GR-65630

The effects of alcohol monoterpene menthol, a major active ingredient of the peppermint plant, were tested on the function of human 5-hydroxytryptamine type 3 (5-HT3) receptors expressed in Xenopus laevis oocytes. 5-HT (1 μM)-evoked currents recorded by two-electrode voltage-clamp technique were reversibly inhibited by menthol in a concentration-dependent (IC50 = 163 μM) manner. The effects of menthol developed gradually, reaching a steady-state level within 10-15 minutes and did not involve G-proteins, since GTPγS activity remained unaltered and the effect of menthol was not sensitive to pertussis toxin pretreatment. The actions of menthol were not stereoselective as (-), (+), and racemic menthol inhibited 5-HT3 receptor-mediated currents to the same extent. Menthol inhibition was not altered by intracellular 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid injections and transmembrane potential changes. The maximum inhibition observed for menthol was not reversed by increasing concentrations of 5-HT. Furthermore, specific binding of the 5-HT3 antagonist [(3)H]GR65630 was not altered in the presence of menthol (up to 1 mM), indicating that menthol acts as a noncompetitive antagonist of the 5-HT3 receptor. Finally, 5-HT3 receptor-mediated currents in acutely dissociated nodose ganglion neurons were also inhibited by menthol (100 μM). These data demonstrate that menthol, at pharmacologically relevant concentrations, is an allosteric inhibitor of 5-HT3 receptors.

BACKGROUND AND PURPOSE Morphine is an antagonist at 5‐HT3A receptors. 5‐HT3 and opioid receptors are expressed in many of the same neuronal pathways where they modulate gut motility, pain and reinforcement. There is increasing interest in the 5‐HT3B subunit, which confers altered pharmacology to 5‐HT3 receptors. We investigated the mechanisms of inhibition by morphine of 5‐HT3 receptors and the influence of the 5‐HT3B subunit.EXPERIMENTAL APPROACH 5‐HT‐evoked currents were recorded from voltage‐clamped HEK293 cells expressing human 5‐HT3A subunits alone or in combination with 5‐HT3B subunits. The affinity of morphine for the orthosteric site of 5‐HT3A or 5‐HT3AB receptors was assessed using radioligand binding with the antagonist [3H]GR65630.KEY RESULTS When pre‐applied, morphine potently inhibited 5‐HT‐evoked currents mediated by 5‐HT3A receptors. The 5‐HT3B subunit reduced the potency of morphine fourfold and increased the rates of inhibition and recovery. Inhibition by pre‐applied morphine was insurmountable by 5‐HT, was voltage‐independent and occurred through a site outside the second membrane‐spanning domain. When applied simultaneously with 5‐HT, morphine caused a lower potency, surmountable inhibition of 5‐HT3A and 5‐HT3AB receptors. Morphine also fully displaced [3H]GR65630 from 5‐HT3A and 5‐HT3AB receptors with similar potency.CONCLUSIONS AND IMPLICATIONS These findings suggest that morphine has two sites of action, a low‐affinity, competitive site and a high‐affinity, non‐competitive site that is not available when the channel is activated. The affinity of morphine for the latter is reduced by the 5‐HT3B subunit. Our results reveal that morphine causes a high‐affinity, insurmountable and subunit‐dependent inhibition of human 5‐HT3 receptors.