Agonist-stimulated [35S]GTPgammaS autoradiography: optimization for high sensitivity.
The receptor-stimulated accumulation of [35S]GTPgammaS provides a measure of functional coupling of G proteins with receptors. Sensitivity for autoradiographic visualization of [35S]GTPgammaS binding was improved two- to threefold in rat brain sections by optimizing assay conditions. Non-specific (NSB), basal and agonist-stimulated [35S]GTPgammaS binding were measured, using methadone, 5-carboxamidotryptamine and epinephrine for mu-opiate receptors, 5-HT(1A) receptors and alpha(2)-adrenoceptors. Basal and NSB were low in glycylglycine buffer compared to Tris or HEPES buffers, and agonist-stimulated [35S]GTPgammaS binding was more easily observed. Further optimization using glycylglycine buffer found increased signal-to-noise ratio with inclusion of dithiothrietol, increased [35S]GTPgammaS incubation time (2-4 h) and guanosine 5'-diphosphate (GDP) preincubation (20-30 min), and use of [35S]GTPgammaS at 0.1 nM. Improved sensitivity was due to decreased NSB and basal [35S]GTPgammaS binding and agonist-stimulated binding were similarly affected for each receptor system. The assay conditions described should extend the use of agonist-stimulated [35S]GTPgammaS autoradiography to receptors, which produce low levels of [35S]GTPgammaS binding and to the measurement of changes in receptor-G protein coupling.
Happe
H K
HK
Department of Pharmacology, University of Nebraska Medical Center, 986260 Nebraska Medical Center, 600 South 42nd Street, Omaha, NE 68198-6260, USA.
Bylund
D B
DB
Murrin
L C
LC
D000322:Adrenergic Agonists / Q000494:pharmacology*; D058647:Adrenergic alpha-2 Receptor Agonists; D000818:Animals; D001345:Autoradiography; D001667:Binding, Competitive / Q000187:drug effects; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D002021:Buffers; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004837:Epinephrine / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008691:Methadone / Q000494:pharmacology; D058915:Purinergic P1 Receptor Antagonists; D051381:Rats; D017207:Rats, Sprague-Dawley; D011985:Receptors, Serotonin / Q000187:drug effects; D044263:Receptors, Serotonin, 5-HT1; D012680:Sensitivity and Specificity; D012701:Serotonin / Q000031:analogs & derivatives / Q000494:pharmacology; D017366:Serotonin Receptor Agonists / Q000494:pharmacology*; D012965:Sodium Chloride / Q000494:pharmacology; D013462:Sulfur Radioisotopes; D013997:Time Factors; D014970:Xanthines / Q000494:pharmacology
D000322:Adrenergic Agonists; D058647:Adrenergic alpha-2 Receptor Agonists; D002021:Buffers; D058915:Purinergic P1 Receptor Antagonists; D011985:Receptors, Serotonin; D044263:Receptors, Serotonin, 5-HT1; D017366:Serotonin Receptor Agonists; D013462:Sulfur Radioisotopes; D014970:Xanthines; D006153:Guanosine Diphosphate; D012701:Serotonin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012965:Sodium Chloride; C047087:5-carboxamidotryptamine; C051360:1,3-dipropyl-8-cyclopentylxanthine; D008274:Magnesium; D004229:Dithiothreitol; D008691:Methadone; D004837:Epinephrine