Characterization of 5-HT1A receptor-mediated [35S]GTPγS binding in rat hippocampal membranes

Stimulation of [S-35]GTP gamma S binding by serotonin (5-hydroxytryptamine, 5-HT) receptor ligands was characterized in rat hippocampal membranes. The optimized assay contained 30-50 mu g protein, 300 mu M GDP and 0.1 nM [S-35]GTP gamma S, incubated at 37 degrees C for 20 min. At 10 mu M, the 5-HT1A receptor agonist R(+)-8-hydroxy-2-(di-n-propylamino)tetralin [R(+)-8-OH-DPAT] stimulated GTP gamma S binding from 27.1 +/- 2.5 to 45.7 +/- 4.2 fmol/mg protein. Increasing the protein concentration did not affect the absolute difference between basal and maximal GTP gamma S binding nor the EC50, but decreased the percent stimulation. The non-selective agonists serotonin and 5-carboxamidotryptamine were 30-35% more efficacious, whereas the partial agonists buspirone and S(-)-8-hydroxy-2-(di-n-propylamino)tetralin stimulated GTP gamma S binding by 19 +/- 1 and 43 +/- 3%, respectively, compared to R(+)-8-OH-DPAT. Neither the 5-HT2 receptor agonist [(+/-)1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl] (DOI) nor the 5-HT1A receptor antagonists WAY 100,635 (n-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-n-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride) and spiperone altered basal GTP gamma S binding. WAY 100,635 abolished the effect of R(+)-8-OH-DPAT, but only reduced the effect of serotonin by 88 +/- 3%. Finally, methiothepin antagonized R(S)-8-OH-DPAT-stimulated GTP gamma S binding and reduced basal GTP gamma S binding by itself. The reduction was not affected by WAY 100,635. We have characterized a method to assess functional activity at 5-HT1A receptors in rat hippocampal membranes by measuring agonist-induced [S-35]GTP gamma S binding. (C) 1998 Elsevier Science B.V.