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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 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.
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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.

Mentions: To examine which type of transporters were responsible for 5-HT uptake in HBVSMCs, the effects of various inhibitors was studied. Citalopram (a specific SERT inhibitor), desipramine (a specific norepinephrine transporter (NET) inhibitor), and GBR12935 (a specific dopamine transporter (DAT) inhibitor) completely inhibited 5-HT uptake in HBVSMCs with IC50 values of 97.03 ± 40.10, 10.49 ± 5.98, and 2.80 ± 1.04 μM, respectively (Figure 3). The IC50 value for citalopram was significantly different from the that for GBR12935 (P < 0.05) but not for desipramine. The IC50 values for desipramine and GBP12935 were not statistically different. Corticosterone (a specific OCT-3 inhibitor) could only inhibited 5-HT uptake in HBVSMCs by 27%, with the threshold concentration between 10 and 100 nM.


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

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

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.
Mentions: To examine which type of transporters were responsible for 5-HT uptake in HBVSMCs, the effects of various inhibitors was studied. Citalopram (a specific SERT inhibitor), desipramine (a specific norepinephrine transporter (NET) inhibitor), and GBR12935 (a specific dopamine transporter (DAT) inhibitor) completely inhibited 5-HT uptake in HBVSMCs with IC50 values of 97.03 ± 40.10, 10.49 ± 5.98, and 2.80 ± 1.04 μM, respectively (Figure 3). The IC50 value for citalopram was significantly different from the that for GBR12935 (P < 0.05) but not for desipramine. The IC50 values for desipramine and GBP12935 were not statistically different. Corticosterone (a specific OCT-3 inhibitor) could only inhibited 5-HT uptake in HBVSMCs by 27%, with the threshold concentration between 10 and 100 nM.

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