Agonist-stimulated [ 35S]GTPγS autoradiography: optimization for high sensitivity

• Published in: European journal of pharmacology 2001-06, Vol.422 (1), p.1-13
• Main Authors: Happe, H.Kevin, Bylund, David B., Murrin, L.Charles
• Format: Article
• Language: English
• Subjects: 5-HT 1A receptor; Biological and medical sciences; Cell receptors; Cell structures and functions; Fundamental and applied biological sciences. Psychology; G protein-coupled receptor; General pharmacology; Medical sciences; Miscellaneous; Molecular and cellular biology; Pharmaceutical technology. Pharmaceutical industry; Pharmacology. Drug treatments; Signal transduction; α 2 adrenergic receptor; μ-Opiate receptor
• ISSN: 0014-2999; 1879-0712
• DOI: 10.1016/S0014-2999(01)01043-3

The receptor-stimulated accumulation of [ 35S]GTPγS provides a measure of functional coupling of G proteins with receptors. Sensitivity for autoradiographic visualization of [ 35S]GTPγS binding was improved two- to threefold in rat brain sections by optimizing assay conditions. Non-specific (NSB), basal and agonist-stimulated [ 35S]GTPγS binding were measured, using methadone, 5-carboxamidotryptamine and epinephrine for μ-opiate receptors, 5-HT 1A receptors and α 2-adrenoceptors. Basal and NSB were low in glycylglycine buffer compared to Tris or HEPES buffers, and agonist-stimulated [ 35S]GTPγS binding was more easily observed. Further optimization using glycylglycine buffer found increased signal-to-noise ratio with inclusion of dithiothrietol, increased [ 35S]GTPγS incubation time (2–4 h) and guanosine 5′-diphosphate (GDP) preincubation (20–30 min), and use of [ 35S]GTPγS at 0.1 nM. Improved sensitivity was due to decreased NSB and basal [ 35S]GTPγS binding and agonist-stimulated binding were similarly affected for each receptor system. The assay conditions described should extend the use of agonist-stimulated [ 35S]GTPγS autoradiography to receptors, which produce low levels of [ 35S]GTPγS binding and to the measurement of changes in receptor–G protein coupling.