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International audience; Long-term consumption of tobacco by smokers causes addiction and increases the level of neuronal nicotinic acetylcholine receptors (nAChRs) in the brain, a phenomenon known as up-regulation. Here, we show that up-regulation of specific nAChR subunits takes place in white blood cells (WBCs) of smokers and mice subjected to long-term administration of nicotine. The basal level of alpha-bungarotoxin binding site, which corresponds to the homomeric alpha7 nAChR subtype, was not affected in WBCs of both smokers and mice administered nicotine. In contrast, epibatidine (EB) binding sites, which correspond to heteromeric nAChR subtypes, were detected in WBCs of smokers but not in WBCs of nonsmokers. The number of EB binding sites significantly decreased after incubation of the smokers' WBCs for 3 days in nicotine-free culture medium. In WBCs of wild-type mice, basal level of EB binding sites was detected before nicotine administration. This basal level is reduced by approximately 60% in knockout mice lacking the genes encoding either the beta2 or the alpha4 receptor subunits. Additional analysis of knockout mice revealed that the remaining approximately 40% do not undergo up-regulation, indicating that the alpha4/beta2 subunits comprise the up-regulated nAChRs. We further found that upregulation in mouse WBCs is accompanied by a significant decrease in the capacity of the up-regulated receptor channels to convey calcium ions. The phenomenon of nAChR up-regulation in WBCs provides a simple tool to evaluate and study tobacco addiction.

Background. The renal medulla is particularly sensitive to oxidant stress and to ischaemia-reperfusion injury (IRI). In organ transplantation, delayed graft function is an important problem and cold ischaemia is thought to be the most important factor in short- and long-term complications. Our aim was to study cold-induced damage in proximal tubular segments and renal medulla osmolite excretion during use of various preservation solutions, and to clarify the role of trimetazidine (TMZ) in limiting renal dysfunction. Methods. Using an autotransplanted pig kidney model, we assessed renal tubule function, medullary osmolite excretion and renal damage between day 1 and week 2 after 24 or 48 h cold storage in University of Wisconsin solution (UW), Celsior and ECPEG (two new high Na + preservation solutions) or the Hopital Edouard Herriot solution (HEH; a high Na + version of UW). In additional groups, TMZ was added to these preservation solutions for 24 and 48 h cold storage. Results. Renal function was reduced under these preservation conditions. Tubular injury was associated with aminoaciduria and with a limited Na + reabsorbtion. Medullary damage led to the early appearance of trimethylamine-N-oxide and dimethylamine in urine. However, renal damage was modulated by preservation conditions. In addition, TMZ added to each of the solutions efficiently protected against IRI even after prolonged preservation. Conclusion. TMZ efficiently protected kidneys against damage when added to the HEH and particularly ECPEG solutions, even after 24 h cold storage. These findings point to a role for drugs that target mitochondria, and demonstrate that TMZ may provide a valuable therapeutic tool against IRI and could be included in therapeutic protocols.

International audience; The binding profile of [3H]BHDP [3H]N-benzyl-N'-(2-hydroxy-3,4-dimethoxybenzyl)-piperazine) was evaluated. [3H]BHDP labelled a single class of binding sites with high affinity (Kd = 2-3 nM) in rat liver mitochondria and synaptic membranes. The pharmacological characterization of these sites using δ reference compounds revealed that these sites are δ receptors and, more particularly, δ1 receptors. Indeed, BHDP inhibited [3H]pentazocine binding, a marker for δ1 receptors, with high affinity in a competitive manner. BHDP is selective for δ1 receptors since it did not show any relevant affinity for most of the other receptors, ion channels or transporters tested. Moreover, in an in vitro model of cellular hypoxia, BHDP prevented the fall in adenosine triphosphate (ATP) levels caused by 24 h hypoxia in cultured astrocytes. Taken together, these results demonstrate that [3H]BHDP is a potent and selective ligand for δ1 receptors showing cytoprotective effects in astrocytes.

In this study, we investigated the effects of nicotine on rat brain mitochondria. The polarographic studies determined the effects on the respiratory chain, whereas enzymatic assays and [3H]-nicotine binding allowed us to precisely identify its target and site of action. The measurements of oxygen consumption showed a significantly concentration-dependent inhibition by nicotine (EC50 was 4.95x10(-11) M), and a maximal decrease of 23.90% at 10(-7) M. Nicotine bound to complex I of the respiratory chain and inhibited the NADH-Ubiquinone reductase activity. We also showed that nicotine and NADH were competitive on complex I. Effects of cotinine, the main nicotine metabolite, and nornicotine, were also investigated: nornicotine inhibited the mitochondrial respiration whereas cotinine did not. Because the complex I generates superoxide anion, we investigated the effects of nicotine, following NBT oxidation, and showed that nicotine was able to inhibit this reactive oxygen species (ROS) generation by 15.74% with an EC50 of 2.02x10(-11) M. In conclusion, the present study shows that nicotine interacts with the complex I of brain mitochondrial respiratory chain and decreases ROS generation. This may explain a part of the beneficial and protective effects of nicotine in few neurodegenerative diseases, as suggested by many epidemiological studies.

The effects of hydrocortisone, triamcinolone, prednisolone and dexamethasone have been investigated in vitro using mitochondria isolated from rat brain. Respiratory control ratio (RCR), oxygen consumption, ATP synthesis, enzymatic activities of involved complexes and superoxide anion generation have been measured to assess the effects of these drugs. Our data showed that the decrease of RCR induced by glucocorticoids was due to a common inhibition of oxidative phosphorylation (State 3) and of complex V activity and a modification of the proton-fluxes through the mitochondrial inner membrane. These effects were quantitatively limited, since they occurred at concentrations lower than 2 nM. Dexamethasone was the only one able to induce a specific inhibition of complex I activity and to decrease the superoxide anion radical generation. Inhibition of complex V and partial reversion of uncoupling seem to be common properties of glucocorticoids. The theoretical consequence of these inhibitions could be the modulation of the mitochondrial function, oxygen consumption rate, ATP synthase activity and superoxide anion radical production, involved in many patho-physiological phenomena.

The effects of tacrolimus (FK 506) on brain phosphorylation have been investigated in vitro using mitochondria isolated from rat brain. Respiratory control ratio (RCR), oxygen consumption, ATP synthesis and enzymatic activities of involved complexes have been measured to assess the mechanisms of action of tacrolimus. Our data show that this drug decreases RCR and ATP synthesis. This effect is quantitatively limited after a single application of the drug (14%), concentration-dependent and biphasic, the respective effect 50%-concentration (EC50) being 0.129 and 247 nM, each step corresponding to 50% of the total oxygen consumption inhibition. Tacrolimus acts mainly as an inhibitor of ubiquinol-cytochrome c reductase (complex III), competing at least partly with antimycin A or myxothiazol, the corresponding EC50 being 0.27 and 103 nM respectively. Tacrolimus inhibits also complex V i.e. ATPase activity (40%) and ATP synthase activity (30%) in a concentration-dependent manner, the relevant EC50 being 78 and 394 nM respectively. These data may be relevant for the protective effect of tacrolimus observed in ischemia-reperfusion, which may be due to its inhibition of both complex III, where Reactive Oxygen Species (ROS) are generated, and complex V, where ATP is depleted by ATPase activation. It may also be related to neurotoxicity occurring along chronic administration of tacrolimus in humans.

IN RENAL transplantation, delayed graft function remains a major problem and increases the posttransplant morbidity and hospital costs. Trimetazidine (TMZ), an antiischemic drug, was introduced several years ago and used as an antianginal agent whose main effect is to be devoid of hemodynamic effects. TMZ is a cytoprotective drug that counteracts the metabolic disorders occurring at the level of ischemic cells. Several experimental results have suggested direct actions of the drug on mitochondrial function, intracellular acidosis, and calcium intracellular accumulation. 1,2 The aim of this study was to assess beneficial effect of TMZ during cold storage (CS) with Euro-Collins solution (EC) and normothermic reperfusion on energetic status of the kidneys.

Eight days' desipramine administration (16 mg/kg per day i.p.) to rats resulted in a significant decrease in the density of beta-adrenoceptors in neuronal and astroglial cells from rat forebrain and cerebellum without modification of their corresponding affinity. beta-Adrenoceptor subtypes, beta 1 and beta 2, which coexist in neurons and astrocytes, are differently distributed in the brain and differently modified by desipramine administration which down-regulates beta 1-adrenoceptor in forebrain neurons and astrocytes and beta 2-adrenoceptor in cerebellum neurons. This down-regulation affects the predominant subtype, beta 1 or beta 2, of the relevant structure. Astroglial and neuronal beta-adrenoceptors are differently coupled to G-proteins. Only neuronal cells contain the high-affinity conformational state of the beta-adrenoceptors which is sensitive to GTP. The percentage of neuronal receptors in the high-affinity state differs according to brain area. Desipramine treatment decreases the neuronal density of both cerebellar high- and low-affinity sites and only the forebrain high-affinity site. The desipramine effects are thus subtype-dependent and differ between the two brain areas selected.