Rahima Chennoufi1, Aimeric Cabrié1, Nhi Ha Nguyen2, Nicolas Bogliotti2, Françoise Simon1, Bertrand Cinquin1, Patrick Tauc1, Jean-Luc Boucher3, Anny Slama-Schwok4, Juan Xie2, Eric Deprez5. 1. LBPA, CNRS UMR8113, IDA FR3242, ENS Paris-Saclay, Université Paris-Saclay, F-94235 Cachan, France. 2. PPSM, CNRS UMR8531, IDA FR3242, ENS Paris-Saclay, Université Paris-Saclay, F-94235 Cachan, France. 3. Laboratoire de "Chimie et Biochimie Pharmacologiques et Toxicologiques", CNRS UMR8601, Université Paris Descartes, 75270 Paris, France. 4. Laboratoire de "Stabilité Génétique et Oncogénèse", CNRS UMR8200, Gustave Roussy, Université Paris-Saclay, 94607 Villejuif, France. 5. LBPA, CNRS UMR8113, IDA FR3242, ENS Paris-Saclay, Université Paris-Saclay, F-94235 Cachan, France. Electronic address: deprez@lbpa.ens-cachan.fr.
Abstract
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.
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.