Monitoring of cell oxygenation and responses to metabolic stimulation by intracellular oxygen sensing technique

Abstract

I.; Taylor, Cormac T.; et al. Publication date 2010 Publication informationIntegrative Biology, 2 (9): Publisher The Royal Society of ChemistryItem record/more information http://hdl.handle.net/10197/5029Publisher's version (DOI) 10.1039/c0ib00021cDownloaded 2019-04-05T04:17:49ZThe UCD community has made this article openly available. Please share how this access benefits you. Your story matters! (@ucd_oa) Some rights reserved. For more information, please see the item record link above. Running title: Oxygen Gradients in Respiring SamplesAbstract Changes in cellular O 2 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 O 2 sensing technique, we monitored O 2 gradients in static cultures of adherent PC12 cells exposed to graded atmospheric O 2 at rest and upon metabolic stimulation. Under high atmospheric O 2 (10-21%) the respiration of resting cells dictated that local O 2 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 O 2 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 O 2 levels and oxygen-sensitive signaling pathways are the atmospheric O 2 , sample geometry, cell density, respiration rate and its dynamics. Changes in any of these parameters can significantly alter the O 2 levels experienced by the cells and the subsequently activated signaling pathways. A model commonly used in in vitro studies is a static cell culture maintained at a constant external (atmospheric) pO 2 . For such a system, O 2 consumption rates and diffusion processes are important factors which contribute to O 2 transport to the cells from gaseous macro-phase 24,25 . Careful control of cellular O 2 in such experiments is still rare, and partial or complete deoxygenation and hypoxia-specific metabolic rearrangements may occur 17 and contribute to the observed biological effects. In this work, using quenchedphosphorescence O 2 sensing technique which allows real-time monitoring of both intra-and extracellular O 2 concentration 22,26,27 we investigated O 2 gradients in dense populations of neurosecretory pheochromocytoma PC12 cells cultured and differentiated under standard conditions in 96-well plates (dPC12). dPC12 cells possess active oxidative phosphorylation and glycolysis, produce robust responses to excitatory stimulation 22,26 , and they represent a convenient model for studies of brain function and pathologies 28 . For such a system with resting respiring dPC12 cells, steady state O 2 gradients at different...

O2 Analysis on a Fluorescence Spectrometer or Plate Reader

“…2.18a) without significant effect on cellular ATP levels. Since relative deoxygenation of respiring cells increases with decreased O 2 availability [59], in agreement with this rule at 6 % atmospheric O 2 the effect of Baf became more pronounced and detectable already at concentration 0.06 lM (Fig. 2.18b), while 0.2-0.5 lM Baf caused a sustained decrease in icO 2 down to 1-5 lM.…”

“…In the presence of NO the cells cannot deoxygenate themselves below a certain level. This is because NO effectively competes with O 2 for cytochrome c oxidase (mitochondrial complex IV) and inhibits its activity and at reduced O 2 availability decreases cell respiration to a certain level in a manner dependent on cellular O 2 and NO concentrations [59]. inhibited when O 2 levels drop to about 20-25 lM.…”

“…Even a short, transient increase in respiration may lead to sustained deoxygenation, imposing an energy stress which can in turn trigger cell adaptation to hypoxia. The latter can be seen, for example, as a shift in energy production from OxPhos to glycolysis, activation of hypoxia inducible factor (HIF) pathway and change in gene expression profile [59]. When measured in icO 2 [17].…”

“…As icO 2 concentration is a function of O 2 availability, conditions of mass exchange and cell respiratory activity, its levels may be affected significantly by rapid metabolic stimulation whereby a transient imbalance between O 2 supply and demand leads to a rapid local decrease in icO 2 ('overshoot effect' [59]). Even a short, transient increase in respiration may lead to sustained deoxygenation, imposing an energy stress which can in turn trigger cell adaptation to hypoxia.…”

“…2.13), O 2 concentration in the solution layer (Cs) and within the cell layer (Cc) can be described by simple mathematical equations. Using a number of valid assumptions, one can work out that the profiles of Cs and Cc within the sample obey the linear and quadratic functions of the distance from the headspace which serves as an O 2 reservoir, and the thickness of the Ls and Lc layers [59]:…”