Abdallah Gharib

Voltammetric detection of the release of 5-hydroxyindole compounds throughout the sleep-waking cycle of the rat

SummaryIn the present work, differential pulse voltammetry (DPV) measurements of the extracellular fraction of 5-hydroxyindole compounds were performed in rats under long-term chronic conditions. In the nucleus Raphe Dorsalis (n.RD), the voltammetric signal measured at +300 mv (peak 3) disappeared completely 70 to 90 min after injection of Clorgyline (10 mg/kg), a monoamine oxidase inhibitor type A (MAOI-A); the signal measured in such conditions is thus dependent upon extracellular 5-hydroxyindoleacetic acid (5-HIAA peak 3). Deprenyl, an MAOI type B, at the same dose, induced only a slight increase in peak 3 height; according to the fact that MAO-B is selectively located in the 5-HT neurons and since their inhibition does not decrease 5-HIAA peak 3 nor the endogenous 5-HIAA content as measured with High Performance Liquid Chromatography (HPLC), 5-HIAA measured with DPV in the extracellular fluid of untreated animals might come from 5HT released and metabolized by MAO-A outside the 5-HT neurons. In animals implanted for measurements of both voltammetric and polygraphic parameters, the 5-HIAA peak 3 measured mainly in the anterior and ventral part of the n.RD exhibited large increases in its height during slow-wave sleep (SWS: +39%) and paradoxical sleep (PS=+71%) as compared to the waking state (W=100%); these variations could reflect the dendritic release of 5-HT. In the Caudate nucleus (n.Cd) the same voltammetric signal presented reverse fluctuations, i.e. an increase during W and a decrease during SWS and PS. Intracerebroventricular administration of Corticotropin-Like Intermediate lobe Peptide (CLIP, 10 ng/2 μl) induced an increase in PS duration (+51%) preceded and accompanied by an increase in the n.RD 5-HIAA peak 3 height (+50%).

Inhibition of complex I regulates the mitochondrial permeability transition through a phosphate-sensitive inhibitory site masked by cyclophilin D

Inhibition of the mitochondrial permeability transition pore (PTP) has proved to be an effective strategy for preventing oxidative stress-induced cell death, and the pore represents a viable cellular target for drugs. Here, we report that inhibition of complex I by rotenone is more effective at PTP inhibition than cyclosporin A in tissues that express low levels of the cyclosporin A mitochondrial target, cyclophilin D; and, conversely, that tissues in which rotenone does not affect the PTP are characterized by high levels of expression of cyclophilin D and sensitivity to cyclosporin A. Consistent with a regulatory role of complex I in the PTP-inhibiting effects of rotenone, the concentrations of the latter required for PTP inhibition precisely match those required to inhibit respiration; and a similar effect is seen with the antidiabetic drug metformin, which partially inhibits complex I. Remarkably (i) genetic ablation of cyclophilin D or its displacement with cyclosporin A restored PTP inhibition by rotenone in tissues that are otherwise resistant to its effects; and (ii) rotenone did not inhibit the PTP unless phosphate was present, in striking analogy with the phosphate requirement for the inhibitory effects of cyclosporin A [Basso et al. (2008) J. Biol. Chem. 283, 26307-26311]. These results indicate that inhibition of complex I by rotenone or metformin and displacement of cyclophilin D by cyclosporin A affect the PTP through a common mechanism; and that cells can modulate their PTP response to complex I inhibition by modifying the expression of cyclophilin D, a finding that has major implications for pore modulation in vivo.

p53-PGC-1α Pathway Mediates Oxidative Mitochondrial Damage and Cardiomyocyte Necrosis Induced by Monoamine Oxidase-A Upregulation: Role in Chronic Left Ventricular Dysfunction in Mice

AIMS Oxidative stress and mitochondrial dysfunction participate together in the development of heart failure (HF). mRNA levels of monoamine oxidase-A (MAO-A), a mitochondrial enzyme that produces hydrogen peroxide (H(2)O(2)), increase in several models of cardiomyopathies. Therefore, we hypothesized that an increase in cardiac MAO-A could cause oxidative stress and mitochondrial damage, leading to cardiac dysfunction. In the present study, we evaluated the consequences of cardiac MAO-A augmentation on chronic oxidative damage, cardiomyocyte survival, and heart function, and identified the intracellular pathways involved. RESULTS We generated transgenic (Tg) mice with cardiac-specific MAO-A overexpression. Tg mice displayed cardiac MAO-A activity levels similar to those found in HF and aging. As expected, Tg mice showed a significant decrease in the cardiac amounts of the MAO-A substrates serotonin and norepinephrine. This was associated with enhanced H(2)O(2) generation in situ and mitochondrial DNA oxidation. As a consequence, MAO-A Tg mice demonstrated progressive loss of cardiomyocytes by necrosis and ventricular failure, which were prevented by chronic treatment with the MAO-A inhibitor clorgyline and the antioxidant N-acetyl-cystein. Interestingly, Tg hearts exhibited p53 accumulation and downregulation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a master regulator of mitochondrial function. This was concomitant with cardiac mitochondrial ultrastructural defects and ATP depletion. In vitro, MAO-A adenovirus transduction of neonatal cardiomyocytes mimicked the results in MAO-A Tg mice, triggering oxidative stress-dependent p53 activation, leading to PGC-1α downregulation, mitochondrial impairment, and cardiomyocyte necrosis. INNOVATION AND CONCLUSION We provide the first evidence that MAO-A upregulation in the heart causes oxidative mitochondrial damage, p53-dependent repression of PGC-1α, cardiomyocyte necrosis, and chronic ventricular dysfunction.

Detection of the release of 5-hydroxyindole compounds in the hypothalamus and the n. raphe dorsalis throughout the sleep-waking cycle and during stressful situations in the rat: a polygraphic and voltammetric approach

SummaryIn the present work, voltammetric method combined with polygraphic recordings were used in animals under long-term chronic conditions; the extracellular concentrations of 5-hydroxyindole compounds (5-OHles) and in particular 5-hydroxyindoleacetic acid (5-HIAA) were measured in the hypothalamus and in the nucleus Raphe Dorsalis (n.RD). The hypothesis that extracellular detection of 5-HIAA, in animals under physiological conditions, might reflect serotonin (5-HT) release is suggested by the following observations: — serotoninergic neurons are reported to contain only monoamine oxidase type B (MAO-B); — an inhibitor of such an enzyme, MDL 72145 (1 mg/kg), fails to decrease the extracellular 5-HIAA peak 3 height; — MAO type A is contained in non-5-HT cells or neurons; — only the inhibitor of this last type of enzyme (Clorgyline 2.5 mg/kg) induces a complete disappearance of the voltammetric signal. The 5-HIAA measured in the extracellular space thus comes from the 5-HT released and metabolized outside the 5-HT neurons. Throughout the sleep-waking cycle, 5-OHles release occurs following two different modes: 1 — during sleep, in the vicinity of the 5-HT cellular bodies in the n.RD; this release might come from dendrites and be responsible for the 5-HT neuronal inhibition occurring during sleep; 2 — during waking, at the level of the axonal nerve endings impinging on the hypothalamus; this release might be related to the synthesis of “hypnogenic factors”. Finally, we have observed that in the hypothalamus, 30 min. of immobilization-stress (IS) induces a larger increase of the voltammetric signal (+ 80%) than a painful stimulation of the same duration (+ 30%); the possible link between the 5-OHles release occurring in this area during an IS and the subsequent paradoxical sleep rebound is discussed.

Inhaled Nitric Oxide Reduces Brain Damage by Collateral Recruitment in a Neonatal Stroke Model

Background and Purpose— We recently demonstrated that endogenous nitric oxide (NO) modulates collateral blood flow in a neonatal stroke model in rats. The inhalation of NO (iNO) has been found to be neuroprotective after ischemic brain damage in adults. Our objective was to examine whether iNO could modify cerebral blood flow during ischemia–reperfusion and reduce lesions in the developing brain. Methods— In vivo variations in cortical NO concentrations occurring after 20-ppm iNO exposure were analyzed using the voltammetric method in P7 rat pups. Inhaled NO-mediated blood flow velocities were measured by ultrasound imaging with sequential Doppler recordings in both internal carotid arteries and the basilar trunk under basal conditions and in a neonatal model of ischemia–reperfusion. The hemodynamic effects of iNO (5 to 80 ppm) were correlated with brain injury 48 hours after reperfusion. Results— Inhaled NO (20 ppm) significantly increased NO concentrations in the P7 rat cortex and compensated for the blockade of endogenous NO synthesis under normal conditions. Inhaled NO (20 ppm) during ischemia increased blood flow velocities and significantly reduced lesion volumes by 43% and cellular damage. In contrast, both 80 ppm iNO given during ischemia and 5 or 20 ppm iNO given 30 minutes after reperfusion were detrimental. Conclusions— Our findings strongly indicate that, with the appropriate timing, 20 ppm iNO can be transported into the P7 rat brain and mediated blood flow redistribution during ischemia leading to reduced infarct volume and cell injury.

CHANGES IN THE SLEEP-WAKE CYCLE ARCHITECTURE AND CORTICAL NITRIC OXIDE RELEASE DURING AGEING IN THE RAT

Changes in sleep-wake states and nitric oxide release were examined in aged rats versus young-adult ones. Sleep-wake recordings and nitric oxide measurements were taken from animals chronically equipped with polygraphic and voltametric electrodes. Animals were examined in baseline conditions and in response to a 24-hour paradoxical sleep deprivation. In aged rats, basal amount of paradoxical sleep is decreased during the light phase versus young-adult animals. After paradoxical sleep deprivation, a paradoxical sleep rebound occurs with an amount and intensity that are less marked in aged animals than in young-adult rats. The amplitude of the circadian distribution for wakefulness, slow-wave sleep and paradoxical sleep amounts is reduced with age. Finally, delta-slow-wave sleep and theta-paradoxical sleep power spectra are attenuated either in baseline conditions or after paradoxical sleep deprivation in aged animals. It is also reported that cortical nitric oxide release exhibits a circadian rhythm with higher amplitude in aged rats than in young-adult ones. However, after paradoxical sleep deprivation, a limited overproduction of nitric oxide is obtained compared with young-adult ones. These results, evidencing the dynamics of the nitric oxide changes occurring in relation to the sleep-wake cycle, point out the homeostatic paradoxical sleep regulation as an age-dependent process in which the nitric oxide molecule is possibly involved.

Biochemical and autoradiographic measurements of brain serotonin synthesis rate in the freely moving rat: a reexamination of the alpha-methyl-L-tryptophan method.

Abstract: Biochemical approaches were used in freely moving rats to determine, under steady‐state conditions, the brain/arterial plasma partition coefficients of L‐tryptophan and α‐[3H]methyl‐L‐tryptophan, from which the lumped constant for the α‐methyl‐L‐tryptophan method of estimating the rate of brain serotonin synthesis is calculated. The lumped constants were significantly different in the various structures examined: 0.149 ± 0.003 in the raphe dorsalis, 0.103 ± 0.002 in the raphe centralis, 0.087 ± 0.003 in the reticular formation, and 0.62 ± 0.08 in the pineal gland. From these data we proposed a two‐compartment model to calculate the rate of serotonin synthesis by quantitative autoradiography using a three‐time point experiment. Rates of synthesis for the raphe dorsalis and the reticular formation (620 ± 57 and 80 ± 35 pmol/g of tissue/min, respectively) were similar to those measured simultaneously by biochemical means, but rates were 50% higher for the raphe centralis (568 ± 90 vs. 381 ± 31 pmol/g of tissue/min). The lack of dynamic equilibrium of the tracer between plasma and tissue pools may explain the discrepancy between the two methods. Our findings did not confirm previous data, indicating that the application of the autoradiographic method to measure the rate of brain serotonin synthesis using α‐methyl‐L‐tryptophan as tracer has limitations.

Inhibition of myocardial reperfusion injury by ischemic postconditioning requires sirtuin 3-mediated deacetylation of cyclophilin D

RATIONALE How ischemic postconditioning can inhibit opening of the mitochondrial permeability transition pore (PTP) and subsequent cardiac myocytes death at reperfusion remains unknown. Recent studies have suggested that de-acetylation of cyclophilin D (CyPD) by sirtuin 3 (SIRT3) can modulate its binding to the PTP. OBJECTIVE The aim of the present study was to examine whether ischemic postconditioning (PostC) might activate SIRT3 and consequently prevent lethal myocardial reperfusion injury through a deacetylation of CyPD. METHODS AND RESULTS Using hypoxia-reoxygenation (H/R) in H9C2 cells, we showed that SIRT3 overexpression prevented CyPD acetylation, limited PTP opening and reduced cell death by 24%. In vitro modification of the CyPD acetylation status in MEFs by site-directed mutagenesis altered capacity of PTP opening by calcium. Calcium Retention Capacity (CRC) was significantly decreased with CyPD-KQ that mimics acetylated protein compared with CyPD WT (871 ± 266 vs 1193 ± 263 nmoles Ca(2+)/mg protein respectively). Cells expressing non-acetylable CyPD mutant (CyPD-KR) displayed 20% decrease in cell death compared to cells expressing CyPD WT after H/R. Correspondingly, in mice we showed that cardiac ischemic postconditioning could not reduce infarct size and CyPD acetylation in SIRT3 KO mice, and was unable to restore CRC in mitochondria as it is observed in WT mice. CONCLUSIONS Our study suggests that the increased acetylation of CyPD following myocardial ischemia-reperfusion facilitates PTP opening and subsequent cell death. Therefore ischemic postconditioning might prevent lethal reperfusion injury through an increased SIRT3 activity and subsequent attenuation of CyPD acetylation at reperfusion.

Regional age-related changes in neuronal nitric oxide synthase (nNOS), messenger RNA levels and activity in SAMP8 brain

BackgroundNitric oxide (NO) is a multifunctional molecule synthesized by three isozymes of the NO synthase (NOSs) acting as a messenger/modulator and/or a potential neurotoxin. In rodents, the role of NOSs in sleep processes and throughout aging is now well established. For example, sleep parameters are highly deteriorated in senescence accelerated-prone 8 (SAMP8) mice, a useful animal model to study aging or age-associated disorders, while the inducible form of NOS (iNOS) is down-regulated within the cortex and the sleep-structures of the brainstem. Evidence is now increasing for a role of iNOS and resulting oxidative stress but not for the constitutive expressed isozyme (nNOS). To better understand the role of nNOS in the behavioural impairments observed in SAMP8 versus SAMR1 (control) animals, we evaluated age-related variations occurring in the nNOS expression and activity and nitrites/nitrates (NOx-) levels, in three brain areas (n = 7 animals in each group). Calibrated reverse transcriptase (RT) and real-time polymerase chain reaction (PCR) and biochemical procedures were used.ResultsWe found that the levels of nNOS mRNA decreased in the cortex and the hippocampus of 8- vs 2-month-old animals followed by an increase in 12-vs 8-month-old animals in both strains. In the brainstem, levels of nNOS mRNA decreased in an age-dependent manner in SAMP8, but not in SAMR1. Regional age-related changes were also observed in nNOS activity. Moreover, nNOS activity in hippocampus was found lower in 8-month-old SAMP8 than in SAMR1, while in the cortex and the brainstem, nNOS activities increased at 8 months and afterward decreased with age in SAMP8 and SAMR1. NOx- levels showed profiles similar to nNOS activities in the cortex and the brainstem but were undetectable in the hippocampus of SAMP8 and SAMR1. Finally, NOx- levels were higher in the cortex of 8 month-old SAMP8 than in age-matched SAMR1.ConclusionConcomitant variations occurring in NO levels derived from nNOS and iNOS at an early age constitute a major factor of risk for sleep and/or memory impairments in SAMP8.

Brain Protein Synthesis in the Conscious Rat Using L-[35S]Methionine: Relationship of Methionine Specific Activity Between Plasma and Precursor Compartment and Evaluation of Methionine Metabolic Pathways

Abstract: The method previously developed for the measurement of rates of methionine incorporation into brain proteins assumed that methionine derived from protein degradation did not recycle into the precursor pool for protein synthesis and that the metabolism of methionine via the transmethylation pathway was negligible. To evaluate the degree of recycling, we have compared, under steady‐state conditions, the specific activity of L‐[35S]methionine in the tRNA‐bound pool to that of plasma. The relative contribution of methionine from protein degradation to the precursor pool was 26%. Under the same conditions, the relative rate of methionine flux into the transmethylation cycle was estimated to be 10% of the rate of methionine incorporation into brain proteins. These results indicate the following: (a) there is significant recycling of unlabeled methionine derived from protein degradation in brain; and (b) the metabolism of methionine is directed mainly towards protein synthesis. At normal plasma amino acid levels, methionine is the amino acid which, to date, presents the lowest degree of dilution in the precursor pool for protein synthesis. L‐[35S]‐Methionine, therefore, presents radiobiochemical properties required to measure, with minimal underestimation, rates of brain protein synthesis in vivo.