Indicator Of Mitochondrial Membrane Potential Research Articles

Mitochondrial Depolarization in Glutamate-Stimulated Neurons: An Early Signal Specific to Excitotoxin Exposure

A brief exposure to high concentrations of glutamate kills cultured forebrain neurons by an excitotoxic process that is dependent on Ca2+ influx through the NMDA receptor. In this study, we have measured striking changes in mitochondrial function during and immediately after intense glutamate receptor activation. Using indo-1 microfluorometry and a specific inhibitor of the mitochondrial Na+/Ca2+ exchanger, CGP-37157, we have demonstrated that mitochondria accumulate large quantities of Ca2+ during a toxic glutamate stimulus and further that Ca2+ efflux from mitochondria contributes to the prolonged [Ca2+]i elevation after glutamate removal. We then used JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine+ ++ iodide), a ratiometric indicator of mitochondrial membrane potential (delta psi), to show that Ca2+ accumulation within the organelle dissipates delta psi. The abrupt loss of delta psi after glutamate stimulation did not occur in the presence of MK801 or in the absence of extracellular Ca2+. The mitochondrial depolarization was also cyclosporin A-sensitive, indicating a probable role for the permeability transition pore. Hence mitochondrial Ca2+ accumulation and the subsequent permeability transition may be a critical early event specific to the NMDA receptor-mediated excitotoxic cascade.

Further evidence that lucigenin-derived chemiluminescence monitors mitochondrial superoxide generation in rat alveolar macrophages

Lucigenin is well recognized for its ability to react with superoxide, yielding a product that emits chemiluminescence. Accordingly, lucigenin-derived chemiluminescence (LDCL) has been widely used to assess the generation of superoxide by the NADPH oxidase of leukocytes. Previously, we suggested that lucigenin could interact with mitochondrial-derived superoxide in alveolar macrophages. The purpose of this study was to further demonstrate that LDCL is in fact a probe of mitochondrial superoxide generation. Using fluorescence microscopy, we have observed that lucigenin accumulates at the mitochondria of alveolar macrophages and exhibits a localization similar to that of rhodamine 123, a fluorescent indicator of mitochondrial membrane potential. We have also compared the effects of a spectrum of agents known to modulate mitochondrial respiration on both LDCL and cellular oxygen consumption. The agents examined included a Complex I inhibitor, rotenone; a Complex III inhibitor, antimycin a; and a Complex IV inhibitor, KCN. While these compound all inhibited oxygen consumption, only those that exert an effect prior to (rotenone) or at (antimycin a) the point of mitochondrial superoxide formation inhibited LDCL. KCN exhibits effects that are concetration dependent. At low concentrations (1–100 μM), a slight enhancement of LDCL is observed, while at higher concentrations (1–10 mM) both LDCL and oxygen consumption are inhibited. The ATP synthase inhibitor, oligomycin, was also observed to correspondingly inhibit oxygen consumption and LDCL. These inhibitor studies, taken together with the observation that lucigenin localizes to the mitochondria of alveolar macrophages, provides strong evidence that LDCL can be used to qualitatively asses the modulation of mitochondrial superoxide generation in mononuclear cells.

Mitochondrial membrane potential monitored in situ within isolated guinea pig brown adipocytes by a styryl pyridinium fluorescent indicator

2-(Dimethylaminostyryl)-1-methyl pyridinium, a fluorescent indicator of mitochondrial membrane potential, is accumulated up to 400-fold by intact, isolated brown adipocytes from cold-adapted guinea pigs. The indicator can be optically localized within the in situ mitochondria. Ionophores which would be predicted to lower or increase the in situ mitochondrial membrane potential lead to decreased and increased uptake, respectively, of the radio-labelled indicator. Noradrenaline induces a 15 mV decrease in the sum of mitochondrial and plasma membrane potentials. The results indicate that the mitochondria within isolated brown adipocytes maintain a substantial membrane potential in the resting state, rather than being artifactually depolarized.

Uptake and toxicity of safranine and triphenylmethylphosphonium ion in human tumour cells.

Despite having lower cell membrane potentials, four human melanoma cell lines and HeLa cells accumulated the membrane probe triphenylmethylphosphonium ion (TPMP, 2 microM) 2-10 fold more rapidly than human fibroblasts, and did not reach equilibrium during the 2 h incubation period studied. The difference was not due to drug toxicity, although at much higher levels of TPMP (greater than 20 microM) selective killing of melanoma cells compared with fibroblasts was observed. Some dependence of TPMP accumulation upon membrane potential was indicated by inhibition of uptake in fibroblasts depolarised by culture in calcium-deficient medium. Melanoma cells exhibited no change in TPMP uptake or membrane potential under these conditions. Uptake of safranine, an indicator of mitochondrial membrane potential, did not follow the TPMP pattern. Thus, the anomalous accumulation of TPMP by human tumour cells appeared to result from either carrier-mediated transport or rapid intracellular metabolism and could not be used to determine cell membrane potentials.