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

and the pellet resuspended in stimulation buffer. A standard curve of known cAMP concentrations was constructed and pipetted into the designated wells. Cells (50 μl) were added to the remaining wells containing either water or test drug to give a final volume of 100 μl, and the plate incubated at 37C for 15 mins. The reaction was then terminated by addition of 100 μl of detection buffer containing 0.018 μCi of 125I-cAMP and membrane permeabilising reagent (provided with the FlashPlate kit). After 2 hrs. at room temperature, the FlashPlate was placed onto a Packard TopCount Microplate Scintillation Counter and the level of bound radioactivity measured.

Results

-2 adrenoreceptors are positively coupled to adenylyl cyclase and so stimulate the production of cAMP. It was important to establish the number of cells per well that would produce measurable basal and stimulated levels of cAMP, i.e., that would lie on the standard curve. Figure 1 shows a linear relationship between the number of cells added per well and basal levels of cAMP produced. The receptor agonist, isoprenaline (10 nM), stimulated cAMP levels approximately 5 fold for 1 x 105 and 5 x 105 cells per well, but could not be measured from the standard curve for 1 x 106 cells per well. These results suggest that a range from 1 x 105 to 5 x 105 cells per well could be used in this assay system. Because this assay is homogeneous, i.e., a uniform cell suspension is added to each well, we would expect very little intra-assay variation, as shown in Figure 2. Mean basal cAMP levels over 10 wells = 1.34 0.04 and isoprenaline-stimulated levels = 5.93 0.12, thus showing approximately 2% error between wells.

Isoprenaline stimulated cAMP accumulation (Fig. 3) with a pEC50 = 7.8 0.1 and could be dosedependently blocked by the receptor antagonist pind
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