期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 105, 期 1, 页码 365-370出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0710387105
关键词
fluorescent biosensors; natriuretic peptides; nitric oxide; confocal microscopy; cGMP-dependent protein kinase
资金
- NHLBI NIH HHS [HL77378, HL68891, HL44455, R01 HL068891, P01 HL077378, R01 HL068991, R01 HL044455] Funding Source: Medline
- NIDDK NIH HHS [R01 DK065947, DK65947, R37 DK053832, R01 DK053832, DK53832] Funding Source: Medline
Here, we report the design of unprecedented, non-FRET based cGMP-biosensors, named FlincGs, to assess the dynamics of nitric oxide (NO) and atrial natriuretic peptide (ANP) induced synthesis of intracellular cGMP, [cGMP](i). Regulatory fragments of PKG I alpha, PKG I beta, and an N-terminal deletion mutant of PKG I alpha were fused to circular permutated EGFP to generate alpha-, beta-, and delta-FlincG, with high dynamic ranges and apparent KD,CGMP values of 35 nM, 1.1 mu M, and 170 nM, respectively. All indicators displayed significant selectivity for cGMP over cAMP, and 1.5- to 2.1-fold increases in fluorescence intensity at 510 nm when excited at 480 nm. Surprisingly, FlincGs displayed an additional excitation peak at 410 nm. delta-FlincG permitted ratiometric (480/410 nm) measurements, with a cGMP-specific 3.5-fold ratio change. In addition, delta-FlincG presented cGMP association and dissociation kinetics sufficiently fast to monitor rapid changes of [cGMP](i) in intact cells. In unpassaged, adenoviral transfected vascular smooth muscle (VSM) cells, delta-FlincG had an EC50.cGMP of 150 nM, and revealed transient global cGMP elevations to sustained physiological NO (EC50,(DEA/NO) = 4 nM), and the decay phase depended on the activity of PDE-5. In contrast, ANP elicited sustained submembrane elevations in [cGMP](i), which were converted to global cGMP elevations by inhibition of PDE-5 by sildenafil. These results indicate that FlincGs is an innovative tool to elucidate the dynamics of a central biological signal, cGMP, and that NO and natriuretic peptides induce distinct cGMP patterning under the regulation of PDE-5, and therefore likely differentially engage cGMP targets.
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