Production of cAMP is regulated through AC — an effector enzyme whose activity can be modulated by different GTP binding proteins. Ten different isoforms of AC have been identified, based on primary sequence, tissue distribution, and regulation.38 Forskolin, a diterpene, can be used to directly activate AC to increase cAMP production. AC enzymes can be regulated by a variety of input signals (Ca2+, calmodulin, GaS, Gai, PKC) and should be considered as signal integrators. Subsequent to Gs-coupled receptor activation, an increase in AC activity converts ATP to cAMP and inorganic pyrophosphate. Gi-coupled receptor activation results in the inhibition of AC activity. Gs proteins activate all isoforms of AC, whereas Gi proteins only inhibit a subset of isoforms. Therefore, measurement of Gi-coupled receptor-mediated inhibition of AC can prove challenging.
Many methods are available to determine the concentrations of cAMP in cells or membranes; the easiest is the detection of cAMP formation. Alternatively, one could assess AC activity rather than cAMP production. The adenine nucleotides of whole cells can be labeled with [3H] adenine and then the amount of radiolabeled cAMP present in cells can be determined. The same assay can be conducted using cell membranes or homogenates to assess the conversion of radiolabeled [32P] ATP to cAMP. These methods employ column chromatography to isolate the radiolabeled cAMP product. They are tedious and not suitable for HTS applications. cAMP detection methods in HTS and its advantages and disadvantages have been exten sively reviewed by Williams.39 Although a large variety of cAMP detection technologies employ antibodies, a number of available assays do not rely on antibodies. The sensitivity of a kit that relies on antibody detection is determined by the detection technology and the quality of the antibody. Antibody-free cAMP technologies such as gene reporter assays and Ca2+-based assays are extensively used in HTS labs.
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