The second messengers adenosine 3’, 5’-cyclic monophosphate (cyclic AMP; cAMP) and guanosine 3’, 5’-cyclic monophosphate (cyclic GMP; cGMP) are important intracellular regulators downstream from GPCRs.

cAMP is also involved in regulating neuronal, glandular, cardiovascular, immune and other functions. A number of hormones are known to activate cAMP through the action of the enzyme adenylate cyclase which converts ATP to cAMP. The diversity of receptors known to impact cAMP-mediated signaling includes those known to affect cardiovascular and nervous systems, immune mechanisms, cell growth, differentiation, and general metabolism. Thus, there remains considerable interest in the measurement of intracellular cAMP concentrations in tissues and cell culture.

cGMP is typically present at levels 10 to 100 fold lower than cAMP in most tissues and is formed by the action of the enzyme guanylate cyclase on GTP. It is most well recognized as a key signaling trigger in the relaxation of smooth muscle cells (resulting in vasodilation) in response to nitric oxide, and as a sodium ion channel regulator (via its degradation by phosphodiesterases) critical to phototransduction. Vasodilators and hormones, such as acetylcholine, insulin and oxytocin, as well as serotonin and histamine, cause an increase in cGMP levels.

In addition to measuring cAMP and cGMP, detection of downstream GPCR signaling events such as activation of Erk1/2, Akt, Src and Stat3 are routinely assayed using phospho-specific antibodies to distinguish phosphorylation of signaling proteins at specific activating or inactivating serine, threonine, and tyrosine residues.