safranine-t and malic-acid

Mitochondrial calcium-activated K(+) (mitoK(Ca)) channels have been described as channels that are activated by Ca(2+), inner mitochondrial membrane depolarization and drugs such as NS-1619. NS-1619 is cardioprotective, leading to the assumption that this effect is related to the opening of mitoK(Ca) channels. Here, we show several weaknesses in this hypothesis.. Isolated mitochondria from rat hearts were tested for evidence of mitoK(Ca) activity by analyzing functional parameters in K(+)-rich and K(+)-free media.. NS-1619 promoted mitochondrial depolarization both in K(+)-rich and K(+)-free media. Respiratory rate increments were also seen in the presence of NS-1619 for both media. In parallel, NS-1619 promoted respiratory inhibition, as evidenced by respiratory measurements in state 3. Mitochondrial volume measurements conducted using light scattering showed that NS-1619 led to swelling, in a manner unaltered by inhibitors of mitoK(Ca) channels, antagonists of adenosine triphosphate-sensitive potassium channels or inhibitors of the permeability transition. Swelling was also maintained when K(+) in the media was substituted with tetraethylammonium (TEA(+)), which is not transported by any known K(+) carrier. Electron microscopy experiments gave support to the idea that NS-1619-induced mitochondrial swelling took place in the absence of K(+). In addition to testing the pharmacological effects of NS-1619, we attempted, unsuccessfully, to promote mitoK(Ca) activity by altering Ca(2+) concentrations in the medium and inducing mitochondrial uncoupling.. Our data indicate that NS-1619 promotes non-selective permeabilization of the inner mitochondrial membrane to ions, in addition to partial respiratory inhibition. Furthermore, we found no specific K(+) transport in isolated heart mitochondria compatible with mitoK(Ca) opening, whether by pharmacological or physiological stimuli. Our results indicate that NS-1619 has extensive mitochondrial effects unrelated to mitoK(Ca) and suggest that tissue protection mediated by NS-1619 may occur through mechanisms other than activation of these channels.

Topics: Animals; Benzimidazoles; Calcium; Culture Media; Glutamic Acid; Ion Channel Gating; Malates; Membrane Potentials; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Mitochondria, Heart; Mitochondrial Membranes; Mitochondrial Swelling; NADP; Oligomycins; Oxidation-Reduction; Oxygen Consumption; Phenazines; Potassium; Potassium Channels, Calcium-Activated; Rats; Rats, Sprague-Dawley; Rotenone; Sodium; Succinic Acid; Tetraethylammonium; Tissue Culture Techniques; Uncoupling Agents

In the light of the occurrence of L-lactate dehydrogenase inside the mitochondrial matrix, we looked at whether isolated rat liver mitochondria can take up and metabolize L-lactate, and provide oxaloacetate outside mitochondria, thus contributing to a partial reconstruction of gluconeogenesis in vitro. We found that: (1) L-lactate (10 mM), added to mitochondria in the presence of a cocktail of glycolysis/gluconeogenesis enzymes and cofactors, can lead to synthesis of glyceraldehyde-3-phosphate at a rate of about 7 nmol/min per mg mitochondrial protein. (2) Three novel translocators exist to mediate L-lactate traffic across the inner mitochondrial membrane. An L-lactate/H+ symporter was identified by measuring fluorimetrically the rate of endogenous pyridine nucleotide reduction. Consistently, L-lactate oxidation was found to occur with P/O ratio=3 (where P/O ratio is the ratio of mol of ATP synthesized to mol of oxygen atoms reduced to water during oxidative phosphorylation) and with generation of membrane potential. Proton uptake, which occurred as a result of addition of L-lactate to RLM together with electron flow inhibitors, and mitochondrial swelling in ammonium L-lactate solutions were also monitored. L-Lactate/oxaloacetate and L-lactate/pyruvate anti-porters were identified by monitoring photometrically the appearance of L-lactate counter-anions outside mitochondria. These L-lactate translocators, which are distinct from the monocarboxylate carrier, were found to differ from each other in V(max) values and in inhibition and pH profiles, and proved to regulate mitochondrial L-lactate metabolism in vitro. The role of lactate/mitochondria interactions in gluconeogenesis is discussed.

Topics: Animals; Antimycin A; Biological Transport; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell-Free System; Cyanides; Gluconeogenesis; Glyceraldehyde 3-Phosphate; Humans; L-Lactate Dehydrogenase; Lactic Acid; Malates; Male; Mitochondria, Liver; Models, Biological; Monocarboxylic Acid Transporters; Osmotic Pressure; Oxaloacetic Acid; Oxidation-Reduction; Phenazines; Protons; Pyruvic Acid; Rats; Rats, Wistar; Rotenone; Succinates; Tetramethylphenylenediamine; Uncoupling Agents