( adrenaline salbutamol terbutaline an...)Development of a sensitive enzyme fragment complementation assay for cyclic adenosine 3', 5' monophosphate and its validation for pharmacological screening at G protein-coupled receptors

adrenaline, salbutamol, terbutaline, and fenoterol (10^-9 to 10^-4 M) induced cAMP generation from A431 cells were carried out using the cAMP II HitHunter Chemiluminescence Assay. Standards (2 µl, 1–770 nM) or cells, prepared in Krebs-Henseleit buffer (2 µl, 5 ×10⁵ cells/ml), were incubated at room temperature (RT) for 30 min in the presence of target agonist, before addition of the EFC detection reagents. Plates were imaged for 30 s on the LEADseeker Multimodality Imaging System.

Results
There was a dose-dependent increase in cAMP generation with the β2 adrenoreceptor agonist isoproterenol (EC₅₀ = 4.3 nM), adrenaline (EC₅₀ = 24.2 nM), noradrenaline (EC₅₀ = 482.9 nM), salbutamol (EC₅₀ = 255.9 nM), terbutaline (EC₅₀ = 2142 nM), and fenoterol (EC₅₀ = 221.5 nM) (Fig 5). EC₅₀ values were broadly similar to those reported previously when SPA and fluorescence polarization techniques were used to measure cAMP induction with several of these agonists (3).

The response of A431 cells to isoproterenol was clearly inhibited by the b2 adrenoreceptor antagonist propranolol (IC₅₀ = 12.99 nM) (Fig 6). Briefly, A431 cells (2 µl, 5 ×10⁵ cells/ml) were pre-incubated (30 min, RT) with propranolol (1–300 nM) before addition of isoproterenol (2.5 µM, 30 min, RT), and EFC reagents (2 ×2 µl). Plates were incubated overnight before imaging on LEADseeker Multimodality Imaging system for 30 s.

Pharmacological screening
Here we describe an approach that employed twenty 96-well compound library plates reformatted and dispensed into five 384-well source plates consisting 2 ×384 Library of Pharmacologically Acti
'"/>

Source:

Page: All 1 2 3 4 5 6
Related biology technology :

1. The let-7 microRNA Family Regulates RAS: Implications for Development and Oncogenesis
2. Rapid Method Development With the BioLogic DuoFlow Chromatography System for the Purification of His-Tagged Proteins
3. A Multiple Mutation Model System as a Test Development and Training Tool for Denaturing Gradient Gel Electrophoresis
4. Development of a Multiplex Bead-Based Assay for Antibody Screening of a Nonhuman Primate Colony on the Bio-Plex System
5. Development of a histamine H1 receptor binding assay using the LEADseeker Multimodality Imaging System
6. Development of an NK2 neuropeptide receptor binding assay using LEADseeker Multimodality Imaging System
7. Development of an adrenergic α2a receptor binding assay using the LEADseeker Multimodality Imaging System
8. Development of a CRF1 neuropeptide receptor binding assay using LEADseeker Multimodality Imaging System
9. Development of Radioligand Binding Assays for the Motilin Receptor Using ScreenReady Targets.
10. Screening for β2-adrenergic receptor agonists using the pH-sensitive dye,CypHer5, and the IN Cell Analyzer 3000
11. The combination of monolithic columns and modern HPLC/MS/MS instrumentation for high chromatographic throughput and sensitive detection of beta-agonists