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Differential patterning of cGMP in vascular smooth muscle cells revealed by single GFP-linked biosensors

Nausch L, Ledoux J, Bonev A, Nelson MT, Dostmann W

National Academy of Sciences. Proceedings. 2008;105(1):365-370.

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Abstract

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 K(D,cGMP) values of 35 nM, 1.1 microM, 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 EC(50,cGMP) of 150 nM, and revealed transient global cGMP elevations to sustained physiological NO (EC(50,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 FlincG 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.

Bibliographic metadata

Type of resource:
Content type:
Publication status:
Accepted
Publication form:
Published date:
Language:
eng
Abbreviated journal title:
Alternative journal title:
Proceedings of the National Academy of Sciences
ISSN:
Place of publication:
United States
Volume:
105
Issue:
1
Start page:
365
End page:
370
Total:
5
Pagination:
365-370
Digital Object Identifier:
10.1073/pnas.0710387105
Attached files embargo period:
Immediate release
Attached files release date:
2nd October, 2014
Access state:
Active

Institutional metadata

University researcher(s):

Record metadata

Manchester eScholar ID:
uk-ac-man-scw:1d20819
Created:
30th August, 2009, 15:51:44
Last modified by:
Nelson, Mark
Last modified:
2nd October, 2014, 19:28:39

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