Real-time monitoring of the PDE2 activity of live cells: hormone-stimulated cAMP hydrolysis is faster than hormone-stimulated cAMP synthesis

J Biol Chem. 2005 Jan 21;280(3):1716-9. doi: 10.1074/jbc.C400505200. Epub 2004 Nov 22.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Atrial Natriuretic Factor / pharmacology
  • Cattle
  • Colforsin / pharmacology
  • Cyclic AMP / metabolism*
  • Enzyme Activation
  • Fluorescence Resonance Energy Transfer
  • Hydrolysis
  • Phosphoric Diester Hydrolases / metabolism*
  • Zona Glomerulosa / cytology
  • Zona Glomerulosa / drug effects
  • Zona Glomerulosa / enzymology

Substances

  • Colforsin
  • Atrial Natriuretic Factor
  • Cyclic AMP
  • Phosphoric Diester Hydrolases