In cellular redox biology, the intricate interplay between nitric oxide (NO) and hydrogen peroxide (H2O2) has remained a long-standing challenge. The elusive nature of these reactive molecules, coupled with their transient existence within biological systems, has historically posed a challenge for researchers to understand their roles and regulatory mechanisms [1,2]. Recent advances have been catalyzed by the application of genetically encoded biosensors, which empower real-time visualization of reactive oxygen and nitrogen species (ROS/RNS) in cellular models. These cutting-edge tools have brought us closer to understanding the dynamic behavior of NO and H2O2. However, unraveling the relationship between these two signaling molecules is not straightforward, as technical complexities inherent to in vitro systems continue to pose challenges in interpreting the nuanced interactions between these second messengers.

The introduction of HyPer biosensors in 2006 marked a significant turning point in the field of redox biology, revolutionizing our ability to unravel H2O2 dynamics [3] and significantly advancing our understanding of H2O2 dynamics within cellular systems. These biosensors have allowed us to define intricate processes governing the transport and subcellular localization of H2O2 [4,5]. Furthermore, the ability to visualize this reactive molecule has uncovered its characteristics as a second messenger and its modulation of redox signaling [6]. This has been instrumental in unraveling complex antioxidant response mechanisms and shedding light on the pivotal role of H2O2 in cancer [2]. The application of chemogenetic tools for manipulating the redox status of cells has enabled researchers to precisely modulate cellular redox states [[7], [8], [9], [10], [11]]. Thus, the utilization of HyPer biosensors has undeniably played a pivotal role in understanding cellular H2O2 dynamics.

A decade after the introduction of HyPer biosensors, we developed a biosensor for NO, geNOps, to monitor NO dynamics within subcellular locales [12]. The distinctive spectral properties of geNOps biosensors enabled multispectral imaging of subcellular NO levels [[12], [13], [14], [15], [16], [17]]. We have obtained critical insights, including the diffusion of NO across biological membranes, the direct relationship between NO and calcium ions (Ca2+), and the bioavailability of various NO donors and drugs metabolized to NO within cellular environments [14,15,[18], [19], [20]].

More recently, we provided novel insights into geNOps signaling under controlled pericellular oxygen (O2) conditions encountered by cells in vivo [21]. We reported the first evidence that NO bioavailability was significantly increased in cells adapted to physiological normoxia (5 kPa O2) compared to standard cell culture under atmospheric O2 levels. Notably, our findings provided novel and critical insights: firstly, the susceptibility of fluorescent proteins to pericellular O2 levels emerged, as FPs rely on O2 for the maturation of their chromophore [21]. Under physiological normoxia, the significant increase in NO bioavailability was paralleled by substantial alterations in redox signaling and a dramatic disruption in ferrous iron uptake. Notably, the influence of oxygen extends beyond its modulation of ROS and RNS levels, as it exerts a key role in shaping the properties of the fluorescent proteins themselves, a factor that warrants careful experimental design [22]. Furthermore, we acknowledge that others have demonstrated the profound impact of pericellular O2 culture conditions on key cellular parameters, including endothelial nitric oxide synthase (eNOS) levels and activity, antioxidant defense proteins, and Ca2+ signaling, all of which are intrinsically linked with and substantially influence the roles of H2O2 and NO within cellular systems [[23], [24], [25], [26]]. These findings collectively emphasize the importance of the interplay between intracellular O2 levels and the redox signaling landscape.

This study explores, for the first time, the direct relationship between H2O2 and NO in endothelial cells utilizing genetically encoded biosensors, within controlled atmospheric oxygen conditions with an emphasis on the optimum experimental settings.