Evidence for significant contribution of a newly identified monoamine transporter (PMAT) to serotonin uptake in the human brain

Abstract

The high affinity serotonin transporter (SERT) constitutes the principal pathway for removal of serotonin (5-HT) from extracellular fluid of brain, but evidence indicates that other transporters may also be involved in this process. We recently reported the cloning of a novel plasma membrane monoamine transporter (PMAT), which is abundantly expressed in the human brain and avidly transports 5-HT [Engel K, Zhou M, Wang J. Identification and characterization of a novel monoamine transporter in the human brain. J Biol Chem 2004;279:50042-9]. In this study, we evaluated whether PMAT contributes to total human brain uptake of 5-HT using a hybrid depletion approach in Xenopus laevis oocytes. We also examined whether PMAT interacts with selective serotonin reuptake inhibitors (SSRIs) using MDCK cells stably expressing recombinant human PMAT. Microinjection of total human brain poly(A)(+) mRNA into oocytes elicited approximately 2.5-3-fold increase in 5-HT uptake. Pre-hybridization of poly(A)(+) mRNA with PMAT or SERT antisense oligonucleotides significantly reduced mRNA-induced 5-HT uptake. An additive inhibitory effect was observed when poly(A)(+) mRNA was co-hybridized with both PMAT and SERT antisense oligonucleotides. In contrast, mRNA-induced 5-HT uptake was not affected by pre-hybridization with sense oligonucleotides. These data suggest that functional transcripts of PMAT are present in the human brain, and the PMAT transporter may be significantly involved in brain uptake of 5-HT. All five tested SSRIs inhibited PMAT with IC(50) values ranging from 11 to 116 microM, which are much greater than clinically encountered concentrations, suggesting that PMAT activity is minimally affected by SSRI therapies.

Figure 1

Illustration of positions of PMAT and SERT oligos used in hybrid depletion study.

Figure 2. Effect of PMAT and SERT sense and antisense oligos on 5-HT uptake in cRNA-injected oocytes

(a) Oocytes were injected with 50 nL water (open bars), 40 ng PMAT cRNA only (solid bars), 40 ng PMAT cRNA pre-hybridized with 1.5 ng sense oligos (shaded bars) or 40 ng PMAT cRNA pre-hybridized with 1.5 ng antisense oligos (gray bars). Uptake of 1 μM [³H]5-HT was performed after 1 hr incubation at 25° C. (b) Oocytes were injected with 50 nL water (open bars), 40 ng SERT cRNA only (solid bars), 40 ng SERT cRNA pre-hybridized with 1.5 ng sense oligos (shaded bars) or 40 ng SERT cRNA pre-hybridized with 1.5 ng antisense oligos (gray bars). Uptake of 20 nM [³H]5-HT was performed after 20 min incubation in Na⁺/Cl⁻ buffer or mannitol buffer at 25° C Each bar represents the Mean ± S.E. (n=8–10). #, significantly different from water injected oocytes (p<0.001); *, significantly different from cRNA injected oocytes (p<0.001).

Figure 3. Effect of sense and antisense oligos on 5-HT uptake in human brain poly(A)⁺mRNA-injected oocytes.

(a) Oocytes were injected with 50 nL water (open bars), 40 ng human brain poly(A)⁺ mRNA only (solid bars), 40 ng human brain poly(A)⁺ mRNA pre-hybridized with 1.5 ng PMAT sense oligos (shaded bars) or 40 ng human brain poly(A)⁺ mRNA pre-hybridized with 1.5 ng PMAT antisense oligos (gray bars). Uptake of 1 μM [³H] 5-HT were performed during 1h exposure at 25° C. (b) Oocytes were injected with 50 nL water (open bars), 40 ng human brain poly(A)⁺ mRNA only (solid bars), 40 ng human brain poly(A)⁺ mRNA pre-hybridized with 1.5 ng SERT sense oligos (shaded bars) or 40 ng human brain poly(A)⁺ mRNA pre-hybridized with 1.5 ng SERT antisense oligos (gray bars). Uptake of 1μM [³H]5-HT was performed during 1hr exposure at 25° C. For the double hybrid depletion, oocytes were injected with 40 ng human brain poly(A)⁺ mRNA pre-hybridized with 1.5 ng SERT antisense oligo E and 1.5 ng PMAT antisense oligo B. Each bar represents the Mean ± S.E. (n=8–10). #, significantly different from water injected oocytes (p<0.001); *, significantly different from mRNA injected oocytes (p<0.001); +, significantly different from oocytes injected with mRNA hybridized with single antisense oligo (p<0.01).

Figure 4. Effect of SSRIs on PMAT-mediated [³H]MPP⁺uptake in MDCK cells

Transport was measured in PMAT-transfected cells and vector-transfected cells (control) with 0.1 μM [³H]MPP⁺. Inhibitors were present during 15 min preincubation and 1 min incubation periods. The PMAT-specific uptake was calculated by subtracting the transport activity in the control cells. Each value represents the mean ± S.D. (n=3).