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

Abstract

Serotonin (5-hydroxytryptamine; 5-HT) receptor ligands were used to assess agonist-stimulated [(35)S]GTPgammaS binding in rat and guinea pig striatal membranes. The assay contained 45-60 microgram protein, 300 microM GDP and 0.1 nM [(35)S]GTPgammaS, incubated at 37 degrees 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 [(35)S]GTPgammaS 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 [(35)S]GTPgammaS 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 [(35)S]GTPgammaS 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 [(35)S]GTPgammaS binding. The ability of cyanopindolol or spiperone (a 5-HT(1A/1D) receptor antagonist) to alter CP 93,129-stimulated [(35)S]GTPgammaS 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 [(35)S]GTPgammaS binding. In conclusion, these studies indicate that 5-HT(1B) receptor function can be assessed using agonist-stimulated [(35)S]GTPgammaS binding in rat striatal membranes using CP 93,129, 5-HT or 5-CT, but not L-694, 247.