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Comparative Study
. 2012 Apr;341(1):251-62.
doi: 10.1124/jpet.111.188946. Epub 2012 Jan 23.

Studies of the biogenic amine transporters. 14. Identification of low-efficacy "partial" substrates for the biogenic amine transporters

Richard B Rothman  1 , John S Partilla, Michael H Baumann, Catrissa Lightfoot-Siordia, Bruce E Blough
Affiliations
Comparative Study

Studies of the biogenic amine transporters. 14. Identification of low-efficacy "partial" substrates for the biogenic amine transporters

Richard B Rothman et al. J Pharmacol Exp Ther. 2012 Apr.

Abstract

Several compounds have been identified that display low-efficacy, "partial substrate" activity. Here, we tested the hypothesis that the mechanism of this effect is a slower rate of induced neurotransmitter efflux than that produced by full substrates. Biogenic amine transporter release assays were carried out in rat brain synaptosomes and followed published procedures. [(3)H]1-methyl-4-phenylpyridinium (MPP(+)) was used to assess release from dopamine (DA) and norepinephrine nerve terminals, whereas [(3)H]5-hydroxytryptamine (5-HT) was used to assess release from 5-HT nerve terminals. A detailed time-course evaluation of DA transporter (DAT)-mediated efflux was conducted by measuring the efflux of [(3)H]MPP(+) after the addition of various test compounds. In vivo microdialysis experiments compared the effects of the full substrates [(±)-1-(2-naphthyl)propan-2-amine (PAL-287) and (S)-N-methyl-1-(2-naphthyl)propan-2-amine (PAL-1046)], to that of a partial DAT/5-HT transporter substrate [(S)-N-ethyl-1-(2-naphthyl)propan-2-amine (PAL-1045)] on extracellular DA and 5-HT in the nucleus accumbens of the rat. The in vitro release assays demonstrated that partial substrate activity occurs at all three transporters. In the DAT efflux experiments, D-amphetamine (full substrate) promoted a fast efflux (K1 = 0.24 min(-1)) and a slow efflux (K2 = 0.008 min(-1)). For the partial DAT substrates, K1 = ∼0.04 min(-1), and K2 approximated zero. The in vivo microdialysis experiments showed that the partial substrate (PAL-1045) was much less effective in elevating extracellular DA and 5-HT than the comparator full substrates. We conclude that low-efficacy partial DAT substrates promote efflux at a slower rate than full substrates, and "partiality" reflects the ultra-slow K2 constant, which functionally limits the ability of these compounds to increase extracellular DA. We speculate that partial biogenic amine transporter substrates bind to the transporter but are less effective in inducing conformational changes required for reverse transport activity.

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Figures

Fig. 1.
Fig. 1.
Effect of d-amphetamine (A) and PAL-1046 (B) on DAT-mediated [3H]MPP+ efflux. Efflux experiments were conducted as described under Materials and Methods. The data were fit to a biexponential decay equation, and the best-fit estimates of the four parameters are shown in Table 3. Each point is the mean ± S.D. (n = 3).
Fig. 2.
Fig. 2.
Effect of PAL-1045 (A) and PAL-738 (B) on DAT-mediated [3H]MPP+ efflux. Efflux experiments were conducted as described under Materials and Methods. The data were fit to a biexponential decay equation, and the best-fit estimates of the four parameters are shown in Table 3. Each point is the mean ± S.D. (n = 3).
Fig. 3.
Fig. 3.
Effect of PAL-192 (A) and PAL-193 (B) on DAT-mediated [3H]MPP+ efflux. Efflux experiments were conducted as described under Materials and Methods. The data were fit to a biexponential decay equation, and the best-fit estimates of the four parameters are shown in Table 3. Each point is the mean ± S.D. (n = 3).
Fig. 4.
Fig. 4.
Effect of time on PAL-192- and PAL-193-induced DAT-mediated [3H]MPP+ efflux. Release experiments were conducted as described under Materials and Methods. Samples were filtered at the standard time (30 min) and 60 min. The data were fit to the dose-response curve equation for the best-fit estimates of the EC50 and EMAX values. Each value is ± S.D. (n = 3).
Fig. 5.
Fig. 5.
Effect of time on PAL-153- and PAL-175-induced SERT-mediated [3H]5-HT efflux. Release experiments were conducted as described under Materials and Methods. Samples were filtered at the standard time (5 min) and 15 min. The data were fit to the dose-response curve equation for the best-fit estimates of the EC50 and EMAX values. Each value is ± S.D. (n = 3).
Fig. 6.
Fig. 6.
Effect of time on PAL-218-induced NET-mediated [3H]MPP+ efflux. Release experiments were conducted as described under Materials and Methods. Samples were filtered at the standard time (30 min) and 60 min. The data were fit to the dose-response curve equation for the best-fit estimates of the EC50 and EMAX values. Each value is ± S.D. (n = 6).
Fig. 7.
Fig. 7.
Effect of cocaine and GBR12909 on d-amphetamine-mediated DAT release. d-Amphetamine dose-response curves were generated in the absence and presence of cocaine (1 μM) and GBR12909 (0.5 nM). The data were fit to the dose-response equation for the best-fit estimates of the EC50 and EMAX values (Table 3). Each value is ± S.D. (n = 3).
Fig. 8.
Fig. 8.
Effect of drug administration on extracellular dopamine in the nucleus accumbens. SAL, PAL-1045, PAL-1046, and PAL-287 were administered intravenously (1 mg/kg at time 0; 3 mg/kg at 60 min, see arrows). A, the time course. Two-way ANOVA revealed a highly significant elevation of extracellular DA (Fdrug effect = 32.9, p < 0.001; Ftime= 18.8, p < 0.001; Finteraction = 4.77, p < 0.001). B, the mean effect observed in the three samples after each injection. Each value is the mean ± S.E.M. (n = 7–8). *, p < 0.01 compared with control (one-way ANOVA followed by Newman-Keul's post hoc test). #, p < 0.01 compared with PAL-1046 (one-way ANOVA followed by Newman-Keul's post hoc test).
Fig. 9.
Fig. 9.
Effect of drug administration on extracellular 5-HT in the nucleus accumbens. SAL, PAL-1045, PAL-1046, and PAL-287 were administered intravenously (1 mg/kg at time 0; 3 mg/kg at 60 min, see arrows). A, the time course. Two-way ANOVA revealed a highly significant elevation of extracellular DA (Fdrug effect = 59.9, p < 0.001; Ftime = 44.1, p < 0.001; Finteraction = 7.97, p < 0.001). B, the mean effect observed in the three samples after each injection. Each value is the mean ± S.E.M. (n = 7–8). *, p < 0.01 compared with control (one-way ANOVA followed by Newman-Keul's post hoc test). #, p < 0.01 compared with PAL-287 and PAL-1046 (one-way ANOVA followed by Newman-Keul's post hoc test).
Fig. 10.
Fig. 10.
Effect of drug administration on horizontal locomotor activity. These data were gathered concurrently with the experiments shown in Figs. 8 and 9. A, the time course. Arrows indicate time of drug administration. Two-way ANOVA revealed a highly significant increase in horizontal locomotor activity (HAL) (Fdrug effect = 39.9, p < 0.001; Ftime = 20.0, p < 0.001; Finteraction = 4.84, p < 0.001). B, the mean effect observed in the three samples after each injection. Each value is the mean ± S.E.M. (n = 7–8). *, p < 0.01 compared with control (one-way ANOVA followed by Newman-Keul's post hoc test). #, p < 0.01 compared with PAL-287 and PAL-1046 (one-way ANOVA followed by Newman-Keul's post hoc test).
Fig. 11.
Fig. 11.
Effect of drug administration on stereotypy. These data were gathered concurrently with the experiments shown in Figs. 8 and 9. A, the time course. Arrows indicate time of drug administration. Two-way ANOVA revealed a highly significant increase in stereotypy (Fdrug effect = 23.0, p < 0.001; Ftime = 14.0, p < 0.001; Finteraction = 2.71, p < 0.001). B, the mean effect observed in the three samples after each injection. Each value is the mean ± S.E.M. (n = 7–8). *, p < 0.01 compared with control (one-way ANOVA followed by Newman-Keul's post hoc test).
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