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Use of Novel FlashPlate Technology to Measure cAMP Accumulation in Chinese Hamster Ovary Cells Expressing Human -2 Adrenoreceptors

olol. Schild analysis of this data gave a pKb for pindolol = 9.1 0.1 (Fig. 4) which is consistent with its affinity for native receptors (Tsuchihashi et al . 1989). Pindolol also appeared to depress basal levels of cAMP (Fig. 3) which could be due to the presence of endogenous agonist or that pindolol has negative intrinsic activity.

Discussion

We have investigated the use of novel FlashPlate technology to directly measure cAMP accumulation in CHO cells expressing h-2 receptors. Previous assay methods involve a 2-step process: 1) generation of cAMP and 2) measurement of cAMP levels, whereas this new method is comprised of a single step process that can be carried out in either 96-well or 384-well FlashPlates.

Our studies revealed that 1 x 105 cells per well produced a suitable signal, although this may change depending on the cell line, receptor type and/or G protein-coupling involved. It is therefore essential to validate each new system that is to be used. Studies on assay variability showed the production of cAMP to be robust within plates, and measurement of pharmacological parameters of standard compounds (pEC50 for isoprenaline and pKb for pindolol) showed robust responses between plates.

In conclusion, Adenylyl Cyclase Activation FlashPlate Assay can be used to directly measure the production of cAMP from CHO cells expressing human -2 adrenoreceptors. This provides a simple method that can be easily automated and hence be used as a high-throughput assay.

References

P.A. Kasila. A Novel Adenylyl Cyclase Activation Assay on FlashPlate, FlashPlate File #1 , PerkinElmer Life Sciences (1998).

Tsuchihashi et al . Binding Characteristics of 3H-CGP12177 to -adrenoreceptors in Rat Myocardial Membranes. Japan. J. Pharmacol. 49, 11-19
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