Light-induced formation of NO in endothelial cells by photoactivatable NADPH analogues targeting nitric-oxide synthase

Background: Nitric-oxide synthases (NOS) catalyze the formation of NO using NADPH as electron donor. We have recently designed and synthesized a new series of two-photon absorbing and photoactivatable NADPH analogues (NT). These compounds bear one or two carboxymethyl group(s) on the 2'- or/and 3'-position(s) of the ribose in the adenosine moiety, instead of a 2'-phosphate group, and differ by the nature of the electron donor in their photoactivatable chromophore (replacing the nicotinamide moiety). Here, we addressed the ability of NTs to photoinduce eNOS-dependent NO production in endothelial cells. Methods: The cellular fate of NTs and their photoinduced effects were studied using multiphoton fluorescence imaging, cell viability assays and a BODIPY-derived NO probe for NO measurements. The eNOS dependence of photoinduced NO production was addressed using two NOS inhibitors (NS1 and L-NAME) targeting the reductase and the oxygenase domains, respectively. Results: We found that, two compounds, those bearing a single carboxymethyl group on the 3'-position of the ribose, colocalize with the Golgi apparatus (the main intracellular location of eNOS) and display high intracellular two-photon brightness. Furthermore, a eNOS-dependent photooxidation was observed for these two compounds only, which is accompanied by a substantial intracellular NO production accounting for specific photocytotoxic effects. Conclusions: We show for the first time that NT photoactivation efficiently triggers electron flow at the eNOS level and increases the basal production of NO by endothelial cells. General significance: Efficient photoactivatable NADPH analogues targeting NOS could have important implications for generating apoptosis in tumor cells or modulating NO-dependent physiological processes.