title: Differential patterning of cGMP in vascular smooth muscle cells revealed by single GFP-linked biosensors.
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.
authors.affiliation: Department of Pharmacology, University of Vermont College of Medicine, Burlington, VT 05405, USA.
authors.forename: Lydia W M
authors.initials: LW
authors.lastname: Nausch
authors.forename: Jonathan
authors.initials: J
authors.lastname: Ledoux
authors.forename: Adrian D
authors.initials: AD
authors.lastname: Bonev
authors.forename: Mark T
authors.initials: MT
authors.lastname: Nelson
authors.forename: Wolfgang R
authors.initials: WR
authors.lastname: Dostmann
mesh_terms: D000818:Animals; D001011:Aorta / Q000473:pathology; D015374:Biosensing Techniques; D002138:Calibration; D006152:Cyclic GMP / Q000378:metabolism*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D007700:Kinetics; D018613:Microscopy, Confocal / Q000379:methods; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology*; D045265:Natriuretic Peptides / Q000737:chemistry; D009569:Nitric Oxide / Q000737:chemistry; D051381:Rats
chemical_list: D045265:Natriuretic Peptides; D049452:Green Fluorescent Proteins; D009569:Nitric Oxide; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP