Serotonin 5-HT 1B/1D agonist-stimulated [ 35S]GTPγS binding in rat and guinea pig striatal membranes

Abstract

Serotonin (5-hydroxytryptamine; 5-HT) receptor ligands were used to assess agonist-stimulated [ 35S]GTPγS binding in rat and guinea pig striatal membranes. The assay contained 45–60 μg protein, 300 μM GDP and 0.1 nM [ 35S]GTPγS, incubated at 37°C for 20 min. The non-selective agonists 5-HT, 5-CT (5-carboxyamidotryptamine), and L-694,247, and the selective 5-HT 1B receptor agonist CP 93,129 produced concentration-dependent increases in [ 35S]GTPγS binding in rat striatum, whereas the selective 5-HT 1A receptor agonist R(+)-8-OH-DPAT [R(+)-8-hydroxy-2-(di- n-propylamino)tetralin] was inactive. Cyanopindolol, a 5-HT 1A/1B receptor antagonist, completely blocked the effect of 5-HT. Methiothepin, yohimbine and cyanopindolol also blocked 5-CT-stimulated [ 35S]GTPγS binding with the following rank order of potency: cyanopindolol ≥ methiothepin >>> yohimbine, consistent with rat 5-HT 1B receptor pharmacology. Neither cyanopindolol nor methiothepin altered basal [ 35S]GTPγS binding by themselves while yohimbine had weak partial agonist activity. Furthermore, cyanopindolol shifted the 5-CT concentration-response curve rightward, increasing the EC 50 and decreasing the maximal response, but did not affect L-694,247-stimulated [ 35S]GTPγS binding. The ability of cyanopindolol or spiperone (a 5-HT 1A/1D receptor antagonist) to alter CP 93,129-stimulated [ 35S]GTPγS binding was determined. Cyanopindolol produced a rightward shift in the CP 93,129 concentration-response curve, while spiperone had no affect. Finally, in guinea pig striatum and hippocampus, L-694,247 produced a concentration dependent increase in [ 35S]GTPγS binding. In conclusion, these studies indicate that 5-HT 1B receptor function can be assessed using agonist-stimulated [ 35S]GTPγS binding in rat striatal membranes using CP 93,129, 5-HT or 5-CT, but not L-694,247.