Differential ligand binding affinities of human estrogen receptor-α isoforms

Rapid non-genomic effects of 17β-estradiol are elicited by the activation of different estrogen receptor-α isoforms. Presence of surface binding sites for estrogen have been identified in cells transfected with full-length estrogen receptor-α66 (ER66) and the truncated isoforms, estrogen receptor-α46 (ER46) and estrogen receptor-α36 (ER36). However, the binding affinities of the membrane estrogen receptors (mERs) remain unknown due to the difficulty of developing of stable mER-transfected cell lines with sufficient mER density, which has largely hampered biochemical binding studies. The present study utilized cell-free expression systems to determine the binding affinities of 17β-estradiol to mERs, and the relationship among palmitoylation, membrane insertion and binding affinities. Saturation binding assays of human mERs revealed that [3H]-17β-estradiol bound ER66 and ER46 with Kd values of 68.81 and 60.72 pM, respectively, whereas ER36 displayed no specific binding within the tested concentration range. Inhibition of palmitoylation or removal of the nanolipoprotein particles, used as membrane substitute, reduced the binding affinities of ER66 and ER46 to 17β-estradiol. Moreover, ER66 and ER46 bound differentially with some estrogen receptor agonists and antagonists, and phytoestrogens. In particular, the classical estrogen receptor antagonist, ICI 182,780, had a higher affinity for ER66 than ER46. In summary, the present study defines the binding affinities for human estrogen receptor-α isoforms, and demonstrates that ER66 and ER46 show characteristics of mERs. The present data also indicates that palmitoylation and membrane insertion of mERs are important for proper receptor conformation allowing 17β-estradiol binding. The differential binding of ER66 and ER46 with certain compounds substantiates the prospect of developing mER-selective drugs.published_or_final_versio

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

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-HT2 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. [3H]5-HT uptake in HBVSMCs was Na+-independent. Kinetic analyses of [3H]5-HT uptake in HBVSMCs revealed a Km of 50.36 ± 10.2 mM and a Vmax 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 IC50 values of 97.03 ± 40.10, 10.49 ± 5.98, and 2.80 ± 1.04 μM, respectively. These IC50 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

Control of extracellular adenosine levels in inflammation

Conference Theme: Improving the Health of an Aging Populatio

Diabetes- and hypertension-induced upregulation of concentrative nucleoside transporter-2 in endothelial cells

This free access journal suppl. is Proceedings of the 10th International Symposium on Mechanisms of Vasodilatation 2009Adenosine modulates a variety of vascular functions and adenosine homeostasis is controlled by nucleoside transporters. Nucleoside transporters are divided into 2 classes. The equilibrative nucleoside transporters (ENTs) are Na+ -independent and are VXEGLYLGHGLQWR W\SHVEDVHGRQWKHLUVHQVLWLYLWLHVWR > QLWUREHQ]\O WKLRO@ ȕ ' ribofuranosylpurine (NBMPR). The concentrative nucleoside transporters (CNTs) are Na+ -dependent and are subdivided into 3 types based on the substrate selectivity. In this study, the nucleoside transporters in vascular cells were characterized and their changes in diabetes and hypertension were investigated. )XQFWLRQDOVWXGLHVVKRZHGWKDW RIWKH>3 H]adenosine transport in human brain vascular endothelial cells (HBECs) was Na+ -dependent. In the Na+ -independent component, 80% was inhibited by NBMPR (10 nM, which inhibits ENT1 but not ENT2). However, the [3 H]adenosine transport in vascular smooth muscle cells (HBSMCs) was totally Na+ -independent and 95% of it was inhibited by NBMPR (10 nM). RT-PCR revealed the presence of mRNA of ENT1, ENT2 and CNT2 in HBECs, but no CNT2 was found in HBSMCs. The unique presence of CNT2 in endothelial cells was confirmed by the observation that mRNA of ENT1, ENT2, and CNT2 were present in rat basilar arteries but no CNT2 was detected after removal of endothelia. The mRNA expression of CNT2, but not ENT1 and ENT2, was higher in basilar arteries of spontaneously hypertensive rats and streptozotocin-induced diabetic rats compared with those in normal rats. Such increase in CNT2 in hypertension and diabetes may affect the availability of adenosine in the vicinity of adenosine receptors of endothelial cells and, thus, alter vascular functions.link_to_OA_fulltex

Lipid emulsion infusion vs standard cardiac resuscitation in management of ropivacaine cardiac toxicity in pigs

Australian and New Zealand College of Anaesthetists Annual Scientific Meetin

Possible role of organic cation transpporter-3 in serotonin uptake in human brain vascular smooth muscle cells

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Possible role of organic cation transport-3 in serotonin uptake in human brain vascular smooth muscle cells

This free access journal suppl. is Proceedings of the 10th International Symposium on Mechanisms of Vasodilatation 2009Serotonin (5HT) is a vasoconstrictor. It has been reported that 5HT can be taken up by vascular smooth muscle cells of rat aortas through the serotonin transporters (SERT). 7KLV +7XSWDNHPHFKDQLVPSOD\VDFUXFLDOUROHLQ¿QH WXQLQJWKHDYDLODELOLW\RI +7DW its cognate receptors. However, it is unclear if SERT or other transporters are responsible for the 5HT uptake in vascular smooth muscle cells of human resistance arteries. The aim of this work was to characterize the 5HT uptake in human brain vascular smooth muscle cells (HBVSMCs). The [3 H]5HT uptake in HBVSMCs was increased with time and was saturable with a Michaelis-menten constant of 50.36 ± 10.2 mM. The [3 H]5HT uptake was enhanced when the extracellular medium was changed alkaline. Moreover, the [3 H]5HT XSWDNHZDVUHVLVWDQWWRFLWDORSUDP D6(57LQKLELWRU XSWR ȝ0 EXWLWFRXOGEHLQKLELWHG by citalopram at higher concentrations (nearly 40% inhibition by 1 mM). Tetraammonium (TEA, at 1 mM) and 1-methy-4-phenylpyridinium (MPP, at 1 mM), which are substrates of organic cation transporters (OCTs), inhibited 5HT uptake by 17 and 26%, respectively. The ORZDI¿QLW\RI +7WUDQVSRUW S+GHSHQGHQFHDQGLQKLELWLRQE\RUJDQLFFDWLRQVDUHW\SLFDO characteristics of OCTs. In addition, the results of RT-PCR demonstrated the presence of mRNA of OCT-3, but the absence of OCT-1, OCT-2, organic anion transporters (OATs) and SERT. Therefore, we suggest that the 5HT uptake in HBVSMCs is different from that in rat aorta. It is partly mediated by OCT-3, but does not involve SERT.link_to_OA_fulltex