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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

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).
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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).

Mentions: Reverse transcription-polymerase chain reaction (RT-PCR) was used to study the mRNA expressions of different transporters in HBVSMCs. cDNA from human livers and kidneys were used as positive controls because all the transporters studied were expressed in these tissues. The PCR products of OCT-3 and PMAT were amplified by RT-PCR from RNA isolated from HBVSMCs (Figure 4). The molecular sizes of PCR products were the same as expected and the PCR products were confirmed by sequencing, indicating the mRNA expressions of OCT-3 and PMAT in HBVSMCs. In contrast, the PCR product of OCT-1, OCT-2, SERT, NET, and DAT were not detected.


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)

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

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

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).
Mentions: Reverse transcription-polymerase chain reaction (RT-PCR) was used to study the mRNA expressions of different transporters in HBVSMCs. cDNA from human livers and kidneys were used as positive controls because all the transporters studied were expressed in these tissues. The PCR products of OCT-3 and PMAT were amplified by RT-PCR from RNA isolated from HBVSMCs (Figure 4). The molecular sizes of PCR products were the same as expected and the PCR products were confirmed by sequencing, indicating the mRNA expressions of OCT-3 and PMAT in HBVSMCs. In contrast, the PCR product of OCT-1, OCT-2, SERT, NET, and DAT were not detected.

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