Direct inhibition by cannabinoids of human 5-HT3A receptors: probable involvement of an allosteric modulatory site

Abstract

Excised outside-out patches from HEK293 cells stably transfected with the human (h) 5-HT3A receptor cDNA were used to determine the effects of cannabinoid receptor ligands on the 5-HT-induced current using the patch clamp technique. In addition, binding studies with radioligands for 5-HT3 as well as for cannabinoid CB1 and CB2 receptors were carried out. The 5-HT-induced current was inhibited by the following cannabinoid receptor agonists (at decreasing order of potency): 9-THC, WIN55,212-2, anandamide, JWH-015 and CP55940. The WIN55,212-2-induced inhibition was not altered by SR141716A, a CB1 receptor antagonist. WIN55,212-3, an enantiomer of WIN55,212-2, did not affect the 5-HT-induced current. WIN55,212-2 did not change the EC50 value of 5-HT in stimulating current, but reduced the maximum effect. The CB1 receptor ligand [3H]-SR141716A and the CB1/CB2 receptor ligand [3H]-CP55940 did not specifically bind to parental HEK293 cells. In competition experiments on membranes of HEK293 cells transfected with the h5-HT3A receptor cDNA, WIN55,212-2, CP55940, anandamide and SR141716A did not affect [3H]-GR65630 binding, but 5-HT caused a concentration dependent-inhibition. In conclusion, cannabinoids stereoselectively inhibit currents through recombinant h5-HT3A receptors independently of cannabinoid receptors. Probably the cannabinoids act allosterically at a modulatory site of the h5-HT3A receptor. Thus the functional state of the receptor can be controlled by the endogenous ligand anandamide. This site is a potential target for new analgesic and antiemetic drugs.

Figure 1

Influence of WIN55,212-2 on the 5-HT-induced currents (−100 mV holding potential) in excised outside-out patches of HEK 293 cells stably transfected with the h5-HT_3A receptor cDNA. Concentration-response curve of 5-HT in the absence (open symbols) and in the presence (filled symbols) of WIN55,212-2 (300 nM; present 3 min before, during and after the 2 s 5-HT pulse). The currents in the absence and presence of the drug, respectively, are expressed as percentages of the currents evoked by the standard concentration of 30 μM 5-HT without additional drug (determined in all experiments as a reference effect). Shown are means±s.e.m. of 3–10 different patches.

Figure 2

Effects of the enantiomers WIN55,212-2 (upper right panel) and WIN55,212-3 (lower left panel), both at 1 μM, on 5-HT (30 μM; −100 mV)-induced currents obtained in one outside-out patch of a HEK 293 cell stably transfected with the h5-HT_3A receptor cDNA. Drugs were applied for 3 min before, during and after stimulation with 5-HT (upper right and lower left panels). 5-HT (30 μM; −100 mV)-induced currents under control conditions (i.e. absence of a further drug; upper left panel); ‘wash' refers to the control response to 30 μM 5-HT after omission of the drug for 3 min at the end of the experiment (lower right panel).

Figure 3

Influence of cannabinoid receptor ligands on 5-HT (30 μM; −100 mV)-induced currents in patches of HEK 293 cells stably transfected with the h5-HT_3A receptor cDNA. (a) Concentration-dependent inhibition by the cannabinoid receptor agonist WIN55,212-2 in the absence and presence of the CB₁ receptor antagonist SR141716A (at 1 μM) and lack of inhibition by the enantiomer WIN55,212-3 and by SR141716A alone. (b) Concentration-dependent inhibition of 5-HT (30 μM; −100 mV)-induced currents by various cannabinoid receptor ligands. Drugs were present 3 min before, during and after the 2 s 5-HT pulse. Shown are means±s.e.m. of 3–6 different patches.

Figure 4

Effects of the cannabinoid receptor agonist WIN55,212-2 (1 μM) on 5-HT (30 μM, −100 mV)-induced currents obtained in one outside-out patch of a HEK 293 cell stably transfected with the h5-HT_3A receptor cDNA at three different modes of drug application: exclusive co-application of the drug with 5-HT (without preexposure to the drug; upper right panel); application of the drug 3 min before, during and after stimulation with 5-HT (lower left panel); application exclusively for 3 min prior to and after, but not together with, 5-HT (lower right panel); 5-HT-induced current under control conditions (upper left panel); ‘wash' refers to the response to 30 μM 5-HT after omission of the drug for 3 min at the end of the experiment.

Figure 5

Influence of 5-HT and cannabinoid receptor agonists on specific binding of the selective 5-HT₃ receptor ligand [³H-GR65630 to membranes of HEK 293 cells stably transfected with the h5-HT_3A receptor cDNA. (a) Concentration-dependent inhibition by 5-HT of specific [³H-GR65630 binding in the absence (open symbols) and presence of the cannabinoid receptor agonist CP55940 and lack of inhibition by CP55940 alone. (b) No inhibition by WIN55212-2 and (c) no inhibition by anandamide. All points are means±s.e.m., n=3–6 different binding experiments.