Imaging of oxygen gradients in giant umbrella cells: an ex vivo PLIM study

Imaging of oxygen gradients in giant umbrella cells: an ex vivo PLIM study. Am J Physiol Cell Physiol 309: C501-C509, 2015. First published August 5, 2015; doi: 10.1152/ajpcell.00121.2015.-O-2 plays a pivotal role in aerobic metabolism and regulation of cell and tissue function. Local differences and fluctuations in tissue O-2 levels are well documented; however, the physiological significance of O-2 microgradients, particularly at the subcellular level, remains poorly understood. Using the cell-penetrating phosphorescent O-2 probe Pt-Glc and confocal fluorescence microscopy, we visualized O-2 distribution in individual giant (> 100-mu m) umbrella cells located superficially in the urinary bladder epithelium. We optimized conditions for in vivo phosphorescent staining of the inner surface of the mouse bladder and subsequent ex vivo analysis of excised live tissue. Imaging experiments revealed significant (<= 85 mu M) and heterogeneous deoxygenation within respiring umbrella cells, with radial O-2 gradients of up to 40 mu M across the cell, or similar to 0.6 mu M/mu m. Deeply deoxygenated (5-15 mu M O-2) regions were seen to correspond to the areas enriched with polarized mitochondria. Pharmacological activation of mitochondrial respiration decreased oxygenation and O-2 gradients in umbrella cells, while inhibition with antimycin A dissipated the gradients and caused gradual reoxygenation of the tissue to ambient levels. Detailed three-dimensional maps of O-2 distribution potentially can be used for the modeling of intracellular O-2-dependent enzymatic reactions and downstream processes, such as hypoxia-inducible factor signaling. Further ex vivo and in vivo studies on intracellular and tissue O-2 gradients using confocal imaging can shed light on the molecular mechanisms regulating O-2-dependent (patho) physiological processes in the bladder and other tissues.