valinomycin and Lung-Neoplasms

The development and application of a methodology for measurement of oxygen within single mammalian cells are presented, which employ novel macromolecular near infrared (NIR) oxygen probes based on new metalloporphyrin dyes. The probes, which display optimal spectral characteristics and sensitivity to oxygen, excellent photostability, and low cytotoxicity and phototoxicity, are loaded into cells by simple transfection procedures and subsequently analyzed by high-resolution fluorescence microscopy. The methodology is demonstrated by sensing intracellular oxygen in different mammalian cell lines, including A549, Jurkat, and HeLa, and monitoring rapid and transient changes in response to mitochondrial uncoupling by valinomycin and inhibition by antimycin A. Furthermore, the effect of ryanodine receptor-mediated Ca(2+) influx on cellular oxygen uptake is shown by substantial changes in the level of intracellular oxygen. The results demonstrate the ability of this technique to measure small, rapid, and transient changes in intracellular oxygen in response to different biological effectors. Moreover, this technique has wide ranging applicability in cell biology and is particularly useful in the study of low oxygen environments (cellular hypoxia), mitochondrial and cellular (dys)function, and for therapeutic areas, such as cardiovascular and neurological research, metabolic diseases, and cancer.

Topics: Antimycin A; Biosensing Techniques; Cell Line, Tumor; Fluorescence; Fluorescent Dyes; HeLa Cells; Humans; Indoles; Ionophores; Jurkat Cells; Kinetics; Luminescent Agents; Luminescent Measurements; Lung Neoplasms; Metalloporphyrins; Microscopy, Confocal; Microscopy, Fluorescence; Mitochondria; Oxygen; Spectroscopy, Near-Infrared; Valinomycin

The uncoupler-stimulated mitochondrial ATPase of four human tumors, mouse kidney, brain and fetal liver exhibited a characteristic behavior when preincubated with the H+-conducting uncouplers, dinitrophenol, CCCP, S-13 and gramicidin. The ATPase activity was considerably lower with preincubation than without. Preincubation with valinomycin (+ K+), on the other hand, did not result in a significant decrease of the ATPase activity. These results may be contrasted with those obtained with liver or heart mitochondria, the ATPase activity of which did not suffer any loss when preincubated with dinitrophenol. The effect of preincubation with dinitrophenol on the tumor mitochondria could not be accounted for by dinitrophenol-induced Mg2+ efflux, since the differential effects of dinitrophenol and valinomycin (+ K+) remained even when ATPase activity was determined in presence of Mg2+. Small amounts of ATP and ADP in the preincubation mixture containing dinitrophenol protected against the decay of the ATPase activity, implicating the exchangeable adenine nucleotides in the tumor mitochondria. In a model system where liver mitochondria were depleted of their adenine nucleotides, a lower ATPase activity was indeed obtained. However, direct determination of the concentrations of adenine nucleotides in dinitrophenol- and valinomycin-treated tumor mitochondria revealed only slight differences.

Topics: 2,4-Dinitrophenol; Adenosine Triphosphatases; Animals; Astrocytoma; Brain; Carcinoma, Hepatocellular; Carcinoma, Small Cell; Cell Line; Dinitrophenols; Humans; Kidney; Kinetics; Liver; Liver Neoplasms; Lung Neoplasms; Melanoma; Mice; Mitochondria; Myocardium; Neoplasms; Uncoupling Agents; Valinomycin