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 Gα q/11-coupled muscarinic M 3 receptors facilitate Ca 2+ signaling by Gα s-coupled β 2-adrenoceptors. Measurement of changes in intracellular [Ca 2+] demonstrated that noradrenaline released Ca 2+ from thapsigargin-sensitive intracellular stores only during activation of muscarinic receptors. Agonists with low efficacy for muscarinic receptor-mediated Ca 2+ responses facilitated cross-talk more effectively than full agonists. The cross-talk required Gα s and was dependent upon intracellular Ca 2+ release channels, particularly inositol (1,4,5)-trisphosphate receptors. However, β 2-adrenoceptor-mediated Ca 2+ 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 2+ signaling to noradrenaline. Thus, activation of muscarinic M 3 receptors profoundly influences the magnitude and oscillatory behavior of intracellular Ca 2+ signaling by β 2-adrenoceptors. Although these receptor subtypes are often coexpressed and mediate contrasting acute physiological effects, altered oscillatory Ca 2+ signaling suggests that cross-talk could influence longer term events through, for example, regulating gene transcription.
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Materials. Cell culture reagents were from Invitrogen (Paisley, UK). Cell culture plastics were from Nalgene (Hereford, UK). Poly-d-lysine-coated 96-well plates for fluorescence imaging plate reader (FLIPR) and other plate reader assays were from BD Biosciences (Oxford, UK). Cholera toxin (CTX), cAMP, Ins(1,4,5)P 3, pertussis toxin, fluo-3-acetoxymethyl ester (AM), fluo-4-AM, mouse γ-tubulin antibody, horseradish peroxidase-conjugated secondary antibodies, and the protein kinase inhibitor H89
Demonstration of Cross-Talk. Challenge of HEK 293 cells with the muscarinic receptor agonist methacholine (1 mM) resulted in an increase in [Ca 2+]i, consisting of a rapid transient peak, followed by a more sustained plateau phase. Addition of noradrenaline (10 μM) failed to elevate [Ca 2+]i (Fig. 1). However, challenge of cells with noradrenaline (10 μM) in the continued presence of methacholine (1 mM) resulted in a rapid elevation of [Ca 2+]i, which subsided over the subsequent few minutes (Fig.
Ca 2+ responses to muscarinic receptor agonists in our HEK 293 cells are mediated by Gα q/11-coupled muscarinic M 3 receptors. Furthermore, noradrenaline elevates [Ca 2+]i in these cells via Gα s-coupled β 2-adrenoceptors only in the presence of muscarinic receptor activation. This β 2-adrenoceptor-mediated Ca 2+ signaling is facilitated by both full and partial muscarinic receptor agonists, with partial agonists often being more effective than full agonists. Ca 2+ responses to noradrenaline, although
This work was supported in part by AstraZeneca (UK); and the Research Councils UK [Grant BBS/S/N/2006/13051] (Biotechnology and Biological Sciences Research Council).
doi:10.1124/jpet.109.153619.
GPCR, G protein-coupled receptor; PLC, phospholipase C; Ins(1,4,5)P 3, inositol 1,4,5-trisphosphate; FLIPR, fluorescent imaging plate reader; AM, acetoxymethyl ester; H89, _N_-[2-(_p_-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; PKA, protein kinase A; PKC, protein kinase C; ERK, extracellular signal-regulated kinase; HEK, human embryonic kidney; CTX, cholera toxin; HBSS, Hanks’ balanced salt solution; PAGE, polyacrylamide gel electrophoresis; InsP x, inositol phosphates; ANOVA, analysis of variance; ICI-118,551, (±)-1-[2,3-(dihydro-7-methyl-1 _H_-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride; 2-APB, 2-aminoethoxydiphenyl borane; eGFP, enhanced green fluorescent protein; PH, pleckstrin homology; EPAC, exchange proteins directly activated by cAMP.
