To Activate NAD(P)H Oxidase With a Brief Pulse of Photodynamic Action

NAD(P)H oxidases (NOX) are a major source of reactive oxygen species (ROS), to regulate multiple physiological functions. Much effort has been made to develop different NOX inhibitors, but technologies for controlled NOX activation are not available. Ultraviolet A (UVA) irradiation is known to activate NOX2 in rodent mast cells, to elicit persistent calcium spikes. NOX2 is composed of both membrane and cytosolic subunits, making its studies rather complicated. Here we show that the catalytic subunit only NOX5, when expressed ectopically in CHO-K1 cells, is activated by UVA irradiation, inducing repetitive calcium spikes, as monitored by Fura-2 fluorescent calcium imaging. UVA-elicited calcium oscillations are inhibited by pre-incubation with NOX inhibitor diphenyleneiodonium chloride (DPI), and blocked by singlet oxygen ( 1 O 2 ) quencher Trolox-C. A brief pulse of photodynamic action (1.5 min) with photosensitizer sulphonated aluminium phthalocyanine (SALPC 2 μM, 675 nm, 85 mW.cm -2 ) in NOX5-CHO-K1 cells, or with genetically encoded protein photosensitizer miniSOG tagged to N-terminus of NOX5 (450 nm, 85 mW.cm -2 ) in miniSOG-NOX5-CHO-K1 cells, induces persistent calcium oscillations, which are blocked by DPI. In the presence of Trolox-C, miniSOG photodynamic action no longer induces any calcium increases in miniSOG-NOX5-CHO-K1 cells. DUOX2 in human thyroid follicular cells SW579 and ectopically expressed in DUOX2-CHO-K1 cells are similarly activated by UVA irradiation and SALPC photodynamic action. These data together suggest that NOX is activated with a brief pulse of photodynamic action. Supported by grant from The Natural Science Foundation of China (Grant No. 32271278). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.