Alpha-lipoic acid preconditioning reduces ischemia-reperfusion injury of the rat liver via the PI3-kinase/Akt pathway

In liver resection and transplantation ischemia-reperfusion injury (IRI) is one of the main causes of organ dys- or nonfunction. The aim of the present study was to determine whether alpha-lipoic acid (LA) is able to attenuate IRI. Rat livers were perfused with Krebs-Henseleit buffer with or without LA (+/-wortmannin), followed by ischemia (1 h, 37 degrees C) and reperfusion (90 min). Efflux of lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP) and hepatic ATP content were determined enzymatically. Activation of NF-kappaB and activating protein 1 (AP-1) was examined by EMSA, and protein phosphorylation was examined by Western blot. Caspase-3-like activity served as an indicator for apoptotic processes. Animals treated intravenously with 500 micromol LA were subjected to 90 min of partial no-flow ischemia followed by reperfusion for up to 7 days. Preconditioning with LA significantly reduced LDH and PNP efflux during reperfusion in isolated perfused rat livers. ATP content was significantly increased in LA-treated livers. Postischemic activation of NF-kappaB and AP-1 was significantly reduced in LA-pretreated organs. Preconditioning with LA significantly enhanced Akt phosphorylation. It showed neither effect on endothelial nitric oxide synthase nor on Bad phosphorylation. Importantly, simultaneous administration of wortmannin, an inhibitor of the phosphatidylinositol (PI)3-kinase/Akt pathway, blocked the protective effect of LA on IRI, demonstrating a causal relationship between Akt activation and hepatoprotection by LA. Interestingly, despite activation of Akt, LA did not reduce postischemic apoptotic cell death. The efficacy of LA treatment in vivo was shown by reduced GST plasma levels and improved liver histology of animals pretreated with LA. This study shows for the first time that the PI3-kinase/Akt pathway plays a central protective role in IRI of the rat liver and that LA administration attenuates IRI via this pathway.     

Involvement of oxygen free radicals in the respiratory uncoupling induced by free calcium and ADP-magnesium in isolated cardiac mitochondria: Comparing reoxygenation in cultured cardiomyocytes

Recently, we have observed that the simultaneous application of free calcium (fCa) and ADP-magnesium (Mg) reduced the ADP:O ratio in isolated cardiac mitochondria. The uncoupling was prevented by cyclosporin A, an inhibitor of the permeability transition pore. The purpose of this study was to know if the generation of oxygen free radicals (OFR) is involved in this phenomenon and if it occurs during reoxygenation (Reox) of cultured cardiomyocytes. Cardiac mitochondria were harvested from male Wistar rats. Respiration was assessed in two media with different fCa concentrations (0 or 0.6 M) with palmitoylcarnitine and ADP-Mg as respiration substrates. The production of Krebs cycle intermediates (KCI) was determined. Without fCa in the medium, the mitochondria displayed a large production of citrate + isocitrate + -ketoglutarate. fCa drastically reduced these KCI and promoted the accumulation of succinate. To know if OFR are involved in the respiratory uncoupling, the effect of 4OH-TEMPO (250 M), a hydrosoluble scavenger of OFR, was tested. 4OH-TEMPO completely abolished the fCa- and ADP-Mg-induced uncoupling. Conversely, vitamin E contributed to further decreasing the ADP:O ratio. Since no hydrosoluble electron acceptor was added in our experiment, the oxygen free radical-induced oxidized vitamin E was confined near the mitochondrial membranes, which should reduce the ADP:O ratio by opening the permeability transition pore. The generation of OFR could result from the matrix accumulation of succinate. Taken together, these results indicate that mitochondrial Ca uptake induces a slight increase in membrane permeability. Thereafter, Mg enters the matrix and, in combination with Ca, stimulates the isocitrate and/or -ketoglutarate dehydrogenases. Matrix succinate favors oxygen free radical generation that further increases membrane permeability and allows respiratory uncoupling through proton leakage. To determine whether the phenomenon takes place during Reox, cultured cardiomyocytes were subjected to hypoxia and Reox. ¹⁴C-palmitate was added during Reox to determine the KCI profile. Succinate had not increased during Reox. In conclusion, calcium- and ADP-Mg-induced respiratory uncoupling is due to oxygen free radical generation through excess matrix accumulation of succinate. The phenomenon does not occur during reoxygenation because of a total restoration of mitochondrial magnesium and/or ADP concentration.     

Increased nitric oxide levels as an early sign of premature aging in diabetes

The levels of different reactive species, especially those of nitric oxide and peroxynitrite, were determined in streptozotocin-induced diabetic rat tissues, before the development of histopathological damages. Significantly higher steady state free radical concentrations were found in the liver 3 weeks after the onset of diabetes compared to age-matched control groups. Increased nitric oxide levels in diabetic vasculature and kidney decreased the production of detectable reactive oxygen species. High peroxynitrite generation suggested the onset of processes characteristic to premature aging of the endothelium. According to the histopathological results, there were no signs of late complications in the tissues up to 7 weeks after induction of diabetes. These results support the idea that oxidative stress is increased at a very early stage of diabetes and, in particular, that high levels of nitric oxide and peroxynitrite could play a decisive role in the development of late complications in the diabetic vasculature and kidney.     

Functional specialization of presynaptic Cav2.3 Ca2+ channels

Ca2+ influx into presynaptic terminals via voltage-dependent Ca2+ channels triggers fast neurotransmitter release as well as different forms of synaptic plasticity. Using electrophysiological and genetic techniques we demonstrate that presynaptic Ca2+ entry through Cav2.3 subunits contributes to the induction of mossy fiber LTP and posttetanic potentiation by brief trains of presynaptic action potentials while they do not play a role in fast synaptic transmission, paired-pulse facilitation, or frequency facilitation. This functional specialization is most likely achieved by a localization remote from the release machinery and by a Cav2.3 channel-dependent facilitation of presynaptic Ca2+ influx. Thus, the presence of Cav2.3 channels boosts the accumulation of presynaptic Ca2+ triggering presynaptic LTP and posttetanic potentiation without affecting the low release probability that is a prerequisite for the enormous plasticity displayed by mossy fiber synapses.     

Secretion of endogenous kallikreins 2 and 3 by androgen receptor-transfected PC-3 prostate cancer cells

Androgen independent PC-3 cells lack androgen receptor (AR) expression and do not produce kallikrein 2 (hK2) or 3 (prostate-specific antigen, PSA). In this paper, we examined the ability of androgens to stimulate PSA and hK2 production in AR transfected PC-3 cells (PC-3(AR)) and compared this to LNCaP cells. PSA and hK2 were measured in the culture medium and cell lysates using an ELISA-based immunofluorometric assay. Only androgens were able to induce PSA and hK2 secretion in PC-3(AR) cells in a dose- and time-dependent manner depending on the level of AR present. The level of androgen-induced PSA and hK2 secretion in PC-3(AR) cells was approximately 1.5 and 0.9% that induced in LNCaP cells, respectively. Insulin-like growth factor-I (IGF-I), which has been shown to activate AR in the absence of ligand, did not activate PSA secretion in the absence of androgen, but further increased the dihydrotestosterone-induced PSA secretion in PC-3(AR) cells. The lack of PSA and hK2 production in parental PC-3 cells is thus a result of their lack of AR expression. PSA and/or hK2 production in PC-3(AR) cells can thus serve as an endogenous reporter system to investigate AR action or to screen putative endocrine disrupters.