell

Summary The energy charge of the adenylate system increases in the liver cells of normal rats 30–120 minutes after the intra — peritoneal injection of adenosine (200 mg/Kg body weight). Simultaneously, in the liver there is an enhancement of glycogen biosynthesis and a diminution in fatty acid catabolism. These metabolic changes correspond to what could be expected from an increase in the energy charge. Thus, a regulatory role of the adenylate system under in vivo conditions is substantiated.

Possible role of cell redox state on collagen metabolism in carbon tetrachloride-induced cirrhosis as evidenced by adenosine administration to rats

Marked changes in the redox state of liver cells in carbon tetrachloride (CCl4)-induced cirrhosis after chronic treatment with the hepatotoxin (4-8 weeks) were observed. A shift of the redox state towards the reduced side is noticed in both compartments, cytosol and mitochondria. At 8 weeks of treatment an imbalance between these two compartments was evident. The alteration produced by the CCl4 treatment in the cell redox state might be related to the mitochondrial damage elicited by the hepatotoxin. Adenosine treatment to CCl4-poisoned rats was able to counteract the effect of the hepatotoxin on the redox equilibrium; hence, it could be linked to the beneficial action of the nucleoside in the maintenance of mitochondrial function. The changes in the hepatocyte redox state, induced by CCl4 and/or adenosine, seem to modify collagen and nitrogen metabolism, indicating a linear correlation between the redox state and the collagen synthesis rate, whereas an inverse relationship was observed with collagenase activity. The possible role of the changes in cell redox state as signals for communication between parenchymal and mesenchymal liver cells is discussed. The results suggest an important correlation among mitochondrial function, cellular redox state, and regulation of collagen metabolism that could be relevant for the physio-pathology of this model of experimental cirrhosis.

On the mechanism of ethanol-induced fatty liver and its reversibility by adenosine

Abstract The administration of adenosine partially prevented and reverted the ethanol-induced fatty liver. The hepatic α-glycerophosphate concentration and the α-glycerophosphate/dihydroxyacetone phosphate ratio were significantly increased after ethanol administration. The nucleoside decreased with ratio and enhanced the oxidation of ethanol. A strong correlation between the cytoplasmic redox state and the amount of triacylglycerols in the liver was found (8 h after treatments) stressing the paramount importance of the redox state in the pathogenesis of ethanol-induced fatty liver. As previously reported, the nucleoside expanded the adenine nucleotide pool size and the hepatic ATP level. Ethanol potentiated these effects. It is suggested that adenosine ameliorated the ethanol-induced fatty liver through an increased utilization of reducing equivalents by the mitochondria. An interdependence of these effects is proposed and discussed.

Circadian variations of adenosine level in blood and liver and its possible physiological significance

The role of adenosine as a possible physiological modulator was explored by measuring its concentration in different tissues during a 24-hour period. Initially the circadian variations of adenosine and other purine compounds such as inosine, hypoxanthine, uric acid and adenine nucleotides were studied in the rat blood. A daily cyclic response was observed, with low levels of adenosine from 08.00 - 20.00 h, followed by an increase from this time on. Inosine and hypoxanthine levels were elevated during the day and low at night. The uric acid changes observed indicate that the decrease in purine catabolism coincides with a decrease in inosine and hypoxanthine levels and an increase in adenosine. The blood adenine nucleotides, energy charge and phosphorylation potential remained constant during the day and showed oscillatory changes during the night. Similar studies were made in the liver, a primary source of circulating purines. Liver adenosine was high during the night while inosine and hypoxanthine remained low along the 24 hours. The results suggest that liver purine metabolism might participate in the maintenance and renewal of the blood purine pool and in the energy state of erythrocytes in vivo.

Inhibitory effect of vitamin E administration on the progression of liver regeneration induced by partial hepatectomy in rats

We have proposed that controlled peroxidative modifications of membranes could be playing a role in the early steps of liver regeneration. Hence, lipid peroxidation (LP) was modified in vivo by treatment with vitamin E in rats subjected to partial hepatectomy (PH), and its influence on liver regeneration was evaluated. Our results, using several methods to monitor LP, indicate that vitamin E administration promoted a decreased LP rate in liver subcellular membranes. Vitamin E drastically diminished cytosolic LP, shifting earlier increased LP in plasma membranes, and promoted a higher increase of nuclear LP in animals subjected to PH. Pretreatment with vitamin E induced a striking reduction of liver mass recovery and nuclear bromodeoxyuridine labeling (clearly shown at 24 hours after surgery), as well as promoted a decreased expression of cyclin D1 and of the proliferating cell nuclear antigen after PH. These effects seem to lead to a decreased mitotic index at 48 hours after PH. Vitamin E pretreatment also diminished PH-induced hypoglycemia but elevated serum bilirubin level, which was not observed in PH animals without vitamin treatment. In conclusion, an enhanced but controlled LP seems to play a critical role during the early phases of liver regeneration. Decreasing magnitude or time course of the PH-promoted enhanced LP (at early post-PH stages) by in vivo treatment with vitamin E could promote an early termination of preparative cell events, which lead to the replicative phase, during PH-promoted liver proliferation. The latter could have a significant implication in the antitumorigenic effect ascribed to the treatment with vitamin E.

Correlation between oxidative stress and alteration of intracellular calcium handling in isoproterenol-induced myocardial infarction

Myocardial Ca2+ overload and oxidative stress are well documented effects associated to isoproterenol (ISO)-induced myocardial necrosis, but information correlating these two issues is scarce. Using an ISO-induced myocardial infarction model, 3 stages of myocardial damage were defined: pre-infarction (0–12 h), infarction (12–24 h) and post-infarction (24–96 h). Alterations in Ca2+ homeostasis and oxidative stress were studied in mitochondria, sarcoplasmic reticulum and plasmalemma by measuring the Ca2+ content, the activity of Ca2+ handling proteins, and by quantifying TBARs, nitric oxide (NO) and oxidative protein damage (changes in carbonyl and thiol groups). Free radicals generated system, antioxidant enzymes and oxidative stress (GSH/GSSG ratio) were also monitored at different times of ISO-induced cardiotoxicity. The Ca2+ overload induced by ISO was counterbalanced by a diminution in the ryanodine receptor activity and the Na+-Ca+2 exchanger as well as by the increase in both calcium ATPases activities (vanadate- and thapsigargine-sensitive) and mitochondrial Ca2+ uptake during pre-infarction and infarction stages. Pro-oxidative reactions and antioxidant defences during the 3 stages of cardiotoxicity were observed, with maximal oxidative stress during the infarction. Significant correlations were found among pro-oxidative reactions with plasmalemma and sarcoplasmic reticulum Ca2+ ATPases, and ryanodine receptor activities at the onset and development of ISO-induced infarction. These findings could be helpful in the design of antioxidant therapies in this pathology.

Effects of adenosine on liver cell damage induced by carbon tetrachloride

Adenosine administration delayed the fatty liver and cell necrosis induced by carbon tetrachloride without affecting the action of the hepatotoxin on protein synthesis and liver triacylglycerol release. Adenosine produced a drastic antilipolytic effect accompanied by a decrease in the incorporation of [1-14C]palmitic acid into triacylglycerols and free fatty acids of the liver. Furthermore, a decrease in the serum levels of ketone bodies was observed at early times. The nucleoside also avoided the release of intracellular enzymes and prevented the lipid peroxidation produced by carbon tetrachloride during the 4 hr of treatment. The protective action of adenosine was transient, lasting 3-4 hr, probably the time required to be metabolized. The results suggest that the antilipolytic effect of the nucleoside, the inhibition of hepatic fatty acid metabolism, and the decrease in carbon tetrachloride-induced lipoperoxidation that it produced are involved in the delayed acute hepatotoxicity induced by carbon tetrachloride.

Prevention of in vitro hepatic stellate cells activation by the adenosine derivative compound IFC305.

We have previously shown that adenosine and the aspartate salt of adenosine (IFC305) reverse pre-established CCl(4)-induced cirrhosis in rats. However, their molecular mechanism of action is not clearly understood. Hepatic stellate cells (HSC) play a pivotal role in liver fibrogenesis leading to cirrhosis, mainly through their activation, changing from a quiescent adipogenic state to a proliferative myofibrogenic condition. Therefore, we decided to investigate the effect of IFC305 on primary cultured rat HSC. Our results reveal that this compound suppressed the activation of HSC, as demonstrated by the maintenance of a quiescent cell morphology, including lipid droplets content, inhibition of α-smooth muscle actin (α-SMA) and collagen α1(I) expression, and up-regulation of MMP-13, Smad7, and PPARγ expression, three key antifibrogenic genes. Furthermore, IFC305 was able to repress the platelet-derived growth factor (PDGF)-induced proliferation of HSC. This inhibition was independent of adenosine receptors stimulation; instead, IFC305 was incorporated into cells by adenosine transporters and converted to AMP by adenosine kinase. On the other hand, addition of pyrimidine ribonucleoside as uridine reversed the suppressive effect of IFC305 on the proliferation and activation of HSC, suggesting that intracellular pyrimidine starvation would be involved in the molecular mechanism of action of IFC305. In conclusion, IFC305 inhibits HSC activation and maintains their quiescence in vitro; these results could explain in part the antifibrotic liver beneficial effect previously described for this compound on the animal model.

Effect of (NH4)2 SO4 and glycerol on the preservation of the “NAD+-independent” activity of d-glucose-6-phosphate, l-myo-inositol-1-phosphate cycloaldolase from Neurospora crassa

Abstract Partially purified preparations of glucocycloaldolase from Neurospora crassa show an absolute requirement for NAD+. The experimental findings in this paper indicate that the conditions of isolation used determine whether or not glucocycloaldolase activity can be detected in the absence of added NAD+. The protective action of glycerol and (NH4)2SO4 on this so-called “NAD+-independent” activity was studied. Extraction of the enzyme in the presence of either 25% glycerol or 0.19 m (NH4)2SO4 preserved 80–90% of the “NAD+-independent” activity. Precipitation of the enzyme between 50–66.6% (NH4)2SO4 and dialysis of this fraction for 13 hr against Tris, Tris plus 25% glycerol or Tris plus 0.19 m (NH4)2SO4 preserved, respectively, 0, 30, and 70% of the “NAD+-independent” activity. Other salts containing either NH4+ or SO4−2 ions were not as effective as (NH4)2SO4 itself. Exogenously added NAD+ could be coprecipitated with glucocycloaldolase which was completely dependent upon added NAD+, to give rise to a preparation which was 41% “NAD-independent”. Studies of a purified glucocycloaldolase preparation, which gives only three bands on acrylamide gel electrophoresis, showed that addition of exogenous NAD+, followed by dialysis, gave 50% of the “NAD+-independent activity” provided the dialysis was effected against Tris containing 0.19 m (NH4)2SO4. Only 10% was recovered when Tris alone was used.