Mauricio Díaz-Muñoz

Persistence of metabolic rhythmicity during fasting and its entrainment by restricted feeding schedules in rats

The presence of a food-entrainable oscillator (FEO) independent from the SCN is now well established, but until now its location and characterization have been elusive. Because its expression requires priming of the animals metabolism toward a catabolic state, it is possible that metabolic rhythms may be related to FEO. The present study was designed to determine whether metabolic rhythms persist during fasting and whether such rhythms could be entrained to a restricted feeding schedule. The results indicate persistent rhythms of triacylglycerides, free fatty acids, glucose, and proteins during fasting, whereas ketone bodies and liver glycogen changed their concentration as a function of fasting. Daily food pulses of 2 h entrained the rhythms of triacylglycerides and free fatty acids and restored ketone bodies and liver glycogen to similar levels as controls. Neither glucose nor proteins were affected by the food pulse. These results indicate the relevance of lipid metabolism as a phenomenon associated with the FEO.The presence of a food-entrainable oscillator (FEO) independent from the SCN is now well established, but until now its location and characterization have been elusive. Because its expression requires priming of the animals metabolism toward a catabolic state, it is possible that metabolic rhythms may be related to FEO. The present study was designed to determine whether metabolic rhythms persist during fasting and whether such rhythms could be entrained to a restricted feeding schedule. The results indicate persistent rhythms of triacylglycerides, free fatty acids, glucose, and proteins during fasting, whereas ketone bodies and liver glycogen changed their concentration as a function of fasting. Daily food pulses of 2 h entrained the rhythms of triacylglycerides and free fatty acids and restored ketone bodies and liver glycogen to similar levels as controls. Neither glucose nor proteins were affected by the food pulse. These results indicate the relevance of lipid metabolism as a phenomenon associated with the FEO.

Inhibition of N-methyl-N-nitrosourea-induced mammary carcinogenesis by molecular iodine (I2) but not by iodide (I − ) treatment Evidence that I2 prevents cancer promotion

We analyzed the effect of molecular iodine (I2), potassium iodide (KI) and a subclinical concentration of thyroxine (T4) on the induction and promotion of mammary cancer induced by N-methyl-N-nitrosourea. Virgin Sprague-Dawley rats received short or continuous treatment. Continuous I2 treated rats exhibited a strong and persistent reduction in mammary cancer incidence (30%) compared to controls (72.7%). Interruption of short or long term treatments resulted in a higher incidence in mammary cancer compared to the control groups. The protective effect of I2 was correlated with the highest expression of the I-/Cl- transporter pendrin and with the lowest levels of lipoperoxidation expression in mammary glands. Triiodothyronine serum levels and Na+/I- symporter, lactoperoxidase, or p53 expression did not show any changes. In conclusion continuous I2 treatment has a potent antineoplastic effect on the progression of mammary cancer and its effect may be related to a decrease in the oxidative cell environment.

Biochemical characterization, distribution and phylogenetic analysis of Drosophila melanogaster ryanodine and IP3 receptors, and thapsigargin-sensitive Ca2+ ATPase

We characterized the biochemistry, distribution and phylogeny of Drosophila ryanodine (RyR) and inositol triphosphate (IP3R) receptors and the endoplasmic reticulum Ca2+-ATPase (SERCA) by using binding and enzymatic assays, confocal microscopy and amino acid sequence analysis. [3H]-ryanodine binding in total membranes was enhanced by AMP-PCP, caffeine and xanthine, whereas Mg2+, Ruthenium Red and dantrolene were inhibitors. [3H]-ryanodine binding showed a bell-shaped curve with increasing free [Ca2+], without complete inhibition at millimolar levels of [Ca2+]. [3H]-IP3 binding was inhibited by heparin, 2-APB and xestospongin C. Microsomal Ca2+-ATPase activity was inhibited by thapsigargin. Confocal microscopy demonstrated abundant expression of ryanodine and inositol triphosphate receptors and abundant Ca2+-ATPase in Drosophila embryos and adults. Ryanodine receptor was expressed mainly in the digestive tract and parts of the nervous system. Maximum parsimony and Neighbour Joining were used to generate a phylogenetic classification of Drosophila ryanodine and insitol triphosphate receptors and Ca2+-ATPase based on 48 invertebrate and vertebrate complete sequences. The consensus trees indicated that Drosophila proteins grouped with proteins from other invertebrates, separately from vertebrate counterparts. Despite evolutionary distances, our functional results demonstrate that Drosophila ryanodine and inositol triphosphate receptors and Ca2+-ATPase are reasonably similar to vertebrate counterparts. Our protein expression data are consistent with the known functions of these proteins in the Drosophila digestive tract and nervous system. Overall, results show Drosophila as a valuable tool for intracellular Ca2+ dynamics studies in eukaryotes.

Lipid peroxidation, mitochondrial dysfunction and neurochemical and behavioural deficits in different neurotoxic models: Protective role of S-allylcysteine

Experimental evidence on the protective properties of S-allylcysteine (SAC) was collected from three models exerting striatal toxicity. In the first model, SAC (120mg kg−1×5) prevented lipoperoxidation (LP) and mitochondrial dysfunction (MD) in synaptosomal fractions from 1-methyl-4-phenyl-1,2,3,6-tetrahydropiridinium-treated mice (30mg kg−1), but without complete restoration of dopamine levels. In the second model, SAC (300mg kg−1×3), prevented LP and MD in synaptosomes from rats infused with 6-hydroxydopamine (8µg µl−1) into the substantia nigra pars compacta, but again, without total reversion of depleted dopamine levels. In the third model, SAC (100 mg kg−1×1) prevented MD in synaptosomes from rats injected with 3-nitropropionic acid (10 mg kg−1), but in contrast to the other models, it failed to prevent LP. SAC also prevented the aberrant motor activity patterns evoked by the three toxins. Altogether, the results suggest that the antioxidant properties of SAC are responsible for partial or total preservation of neurochemical, biochemical and behavioural markers, indicating that pro-oxidant reactions underlie the neurotoxicity in these models.

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.

Effects of Nω-nitro-L-arginine and L-arginine on quinolinic acid-induced lipid peroxidation

Abstract The effects of a nitric oxide synthase inhibitor, Nω-nitro-L-arginine (L-NARG), and a nitric oxide precursor, L-arginine (L-ARG), on the lipid peroxidation induced by quinolinic acid (QUIN, an NMDA receptor agonist), were both tested in synaptosomal fractions from whole rat brain. Baseline of lipid peroxidation was found at 2.43±0.24 fluorescence units/mg protein or 14.27±1.24 nmoles of TBARS/mg protein (100%). QUIN (100 μM)-induced lipid peroxidation in synaptosomes (256% and 166% vs. control, as measured by lipid fluorescent products and thiobarbituric acid-reactive substances, respectively) was inhibited by concentrations of 10, 40, 100, 200 and 400 μM of L-NARG (74%, 58%, 56%, 48% and 48% vs. quinolinate value, respectively). Coincubation of synaptosomes with QUIN plus L-ARG (100 μM), which alone resulted a potent pro-oxidant (277% vs. control), increased the lipoperoxidative effect induced by QUIN alone in 120% (290% vs. control). Synaptosomes simultaneously exposed to QUIN (100 μM) plus L-ARG (100 μM) plus L-NARG (200 μM) showed levels of lipid peroxidation similar to those of quinolinate alone. These findings suggest that nitric oxide may contribute to the oxidative damage induced in vitro by QUIN.

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.

Properties of the ryanodine-sensitive release channels that underlie caffeine-induced Ca2+ mobilization from intracellular stores in mammalian sympathetic neurons.

The most compelling evidence for a functional role of caffeine‐sensitive intracellular Ca2+ reservoirs in nerve cells derives from experiments on peripheral neurons. However, the properties of their ryanodine receptor calcium release channels have not been studied. This work combines single‐cell fura‐2 microfluorometry, [3 H]ryanodine binding and recording of Ca2+ release channels to examine calcium release from these intracellular stores in rat sympathetic neurons from the superior cervical ganglion. Intracellular Ca2+ measurements showed that these cells possess caffeine‐sensitive intracellular Ca2+ stores capable of releasing the equivalent of 40% of the calcium that enters through voltage‐gated calcium channels. The efficiency of caffeine in releasing Ca2+ showed a complex dependence on [Ca2+]i. Transient elevations of [Ca2+]i by 50–500 nM were facilitatory, but they became less facilitatory or depressing when [Ca2+]i reached higher levels. The caffeine‐induced Ca2+ release and its dependence on [Ca2+]i was further examined by [3 H]ryanodine binding to ganglionic microsomal membranes. These membranes showed a high‐affinity binding site for ryanodine with a dissociation constant (KD= 10 nM) similar to that previously reported for brain microsomes. However, the density of [3H]ryanodine binding sites (Bmax= 2.06 pmol/mg protein) was at least three‐fold larger than the highest reported for brain tissue. [3 H]Ryanodine binding showed a sigmoidal dependence on [Ca2+] in the range 0.1–10 μM that was further increased by caffeine. Caffeine‐dependent enhancement of [3 H]ryanodine binding increased and then decreased as [Ca2+] rose, with an optimum at [Ca2+] between 100 and 500 nM and a 50% decrease between 1 and 10 μM. At 100 μM [Ca2+], caffeine and ATP enhanced [3 H]ryanodine binding by 35 and 170% respectively, while binding was reduced by >90% with ruthenium red and MgCl2. High‐conductance (240 pS) Ca2+ release channels present in ganglionic microsomal membranes were incorporated into planar phospholipid bilayers. These channels were activated by caffeine and by micromolar concentrations of Ca2+ from the cytosolic side, and were blocked by Mg2+ and ruthenium red. Ryanodine (2 μM) slowed channel gating and elicited a long‐lasting subconductance state while 10 mM ryanodine closed the channel with infrequent opening to the subconductance level. These results show that the properties of the ryanodine receptor/Ca2+ release channels present in mammalian peripheral neurons can account for the properties of caffeine‐induced Ca2+ release. Our data also suggest that the release of Ca2+ by caffeine has a bell‐shaped dependence on Ca2+ in the physiological range of cytoplasmic [Ca2+].

A Procedure for Purification of the Ryanodine Receptor from Skeletal Muscle

In this paper, we describe a simple and reproducible method for purifying large quantities of ryanodine receptor from skeletal muscle membranes. The procedure involves the use of ion exchange chromatography and sucrose gradient centrifugation to purify the protein which has been identified as the calcium release protein of the sarcoplasmic reticulum (Imagawa, T., Smith, J., Coronado, R. and Campbell, K. (1987) J. Biol. Chem. 262:16,636-16,643). Addition of micromolar quantities of unlabeled ryanodine prior to solubilization and throughout the isolation procedure appears to stabilize the tetrameric structure of the ryanodine receptor. The purified receptor, consisting predominantly of a 400K polypeptide on SDS-PAGE, binds [3H]ryanodine with a binding affinity similar to that in membranes. Overall recovery of ryanodine binding activity was 21% of the initial activity with a 30-fold purification of the receptor.

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.