Involvement of organic cation transporter-3 and plasma membrane monoamine transporter in serotonin uptake in human brain vascular smooth muscle cells

Abstract

The serotonin (5-HT) uptake system is supposed to play a crucial part in vascular functions by "fine-tuning" the local concentration of 5-HT in the vicinity of 5-HT(2) receptors in vascular smooth muscle cells. In this study, the mechanism of 5-HT uptake in human brain vascular smooth muscle cells (HBVSMCs) was investigated. [(3)H]5-HT uptake in HBVSMCs was Na(+)-independent. Kinetic analyses of [(3)H]5-HT uptake in HBVSMCs revealed a K(m) of 50.36 ± 10.2 mM and a V(max) of 1033.61 ± 98.86 pmol/mg protein/min. The specific serotonin re-uptake transporter (SERT) inhibitor citalopram, the specific norepinephrine transporter (NET) inhibitor desipramine, and the dopamine transporter (DAT) inhibitor GBR12935 inhibited 5-HT uptake in HBVSMCs with IC(50) values of 97.03 ± 40.10, 10.49 ± 5.98, and 2.80 ± 1.04 μM, respectively. These IC(50) values were 100-fold higher than data reported by other authors, suggesting that those inhibitors were not blocking their corresponding transporters. Reverse transcription-polymerase chain reaction results demonstrated the presence of mRNA for organic cation transporter (OCT)-3 and plasma membrane monoamine transporter (PMAT), but the absence of OCT-1, OCT-2, SERT, NET, and DAT. siRNA knockdown of OCT-3 and PMAT specifically attenuated 5-HT uptake in HBVSMCs. It is concluded that 5-HT uptake in HBVSMCs was mediated predominantly by a low-affinity and Na(+)-independent mechanism. The most probable candidates are OCT-3 and PMAT, but not the SERT.

Keywords: monoamine transporter; organic cation transporter; serotonin; vascular smooth muscle cells.

Figure 1

Time-course of 5-HT uptake in HBVSMCs. [³H]5-HT uptake (1 μM, 2 μCi/mL) was measured in HBVSMCs in the presence or absence of Na⁺ as indicated. Values are means ± SEM of three experiments carried out in triplicate.

Figure 2

Kinetic analyses of 5-HT uptake in HBVSMCs. Concentration dependence of 5-HT (0.1 μM to 50 mM) uptake was determined by measuring [³H]5-HT uptake at room temperature for 30 min. Values are means ± SEM of three experiments carried out in triplicate.

Figure 3

Effects of various transporter inhibitors on 5-HT uptake in HBVSMCs. [³H]5-HT uptake (1 μM, 2 μCi/mL) was measured at room temperature for 30 min in the presence of various concentrations of citalopram (■), desipramine (□), GBR12935 (●), and corticosterone (◯). Values are means ± SEM of three experiments carried out in triplicate.

Figure 4

Reverse transcription-polymerase chain reaction analyses of various 5-HT transporter mRNAs in HBVSMCs. PCR products are seen in reactions using oligonucleotide primer pairs for (A) OCT-3 and (B) PMAT but not for (A) OCT-1, OCT-2, (C) SERT, (D) NET, and (E) DAT. Positive controls with human liver or brain cDNA indicate the expected sizes of amplified fragments: 363 bp (OCT-1), 334 bp (OCT-2), 419 bp (OCT-3), 319 bp (SERT), 400 bp (PMAT), 395 bp (NET), and 370 bp (DAT).

Figure 5

Effects of siRNA knockdown of OCT-3 and PMAT on 5-HT uptake in HBVSMCs. (A) mRNA and (B) protein expressions of OCT-3 and PMAT mRNA in HBVSMCs transfected with siRNA against OCT-3 and PMAT and a control non-silencing sequence. Bar graph showing the amount of (C) mRNA and (D) protein of OCT-3 and PMAT normalized to β-actin. (E) 5-HT uptake in HBVSMCs transfected with OCT-3 and PMAT siRNA and a control non-silencing sequence. Values are means ± SEM of three separate experiments. *P < 0.05 vs. control.