Pharmacological properties of 5-Hydroxytryptamine(4) receptor antagonists on constitutively active wild-type and mutated receptors.

We studied the pharmacological properties of twenty-four 5-hydroxytryptamine (5-HT)(4) receptor ligands known to act as antagonists on 5-HT(4) receptors positively coupled to adenylyl cyclase endogenously expressed in mouse colliculi neurons. In COS-7 cells expressing human or mouse 5-HT(4(a)) receptors (100-8000 fmol/mg of protein), we found neutral antagonists, partial agonists, and inverse agonists. The majority of neutral antagonists belong to the benzodioxanyl ketone class, whereas partial agonists belong to different chemical classes. We found only two inverse agonists, GR 125487 and SB 207266, which are both indoles. Analysis of pharmacological characteristics of the constitutively active wild-type and constitutively active mutated receptors revealed that 1) the ratio between the efficiencies of the full agonist 5-HT and the partial agonist RS 23597 was invariable when the receptor density increased, but was dependent on receptor structure; 2) similarly, the efficacy of the inverse agonist SB 207266 was not dependent on receptor density but was dependent on receptor structure; 3) when the receptor concentration increased, the EC(50) values of the full agonist 5-HT were not modified and the increase in basal constitutive activity, as well as its stimulation by 5-HT, followed a parallel evolution; and 4) the stimulation of basal constitutive activity by 5-HT was not modified by the overexpression of Galphas. All these results indicate that in COS-7 cells, the coupling of the 5-HT(4) receptor to adenylyl cyclase was linear with no indication of spare receptors even at high receptor density (8 pmol/mg). These results are also in accordance with a precoupling between the activated receptor (f(R*)) and adenylyl cyclase. Such observations allowed us to use the two-state model to calculate the constant J, i.e., the equilibrium allosteric constant denoting the ratio of the receptor in the inactive versus active state (J = [R]/[R*]). We found that J was a receptor structural characteristic, independent of receptor density.