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

Authors
Journal
Proceedings of the National Academy of Sciences
0027-8424
Publisher
Proceedings of the National Academy of Sciences
Publication Date
Keywords
  • Animals
  • Pathology: Aorta
  • Biosensing Techniques
  • Calibration
  • Metabolism: Cyclic Gmp
  • Metabolism: Cyclic Gmp-Dependent Protein Kinases
  • Methods: Fluorescence Resonance Energy Transfer
  • Metabolism: Green Fluorescent Proteins
  • Kinetics
  • Methods: Microscopy
  • Confocal
  • Cytology: Muscle
  • Smooth
  • Vascular
  • Cytology: Myocytes
  • Smooth Muscle
  • Chemistry: Natriuretic Peptides
  • Chemistry: Nitric Oxide
  • Rats
Disciplines
  • Biology
  • Design

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.

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