title: Imaging of oxygen gradients in giant umbrella cells: an ex vivo PLIM study.
abstract: O2 plays a pivotal role in aerobic metabolism and regulation of cell and tissue function. Local differences and fluctuations in tissue O2 levels are well documented; however, the physiological significance of O2 microgradients, particularly at the subcellular level, remains poorly understood. Using the cell-penetrating phosphorescent O2 probe Pt-Glc and confocal fluorescence microscopy, we visualized O2 distribution in individual giant (>100-μm) 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 μM) and heterogeneous deoxygenation within respiring umbrella cells, with radial O2 gradients of up to 40 μM across the cell, or ∼0.6 μM/μm. Deeply deoxygenated (5-15 μM O2) regions were seen to correspond to the areas enriched with polarized mitochondria. Pharmacological activation of mitochondrial respiration decreased oxygenation and O2 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 O2 distribution potentially can be used for the modeling of intracellular O2-dependent enzymatic reactions and downstream processes, such as hypoxia-inducible factor signaling. Further ex vivo and in vivo studies on intracellular and tissue O2 gradients using confocal imaging can shed light on the molecular mechanisms regulating O2-dependent (patho)physiological processes in the bladder and other tissues.
authors.affiliation: School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland; a.zhdanov@ucc.ie.
authors.forename: A V
authors.initials: AV
authors.lastname: Zhdanov
authors.affiliation: Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland; and.
authors.forename: A V
authors.initials: AV
authors.lastname: Golubeva
authors.affiliation: School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland;
authors.forename: I A
authors.initials: IA
authors.lastname: Okkelman
authors.affiliation: Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland; and Department of Anatomy and Neuroscience, University College Cork, Cork, Ireland.
authors.forename: J F
authors.initials: JF
authors.lastname: Cryan
authors.affiliation: School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland;
authors.forename: D B
authors.initials: DB
authors.lastname: Papkovsky
mesh_terms: D000818:Animals; D019069:Cell Respiration / Q000502:physiology*; D004847:Epithelial Cells / Q000378:metabolism*; D008163:Luminescent Measurements; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D013194:Staining and Labeling; D001743:Urinary Bladder / Q000166:cytology / Q000378:metabolism*; D019459:Urothelium / Q000166:cytology / Q000378:metabolism*
chemical_list: D010100:Oxygen
keywords: confocal phosphorescence lifetime imaging microscopy; giant umbrella cells; intracellular oxygen gradients; phosphorescent oxygen-sensitive probe; tissue oxygen imaging; urinary bladder