Prolonged treatment with ligands affects ligand binding to the human serotonin(1A) receptor in Chinese hamster ovary cells

Abstract

1. The serotonin(1A) receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins, and appear to be involved in several behavioral and cognitive functions. 2. We monitored the effect of prolonged treatment of the human serotonin(1A) receptor expressed in Chinese hamster ovary (CHO) cells with pharmacologically well-characterized ligands on its binding to the agonist 8-hydroxy-2(di-N-propylamino)tetralin (8-OH-DPAT) and antagonist 4-(2'-methoxy)-phenyl-1-[2'-(N-2''-pyridinyl)-p-fluorodobenzamido]ethyl-piperazine (p-MPPF). 3. Our results indicate that prolonged treatment with the specific agonist (8-OH-DPAT) differentially affects subsequent binding of the agonist and antagonist to the receptor in a manner independent of receptor-G-protein coupling. Importantly, our results show that prolonged treatment with the commonly used antagonist p-MPPF, and its iodinated analogue 4-(2'-methoxy)-phenyl-1-[2'-(N-2''-pyridinyl)-p-iodobenzamido]ethyl-piperazine (p-MPPI), which have earlier been reported to display similar binding properties to serotonin(1A) receptors, induces significantly different effects on the ligand binding function of serotonin(1A) receptors.

Fig. 1.

Specific binding of the (A) agonist [³H]8-OH-DPAT and (B) antagonist [³H]p-MPPF to membranes isolated from CHO cells stably expressing the human serotonin_1A receptor that were treated with either the agonist 8-OH-DPAT (10 μM), or the antagonist p-MPPF (1 μM) for 18 h. Values are expressed as percentages of specific radioligand binding obtained in membranes isolated from untreated cells (control). The data shown are the means ± SE of duplicate points from at least six independent experiments. See Materials and Methods section for other details.

Fig. 2.

Effect of increasing concentrations of GTP-γ-S on specific [³H]8-OH-DPAT binding to human serotonin_1A receptors in membranes isolated from control (—○—) and 8-OH-DPAT-treated (—●—) cells. The conditions for 8-OH-DPAT treatment are as described in Fig. 1. Values are expressed as percentages of the specific [³H]8-OH-DPAT binding obtained in the absence of GTP-γ-S. The curves are non-linear regression fits to the experimental data using Eq. (1). Data represent the means ± SE of duplicate points from five independent experiments. See Materials and Methods section for other details.

Fig. 3.

Specific binding of the agonist [³H]8-OH-DPAT (white bars) and antagonist [³H]p-MPPF (gray bars) to membranes isolated from CHO cells stably expressing the human serotonin_1A receptor that were treated with p-MPPI (1 μM) for 18 h. Values are expressed as percentages of specific radioligand binding obtained in membranes isolated from untreated cells (control). Data represent the means ± SE of duplicate points from at least six independent experiments. See Materials and Methods section for other details.