Reduced PO 2 alters the behavior of Fura-2 and Indo-1 in bovine pulmonary artery endothelial cells

Abstract

Calcium-sensitive fluorophores are used to estimate cytosolic free Ca 2+ in many cell types under various conditions. We tested the effect of reduced PO 2 on the behavior of Fura-2 and Indo-1 in cultured bovine pulmonary artery endothelial cells. Reduced PO 2 (PO 2 25–35 mmHg) caused a significant upward shift of in vivo calibration curves for both fluorophores. The in vivo emission spectrum of Fura-2 indicated that the effect was principally due to attenuated emission at the Ca 2+-unbound 380 nm wavelength, with no shift in position of the emission maxima for either Ca 2+-bound or unbound forms of the fluorophore. Reduced PO 2 did not directly alter the behavior of the dyes, as no shift of in vitro calibration curves was seen. Neither decreased photobleaching nor altered autofluorescence accounted for the shift. We investigated several potential indirect effects, including cellular acidification, reduced viscosity, inhibition of oxidative energy production and reductive stress. In contrast to lowered PO 2, acidification in vitro produced a leftward but not an upward shift. Estimation of intracellular pH with SNAFL-calcein under reduced PO 2 showed no apparent acidification in these cells, further strengthening the argument that altered intracellular pH was not causing the shift. Others have shown that decreases in viscosity in vitro may shift the calibration curve for Fura-2 upward, similar to our finding with reduced PO 2. However, for Indo-1 we found that decreased viscosity in vitro attenuated fluorescence emission at the Ca 2+-bound 405 nm wavelength, thus producing the opposite effect on fluorescence ratio and indicating that reduced PO 2 was not acting through changes in cellular microviscosity. Because reduced PO 2 could be acting through either reduced energy production or redox, we tested the effects of the metabolic inhibitor rotenone (100 nM), and the reducing agent glutathione (50 mM), on the calibration curves. Neither intervention changed Fura-2 fluorescence in vivo, suggesting that these mechanisms were not responsible for the shift in in vivo calibration under reduced PO 2. We conclude that in pulmonary artery endothelial cells, reduced PO 2 alters the behavior of Ca 2+-sensitive fluorophores through an unidentified, indirect mechanism, and recommend that investigators utilizing this technique to study oxygen-dependent effects employ PO 2-specific in vivo calibration curves to avoid errors in interpretation.