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Efficacy of inverse agonists in cells overexpressing a constitutively active beta2-adrenoceptor and type II adenylyl
1Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Scotland.
Abstract:
1 Maximal stimulant output from the adenylyl cyclase cascade in neuroblastoma x glioma hybrid, NG108-15, cells is limited by the levels of expression of isoforms of adenylyl cyclase. Stable expression in these cells of a constitutively active mutant (CAM) version of the human beta2-adrenoceptor resulted in higher basal adenylyl cyclase activity than following expression of the human wild type beta2-adrenoceptor. Isoprenaline acted as a full agonist in membranes from both wild type and CAM beta2-adrenoceptor expressing clones. 2 Expression of type II adenylyl cyclase resulted in a substantially elevated capacity of isoprenaline to stimulate [3H]-forskolin binding, whereas in CAM beta2-adrenoceptor expressing cells the basal high affinity [3H]-forskolin binding represented a markedly greater % of the maximal effect which could be produced by addition of isoprenaline, and the EC50 for isoprenaline was some 10 fold lower than in cells expressing the wild type beta2-adrenoceptor. 3 Further transfection of the CAM beta2-adrenoceptor expressing cells with type II adenylyl cyclase greatly increased both absolute basal and agonist-stimulated levels of adenylyl cyclase activity. 4 Betaxolol, ICI 118,551, sotalol and timolol acted as inverse agonists with varying degrees of efficacy, whereas propranolol functioned as a neutral antagonist and alprenolol as a partial agonist. 5 Pretreatment of the CAM beta2-adrenoceptor and type II adenylyl cyclase expressing clones with the irreversible alkylating agent BAAM (1 microM) did not reduce the efficacy of isoprenaline but eliminated efficacy from all the inverse agonist ligands. This effect was dependent upon the concentration of BAAM employed, with half-maximal effects being produced between 10 nM and 100 nM of the alkylating agent, which is similar to the concentrations required to prevent subsequent ligand access to some 50% of the CAM beta2-adrenoceptor population. 6 These data demonstrate that inverse agonist efficacy can be modulated by receptor availability and also indicate why in physiological systems, inverse agonism can be difficult to detect.
Insights
This study shows that receptor availability influences inverse agonist efficacy in the adenylyl cyclase cascade. Understanding this modulation is key to detecting inverse agonism in physiological systems.
Area of Science:
- Pharmacology
- Molecular Biology
- Cell Signaling
Background:
- Maximal adenylyl cyclase cascade output is limited by adenylyl cyclase isoform expression levels.
- A constitutively active mutant (CAM) beta2-adrenoceptor increases basal adenylyl cyclase activity compared to wild-type receptors.
Purpose of the Study:
- To investigate the impact of adenylyl cyclase type II expression on beta2-adrenoceptor signaling.
- To characterize the agonist and inverse agonist activity at wild-type and CAM beta2-adrenoceptors.
- To explore the role of receptor availability in modulating inverse agonist efficacy.
Main Methods:
- Stable expression of wild-type and CAM human beta2-adrenoceptors in NG108-15 cells.
- Transfection with adenylyl cyclase type II to assess its effect on signaling.
- Ligand binding assays using [3H]-forskolin and various agonists/antagonists.
- Treatment with the irreversible alkylating agent BAAM to probe receptor accessibility.
Main Results:
- Expression of adenylyl cyclase type II enhanced isoprenaline-stimulated signaling.
- CAM beta2-adrenoceptors showed lower EC50 for isoprenaline and higher basal activity.
- Inverse agonists (betaxolol, ICI 118,551, sotalol, timolol) lost efficacy after BAAM treatment, while the full agonist isoprenaline retained it.
- BAAM's effect on inverse agonists was concentration-dependent, correlating with receptor occupancy.
Conclusions:
- Inverse agonist efficacy is significantly modulated by receptor availability.
- The study provides insights into why inverse agonism can be challenging to detect in physiological settings.
- Receptor accessibility plays a critical role in determining the observed efficacy of inverse agonists.