1. cAMP analogues
Used because have increased membrane permeability cf normal cAMP. In fact only 3-4 fold more permeable and therefore have to be used at high concentrations, usually in the 0.5-2mM range.
Some people use 8-bromo cAMP rather than dibutyrate cAMP, as the latter is metabolized in cells to mono-butyrate cAMP and the free butyrate can have non specific effects on cells, including effects on butyrate sensitive ion chanels. 8-bromo does not produce free butyrate.
Use the Na salt as the free acid is very insoluble (need 1M NaOH). The Na salt is aqueous soluble, prepared in appropriate buffer (eg. DMEM) as a 200mM stock and stored at -20¡, where it is very stable (100-400 X stock).
cAMP are full agonists of all mammalian PKA but also effects cAMP dependant ion chanels. However the EC50 for the ion chanels is 50-100 higher (patch clamp experiments) than for PKA.
There is apparently not much difference between cells in their dose response to cAMP.
2. Forskolin.
Also elevates cAMP. At high concentration non-specific effects because can elevate cAMP to non-physiological levels.
The cellular responses they investigate are maximal at 10uM and they use it in the range 1-10uM. Note that elevation in cAMP not maximal even at 100uM. ie. enormous capacity to increase cAMP.
Stocks prepared in ethanol (20mM), diluted in ethanol to 100-200 X and then this stock diluted by vortexing into aqueous media. Ethanol stocks very stable at -20¡.
Used as an additional control with cAMP analogues for specificity (ie. PKA mediated) of cellular response measured. Other controls can be stimualtion of an adenylate cyclase (serpentine) coupled receptor (must have right cells and ligand available)