Article Figures & Data
Figures
- Fig. 1.
System dependence of agonist-induced M2 receptor activation. Shown are simplified illustrations of the tested systems (upper panels) and the corresponding concentration–effect curves of OxoM, Pilo and I-6-p (lower panels). The green triangles represent muscarinic agonists, and the red-labeled terms are the detected readouts of the corresponding assays. All data are mean ± S.E.M. of n independent experimental days. (A, D) G protein activation was quantified in [35S]GTPγS binding experiments. (B, E) Inhibition of acetylcholine release was detected in adult murine hippocampal brain slices. Pilo (1 mM) was tested on two brain slices obtained from the same preparation (0.79, 0.81). (C) Modified from Schulte et al., 2012. (E, F) Reduction of spontaneous beating was measured in isolated embryonic cardiomyocytes isolated from murine atria.
- Fig. 2.
Chronotropic response to muscarinic agonists in spontaneously beating murine embryonic atrial cardiomyocytes (eCM). Increasing concentrations of the muscarinic agonists were applied cumulatively and normalized to baseline frequency. All experiments were performed on at least three independent days; a minimum of three data points was conducted per concentration. (A, B) Representative real-time recordings of spontaneously beating cells and their response to I-6-p under basal conditions (A) and in presence of sympathetic stimulation (B). The dotted lines represent cardiac arrest (0 bpm). The washout period is marked with gray arrows. (C, D) Increasing concentrations of I-6-p were applied under basal conditions (C) and in presence of 100 nM ISO and 100 µM IBMX (D). The dotted lines represent the mean basal frequency: 140 ± 4 bpm (n = 34) and 139 ± 7 bpm (n = 15) in case of C and D, respectively. The washout effect of each tested cell is marked in gray. The response of each individual cell to ISO and IBMX is visualized by a connecting line in D.
- Fig. 3.
Effects of OxoM and I-6-p on blood pressure and heart rate in anesthetized mice. (A, B) Original traces of left ventricular systolic blood pressure (LVSP) and heart rate (HR) recorded in response to OxoM (0.4 µmol/kg). (C, D) Original traces of LVSP and HR recorded in response to I-6-p (0.4 µmol/kg). (E, F) Statistical analysis of changes in LVSP and HR, respectively, in response to either metoprolol (3 mg/kg), OxoM, or I-6-p. P < 0.05 was considered statistically significant according to Student’s t test (***P < 0.001). The paired t test was used to check the effect of metoprolol and the unpaired t test to compare the interindividual effects of both test compounds: OxoM versus I-6-p. Indicated data are mean ± S.E.M. of n independent experiments.
- Fig. 4.
DMR response of CHO-hM2, CHO-hM2 after forskolin-pretreatment (+FSK), and MRC-5. (A, D, G) Indicated are representative real-time recordings of muscarinic agonist-induced DMR traces. The arrow marks the addition of the test compound after baseline read. (B, E, H) Bar graphs show mean maximal DMR responses of supramaximal agonist concentrations (OxoM: 100 µM; I-6-p: 100 µM; I-8-p: 10 µM; Pilo: 100 µM). Compared with OxoM, all ligands were tested for partial agonism; P < 0.05 was considered statistically significant according to one-way analysis of variance with Dunnett’s correction for comparison with OxoM (**P < 0.01). Indicated data are mean ± S.E.M. of at least n independent experiments. Experiments were performed in triplicate or quadruplicate. (C, F, I) Concentration–effect curves of the tested ligands resulting from DMR assays. The dashed lines mark the inflection points (pEC50) of the tested ligands in case of CHO-hM2. Indicated data are mean ± S.E.M. of at least three independent experiments. Experiments were performed in triplicate or quadruplicate. Potencies are listed in Supplemental Table 2 for CHO-hM2, CHO-hM2 + FSK, and MRC-5.
- Fig. 5.
Context-sensitive Gi/o-signaling of muscarinic M2 receptor agonists. Upper panel: Coupling efficiency of agonist-bound receptors to induce dynamic mass redistribution in living cells is reflected by log τ-values for CHO-hM2 cells under basal conditions, for CHO-hM2 after FSK pretreatment to elevate intracellular cAMP, and for MRC-5 fibroblasts with high endogenous cAMP and a minor receptor reserve. Test compounds are designated by the indicated color code. Inclined stippled lines visualize context-dependent shifts of respective test compound coupling efficiency. Coupling efficiencies (log τ) were calculated according to eq. 1 with KA fixed to the agonist’s dissociation binding constant (Supplemental Table 3). Lower panel: Maximum effects (Emax) of the depicted agonists under the respective conditions (CHO-hM2, CHO-hM2 + FSK, MRC-5) expressed as a fraction of the respective system’s maximum response set to Emax = 1. Emax values of the tested compounds were taken from DMR recordings (Fig. 4B: CHO-hM2, 4E: CHO-hM2 + FSK, 4H: MRC-5). Coupling efficiencies correspond to log τ-values shown in the upper panel. Emax and log τ-values are mean values ± S.E.M. The curve indicates the theoretical dependence of the maximum effect Emax from the coupling efficiency “log τ” calculated on the basis of eq. 2. Of note, steepness and thus context-sensitivity of agonist action is highest at the inflection point of the Emax/log τ-relationship. Indicated are mean ± S.E.M. of at least three independent experimental days; further details are listed in Supplemental Table 3.
Additional Files
Data Supplement
Files in this Data Supplement:
- Supplemental Data - 5 supplemental figures, 3 tables.
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Context-Sensitive Agonism through a GPCR
