To improve selectivity and specificity of cell membrane chromatography (CMC), the chromatography affinities of nine ligands of a1-adrenergic receptor(AR)to a1D-AR subtype were investigated. The human embryonic kidney ...To improve selectivity and specificity of cell membrane chromatography (CMC), the chromatography affinities of nine ligands of a1-adrenergic receptor(AR)to a1D-AR subtype were investigated. The human embryonic kidney (HEK) 293 cells expressed by cDNA of a1D-AR subtypes were cultured and cell membrane stationary phase (CMSP) was prepared. Then the interactions between ligands and a1D-AR in CMSP were investigated using CMC. The affinity rank order to a1D-AR subtype obtained from CMC for the nine a1-adrenoceceptor ligands is: prazosin, BMY7378, phentolamine, oxymetazoline, 5-methylurapidil, norepinephrine, phenyle- phrine, methoxamine, RS-17053. The affinity rank order is similar and correlates well with that obtained from others radioligand binding assays (RBA). CMSP prepared by transfected HEK293 cells with 1D-adrenoceptor cDNA and CMC method could be used to evaluate affinities of drug-receptor and drug-receptor subtypes and to screen drugs selective to a1D-AR.展开更多
Chiral drug naftopidil(NAF), a specific α1D-adrenoceptor(AR) antagonist for the treatment of benign prostatic hyperplasia, was used in racemic form for several decades. Our recent work declared that NAF enantiomers s...Chiral drug naftopidil(NAF), a specific α1D-adrenoceptor(AR) antagonist for the treatment of benign prostatic hyperplasia, was used in racemic form for several decades. Our recent work declared that NAF enantiomers showed the same antagonistic effects on the α1D-AR, but the binding mechanism of these two stereochemical NAF isomers to the α1D receptor remained unclear. Herein, we reported the crystallographic structures of optically pure NAF stereoisomers for the first time and unambiguously determined their absolute configurations. The crystal data of R and S enantiomers matched satisfactorily the pharmacophore model for α1D-selective antagonists. Based on the constructed α1D homology model,molecular docking studies shed light on the molecular mechanism of NAF enantiomers binding to α1D-AR. The results indicated that NAF enantiomers exhibited the very similar binding poses and occupied the same binding pocket.展开更多
We compared the norepinephrine (NE) induced α1B-adrenoceptor (α1B-AR) expression modulation between two transfected human embryonic kidney (HEK) 293 cell lines in which α1B-AR densities were (6 336 ±913) and (...We compared the norepinephrine (NE) induced α1B-adrenoceptor (α1B-AR) expression modulation between two transfected human embryonic kidney (HEK) 293 cell lines in which α1B-AR densities were (6 336 ±913) and (773±164) fmol · mg -1, respectively. Treatment of cells with NE (10 μmol · L1) for 48 h decreased high-level expressed α1B-AR density, but increased low-level expressed α1B-AR density. The protein kinase C inhibitor Calphostin C or Ro-31-8220 reversed, and its activator PMA mimicked the NE-induced down-regulation of high-level expressed α1B-AR. Moreover, PMA induced a down-regulation of low-level expressed α1B-AR. The endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) and the calcium chelator BAPTA/AM did not affect the down-regulation of high-level expressed α1B-AR, but inhibited the up-regulation of low-level expression α1B-AR induced by NE. These results suggest that α1B-adrenoceptor densities at different initial expression levels are differentially regulated展开更多
文摘To improve selectivity and specificity of cell membrane chromatography (CMC), the chromatography affinities of nine ligands of a1-adrenergic receptor(AR)to a1D-AR subtype were investigated. The human embryonic kidney (HEK) 293 cells expressed by cDNA of a1D-AR subtypes were cultured and cell membrane stationary phase (CMSP) was prepared. Then the interactions between ligands and a1D-AR in CMSP were investigated using CMC. The affinity rank order to a1D-AR subtype obtained from CMC for the nine a1-adrenoceceptor ligands is: prazosin, BMY7378, phentolamine, oxymetazoline, 5-methylurapidil, norepinephrine, phenyle- phrine, methoxamine, RS-17053. The affinity rank order is similar and correlates well with that obtained from others radioligand binding assays (RBA). CMSP prepared by transfected HEK293 cells with 1D-adrenoceptor cDNA and CMC method could be used to evaluate affinities of drug-receptor and drug-receptor subtypes and to screen drugs selective to a1D-AR.
基金supported by grants from the National Science Foundation of Guangdong Province (No. S2013040014088)the Postdoctoral Science Foundation of Guangzhou City (Q188)partially supported by the Guangdong Major Scientific and Technological Special Project for New Drug Development (2013A022100029)
文摘Chiral drug naftopidil(NAF), a specific α1D-adrenoceptor(AR) antagonist for the treatment of benign prostatic hyperplasia, was used in racemic form for several decades. Our recent work declared that NAF enantiomers showed the same antagonistic effects on the α1D-AR, but the binding mechanism of these two stereochemical NAF isomers to the α1D receptor remained unclear. Herein, we reported the crystallographic structures of optically pure NAF stereoisomers for the first time and unambiguously determined their absolute configurations. The crystal data of R and S enantiomers matched satisfactorily the pharmacophore model for α1D-selective antagonists. Based on the constructed α1D homology model,molecular docking studies shed light on the molecular mechanism of NAF enantiomers binding to α1D-AR. The results indicated that NAF enantiomers exhibited the very similar binding poses and occupied the same binding pocket.
文摘We compared the norepinephrine (NE) induced α1B-adrenoceptor (α1B-AR) expression modulation between two transfected human embryonic kidney (HEK) 293 cell lines in which α1B-AR densities were (6 336 ±913) and (773±164) fmol · mg -1, respectively. Treatment of cells with NE (10 μmol · L1) for 48 h decreased high-level expressed α1B-AR density, but increased low-level expressed α1B-AR density. The protein kinase C inhibitor Calphostin C or Ro-31-8220 reversed, and its activator PMA mimicked the NE-induced down-regulation of high-level expressed α1B-AR. Moreover, PMA induced a down-regulation of low-level expressed α1B-AR. The endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) and the calcium chelator BAPTA/AM did not affect the down-regulation of high-level expressed α1B-AR, but inhibited the up-regulation of low-level expression α1B-AR induced by NE. These results suggest that α1B-adrenoceptor densities at different initial expression levels are differentially regulated
