Intracellular cAMP measurement

Introduction

Cyclic adenosine 3', 5'-monophosphate (cAMP) is one of the most important second messengers involved in the physiological responses of neurotransmitters, hormones and drugs.
cAMP is produced from adenosine triphosphate (ATP) by membrane adenylate cyclase. Regulation of intracellular cAMP concentration is controlled by the balance between its synthesis from ATP and its rapid degradation to 5'-AMP by a phosphodiesterase (PDE). Some GPCR receptors can control cAMP production by acting via the activation of specific G proteins, which can stimulate (Gs) or inhibit (Gi) its production.

The measurement of intracellular cAMP is therefore a method for measuring the effect of compounds on some GPCRs.

Principle

The assay is based on competition between europium-labelled tracer cAMP and sample cAMP for binding to ULight™ dye-labelled anti-cAMP antibodies.
In the absence of free cAMP, the antibodies bind to the tracer cAMP, the energy emitted by the europium excited at 320 or 340nm is transferred by FRET to the ULight™ molecules which emit at 665nm, the TR-FRET signal is maximal. In the presence of free cAMP, there is competition for antibody binding, so the TR-FRET signal is decreased.

Protocol

Format384-well format
Cellular modelHEK293 in suspension   Possibility to work on other cell lines
ReceptorGαs coupled GPCR

Distribution of cells in the plate

Stimulation with compounds of interest

Addition of cAMP tracer and anti-AMPc-ULight™ antibody solutions
DetectionExcitation wavelength : 320 nm
Emission wavelength 1 : 665 nm

Donor fluorophore emission
Emission wavelength 2 : 615 nm
Emission of the acceptor fluorophore

 

Notes :

  • Quantity of cells expressing the receptor of interest (transient or stable line) to be defined during assay validation
  • Allow 2 to 3 mg of compounds to be tested

Recommendation :

  • Ensure solubility of the sample in a solvent compatible with cell culture

Stimulation of the vasopressin receptor and measurement of the associated cAMP signal

Format : 384 wells
Cells  : HEK293 overexpressing the vasopressin receptor AVPR2

 

Agonist assay EC50 = 0.01 nM
Antagonist assay IC50 = 5.1 nM

All the models can be adapted to your needs.