H(2)O(2) disposal in cardiolipin-enriched brain mitochondria is due to increased cytochrome c peroxidase activity.

The mitochondrial electron transport chain is a source of oxygen superoxide anion (O(2)(-)) that is dismutated to H(2)O(2). Although low levels of ROS are physiologically synthesized during respiration, their increase contributes to cell injury. Therefore, an efficient machinery for H(2)O(2) disposal is essential in mitochondria. In this study, the ability of brain mitochondria to acquire cardiolipin (CL), phosphatidylglycerol (PG), and phosphatidylserine (PS) in vitro through a fusion process was exploited to investigate lipid effects on ROS. MTT assay, oxygen consumption, and respiratory ratio indicated that the acquired phospholipids did not alter mitochondrial respiration and O(2)(-) production from succinate. However, in CL-enriched mitochondria, H(2)O(2) levels where 27% and 47% of control in the absence and in the presence of antimycin A, respectively, suggesting an increase in H(2)O(2) elimination. Concomitantly, cytochrome c (cyt c) was released outside mitochondria. Since free oxidized cyt c acquired peroxidase activity towards H(2)O(2) upon interaction with CL in vitro, a contribution of cyt c to H(2)O(2) disposal in mitochondria through CL conferred peroxidase activity is plausible. In this model, the accompanying CL peroxidation should weaken cyt c-CL interactions, favouring the detachment and release of the protein. Neither cyt c peroxidase activity was elicited by PS in vitro, nor cyt c release was observed in PS-enriched mitochondria, although H(2)O(2) levels were significantly decreased, suggesting a cyt c-independent role of PS in ROS metabolism in mitochondria.

Cytochrome c redox state influences the binding and release of cytochrome c in model membranes and in brain mitochondria.

Cytochrome c (cyt c), a component of the respiratory chain, promotes apoptosis when released into the cytosol. Cyt c anchorage within mitochondria depends on cardiolipin (CL). Detachment and release have been related to CL loss and peroxidation. We report that NaN(3)-dependent complex IV inhibition, accompanied by impairment of respiration, resulted in cyt c release. Contrarily, inhibition of respiration upstream cyt c with complex I and III inhibitors was not accompanied by the release of the protein, despite CL decrease and monolyso-CL increase. No CL changes and H(2)O(2) formation were observed by inhibiting complex IV. In cyt c-CL liposomes, breaching cyt c-CL hydrophilic interactions produced a higher release of the reduced, compared to the oxidized form, suggesting that the hydrophobic component of cyt c-CL binding is prevalent in the oxidized form. Free or liposome-reconstituted cyt c was able to form fatty acid-protein complexes (palmitate < linoleate < oleate) only in its reduced form. We hypothesize that reduced cyt c-fatty acid binding favors the dislocation of the protein from anchoring CL. A mechanism for cyt c release independent of CL peroxidation by H(2)O(2) is feasible. It could weaken the hydrophobic component of cyt c-CL interactions and might function following complex IV inhibition or in oxygen lack, both conditions producing accumulation of reduced cyt c and free fatty acids.

Activation of PAF-synthesizing enzymes in rat brain stem slices after LTP induction in the medial vestibular nuclei.

LysoPAF acetyltransferase (lysoPAF-AT) and PAF-synthesizing phosphocholinetransferase (PAF-PCT) are the two enzymes which catalyze the final reactions for the synthesis of PAF. Their activities, assayed in the homogenate of rat brain stem slices and under their optimal conditions, increased 5 min after high frequency stimulation of vestibular afferents, inducing LTP in the medial vestibular nuclei. The activity of phosphatidylcholine-synthesizing phosphocholinetransferase, was not affected. Sixty minutes from the induction of LTP, PAF-PCT activity, but not that of lysoPAF-AT, was still significantly higher with respect to 5 min test stimulated control. We used AP-5 to verify whether this increase was strictly dependent upon LTP induction, which requires NMDA receptor activation. In AP-5 treated slices, lysoPAF-acetyltransferase and PAF-synthesizing phosphocholinetransferase activities increased, but they were reduced after high frequency stimulation under AP-5. In conclusion, we have demonstrated that the activities of PAF-synthesizing enzymes are activated soon after the induction of LTP and that this effect is linked to the activation of NMDA-receptors. We suggest that the enzyme activation by AP-5, preventing LTP, might be due to glutamate enhancement but, in neurons showing LTP and under normal conditions, the activation of potentiation mechanisms is critical for the enhancement of enzyme activities.