Capsaicin Is a Negative Allosteric Modulator of the 5-HT3 Receptor

Abstract

In this study, effects of capsaicin, an active ingredient of the capsicum plant, were investigated on human 5-hydroxytryptamine type 3 (5-HT₃) receptors. Capsaicin reversibly inhibited serotonin (5-HT)-induced currents recorded by two-electrode voltage clamp method in Xenopus oocytes. The inhibition was time- and concentration-dependent with an IC₅₀ = 62 μM. The effect of capsaicin was not altered in the presence of capsazepine, and by intracellular BAPTA injections or trans-membrane potential changes. In radio-ligand binding studies, capsaicin did not change the specific binding of the 5-HT₃ antagonist [³H]GR65630, indicating that it is a noncompetitive inhibitor of 5-HT₃ receptor. In HEK-293 cells, capsaicin inhibited 5-HT₃ receptor induced aequorin luminescence with an IC₅₀ of 54 µM and inhibition was not reversed by increasing concentrations of 5-HT. In conclusion, the results indicate that capsaicin acts as a negative allosteric modulator of human 5-HT₃ receptors.

Keywords: 5-HT3 receptor; HEK-293 cells; Xenopus oocytes; allosteric modulator; capsaicin; docking; serotonin.

Figure 1

Effects of capsaicin on the function of human 5-HT₃ receptors expressed in Xenopus oocytes. (A) Representative traces of currents activated by 5-HT (1 µM; on the left), coapplication of 5-HT and 100 µM capsaicin after 10 min capsaicin pre-application (middle), 20 min wash-out (right). (B) Effect of capsaicin application on the normalized amplitudes of currents activated by 5-HT (1 µM) at 5 min intervals. Current amplitudes were normalized to first agonist application in each experiment. Solid bar represents application time for capsaicin (100 µM). Data points represent means ± S.E.M. of 7–8 cells. (C) Capsaicin inhibits the function of 5-HT₃ receptor in concentration-dependent manner. For all concentrations used, capsaicin was applied for 10 min. Data points represent mean ± S.E.M. (n = 6–8).

Figure 2

Inhibitory effect of capsaicin on 5-HT₃ receptor increases with pre-application times and independent of TRPV1 receptors and intracellular Ca²⁺ levels. (A) Capsaicin inhibition of 5-HT₃ receptor as a function of pre-incubation time. Exponential decay curve with two time constants τ_fast and τ_slow, shows the best fit for data point in the figure. Each data point represents the means ± SEM from 7 to 8 oocytes. (B) Effects of capsazepine (10 µM) on 5-HT (1 µM) induced currents (n = 5–7). Bars represent the means ± S.E.M. (C) Effect of BAPTA injection on the capsaicin inhibition of 5-HT-induced currents. 5-HT (1 μM)-induced currents were recorded before and after 10 min capsaicin (100 μM) application in oocytes injected with 50 nl distilled-water (controls, n = 5) or 50 nl of BAPTA (200 mM, n = 6). Bars represent the means ± S.E.M.

Figure 3

Effects of membrane potential and subunit combination on capsaicin inhibition of 5-HT-activated currents. (A) Current-voltage relationships of 5-HT (1 μM)-activated currents before and after 10 min pre-application of 100 µM capsaicin. Data points are the means ± SEM (n = 5) measured from 2-s voltage ramps. (B) Inhibition of 5-HT-activated current by 100 µM capsaicin at different membrane potentials. Capsaicin inhibition of 5-HT-activated currents did not change significantly at different membrane potentials (P>0.05, ANOVA; n = 5). (C) The effect of 100 μM capsaicin on human 5-HT_3A, 5-HT_3A and 5-HT_3B receptors co-expressed in subunit ratios 1:1 and 1:2. Currents were activated by 3 µM and 30 µM 5-HT for 5-HT_3A and 5-HT_3AB receptor combinations, respectively. The bar graph shows mean ± SEM from 5 to 7 oocytes.

Figure 4

Effect of capsaicin on 5-HT concentration-response relationship and binding of [³H]GR65630 to 5-HT₃ receptor expressed in Xenopus oocytes. (A) Concentration-response curves for 5-HT-activated currents in the absence and presence of 100 µM capsaicin. Data points represent the mean ± S.E.M. (n = 6–8). The curves depict the best fit of the data to the logistic equation described in the methods. The concentration-response for capsaicin is normalized to maximal control response. (B) Effects of capsaicin on the displacement of specific [³H]GR65630 binding by nonlabeled 5-HT in oocyte membranes. Membrane preparations were pre-incubated 100 μM capsaicin for 1 hour. The concentration of [³H]GR65630 was 1 nM. Data points indicate means ± SEM from 8 to 11 measurements from 3 experiments. (C) Effects of increasing concentrations of capsaicin on the specific binding of [³H]GR65630 (1 nM). Data points indicate means ± S.E.M from 7 to 10 measurements.

Figure 5

Effects of capsaicin on 5-HT-induced Ca²⁺ influx through human 5-HT₃ receptors. (A) Concentration-dependent inhibition of 5-HT₃ receptors by capsaicin. Aequorin luminescence induced by 3 μM 5-HT was recorded as a measure of an increased cytosolic Ca²⁺ concentration in coelenterazine h-loaded HEK-293-AEQ17 cells heterologously expressing human 5-HT₃ receptors. Capsaicin was present 10 min before and during 5-HT application. Data are expressed as percentages of the response to 5-HT in the absence of capsaicin (means ± SEM; n = 5). (B) The effects of capsaicin (50 µM) on aequorin luminescence activated by 3 μM, 10 µM, and 30 μM 5-HT. Bars represent means ± SEM; n = 16.

Figure 6

Docking studies on capsaicin, dihydrocapsaicin, capsazepine, and vanillin. (A) 5HT₃ receptor with best ranking poses of the docked capsaicin, dihydrocapsaicin, capsazepine, and vanillin. Proposed binding site for capsazepine and vanillin is located at interface with transmembrane domain (TMD). Proposed binding site for capsaicin and dihydrocapsaicin is located in the TMD. (B) 3D and 2D binding interactions within capsazepine binding pocket showing potential key residues. Residues that form hydrogen bond with capsazepine are shown in CPK color. (C) 3D and 2D binding interactions within capsaicin binding pocket showing potential key residues. Residue that forms hydrogen bond with capsaicin is shown in CPK color.