Pharmacological characterizations of recombinant human 5-HT1D_1Dα and 5-HT_1Dβ receptor subtypes coupled to adenylate cyclase inhibition in clonal cell lines: apparent differences in drug intrinsic efficacies between human 5-HT_1D subtypes

Abstract

Recombinant human 5-HT_1Dα and 5-HT_1Dβ receptor subtypes were stably expressed in NIH-3T3 fibroblasts (1Dα cell line) and Y-1 adrenocortical tumor cells (1Dβ cell line), respectively, for pharmacological evaluations of serotonergic compounds to inhibit forskolin-stimulated CAMP accumulation (FSCA). [³H]LSD saturation studies indicated that 5-HT_1D receptor expression levels were slightly higher in the 1Dβ cell line (B _max = 1334 ± 134 fmol/mg protein) than in the (1Dα) cell line (B _max = 900 ± 900 fmol/mg protein). 5-HT inhibited FSCA with similar potencies (EC₅₀ ≈ 2 nM) in both assay systems. The rank order of agonist potencies in both clonal cell lines matched their pharmacological profiles previously determined in binding studies: dihydroergotamine >- 5-carboxamidotryptamine (5-CT) > LSD >- 5-HT > sumatriptan > 1-naphthylpiperazine (1-NP) > yohimbine > 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH DPAT) > 1-(2,5-dimethoxy4-iodophenyl)-2-aminopropane (DOI), with K_i/EC₅₀ ratios greater than unity. Methiothepin acted as a silent antagonist at both human 5-HT_1Dα and 5-HT_1Dβ receptors with apparent dissociation constants (K_b values) of 12 ± 1 nM and 3 ± 1 nM, respectively. Whereas GR 127,935, metergoline, DOI, and quipazine acted as full agonists in the 1Dα cell line, these compounds behaved as partial agonists in the 1Dβ cell line.

To determine whether high levels of receptor reserve might mask partial agonist activity in the two second messenger assay systems, studies were performed using the irreversible receptor alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The relationships between receptor occupancy and inhibition of FSCA were determined for 5-HT, sumatriptan, and 1-NP in both clonal cell lines after partial receptor inactivation using Furchgott analysis. Hyperbolic relationships between receptor occupancy and second messenger response were determined for 5-HT in both transfected cell lines. Steep hyperbolic relationships were also found for sumatriptan and 1-NP in the 1Dβ cell line whereas nearly linear relationships were observed for these two compounds in the 1Dα cell line. Moreover, K_A/EC₅₀ ratios of these compounds were significantly larger in the (1Dα)(10–32) as compared to the 1Dα (0.9–2.5) cell line. These data are consistent with the hypothesis that the two heterologous expression systems contain a differential amount of receptor reserve. Despite the presence of an apparently larger receptor reserve in the 1Dβ cell line, GR 127,935, metergoline, DOI, and quipazine behaved as partial agonists.

Although the potencies (EC₅₀ values) of compounds matched their respective affinity constants (K_i values) for the closely-related 5-HT_1D subtypes, differences in intrinsic activities were observed for a few compounds between the two 5-HT_1D receptor expression systems. Since receptor reserve is dependent on the properties of both the assay system and drug, the observed variations in intrinsic activity, although influenced by the variable amounts of receptor reserve in the two transfected cell lines, reflect primarily system-independent differences in the intrinsic efficacy of the tested compounds at the two human 5-HT_1D receptors. Higher intrinsic efficacies of compounds at the human 5-HT_1Dα receptor relative to the human 5-HT_1Dβ subtype may be responsible for the higher intrinsic activities observed in the (1Dα) cell line, even though receptor reserve is apparently lower in this system.