The Determinants of Oxygen Gradients in Respiring Samples and their Impact on Cellular Responses to Hypoxia

Changes in cellular O2 levels elicit adaptive responses which can lead to the activation of oxygen-dependent transcription factors such as the hypoxia-inducible factor (HIF). However, our understanding of the determinants of these processes is still incomplete. Using the new intracellular O2 sensing technique, we monitored O2 gradients in static cultures of adherent PC12 cells exposed to graded atmospheric O2 at rest and upon metabolic stimulation. Under high atmospheric O2 (10-21%) the respiration of resting cells dictated that local O2 was moderately reduced, and at a certain threshold (6% in galactose medium) cell layer became practically anoxic. Furthermore, cell stimulation triggered a major redistribution of O2 and a prominent ‘hypoxic overshoot’ effect mediated by diffusion. The deep, prolonged cell deoxygenation upon stimulation was matched by an increase in nuclear HIF-1α levels. In the presence of nitric oxide the hypoxic overshoot was truncated and HIF-1α stabilization inhibited. Thus, the main determinants which impact upon cellular O2 levels and oxygen-sensitive signaling pathways are the atmospheric O2, sample geometry, cell density, respiration rate and its dynamics. Changes in any of these parameters can significantly alter the O2 levels experienced by the cells and the subsequently activated signaling pathways.