Interactions of metoclopramide and ergotamine with human 5-HT(3A) receptors and human 5-HT reuptake carriers

Abstract

The actions of metoclopramide and ergotamine, drugs which are used as a combined migraine medication, on human (h)5-HT3A receptors and 5-HT reuptake carriers, stably expressed in HEK-293 cells, were studied with patch-clamp- and ([3H]5-HT)-uptake techniques. At clinical concentrations, metoclopramide inhibited peak and integrated currents through h5-HT3A receptors concentration-dependently (IC50 = 0.064 and 0.076 microM, respectively) when it was applied in equilibrium (60 s before and during 5-HT (30 microM) exposure). The onset and offset time constants of metoclopramide action were 1.3 and 2.1 s, respectively. The potency of metoclopramide when exclusively applied during the agonist pulse decreased more than 200-fold (IC50 = 19.0 microM, peak current suppression). Metoclopramide (0.10 microM) did not alter the EC50 of 5-HT-induced peak currents. In contrast to the lack of competitive interaction between metoclopramide and 5-HT in this functional assay, metoclopramide inhibited specific [3H]GR65630 binding to human h5-HT3A receptors in a surmountable manner. This seeming discrepancy between functional studies and radioligand binding experiments may be accounted for by (1) the slow kinetics of inhibition of peak currents by metoclopramide compared with the fast onset and offset kinetics of 5-HT-induced currents and (2) the low efficacy of metoclopramide in inhibiting radioligand binding (e.g. only 20% binding inhibition compared to 79% peak current suppression by 200 nM metoclopramide). At low concentrations (1-10 nM), ergotamine had no effect on 5-HT (30 microM)-induced peak currents. Above clinical concentrations, ergotamine (>3 microM) inhibited them. When both drugs were applied together (0.10 microM metoclopramide +0.001 to 0.01 microM ergotamine), an inhibition of both, peak and integrated current responses was observed. Neither metoclopramide (< or =30 microM) nor ergotamine (< or =30 microM) had an effect on the 5-HT reuptake carrier as they did not alter the citalopram-sensitive [3H]5-HT uptake.

Figure 1

Left: concentration–response curves (−100 mV) of 5-HT at h5-HT_3A receptors stably transfected in HEK-293 cells in the absence (squares) and presence (triangles) of metoclopramide (100 nM; applied in equilibrium mode, ++). The data are normalized to the currents evoked by 30 μM 5-HT in the absence of metoclopramide (=controls). Apparently, the inhibition was unsurmountable by 5-HT. Shown are means±s.d. of 3–10 different patches. Right: effect of metoclopramide (200 nM) on [³H]GR65630 (3, 10 nM) binding. Apparently, the moderate but significant (P<0.01, paired t-test) inhibition of 3 nM [³H]GR65630 binding was surmountable by higher concentrations of [³H]GR65630. Unspecific binding was determined by addition of 100 μM MDL72222 and substracted under each experimental condition. Means±s.d. of three different experiments, performed in duplicates are shown.

Figure 2

Inhibitory effects of metoclopramide on 5-HT (30 μM; −100 mV)-induced peak currents in excised patches of HEK-293 cells stably transfected with h5-HT_3A receptors. The following drug application modes were used (see also legend to Table 1): equilibrium mode (++), closed channel mode (+−), open channel mode (−+). The preapplication of metoclopramide to the patch (either in ++ or +− mode) was a prerequisite of the full inhibitory potency (see also Table 1). Means±s.d. of n⩾4 experiments are shown.

Figure 3

(a) Effect of metoclopramide (50 nM) on 5-HT (30 μM; −100 mV)-induced currents in one excised patch of a HEK-293 cell, stably transfected with h5-HT_3A receptors. Results were obtained using three different modes of drug application (equilibrium=++, closed channel=+−, open channel=−+; see also legend to Table 1). ‘Wash' refers to the response to 5-HT 60 s after metoclopramide application. Data were digitally filtered at 500 Hz, shown are traces from one single patch. (b) Effect of metoclopramide on the onset kinetics (monoexponential fit, τ_ON) of 5-HT (30 μM; −100 mV)-induced currents in excised outside-out patches of HEK-293 cells, stably transfected with h5-HT_3A receptors. Note the different scaling of the Y-axis. Obviously, a concentration-dependent metoclopramide-induced retardation of the current-onset (activation) is present when the drug is applied prior to the agonist (either in ++ or +− mode). This effect is significant when compared to the control experiments in the absence of metoclopramide (***, P<0.001; **, P<0.01; *, P<0.05). Means±s.d. of n⩾4 experiments are shown. The effect is only modest at highest metoclopramide concentrations when the drug is applied exclusively during the 5-HT pulse (−+ mode).

Figure 4

(a) The amplitude of 5-HT (30 μM; −100 mV)-induced peak currents through h5-HT_3A receptor channels as a function of the duration of metoclopramide (100 nM) application prior to the 5-HT pulse (wash-in). (b) The amplitude of 5-HT-induced peak currents through h5-HT_3A receptor channels as a function of the duration of removal of metoclopramide (wash-out). Monoexponential fits with time constants of 1.3 and 2.1 s, respectively are shown; means±s.d. of n⩾4 experiments. (c) Comparison of recovery (open circles) from inhibition by metoclopramide (100 nM applied for 60 s, upper trace) and kinetics of the respective control current induced by 5-HT (30 μM, lower trace).

Figure 5

Effects of ergotamine, applied in equilibrium (++, see Table 1) on 5-HT (30 μM; −100 mV)-induced currents through h5-HT_3A receptor channels. Peak–current responses are shown. Means±s.d. of n⩾4 experiments are shown.

Figure 6

Inhibition by metoclopramide (0.1 μM) of 5-HT (30 μM; −100 mV)-induced peak currents, mediated by h5-HT_3A receptors in the absence and in the presence of ergotamine (0.001, 0.01 μM). Both drugs were applied 60 s prior plus during the 5-HT pulse (=equilibrium condition, ++). Means±s.d. of n=7 experiments are shown.

Figure 7

Metoclopramide and ergotamine effects on the h5-HT carrier-mediated [³H]5-HT uptake (10 nM; 5 min) in stably transfected HEK-293 cells. The specific signal was calculated as difference between the uptake in the absence and presence of citalopram (1 μM). Shown are means±s.d. of 8–10 different experiments.