Nitric oxide biosensor uncovers diminished ferrous iron-dependency of cultured cells adapted to physiological oxygen levels

Abstract

Iron is an essential metal for cellular metabolism and signaling, but it has adverse effects in excess. The physiological consequences of iron deficiency are well established, yet the relationship between iron supplementation and pericellular oxygen levels in cultured cells and their downstream effects on metalloproteins has been less explored. This study investigates the functionality of geNOps - an iron-containing metalloprotein and NO biosensor - in cultured HEK293T epithelial and EA.hy926 endothelial cells adapted to standard room air (18 kPa O2) or physiological normoxia (5 kPa O2). We show that cells in culture require iron supplementation to activate the metalloprotein geNOps and demonstrate that cells adapted to physiological normoxia require significantly lower iron to achieve geNOps signals comparable to those measured in cells adapted to hyperoxia. This study establishes an essential role for recapitulating oxygen levels in vivo and uncovers a previously unrecognized requirement for ferrous iron supplementation under standard cell culture conditions to achieve geNOps functionality.

Simultaneous Manipulation and Imaging of Chemogenetically Induced Hydrogen Peroxide in Hardly Transfectable Endothelial Cells

“…This study has employed the ultrasensitive H2O2 biosensor HyPer7 as a direct read-out for H2O2 -generation. Alternative biosensors such as geNOps for nitric oxide (NO) [38] , GCaMPs [39] for calcium, and MALions [40] for ATP-imaging can be combined with chemogenetic approaches presented in this study to investigate the acute or long-term effects of ultra-local H2O2 with differentially targeted mDAAOs. Notably, these signaling and metabolic parameters are hallmarks of most vascular-derived diseases.…”