Pharmacological analysis of the novel, rapid, and potent inactivation of the human 5-Hydroxytryptamine7 receptor by risperidone, 9-OH-Risperidone, and other inactivating antagonists

Abstract

In a previous publication, using human 5-hydroxytryptamine(7) (h5-HT(7)) receptor-expressing human embryonic kidney (HEK) 293 cells, we reported the rapid, potent inactivation of the h5-HT(7) receptor stimulation of cAMP production by three antagonists: risperidone, 9-OH-risperidone, and methiothepin (Smith et al., 2006). To better understand the drug-receptor interaction producing the inactivation, we 1) expanded the list of inactivating drugs, 2) determined the inactivating potencies and efficacies by performing concentration-response experiments, and 3) determined the potencies and efficacies of the inactivators as irreversible binding site inhibitors. Three new drugs were found to fully inactivate the h5-HT(7) receptor: lisuride, bromocryptine, and metergoline. As inactivators, these drugs displayed potencies of 1, 80, and 321 nM, respectively. Pretreatment of 5-HT(7)-expressing HEK cells with increasing concentrations of the inactivating drugs risperidone, 9-OH-risperidone, methiothepin, lisuride, bromocriptine, and metergoline potently inhibited radiolabeling of the h5-HT(7) receptor, with IC(50) values of 9, 5.5, 152, 3, 73, and 10 nM, respectively. We were surprised to find that maximal concentrations of risperidone and 9-OH-risperidone inhibited only 50% of the radiolabeling of h5-HT(7) receptors. These results indicate that risperidone and 9-OH risperidone may be producing 5-HT(7) receptor inactivation by different mechanisms than lisuride, bromocryptine, metergoline, and methiothepin. These results are not interpretable using the conventional model of G-protein-coupled receptor function. The complex seems capable of assuming a stable inactive conformation as a result of the interaction of certain antagonists. The rapid, potent inactivation of the receptor-G-protein complex by antagonists implies a constitutive, pre-existing complex between the h5-HT(7) receptor and a G-protein.

Fig. 1.

Effect of drug pretreatment on h5-HT₇ receptor-stimulated cAMP production. HEK293 cells stably expressing h5-HT₇ receptors were suspended in isotonic buffer and exposed to 1 μM concentrations of drugs for 30 min. Cells were gently pelleted, buffer was replaced, and the cells were exposed to nondrug-containing buffer. This drug washout procedure was repeated three times. Cells were resuspended and assayed for response to 10 μM 5-HT using the LANCE cAMP Detection kit (PerkinElmer) and time-resolved fluorescence resonance energy transfer. The results are the means ± S.E.M. of three independent experiments performed in triplicate. The following drugs (1 μM) displayed no effect on h5-HT₇-receptor mediated stimulation of cAMP after washout: ICI169369, tenilapine, cyproheptadine, SB269970, 3-trifluoromethylphenylpiperazine, trifluoperazine, methysergide, ritanserin, loxapine, amoxapine, amitriptyline, and LSD. ^*, p < 0.003 versus control.

Fig. 2.

Concentration-response curves for inactivation of h5-HT₇ receptor-stimulated cAMP production. HEK293 cells expressing h5-HT₇ receptors were suspended in isotonic buffer and exposed to buffer (control) or varying concentrations of the six drugs displaying inactivating properties (see Fig. 1). After the drug washout procedure (see Materials and Methods), the cells were exposed to 10 μM 5-HT for 30 min. cAMP levels were determined using the LANCE cAMP Detection kit (PerkinElmer). The results are the means ± S.E.M. of three independent experiments performed in triplicate.

Fig. 3.

Concentration-response curves for loss of specific [³H]5-HT binding to h5-HT₇ receptor after drug removal. HEK293 cells expressing h5-HT₇ receptors were suspended in isotonic buffer and exposed to buffer (control) or varying concentrations of the six drugs displaying inactivating properties (see Fig. 1). After the drug washout procedure (see Materials and Methods), the cells were incubated with 2 nM[³H]5-HT in the presence and absence of 10 μM clozapine. The results are the means ± S.E.M. of three independent experiments performed in triplicate.

Fig. 4.

Affinities of SB-269970 for [³H]5-HT-specific binding after pretreatment with no drug (control), 1 μM risperidone, or 1 μM 9-OH-risperidone. Consistent with Figs. 3 and 5, 50% of the h5-HT₇ receptors are risperidone- and 9-OH-risperidone-resistant. The high affinities displayed by SB269970 indicate the risperidone- and 9-OH-risperidone-resistant radioligand binding signal displays the pharmacological properties of the h5-HT₇ receptor. The results are the means ± S.E.M. of three independent experiments.

Fig. 5.

Saturation analyses of specific [³H]5-HT binding to HEK293 cells expressing the h5-HT₇ receptor after pretreatment with no drug (control), 1 μM risperidone, or 1 μM 9-OH-risperidone. After drug pretreatment and washout procedure, cells were incubated with increasing concentrations of [³H]5-HT in the absence and presence of 10 μM clozapine. The results are the means ± S.E.M. of three independent experiments performed in triplicate. ^*, p < 0.001 versus control B_max values.