Real-time monitoring of phosphodiesterase inhibition in intact cells.
Phosphodiesterases (PDEs) are hydrolytic enzymes, which convert cyclic AMP (cAMP) and cyclic GMP (cGMP) into their corresponding monophosphates. PDE-dependent hydrolysis shape gradients of these second messengers in cells, which may form the basis of their compartmentation and play a key role in a vast number of physiological and pathological processes. Here, we present a novel approach for real-time monitoring of local cAMP and cGMP levels associated with particular PDEs. We used HEK 293 cells expressing genetic constructs encoding a PDE of interest (PDE3A, PDE4A1 or PDE5A) fused to cAMP and cGMP sensors, which allow to directly visualize changes in cyclic nucleotide concentrations in the vicinity of PDE molecules by fluorescence resonance energy transfer (FRET). FRET was detected by imaging of single cells on 96-well plates and demonstrated specific effects of PDE inhibitors on local cyclic nucleotide levels. In addition, this approach reported physiological regulation of PDE3A activity, its activation by PKA-dependent phosphorylation and inhibition by cGMP. In conclusion, our assay provides a unique and highly sensitive method to analyze PDE activity in living cells. It allows to sense cAMP gradients around particular PDE molecules and to study the pharmacological effects of selective inhibitors on localized cAMP signalling.
Herget
Sabine
S
Institute of Pharmacology and Toxicology, University of Würzburg, 97078 Würzburg, Germany.
Lohse
Martin J
MJ
Nikolaev
Viacheslav O
VO
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000032:analysis*; D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000032:analysis*; D002460:Cell Line; D000242:Cyclic AMP / Q000032:analysis; D006152:Cyclic GMP / Q000032:analysis; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3 / Q000032:analysis / Q000235:genetics; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4 / Q000032:analysis / Q000235:genetics; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5 / Q000032:analysis / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D058987:Phosphodiesterase 3 Inhibitors; D058988:Phosphodiesterase 4 Inhibitors; D058986:Phosphodiesterase 5 Inhibitors; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000032:analysis
D058987:Phosphodiesterase 3 Inhibitors; D058988:Phosphodiesterase 4 Inhibitors; D058986:Phosphodiesterase 5 Inhibitors; D010726:Phosphodiesterase Inhibitors; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517287:PDE3A protein, human; C517355:PDE4A protein, human; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517381:PDE5A protein, human; D006152:Cyclic GMP