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

Li RW, Yang C, Kwan YW, Chan SW, Lee SM, Leung GP - Front Pharmacol (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Pharmacy, The University of Hong Kong Pokfulam, Hong Kong.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: Organic cation transporter-3 and PMAT were knocked down specifically using siRNA to verify their contribution to 5-HT transport in HBVSMCs. Transfection of HBVSMCs with siRNA against OCT-3 resulted in a reduction in mRNA and protein expressions of 64.3 and 72.2%, respectively, as well as a reduction in 5-HT uptake of 22.1% in HBVSMCs. Transfection of HBVSMCs with siRNA against PMAT resulted in a reduction in mRNA and protein expressions of 81.2 and 71.5%, respectively, as well as a reduction in 5-HT uptake of 34.2% in HBVSMCs (Figure 5).


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

Li RW, Yang C, Kwan YW, Chan SW, Lee SM, Leung GP - Front Pharmacol (2013)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3569667&req=5

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.
Mentions: Organic cation transporter-3 and PMAT were knocked down specifically using siRNA to verify their contribution to 5-HT transport in HBVSMCs. Transfection of HBVSMCs with siRNA against OCT-3 resulted in a reduction in mRNA and protein expressions of 64.3 and 72.2%, respectively, as well as a reduction in 5-HT uptake of 22.1% in HBVSMCs. Transfection of HBVSMCs with siRNA against PMAT resulted in a reduction in mRNA and protein expressions of 81.2 and 71.5%, respectively, as well as a reduction in 5-HT uptake of 34.2% in HBVSMCs (Figure 5).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Pharmacy, The University of Hong Kong Pokfulam, Hong Kong.

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.

No MeSH data available.


Related in: MedlinePlus