Nicorandil Affects Mitochondrial Respiratory Chain Function by Increasing Complex III Activity and ROS Production in Skeletal Muscle Mitochondria.

Adenosine triphosphate (ATP)-dependent potassium channels openers (KATP) protect skeletal muscle against function impairment through the activation of the mitochondrial KATP channels (mitoKATP). Previous reports suggest that modulators of the mitochondrial KATP channels have additional effects on isolated mitochondria. To determine whether the KATP channel opener nicorandil has non-specific effects that explain its protective effect through the mitochondrial function, chicken muscle mitochondria were isolated, and respiration rate was determined pollarographically. The activity of the electron transport chain (ETC) complexes (I-IV) was measured using a spectrophotometric method. Reactive oxygen species (ROS) levels and lipid peroxidation were assessed using flow cytometry and thiobarbituric acid assay, respectively. Both KATP channel opener nicorandil and KATP channel blocker 5-hydroxydecanoate (5-HD) decreased mitochondrial respiration; nicorandil increased complex III activity and decreased complex IV activity. The effects of nicorandil on complex III were antagonized by 5-HD. Nicorandil increased ROS levels, effect reverted by either 5-HD or the antioxidant N-2-mercaptopropionyl glycine (MPG). None of these drugs affected lipid peroxidation levels. These findings suggest that KATP channel opener nicorandil increases mitochondrial ROS production from complex III. This results by partially blocking electron flow in the complex IV, setting electron carriers in a more reduced state, which is favored by the increase in complex III activity by nicorandil. Overall, our study showed that nicorandil like other mitochondrial KATP channel openers might not act through mitoKATP channel activation.

Nicorandil improves post-fatigue tension in slow skeletal muscle fibers by modulating glutathione redox state.

Fatigue is a phenomenon in which force reduction has been linked to impairment of several biochemical processes. In skeletal muscle, the ATP-sensitive potassium channels (KATP) are actively involved in myoprotection against metabolic stress. They are present in sarcolemma and mitochondria (mitoKATP channels). K+ channel openers like nicorandil has been recognized for their ability to protect skeletal muscle from ischemia-reperfusion injury, however, the effects of nicorandil on fatigue in slow skeletal muscle fibers has not been explored, being the aim of this study. Nicorandil (10 µM), improved the muscle function reversing fatigue as increased post-fatigue tension in the peak and total tension significantly with respect to the fatigued condition. However, this beneficial effect was prevented by the mitoKATP channel blocker 5-hydroxydecanoate (5-HD, 500 µM) and by the free radical scavenger N-2-mercaptopropionyl glycine (MPG, 1 mM), but not by the nitric oxide (NO) synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME, 100 µM). Nicorandil also decreased lipid peroxidation and maintained both reduced glutathione (GSH) levels and an elevated GSH/GSSG ratio, whereas total glutathione (TGSH) remained unaltered during post-fatigue tension. In addition, NO production, measured through nitrite concentrations was significantly increased with nicorandil during post-fatigue tension; this increase remained unaltered in the presence of nicorandil plus L-NAME, nonetheless, this effect was reversed with nicorandil plus MPG. Hence, these results suggest that nicorandil improves the muscle function reversing fatigue in slow skeletal muscle fibers of chicken through its effects not only as a mitoKATP channel opener but also as NO donor and as an antioxidant.

Importance of oxidative damage on the electron transport chain for the rational use of mitochondria-targeted antioxidants.

The electron transport chain (ETC) has become a promising pharmacological target as ETC impairment by reactive oxygen species (ROS) has been detected in several diseases. Therefore, for a better understanding of the actions of mitochondria-targeted antioxidants, it must be considered the interplay between the sources of ROS during disease, the chemical interconversions of ROS and their differential reactivity with ETC components. This review contrasts these aspects with available data about mitochondrial damage in specific diseases to give an insight into the importance of ROS chemistry in the rational use of mitochondria-targeted antioxidants, putting emphasis on the case of MitoQ.

Changes in mitochondrial functionality and calcium uptake in hypertensive rats as a function of age.

We studied whether mitochondrial functions and Ca2+ metabolism were altered in Wistar Kyoto normotensive (WKY) and spontaneous hypertensive rats (SHR). Ca2+ uptake was decreased in SHR compared to WKY rats. Accumulation of Ca2+ was more efficient in WKY than in SHR rats. mDeltaPsi was lower in SHR compared to WKY rats. Basal complex IV activity was higher in SHR than WKY rats, whereas basal L-citrulline production, an indicator of nitric oxide synthesis, was decreased in SHR and dependent on Ca2+ concentration (p<0.05). Impact of Ca2+ was counteracted by EGTA. These data show an age-dependent decreased mitochondrial functions in brain mitochondria during hypertension.

Effects of D-amino acids on lipoperoxidation in rat liver and kidney mitochondria.

The effects of the amino acids D-ser, D-asp, and D-ala on lipoperoxidation under conditions of hypertension, alcoholism, and ammonemia in rat liver and kidney mitochondria were studied. Under normal conditions, D-alanine increased in 54% free radicals production in liver mitochondria (p < 0.05). The D-amino acids had no effect on kidney mitochondria. D-ser and D-ala increased lipoperoxidation in spontaneously hypertensive rats (SHR) as compared with their normotensive genetic control Wistar-Kyoto (WKY) rats (p < 0.05). During hypertension and in oxidative stress in the presence of calcium, only D-ala produced 46% and 29% free radicals in liver and kidney mitochondria (p < 0.05), respectively. During chronic alcoholism, D-ser increased lipoperoxidation in 80% in kidney mitochondria (p < 0.05), as compared to control. During ammonemia, D-ser produced 41% free radicals.

Functional characterization of brain mitochondrial nitric oxide synthase during hypertension and aging.

Nitric oxide (NO*) plays an important role in various physiological processes. The aim of the present study was to investigate if brain mitochondrial nitric oxide synthase (mtNOS) is active and functional during hypertension. L-citrulline production, an indicator of nitric oxide synthesis, was concentration-dependent on L-arginine in all strains and all ages tested, and was inhibited by 7-Nitroindazole (7-NI). Brain mitochondria of 1 month-old (prehypertensive) spontaneously hypertensive rats (SHR) exhibited a significantly (p < 0.05) low basal L-citrulline content as compared to age-matched Wistar (W) and Wistar-Kyoto (WKY) rats. L-citrulline synthesis in SHR rats showed a significant (p < 0.01) low response to L-arginine in 3 and 7 months-old rats. Respiratory rates in states 3 and 4 increased with low L-arginine concentration in all strains and all ages. The results suggest that in rat brain mitochondria, L-citrulline synthesis is constant once age-related hypertension is installed and NO* does not regulate oxidative phosphorylation.

Effect of D-amino acids on some mitochondrial functions in rat liver.

We studied the role of the D-amino acids (D-aa) D-serine, D-alanine, D-methionine, D-aspartate, D-tyrosine and D-arginine on rat liver mitochondria. The stability of D-amino acids, mitochondrial swelling, transmembrane potential and oxygen consumption were studied under oxidative stress conditions in rat liver mitochondria. In the presence of glutamate-malate all D-aas salts increased mitochondrial swelling, while in the presence of succinate plus rotenone only D-ala, D-arg and D-ser, induced mitochondrial swelling. The transmembrane potential (deltapsi) was decreased in the presence of 1 microM Ca(2+). The D-aas inhibited oxygen consumption in state 3. The D-aa studied exerted effects on mitochondria via an increase of free radicals production.