Co-stimulation of muscarinic M3 acetylcholine receptors and β2-adrenoceptors causes a synergistic effect on the release of intracellular Ca2+ ([Ca2+]i) and ERK activation

Caroline Hall2, Alastair Brown1, Graeme Wilkinson1, Gary Willars2

1AstraZeneca, Macclesfield, UK, 2University of Leicester, Leicester, UK

Cells are rarely exposed to a single type of receptor agonist. Crosstalk between different receptors and their signalling pathways provides mechanisms for integrating multiple signalling events and allowing appropriate regulation of cellular responses. Such crosstalk often results in heterologous desensitisation but can also either potentiate or facilitate signalling by alternative pathways. One example of such positive crosstalk is that in which only in the presence of Gαq activation can the activation of Gαi- or Gαs-coupled receptors cause the release of intracellular Ca2+. Here we have explored the ability of Gαq-coupled muscarinic M3 receptors to reveal Ca2+ signalling by Gαs-coupled β2-adrenoceptors and examined the consequences of crosstalk on the activation of extracellular signal-regulated kinase (ERK).

Wild-type HEK 293 cells endogenously expressing muscarinic M3 receptors and β2-adrenoceptors were loaded with fluo-4-acetoxymethylester. An index of [Ca2+]i was obtained by measuring emitted light at >510nm following excitation at 488nm in either populations of cells using a platereader or in single cells by confocal imaging. The muscarinic receptor agonists methacholine or oxotremorine evoked rapid increases in [Ca2+]i but the maximal response to oxotremorine was 25±6% of that of methacholine (data are mean+SEM, n>3). Either methacholine or oxotremorine caused rapid and transient increases in [Ca2+]i in cell populations and generally evoked oscillatory signalling in single cells that was more apparent with the partial agonist. Addition of the adrenoceptor agonist noradenaline (<10μM) did not change [Ca2+]i in either populations of cells or single cells. In contrast, noradrenaline (10μM) added following a maximal concentration of oxotremorine (100μM) elicited a robust Ca2+ response (47±18% of oxotremorine response) that was dependent on continued muscarinic receptor stimulation and thapsigargin-sensitive Ca2+ stores. Noradrenaline, in the presence of an EC50 concentration of oxotremorine (1μM), stimulated a response that was 169±4% of that to oxotremorine. Using 1μM oxotremorine and 10μM noradrenaline we further explored the influence of crosstalk on oscillatory Ca2+ signalling. Approximately 50% of the cells showed oscillatory Ca2+ signalling to oxotremorine and the oscillation frequency decreased slightly over the experiment. However, addition of noradrenaline after oxotremorine significantly (p<0.05) increased both the oscillation frequency (0.19±0.04 vs. 0.51±0.12 oscillations min-1, n=4,) and the area under the oscillations. Oxotremorine (1μM) caused a rapid and transient increase in ERK activation detected by immunoblotting of phospho-ERK, which was maximal at approximately 2 min (2.08±0.824, F/F0-basal) and absent after 20-30 min. Noradrenaline (10μM) did not cause activation of ERK over a 30 min period. Co-stimulation of cells with oxotremorine and noradrenaline resulted in a synergistic activation of ERK (4.94±1.32, F/F0-basal, p<0.05).

Thus, in HEK 293 cells, only during muscarinic receptor stimulation does noradrenaline evoke Ca2+ signalling and contribute to ERK activation. Thus, crosstalk occurs at levels other than Ca2+ signalling, which may be critical in the regulation of cellular activity. However, the mechanisms of crosstalk and the relationship between enhanced Ca2+ signalling and ERK activation remain to be established