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Several new derivatives of the phosphorescent Pt(II)-coproporphyrin (PtCP) were evaluated with respect to the sensing of intracellular oxygen by phosphorescence quenching. Despite the more favorable molecular charge compared to PtCP, self-loading into mammalian cells was rather inefficient for all the dyes, while cell loading by facilitated transport using transfection reagents produced promising results. The PtCP-NH(2) derivative, which gave best loading efficiency and S/N ratio, was investigated in detail including the optimisation of loading conditions, studies of sub-cellular localization, cytotoxicity, oxygen sensitivity and long-term signal stability. Being spectrally similar to the macromolecular MitoXpress™ probe currently used in this application, the PtCP-NH(2) demonstrated higher loading efficiency and phosphorescent signals, suitability for several problematic cell lines and a slightly increased lifetime scale for the physiological range (0-200 μM O(2)). In physiological experiments with different cell types, mitochondrial uncouplers and inhibitors performed on a time-resolved fluorescence plate reader, this probe produced the anticipated profiles of intracellular oxygen concentration and responses to cell stimulation. Therefore, PtCP-NH(2) represents a convenient probe for the experiments and applications in which monitoring of cellular oxygen levels is required.

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dependent mitochondrial impairment and stabilization of hypoxiainducible factor (HIF)-1α, which synergize to activate glycolysis and generate large quantities of ATP. The upregulation of glycolysis is completely dependent on NO and HIF-1α. Furthermore, HIF-1α stabilization is biphasic, with a reactive oxygen species-dependent early phase (1-2 h after activation) and later phase associated with the release of NO. We now demonstrate, using TMRM fluorescence and time-lapse confocal microscopy, that activated MФ maintain a high mitochondrial membrane potential (ΔΨ m ) despite the complete inhibition of respiration by NO. The high ΔΨ m is maintained by utilization of a significant proportion (approx 30%) of total glycolytically-generated ATP and is achieved by the reverse functioning of F O F 1 -ATP synthase and adenine nucleotide translocase (ANT). Treatment of activated MФ with inhibitors of either of these enzymes (oligomycin or bongkrekic acid), but not with inhibitors of the respiratory chain complexes, led to a collapse in ΔΨ m and to an immediate increase in intracellular [ATP]. This collapse of ΔΨ m was biphasic, with a rapid initial drop of approx 30% followed by a gradual decline, and was associated with translocation of Bax from cytosol to the mitochondria, release of cytochrome c into the cytosol, activation of caspases 3 and 9, and apoptotic cell death. Our results indicate that during inflammatory activation "glycolytically competent cells" such as MФ utilize significant amounts of the glycolytically-generated ATP to maintain ΔΨ m and thereby prevent apoptosis. It remains to be investigated whether the diversion of energy for this purpose, which is also likely to occur in tissues, favours or is detrimental to the successful outcome of an inflammatory response.