Abnormalities in neurotransmission within the brain can lead to several disease states, including anxiety, depression or schizophrenia. Extensive research has been carried out to discover the mode of action of various transmitters, in order to understand and combat these diseases. In many cells, binding of the neurotransmitter to its receptor alters the production of the second messenger adenosine 3', 5' cyclic monophosphate (cAMP), through the enzyme adenylyl cyclase. Intracellular changes in the level of cAMP can then initiate a cascade of other cellular events.
We have been investigating changes of adenylyl cyclase activity, induced by receptor stimulation in recombinant cell lines, by measuring the accumulation of cAMP. Until recently, we used the commercially available cAMP [125I] Radioimmunoassay Kit (RIA) from PerkinElmer Life Sciences. (NEK033).
This method has proved highly satisfactory, but the introduction of cAMP [125I] FlashPlate Assay (SMP001) presents a potentially more convenient system for measuring cAMP. The FlashPlate method operates on the same principle as the RIA method, but the assay is performed directly in scintillant-coated microplates where the anti-cAMP antisera has been pre-bound to the wells. We have therefore carried out a study to compare the RIA and FlashPlate methods to investigate the similarities and differences between the two.
Generation of cAMP Source
The method is an adaptation from McHale, et al .1
Chinese Hamster Ovary cells expressing human 5-HT1D receptors were seeded and grown to confluence. They were subsequently harvested and resuspended in medium containing isobutylmethylxanthine (500 μM), paroxetine (1 μM), pargyline (10 μM) and ascorbate (1 μM). After 30 minutes