Full and Partial Agonists of Muscarinic M₃ Receptors Reveal Single and Oscillatory Ca²⁺ Responses by β₂-Adrenoceptors

Abstract

Under physiological circumstances, cellular responses often reflect integration of signaling by two or more different receptors activated coincidentally or sequentially. In addition to heterologous desensitization, there are examples in which receptor activation either reveals or potentiates signaling by a different receptor type, although this is perhaps less well explored. Here, we characterize one such interaction between endogenous receptors in human embryonic kidney 293 cells in which G␣ q/11 -coupled muscarinic M 3 receptors facilitate Ca 2ϩ signaling by G␣ s -coupled ␤ 2 -adrenoceptors. Measurement of changes in intracellular [Ca 2ϩ ] demonstrated that noradrenaline released Ca 2ϩ from thapsigargin-sensitive intracellular stores only during activation of muscarinic receptors. Agonists with low efficacy for muscarinic receptor-mediated Ca 2ϩ responses facilitated cross-talk more effectively than full agonists. The cross-talk required G␣ s and was dependent upon intracellular Ca 2ϩ release channels, particularly inositol (1,4,5)-trisphosphate receptors. However, ␤ 2 -adrenoceptor-mediated Ca 2ϩ release was independent of measurable increases in phospholipase C activity and resistant to inhibitors of protein kinases A and C. Interestingly, single-cell imaging demonstrated that particularly lower concentrations of muscarinic receptor agonists facilitated marked oscillatory Ca 2ϩ signaling to noradrenaline. Thus, activation of muscarinic M 3 receptors profoundly influences the magnitude and oscillatory behavior of intracellular Ca 2ϩ signaling by ␤ 2 -adrenoceptors. Although these receptor subtypes are often coexpressed and mediate contrasting acute physiological effects, altered oscillatory Ca 2ϩ signaling suggests that cross-talk could influence longer term events through, for example, regulating gene transcription.Cells express a range of different receptors able to transduce extracellular signals and ultimately influence cellular behavior. Although receptor activation, intracellular signaling, and functional responses are often studied in isolation, such events in physiological settings are more likely to reflect the integration of signaling mediated by two or more different receptors that are activated either coincidentally or sequentially. For G protein-coupled receptors (GPCRs), such interactions have been explored, and there are many examples in which heterologous desensitization results in the loss of response to the challenge of one receptor type after activation of another receptor type linked to the same or a different signaling pathway. Perhaps less well explored is cross-talk in which activation of one receptor type either reveals or potentiates signaling by a different receptor type. One example of such cross-talk is that in which activation of a G␣ q/11 -coupled GPCR facilitates Ca 2ϩ signaling by either G␣ i/o -or G␣ s -coupled GPCRs (Werry et al., 2003a). In most instances, the facilitated Ca 2ϩ signaling is dependent upon an intracellular store, but the mechanisms through which add...

Muscarinic Receptor Activation Increases hERG Channel Expression through Phosphorylation of Ubiquitin Ligase Nedd4-2

“…HEK 293 cells, both M2 and M3 muscarinic receptors have been reported to be endogenously expressed (Kurian et al, 2009), and our data also confirmed their presence (Fig. 3A, upper panel).…”

Cyclic AMP directs IP3-evoked Ca2+ signalling to different intracellular Ca2+ stores

“…1B), by isoproterenol, which stimulates endogenous b 2 -adrenoceptors (supplementary material Fig. S1A) (Kurian et al, 2009), or by high concentrations of a membranepermeant analogue of cAMP, 8-bromo-cAMP (8-Br-cAMP) (supplementary material Fig. S1B).…”

“…We Tovey et al, 2008;Tovey et al, 2003) and others (Schwindinger et al, 1998) find that in HEK 293 cells stably expressing physiological levels of PTHR1 (HEK-PR1 cells), PTH stimulates AC, but it does not alone evoke an increase in [Ca 2+ ] i . However, PTH (or endogenous b 2 -adrenoceptors) potentiates the Ca 2+ signals evoked by receptors that stimulate PLC (Kurian et al, 2009;Tovey et al, 2008). This effect of PTH is entirely mediated by cAMP, it requires activation of neither cAMP-dependent protein kinase (PKA) nor epacs, and it results from cAMP binding to IP 3 R directly or to a protein that tightly associates with IP 3 R (Tovey et al, 2008;Tovey et al, 2010).…”

Receptor-Mediated Suppression of Potassium Currents Requires Colocalization within Lipid Rafts

“…Activation of these receptors produced accumulation of inositol phosphate, demonstrating the hydrolysis of PIP 2 (Mundell and Benovic, 2000). mRNA for the m3 muscarinic receptor in HEK293 cells had been reported (Ancellin et al, 1999), which has been substantiated by functional pharmacology (Kurian et al, 2009). Therefore, it would be expected that activation of endogenous receptors would evoke suppression of expressed Kv7.2/7.3 currents.…”