Evidence for noncompetitive modulation of substrate-induced serotonin release

Abstract

Prior work indicated that serotonin transporter (SERT) inhibitors competitively inhibit substrate-induced [(3)H]5-HT release, producing rightward shifts in the substrate-dose response curve and increasing the EC(50) value without altering the E(max). We hypothesized that this finding would not generalize across a number of SERT inhibitors and substrates, and that the functional dissociation constant (Ke) of a given SERT inhibitor would not be the same for all tested substrates. To test this hypothesis, we utilized a well-characterized [(3)H]5-HT release assay that measures the ability of a SERT substrate to release preloaded [(3)H]5-HT from rat brain synaptosomes. Dose-response curves were generated for six substrates (PAL-287 [naphthylisopropylamine], (+)-fenfluramine, (+)-norfenfluramine, mCPP [meta-chlorophenylpiperazine], (±)-MDMA, 5-HT) in the absence and presence of a fixed concentration of three SERT inhibitors (indatraline, BW723C86, EG-1-149 [4-(2-(benzhydryloxy)ethyl)-1-(4-bromobenzyl)piperidine oxalate]). Consistent with simple competitive inhibition, all SERT inhibitors increased the EC(50) value of all substrates. However, in many cases a SERT inhibitor decreased the E(max) value as well, indicating that in the presence of the SERT inhibitor the substrate became a partial releaser. Moreover, the Ke values of a given SERT inhibitor differed among the six SERT substrates, indicating that each inhibitor/substrate combination had a unique interaction with the transporter. Viewed collectively, these findings suggest that it may be possible to design SERT inhibitors that differentially regulate SERT function.

Figure 1

Effect of 5-HT_2B receptor ligands on MDMA-induced [³H]5-HT release. MDMA dose-response curves were generated as described in methods. The data of three experiments, expressed as percent [³H]5-HT released, were fit to the dose-response equation for the best fit estimates of the E_MAX and EC₅₀ using KaleidaGraph. Each value is the mean±SEM (n=3).

Figure 2

Effect of SERT inhibitors on [³H]5-HT uptake. Dose-response curves were generated as described in methods. The data of three experiments, expressed as percent inhibition, were fit to the dose-response equation for the best fit estimates of the E_MAX and EC₅₀ using KaleidaGraph. The EC₅₀ values are reported in the Results section. Each value is the mean±SEM (n=3).

Figure 3

Effect of SERT inhibitors on mCPP-induced [³H]5-HT release. mCPP dose-response curves were generated as described in methods. The data, expressed as percent [³H]5-HT released, were fit to the dose-response equation for the best fit estimates of the E_MAX and EC₅₀ using KaleidaGraph. Each value is the mean±SEM (n=3 for indatraline and EG-1-149, n=5 for 5 μM BW723C86, n=9 for 25 μM BW723C86 and n=17 for mCPP).

Figure 4

Effect of SERT inhibitors on 5-HT-induced [³H]5-HT release. 5-HT dose-response curves were generated as described in methods. The data, expressed as percent [³H]5-HT released, were fit to the dose-response equation for the best fit estimates of the E_MAX and EC₅₀ using KaleidaGraph. Each value is the mean±SEM (n=6).

Figure 5

Effect of time on (+)-norfenfluramine-induced [³H]5-HT release. (+)-Norfenfluramine dose-response curves were generated as described in Methods in the absence and presence of 15 μM EG-1-149. The release assays were terminated at 5 min or 15 min. The data, expressed as percent [³H]5-HT released, were fit to the dose-response equation for the best fit estimates of the E_MAX and EC₅₀ using KaleidaGraph. Each value is the mean±SEM (n=9). The parameter values are reported in Table 3.

Figure 6

Effect of time on 5-HT-induced [³H]5-HT release. 5-HT dose-response curves were generated as described in Methods. The release assays were terminated at 5, 10 or 15 min. The data, expressed as percent [³H]5-HT released, were fit to the dose-response equation for the best fit estimates of the E_MAX and EC₅₀ using KaleidaGraph. Each value is the mean±SEM (n=5).