The temporal characterisitics of cAMP production in response to full and weak partial agonists in CHO-K1 cells expressing the human BETA-2-adrenoceptor J.G.Baker, I.P.Hall & S.J.Hill. Institute of Cell Signalling, Queen's Medical Centre, Nottingham NG7 2UH, UK.

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We have previously shown that several "ß-blockers" can stimulate CRE-mediated gene transcription in the absence of a phosphodiesterase inhibitor (IBMX, Baker et al., 2002a). The aim of this study was to see if these partial agonists could stimulate a measurable increase in cAMP accumulation in the absence of a phosphodiesterase inhibitor.

CHO-K1 cells stably expressing the human ß2-adrenoceptor were used (McDonnell et al., 1998). All experiments were performed in serum free media and ³H-cAMP was measured as previously described (Baker et al., 2002b). Where intra- and extracellular cAMP were measured separately, the extra-cellular media was removed, the cells washed twice with 1ml fresh media before measurement of ³H-cAMP.

After 60 minutes incubation with 1mM IBMX, isoprenaline stimulated an increase in total ³H-cAMP production that was 38.1 + 3.6 fold over basal (log EC₅₀ -7.97 + 0.07, n=4). Under the same conditions, salmeterol (log EC₅₀ -9.52 + 0.04, 90.9 + 2.0% isoprenaline max, n=6), alprenolol (log EC₅₀ -9.09 + 0.14, 3.2 + 0.2% isoprenaline max, n=4) and CGP 12177 (log EC₅₀ -9.65 + 0.19, 5.4 + 0.9% isoprenaline max, n=4) also stimulated responses. To determine the time course of cAMP generation, responses were measured in the absence of IBMX, at set times from 0-5hours. Isoprenaline (10µM) stimulated an increase in intracellular cAMP that was maximal (10.3 + 2.9 fold over basal, n=4) within 5 minutes. Salmeterol (100nM) produced a similar rapid increase (96.9 + 1.1% isoprenaline max, n=4). No increase in intracellular cAMP was seen in response to alprenolol (10µM) or CGP 12177 (10µM, n=4).

³H-cAMP was however secreted from cells in a steady time-dependent manner. After 5 hours incubation with 10µM isoprenaline, the extracellular ³H-cAMP was increased from a mean basal at time zero of 1792 to 58799 dpm (p<0.001, n=8, paired t test; extracellular 5hour basal 1.44 + 0.26 fold over time zero basal). Salmeterol (100nM, 82.3 + 1.3% isoprenaline 5 hour secretion) stimulated a similar secretion of ³H-cAMP. Despite the lack of intracellular cAMP detected, alprenolol and CGP 12177 (both 10µM) stimulated a linear secretion of ³H-cAMP such the 5 hour secreted ³H-cAMP responses were 5.69 + 0.09%, n=4 and 6.18 + 0.28% n=4 of the isoprenaline maximum respectively. In the absence of IBMX, 5 hour extracellular cAMP responses were seen in response to isoprenaline (log EC₅₀ -7.80 + 0.05, 14.4 + 1.3 fold over basal) and salmeterol (log EC₅₀ -9.65 + 0.03, 94.3 + 3.0% isoprenaline maximum, n=4). Under these conditions, cAMP responses to alprenolol and CGP 12177 were also seen (log EC₅₀ -9.26 + 0.20 and -9.62 + 0.10; 5.6 + 0.6% and 4.6 + 0.6% isoprenaline maximum, n=4, respectively).

In summary, ³H-cAMP was generated inside cells within minutes of the addition of an efficacious agonist. cAMP was secreted from cells in a steady time-dependent manner. However, responses to very weak agonists, that did not stimulate a measurable increase in intracellular cAMP, could be measured in the absence of IBMX by monitoring secreted cAMP over a longer time.

JGB holds a Wellcome Trust Clinical Training Fellowship.

McDonnell et al., (1998) Br. J. Pharmacol. 125, 717-726.
Baker et al., (2002a) Br. J. Pharmacol. 136, 5P.
Baker et al., (2002b) Br. J. Pharmacol. 137, 400-408.