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.

The effect of AgNPS bio-functionalization on the cytotoxicity of the yeast Saccharomyces cerevisiae.

This work used Sedum praealtum leaf extract to synthesize silver nanoparticles (AgNPs) in a single step. The cytotoxicity of AgNPs was studied with the yeast Saccharomyces cerevisiae W303-1. In addition, the antioxidant activity of the DPPH radical was studied both in the extract of S. praealtum and in the AgNPs. UV-Vis spectroscopy determined the presence of AgNPs by the location of the surface plasmon resonance (SPR) band at 434 nm. TEM and XRD analyzes show AgNPs with fcc structure and hemispherical morphology. Also, AgNPs range in size from 5 to 25 nm and have an average size of 14 nm. 1H NMR, FTIR, and UV-Vis spectroscopy techniques agreed that glycosidic compounds were the main phytochemical components responsible for the reduction and stabilization of AgNPs. In addition, AgNPs presented a maximum of 12% toxicity in yeast attributed to the generation of ROS. Consequently, there was low bioactivity because glycoside compounds cover the biosynthesized AgNPs from S. praealtum. These findings allow applications of AgNPs involving contact with mammals and higher organisms.

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.

Calcium uptake during the cell cycle of Saccharomyces cerevisiae.

Synchronous culture of the budding yeast Saccharomyces cerevisiae was obtained by sucrose density gradient selection with 90-100% of yeast synchronized by using the cells in the bottom. In these adult cells bud emergence is coincident with an increase in calcium uptake at 100 min of the culture, followed by a return to basal values which are maintained until the end of the first cell cycle of study. The phenothiazine derivatives, trifluoperazine and chlorpromazine inhibit bud emergence and trifluoperazine also increases calcium uptake.

Antagonism between the metabolic responses induced by epinephrine and piroxicam on isolated rat hepatocytes.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the most employed therapeutic agents. They have a wide spectrum of biological effects, some of which are independent of cyclooxygenase inhibition, such as the alterations on the components of signal transduction systems. In particular, previous data from our laboratory suggested an antagonism between epinephrine and piroxicam, one of the most prescribed NSAIDs. Thus, this study deals with the epinephrine-piroxicam antagonism recorded for metabolic responses in isolated rat hepatocytes. The obtained results show that epinephrine stimulates lactate and ethanol consumption, stimulates glucose release from lactate only, and has no effect on cellular triacylglycerides content. Otherwise, in a dose-dependent basis, piroxicam stimulates lactate and ethanol consumption accompanied by an increase in triacylglycerides content, without changes in glucose release by hepatocytes. Piroxicam blocks the epinephrine-induced stimulation of glucose release from lactate, and epinephrine blocks the piroxicam-mediated increase in triacylglycerides content from lactate or ethanol. In contrast, the effects of epinephrine and piroxicam, promoting the consumption of lactate and ethanol, are not antagonized or added after the simultaneous administration of both compounds. This last result is probably related to the ability of both compounds to stimulate oxygen consumption. On isolated rat liver mitochondria, micromolar doses of piroxicam partially uncouple oxidative phosphorylation, and paradoxically stimulates an ATP-dependent mitochondrial function as citrullinogenesis. These results show for first time, on isolated rat hepatocytes, an antagonism between the metabolic responses of epinephrine and piroxicam, at the concentration found in plasma after its therapeutical administration.

Effect of hypoxia and reoxygenation on metabolic pathways in rat hepatocytes.

BACKGROUND: The mechanisms whereby rat hepatocytes undergo irreversible injury due to a lack of oxygen have not been established. METHODS: Liver cells were used for reperfusion injury, and four compartmentalized pathways were evaluated during hypoxia (N2/CO2, 19:1) for 30 min followed by oxygen (O2/CO2, 19:1) for 30 min. RESULTS: Cell viability decreased during the hypoxic, but not during the reoxygenation, phase. Glycogenolysis, as measured by glucose release, was significantly increased during hypoxia as compared to controls in oxygen (205 +/- 15 vs. 155 +/- 10 nmol glucose/mg protein/h, respectively), and did not return to normal levels by reoxygenation. Gluconeogenesis was importantly decreased during hypoxia (102 +/- 10 vs. 8 +/- 2 nmol glucose/mg protein/h) with partial recovery during reoxygenation. Ureagenesis diminished in hypoxia, but recovered during reoxygenation. Additionally, 3-hydroxybutyrate formation was augmented by hypoxia, with some recovery when oxygen was present. CONCLUSIONS: These results suggest that compartmentalized pathways are protected from hypoxic injury in isolated hepatocytes, and also suggest it as a model to test the idea that enzymes of those pathways are organized into multienzyme complexes in vivo.

Effects of moderate chronic ethanol consumption on rat liver mitochondrial functions.

The biochemical consequences of moderate chronic ethanol ingestion has been scarcely investigated in spite of the fact that most of the human population drinks ethanol on a moderate basis. This paper describes some metabolic effects produced by moderate ethanol consumption. The substitution of drinking water in rats for a 10% ethanol solution during 4 weeks resulted in: a) a decrease of blood urea and citrulline synthesis in liver mitochondria; b) a slight inhibition in state 3 and state 4 respiration either with glutamate-malate as substrates or succinate as substrate; c) no change in ADP/O ratio with succinate but slight increase with glutamate-malate; d) a reduction of the cytochrome oxidase activity and cytochromes a+a3 content; e) a 42% increase in the succinate dehydrogenase activity and a small but constant increase in the Vmax (no change in the Km) of the adenine nucleotide translocase activity in liver mitochondria. These results show that even moderate, but continuous ethanol ingestion, produces metabolic responses that must be carefully evaluated to define health risk in larger human groups.

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.

Stimulation of L-ornithine uptake and L-citrulline and urea biosynthesis by D-arginine.

The action of D-arginine on isolated cells and mitochondria obtained from rat liver was studied. The D-amino acid at 200 microM stimulated by 40% the rate of urea biosynthesis by isolated hepatocytes. Citrulline formation was increased 60-70% in rat liver mitochondria incubated with 10 microM D-arginine. In these mitochondria, ornithine uptake was enhanced 204% with 1 microM D-arginine. Inhibition in urea and citrulline synthesis and in ornithine uptake was recorded with high concentrations of the D-amino acid. Respiratory control in liver mitochondria with glutamate-malate was inhibited 32% by 100 microM D-arginine. In isolated mitochondria loaded with Fluo-3-acetoxymethyl (AM) ester, 50 microM D-arginine diminished the matrix free calcium concentration.

Ornithine uptake by rat liver mitochondria: effect of calcium and arginine.

The effect of calcium and L-arginine on ornithine uptake by rat liver mitochondria was studied. In the absence of arginine, calcium ions at a concentration of 0.36 microM increase ornithine uptake 218% above control, but at metal concentrations higher than 1.0 microM the amino acid uptake is diminished. Keeping calcium concentration constant in the medium (0.36 microM), ornithine transport was maximal at 5.0 microM L-arginine and decreased at higher concentrations of arginine. The possible role of calcium and arginine as physiological regulators of ornithine transport is discussed.

Control of mitochondrial matrix calcium: studies using fluo-3 as a fluorescent calcium indicator.

Fluo-3, a fluorescent Ca2+ indicator, is sequestered by isolated rat liver mitochondria and is an effective probe for evaluating the concentration and kinetics of change of mitochondrial matrix ionized calcium ([Ca2+]m) under a variety of conditions. At the wavelengths employed, there is no significant interference by auto-fluorescence. There is an insignificant release of the indicator over four hours and the loading and presence of fluo-3 has no effect on respiratory rate or oxidative phosphorylation. The [Ca2+]m steady state can be altered by the assay conditions, i.e. the presence of extra-mitochondrial Ca2+, Mg2+ phosphate and respiratory inhibitors. The total matrix ionized calcium represents a small percent (less than 0.01%) of the total mitochondrial calcium.

Accumulation of D-arginine by rat liver mitochondria.

The permeability of the inner mitochondrial membrane from rat liver to D-arginine was studied. By using safranin as a probe of the membrane potential, depolarization of energized liver mitochondria occurred in a dose-dependent fashion starting at 3.3 mmol/L of D- or DL-arginine. When ethidium bromide fluorescence was employed, a decrease in the membrane potential due to D- or DL-arginine was observed. A parallel significant change in succinate-induced respiration in rat liver mitochondria was found in response to osmotic swelling in 125 mmol/L of D-arginine salts. L-Arginine, L-glutamine, L-asparagine, L-ornithine, D-ornithine, and L-lysine did not modify the membrane potential at the concentrations tested. D-Arginine was not transformed into citrulline, but 1.0 mmol/L of the D-amino acid inhibited, by 42%, the state 3 of mitochondrial respiration using succinate as substrate. When D-arginine was used in combination with nigericin, a 40% inhibition of mitochondrial respiration in state 3 was recorded with succinate and with glutamate-malate as substrates.

Effect of intramitochondrial Mg2+ on citrulline synthesis in rat liver mitochondria.

A correlation was found between mitochondrial matrix free magnesium ([Mg2+]m), measured by means of the fluorescent indicator Mag-Fura-2, and citrulline synthesis in rat liver mitochondria. The variation of [Mg2+]m from 0.05 to 1.7 mM by changes in external Mg2+, induced a 20 +/- 8.5% increase in the rate of citrulline synthesis, whereas a further increase of [Mg2+]m to 3.3 mM induced a return to basal values. The increase in [Mg2+]m, as well as the diminution of external pH, also promoted an elevation of matrix free Ca2+ ([Ca2+]m). An increase in [Ca2+]m, at constant [Mg2+]m and pH, resulted in a 3-fold stimulation of citrulline synthesis. The data suggest that [Mg2+]m may modulate the rate of citrulline synthesis through a direct interaction with carbamoyl-phosphate synthase I (ammonia) and, indirectly, by changing the levels of matrix Ca2+.

Mitochondrial nitric oxide inhibits ATP synthesis. Effect of free calcium in rat heart.

Nitric oxide is a small potentially toxic molecule and a diatomic free radical. We report the interaction of L-arginine, oxygen and calcium with the synthesis of nitric oxide in heart mitochondria. Nitric oxide synthesis is increased in broken rat heart mitochondria compared with intact and permeabilized mitochondria. Intact mitochondria subjected to hypoxia-reoxygenation conditions accumulated nitric oxide that inhibits oxygen consumption and ATP synthesis. ATPase activity is not affected during this augment of nitric oxide. Physiological free calcium concentrations protected mitochondria from the damage caused by the accumulation of nitric oxide. Higher concentrations of the divalent cation increase the damage exerted by nitric oxide.

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.

Regulation of the rate of synthesis of nitric oxide by Mg(2+) and hypoxia. Studies in rat heart mitochondria.

In isolated rat heart mitochondria, L-arginine is oxidized by a nitric oxide synthase (mtNOS) achieving maximal rates at 1 mM L-arginine. The NOS inhibitor N(G)-nitro-L-arginine methyl ester (NAME) inhibits the increase in NO production. Extramitochondrial free magnesium inhibited NOS production by 59% at 3.2 mM. The mitochondrial free Mg(2+) concentration increased to different extents in the presence of L-arginine (29%), the NO donor (S-nitroso-N-acetylpenicillamine) (105%) or the NOS inhibitors L-NAME (48%) or N(G)-nitro-L-arginine methyl ester, N(G)-monomethyl-L-arginine (L-NMMA) (53%). Under hypoxic conditions, mtNOS activity was inhibited by Mg(2+) by up to 50% after 30 min of incubation. Reoxygenation restored the activity of the mtNOS to pre-hypoxia levels. The results suggest that in heart mitochondria there is an interaction between Mg(2+) levels and mtNOS activity which in turn is modified by hypoxia and reoxygenation.

Effect of Ca(2+) and Mg(2+) on the Mn-superoxide dismutase from rat liver and heart mitochondria.

The manganese superoxide dismutase (Mn-SOD) converts superoxide anions to hydrogen peroxide plus oxygen, providing the first line of defense against oxidative stress in mitochondria. Heart mitochondria exhibited higher Mn-SOD activity than liver mitochondria. In mitochondria from both tissues Mn-SOD activity decreased after incubation at low oxygen concentration (hypoxic mitochondria). The effects of free Ca(2+) ([Ca(2+)](f)) and free Mg(2+) ([Mg(2+)](f)) on normoxic and hypoxic mitochondria from either organ were tested. In normoxic mitochondria from either tissue, both [Ca(2+)](f) and [Mg(2+)](f) activated the enzyme, although [Mg(2+)](f) was less efficient as an activator and the effect was lower in heart than in liver mitochondria. When added simultaneously, high [Ca(2+)](f) and [Mg(2+)](f) exhibited additive effects which were more pronounced in heart mitochondria and were observed regardless of whether mitochondria had been incubated under normal or low oxygen. The data suggest that [Ca(2+)](f) plays a role in regulating Mn-SOD in concert with the activation of aerobic metabolism.