کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
5514185 | 1541590 | 2017 | 9 صفحه PDF | دانلود رایگان |
- geNOps are capable to resolve NOS-specific NO generation profiles in single HEK293Â cells.
- Cellular NO generation by Ca2+-activated nNOS strictly follows the Ca2+ signal.
- eNOS-derived NO generation upon cellular Ca2+ signals occur gradually and sustained.
- A novel ratiometric geNOp variant enables detection of high NO levels generated by iNOS.
- L-arginine depletion uncovers the dynamic substrate dependency of iNOS.
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 HEK293Â 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.
Journal: Nitric Oxide - Volume 70, 1 November 2017, Pages 59-67