Detailed pharmacological characterization of 5-HT1A-receptor-mediated [35S]GTP gamma S binding in rat hippocampal membranes.
5-HT-stimulated [(35)S]GTPgammaS binding to rat hippocampal membranes was pharmacologically characterized. Signal/noise ratio or percent increase over basal was optimized with 100 microM GDP, 2-10 mM MgCl(2), and 150-200 mM NaCl. However, we preferred the standard condition (20 microM GDP, 5 mM MgCl(2), and 100 mM NaCl: Condition I) to the alternative one (100 microM GDP, 5 mM MgCl(2), and 150 mM NaCl: Condition II) because 1) absolute values of basal and 5-HT-sensitive bindings decreased with higher concentrations of GDP and NaCl; 2) EC(50) values determined under Condition II were 2 - 6 fold higher than those under Condition I; 3) some partial agonists had less intrinsic activities in the presence of higher concentrations of GDP; and 4) Inhibitory effects of WAY100635 were complete under Condition I, while incomplete under Condition II. Pharmacological profile of concentration-dependent stimulation by a series of 5-HT ligands and concentration-dependent inhibition of 5-HT-stimulated binding by several 5-HT-receptor antagonists clearly indicated that this response under Condition I was mediated solely through 5-HT(1A) receptors. Although caution should be paid especially to the apparent intrinsic activities susceptible to the assay conditions, this method appears useful to investigate functional coupling between 5-HT(1A) receptors and their coupled G proteins in native hippocampal membranes.
Odagaki
Yuji
Y
Department of Psychiatry, Saitama Medical School, Saitama 350-0495, Japan. odagaki@saitama-med.ac.jp
Toyoshima
Ryoichi
R
D000818:Animals; D004305:Dose-Response Relationship, Drug; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006624:Hippocampus / Q000378:metabolism*; D008297:Male; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D044282:Receptor, Serotonin, 5-HT1A / Q000378:metabolism*; D012702:Serotonin Antagonists / Q000378:metabolism; D013462:Sulfur Radioisotopes / Q000378:metabolism
D012702:Serotonin Antagonists; D013462:Sulfur Radioisotopes; D044282:Receptor, Serotonin, 5-HT1A; D016244:Guanosine 5'-O-(3-Thiotriphosphate)