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Key Finding: Intracellular oxygen and not the ambient (atmospheric) oxygen is the important parameter when designing an in vitro model of physiological normoxia.

Photo: Professor Giovanni Mann Kings College London

Although critical for life, oxygen is an inherently toxic molecule with a plethora of damaging effects on our bodies. To combat this, our bodies are designed to control delivery of oxygen to our tissues and maintain a steep gradient between the atmosphere and cells. Despite this, cells grown in the laboratory are routinely maintained at atmospheric oxygen levels (~21% oxygen), which is approximately 4 times higher than the cells would experience in our body. To investigate this, we have grown cells from human blood vessels (endothelium) at more physiologically relevant oxygen levels (5% oxygen) and compared them with equivalent cells cultured under atmospheric oxygen levels. Our findings suggest that cells cultured in this way respond uniquely to stimulation by inflammatory compounds such as histamine, and exhibit characteristics different from cells grown at either atmospheric oxygen levels or under pathologically low O2 levels. These findings using the MitoXpress® Intracellular Oxygen assay add new perspective on how cells should be cultured in the laboratory and we envisage that our findings will inform the development of novel therapeutic compounds to treat diseases like atherosclerosis and hypertension.

Abstract:

Intracellular O2 is a key regulator of NO signaling, yet most in vitro studies are conducted in atmospheric O2 levels, hyperoxic with respect to the physiologic milieu. We investigated NO signaling in endothelial cells cultured in physiologic (5%) O2 and stimulated with histamine or shear stress. Culture of cells in 5% O2 (>5 d) decreased histamine- but not shear stress–stimulated endothelial (e)NOS activity. Unlike cells adapted to a hypoxic environment (1% O2), those cultured in 5% O2 still mobilized sufficient Ca2+ to activate AMPK. Enhanced expression and membrane targeting of PP2A-C was observed in 5% O2, resulting in greater interaction with eNOS in response to histamine. Moreover, increased dephosphorylation of eNOS in 5% O2 was Ca2+-sensitive and reversed by okadaic acid or PP2A-C siRNA. The present findings establish that Ca2+mobilization stimulates both NO synthesis and PP2A-mediated eNOS dephosphorylation, thus constituting a novel negative feedback mechanism regulating eNOS activity not present in response to shear stress. This, coupled with enhanced NO bioavailability, underpins differences in NO signaling induced by inflammatory and physiologic stimuli that are apparent only in physiologic O2 levels. Furthermore, an explicit delineation between physiologic normoxia and genuine hypoxia is defined here, with implications for our understanding of pathophysiological hypoxia.—Keeley, T. P., Siow, R. C. M., Jacob, R., Mann, G. E. A PP2A-mediated feedback mechanism controls Ca2+-dependent NO synthesis under physiological oxygen.

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