Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz

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SHR Neuro Krebs Kardio Lipid Stoffw Microb

Eroglu, E; Hallström, S; Bischof, H; Opelt, M; Schmidt, K; Mayer, B; Waldeck-Weiermair, M; Graier, WF; Malli, R.
Real-time visualization of distinct nitric oxide generation of nitric oxide synthase isoforms in single cells.
Nitric Oxide. 2017; 70(31):59-67 Doi: 10.1016/j.niox.2017.09.001 [OPEN ACCESS]
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Führende Autor*innen der Med Uni Graz: EROGLU Emrah; Hallström Seth
Co-Autor*innen der Med Uni Graz: Bischof Helmut; Graier Wolfgang; Malli Roland; Waldeck-Weiermair Markus
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Abstract:: The members of the nitric oxide synthase (NOS) family, eNOS, nNOS and iNOS, are well-characterized enzymes. However, due to the lack of suitable direct NO sensors, little is known about the kinetic properties of cellular NO generation by the different nitric oxide synthase isoenzymes. Very recently, we developed a novel class of fluorescent protein-based NO-probes, the geNOps, which allow real-time measurement of cellular NO generation and fluctuation. By applying these genetic NO biosensors to nNOS-, eNOS- and iNOS-expressing HEK293 cells we were able to characterize the respective NO dynamics in single cells that exhibited identical Ca²⁺ signaling as comparable activator of nNOS and eNOS. Our data demonstrate that upon Ca²⁺ mobilization nNOS-derived NO signals occur instantly and strictly follow the Ca²⁺ elevation while NO release by eNOS occurs gradually and sustained. To detect high NO levels in cells expressing iNOS, a new ratiometric probe based on two fluorescent proteins was developed. This novel geNOp variant allows the measurement of the high NO levels in cells expressing iNOS. Moreover, we used this probe to study the L-arginine-dependency of NO generation by iNOS on the level of single cells. Our experiments highlight that the geNOps technology is suitable to detect obvious differences in the kinetics, amplitude and substrate-dependence of cellular NO signals-derived from all three nitric oxide synthase isoforms. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.
Find related publications in this database (using NLM MeSH Indexing): Arginine - metabolism; Biosensing Techniques - instrumentation; Calcium - metabolism; Fluorescent Dyes - chemistry; HEK293 Cells - enzymology; Humans -; Isoenzymes -; Kinetics -; Luminescent Proteins - chemistry; Microscopy, Fluorescence -; Nitric Oxide - analysis; Nitric Oxide - biosynthesis; Nitric Oxide - chemistry; Nitric Oxide Synthase Type I - analysis; Nitric Oxide Synthase Type II - analysis; Nitric Oxide Synthase Type III - analysis
Find related publications in this database (Keywords): eNOS; geNOps; iNOS; Live-cell fluorescence imaging; Nitric oxide; nNOS