Real-time monitoring of the PDE2 activity of live cells: hormone-stimulated cAMP hydrolysis is faster than hormone-stimulated cAMP synthesis.
Cyclic nucleotide phosphodiesterases (PDEs) are the enzymes that catalyze the hydrolysis of cAMP and cGMP, thereby restricting the activity of these second messengers in cells. A unique ability to shape gradients of cyclic nucleotides and compartmentalize their signaling implies a high potency and a rapid action of PDEs. However, it has not been demonstrated how fast PDEs can hydrolyze cAMP in a living system. Here we perform a real-time monitoring of PDE2 activity in aldosterone-producing adrenal cells using a recently developed genetically encoded, fluorescent cAMP sensor, which reveals enormously rapid kinetics of cAMP degradation. Activation of PDE2 results in a rapid decrease of intracellular cAMP from high micromolar to the sub-micromolar range within a few seconds. Moreover, the kinetics of atrial natriuretic peptide-stimulated PDE2 activity (measured as decline of cAMP) are much faster than the speed of ACTH and isoprenaline-induced cAMP-synthesis (measured as cAMP accumulation) in the cells, revealing high catalytic activity and fast action of PDEs in regulating cAMP signaling in a physiological system.
Nikolaev
Viacheslav O
VO
Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany.
Gambaryan
Stepan
S
Engelhardt
Stefan
S
Walter
Ulrich
U
Lohse
Martin J
MJ
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000494:pharmacology; D002417:Cattle; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer; D006868:Hydrolysis; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D015384:Zona Glomerulosa / Q000166:cytology / Q000187:drug effects / Q000201:enzymology
D005576:Colforsin; D009320:Atrial Natriuretic Factor; D000242:Cyclic AMP; D010727:Phosphoric Diester Hydrolases