Binding characteristics of SSR180575, a potent and selective peripheral benzodiazepine ligand.

The peripheral benzodiazepine receptor (PBR), has been recently shown to play a key role in the regulation of the mitochondrial process leading to apoptosis, which occurs during cardiac ischemia. The present work shows that SSR180575, a novel PBR ligand of potential interest in pathological cardiovascular indications, irreversibly and specifically binds with high affinity on both rat heart mitochondria and on a cell line transfected with the human PBR (K(d)=1.95+/-0.22 and 4.58+/-0.83nM, respectively). In conclusion, SSR180575 is a specific and potent PBR ligand which irreversible binding to PBR appears of high interest in various therapeutic indications where apoptosis occurs.

Liposomes as fatty acids carriers in isolated rat liver: effect on energy metabolism and on isolated mitochondria activity.

The effects of fatty acids (FA)-carrier, egg-lecithin liposomes (LIPO) as alternative to BSA, on ATP, glycogen and glucose contents in isolated perfused liver of fed rats were non-invasively studied using 31P/13C nuclear magnetic resonance (NMR). Oxidative phosphorylation was studied in isolated mitochondria from the same liver consecutively to the NMR experiments. ATP content decreased slowly and ATP turnover was similar during the perfusion with saline solution (KHB) or LIPO. However, LIPO induced an enhancement of respiratory control ratio in isolated mitochondria. Tissue glycogen and glucose content decreased when FA (linoleate or linolenate) were perfused with defatted BSA (3%) or LIPO (600 mg/l) whereas glucose excretion level was unchanged and lactate excretion tended to increase, reflecting changes in the cytosolic redox state and/or an enhancement of glycolysis. Addition of FA (0.5 or 1.5 mM) to LIPO caused a dramatic fall in liver ATP, a mitochondrial uncoupling and an impairment of the phosphorylation activity. Perfusion with FA (1.5 mM) carried by BSA significantly increased the ATP degradation without change of mitochondrial function. Owing to the higher affinity of BSA than LIPO for FA, these latter could be more easily released from complex LIPO-FA, increasing their uncoupling effect. Hence, the FA concentrations have to be largely decreased from the above currently used concentrations to avoid this effect. It will then be possible to minimize the effector action of FA and to study their more specific metabolic function as fuel. It was concluded that LIPO were appropriate carriers to study the different metabolic effects of FA.

Mitochondrial impairment and recovery after heat shock treatment in a human microglial cell line.

The application of a heat shock on the human microglial cell line (CHME 5) has been shown to cause cytoskeleton modifications and alterations in phosphorylated metabolite content (Macouillard-Poulletier de Gannes et al., 1998a Metabolic and cellular characterization of immortalized human microglial cells under heat stress. Neurochem. Int. 33, 61-73). In this study, we focused on the possible involvement of mitochondria in this heat stress response. The cell respiratory properties were followed during the recovering period and the possible relationships between mitochondria and the cytoskeleton were studied. We observed that the heat shock induced changes in mitochondrial activity due to protein denaturation, rather than mitochondrial loss. Furthermore, these alterations were correlated with cytoskeleton disorganization since vimentine, tubuline and mitochondria shift, simultaneously, to a perinuclear location. The perturbations of the mitochondrial distribution persisted until cytoskeleton networks had recovered. Nevertheless, the respiratory properties recovered rapidly suggesting a renaturation of mitochondrial proteins in connection with mitochondrial cytoplasmic redistribution.

Early transitory rise in intracellular pH leads to Bax conformation change during ceramide-induced apoptosis.

Ceramide can induce apoptosis through a caspase independent pathway. Bax has been described as able to kill cells in the absence of caspase activity, therefore we measured Bax in situ during ceramide-induced apoptosis using anti-Bax antibodies and flow cytometry analysis. An early (<30 min) increase in Bax labeling was observed after the addition of several ceramide species to several hemopoietic-related cell types. On U937, this increase was not due to antigens synthesis or processing, but rather an increased accessibility or reactivity of Bax antigens for antibodies. This increased immuno-reactivity of Bax was not inhibited by Z-VAD-fmk nor leupeptin, and preceded nuclear fragmentation by several hours. Such an increase in immuno-reactivity was also observed after Fas ligation, but it occurred later (>2 h) accompanying nuclear apoptosis, and was inhibited by Z-VAD-fmk. Bax immuno-reactivity was found to be related to intracellular pH (pHi), and C2-Ceramide (C2-Cer) induced a very early (<10 min) transitory increase in pHi. Both Bax immunoreactivity and pHi increases were dependent on the mitochondrial permeability transition pore (PTP) status. It was concluded from these results that C2-Cer induced a transitory increase in pHi in relation to the PTP. This rise in pHi led to conformational changes in Bax which could be responsible for further apoptosis in the C2-Cer pathway while it was a consequence of caspase activation in the Fas pathway.

Flow cytometric analysis of mitochondrial activity in situ: application to acetylceramide-induced mitochondrial swelling and apoptosis.

Changes in mitochondrial matrix volume were studied both on isolated mitochondria and in situ on CHME 5 human microglia and monoblastoid U 937 cells using multiparametric flow cytometric analysis. The use of specific effectors of mitochondrial activity (oligomycin and KCN) allowed the demonstration, on whole cells, of a strict correlation between light scattering and mitochondrial volume changes: mitochondrial swelling induced a concomitant increase in forward scattering, and decrease in side scattering of the cell population. The technique was applied to the study of the early phases of acetyl-ceramide-induced apoptosis, which has been associated with mitochondrial dysfunction in several cellular systems. Acetyl-ceramide caused a marked swelling of isolated rat liver mitochondria. Scatter modifications were also observed in both cell lines during the first hour of incubation with acetylceramide and were accompanied by an increase in DiOC6 (3) fluorescence. The results imply that mitochondrial volume changes can be followed using flow cytometry and eventually used to assist in the interpretation of mitochondrial membrane potential variations obtained from fluorescence measurements. By applying this technique to 2 different cell lines, we demonstrated that mitochondrial swelling occurs during the early phases of acetyl-ceramide treatment, but that the induction of apoptosis is cell type-dependent.

Mitochondrial permeability transition during hypothermic to normothermic reperfusion in rat liver demonstrated by the protective effect of cyclosporin A.

The purpose of this study was to test the hypothesis that mitochondrial permeability transition might be implicated in mitochondrial and intact organ dysfunctions associated with damage induced by reperfusion after cold ischaemia. Energetic metabolism was assessed continuously by 31P-NMR on a model system of isolated perfused rat liver; mitochondria were extracted from the livers and studied by using top-down control analysis. During the temperature transition from hypothermic to normothermic perfusion (from 4 to 37 degrees C) the ATP content of the perfused organ fell rapidly, and top-down metabolic control analysis of damaged mitochondria revealed a specific control pattern characterized by a dysfunction of the phosphorylation subsystem leading to a decreased response to cellular ATP demand. Both dysfunctions were fully prevented by cyclosporin A, a specific inhibitor of the mitochondrial transition pore (MTP). These results strongly suggest the involvement of the opening of MTP in vivo during the transition to normothermia on rat liver mitochondrial function and organ energetics.

Use of spin-traps during warm ischemia-reperfusion in rat liver: comparative effect on energetic metabolism studied using 31P nuclear magnetic resonance.

Detection of free radicals by electron spin resonance (ESR) proves the involvement of reactive oxygen species (ROS) in reperfused organ injuries. Spin-traps are known to ameliorate hemodynamic parameters in an isolated postischemic heart. The effects of 5 mmol/L DMPO (5,5-dimethyl-1-pyrroline-N-oxide) or DEPMPO (5-(diethlphosphoryl)-5-methyl-1-pyrroline N-oxide) on intracellular pH (pHin) and ATP level were evaluated by 31P nuclear magnetic resonance on isolated rat liver submitted to 1 hour of warm ischemia and reperfusion. At the end of the reperfusion period, during which pHin recovered to its initial value (7.16 +/- 0.03) in all groups, the ATP recovery level (expressed in percentage of initial value) was similar in controls and DEPMPO (60% +/- 5%, n = 6 and 54% +/- 4%, n = 6, respectively), but only 37% +/- 1% in DMPO-treated livers (n = 6) (p < 0.05 versus controls and p < 0.05 versus DEPMPO). Oxidative phosphorylation was not affected by an addition of nitrones on isolated mitochondria extracted from livers not submitted to ischemia-reperfusion. In contrast, mitochondria extracted at the end of the ischemia-reperfusion showed an impairment in the phosphorylation parameters, particularly in the presence of DMPO. Mass spectrum of ischemic liver perchloric acid extracts evidenced probable catabolites in treated groups. The differences in the effect of the two nitrones on energetic metabolism may be explained by the production of deleterious catabolites by DMPO as compared to DEPMPO. Even though a specific radical scavenging effect could be operative in the liver, our results indicate that catabolic effects were predominant. The absence of deleterious effects of DEPMPO in contrast to DMPO on the liver energetic metabolism was evidenced, allowing the use of DEPMPO for ESR detection.