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

MTV and MGV: Two Criteria for Nonlinear PCA

ized Variance) are popular criteria for PCA with optimal scaling. They are adopted by the SAS-PRINQUAL procedure and OSMOD (Saito and Otsu,1988). MTV is an intuitive generalization of linear PCA criterion. We will show some proper-ties of nonlinear PCA with these criteria in an application to the data of NLSY79 (Zagorsky,1997), a large panel survey in the U.S., conducted over twenty years. We will show the following. (1) The effectiveness of PCA with optimal scaling as a tool for large social research data analysis. We can obtain useful results when it complements analyses by regression models. (2) Features of MTV and MGV, especially their abilities and deficiencies in real data analysis. 1

Involvement of plasma membrane monoamine transporter in serotonin uptake in vascular smooth muscle cells

Abstracts for Posters: no. P27link_to_OA_fulltex

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

Role of extracellular signal-related kinases in the pathophysiology of vascular dysfunction in aging and hypertension

This free access journal suppl. is Proceedings of the 10th International Symposium on Mechanisms of Vasodilatation 2009Recent evidence suggests that extracellular signal-regulated kinase-1/2 (ERK 1/2) affect vascular tone. The present study examined the role of this enzyme in vascular control under physiological and pathological conditions. Using tissue bath technique, vascular responses were measured in terms of changes in isometric tension in aortic rings that were isolated from normotensive Wistar-Kyoto (WKY) rats and hypertensive spontaneously hypertensive rats (SHR) at different ages, 36-39 week old (adult group) and 72-75 week ROG DJHGJURXS ,QKLELWLRQRI(5. E\8 ȝ0 UHGXFHGSKHQ\OHSKULQH induced contraction in aortae. The inhibitory effect of U0126 on contraction was smaller in aged WKY rats compared to the other groups. While U0126 did not affect relaxation WRDFHW\OFKROLQH ȝ0 LQDGXOW:.< UDWV LWHQKDQFHGDFHW\OFKROLQH LQGXFHGUHOD[DWLRQ in aged WKY rats and in adult and aged SHR. The enhanced relaxation was abolished by / 1$0( DQLWULFR[LGHV\QWKDVHLQKLELWRU ȝ0 LQ:.<DQG6+5RIDOODJHJURXSV DQG UHGXFHGE\LQGRPHWKDFLQ DF\FORR[\JHQDVHLQKLELWRU ȝ0 LQDJHG:.< UDWV Therefore, our results indicate that ERK 1/2 contributes to the contraction induced by phenylephrine to a similar degree in adult rats. On the other hand, ERK 1/2 is activated in aged and hypertensive rats, leading to the suppression of nitric oxide-mediated relaxation. As such, ERK 1/2 appears to contribute to vascular dysfunction in aging and hypertension.link_to_OA_fulltex

Distributions of alpha 2 adrenoceptor subtypes in porcine coronary vasculature

Alpha 2-adrenoreceptor agonists affect vascular tone of coronary vasculature by multiple mechanisms. One of the mechanisms is the direct action on endothelial {alpha}2 adrenoreceptors (mainly {alpha}2A), which induces an endothelial-dependent vasorelaxation via release of nitric oxide. We sought to investigate the subtypes of {alpha}2 adrenoceptors and their relative abundance in coronary blood vessels of different sizes (arteries vs arterioles). Result of RT-PCR clearly demonstrated that mRNA of {alpha}2A and {alpha}2B adrenoceptors was present in coronary arteries but mRNA of {alpha}2C-adrenoceptors was absent. In contrast, mRNA of {alpha}2B and {alpha}2C adrenoceptors was present in subendocardial myocardium (which contains coronary arterioles) but mRNA of {alpha}2A adrenoceptors was not. While mRNA of {alpha}2B adrenoceptors were detected in both coronary arteries and subendocardial myocardium, quantitative analysis revealed that its expression was higher in coronary arteries. Immunohistochemical studies showed that {alpha}2A and {alpha}2B adrenoceptor proteins were located mainly on the endothelial cells of coronary arteries but none was visualized in coronary arterioles. Distributions and cellular localizations of {alpha}2C adrenoceptor proteins had not yet been studied since specific antibody was not available. This preliminary data suggests that coronary blood vessels of different sizes may respond differently to pharmacological agents

Dexmedetomidine relaxes isolated porcine coronary artery via endothelial 2A receptors

Abstract no. A121