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
Clonidine is a classically categorized α2-adrenoceptor (α2-AR) agonist that produces vascular contractions by stimulating arterial smooth muscle α2-ARs. However, clonidine inhibits α1-AR-mediated arterial contract...Clonidine is a classically categorized α2-adrenoceptor (α2-AR) agonist that produces vascular contractions by stimulating arterial smooth muscle α2-ARs. However, clonidine inhibits α1-AR-mediated arterial contractions. Recently, it was suggested that repeated stimulation with clonidine induces desensitization of α2-ARs, thus inhibiting noradrenaline-induced smooth muscle contractions. In the present study, we examined whether clonidine-mediated inhibition of α1-AR contractions involves interactions with α2-ARs in rat thoracic aortae. 1) Clonidine and guanfacine inhibited electrical field stimulation-induced contractions in a concentration-dependent, yohimbine-sensitive manner in isolated rat vas deferens preparations. 2) Clonidine almost completely suppressed phenylephrine-induced sustained contractions of rat thoracic aortae. 3) Clonidine competitively inhibited phenylephrine-induced contractions with a pA2 value of 6.77 at concentrations between 10-7 and 10-6 M. At 10-5 M, clonidine inhibited phenylephrine-induced contractions and dramatically reduced maximum contractions. 4) In contrast, clonidine did not inhibit contractions produced by high KCl or prostaglandin F2α. 5) Inhibition of phenylephrine-induced sustained contractions by clonidine was also produced in the presence of yohimbine. However, guanfacine did not inhibit phenylephrine-induced sustained contractions. These findings suggest that clonidine inhibits phenylephrine-induced contraction of rat thoracic aortae by competitive antagonism of α1-ARs, which is mediated through a mechanism independent of α2-AR stimulation.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.
文摘Clonidine is a classically categorized α2-adrenoceptor (α2-AR) agonist that produces vascular contractions by stimulating arterial smooth muscle α2-ARs. However, clonidine inhibits α1-AR-mediated arterial contractions. Recently, it was suggested that repeated stimulation with clonidine induces desensitization of α2-ARs, thus inhibiting noradrenaline-induced smooth muscle contractions. In the present study, we examined whether clonidine-mediated inhibition of α1-AR contractions involves interactions with α2-ARs in rat thoracic aortae. 1) Clonidine and guanfacine inhibited electrical field stimulation-induced contractions in a concentration-dependent, yohimbine-sensitive manner in isolated rat vas deferens preparations. 2) Clonidine almost completely suppressed phenylephrine-induced sustained contractions of rat thoracic aortae. 3) Clonidine competitively inhibited phenylephrine-induced contractions with a pA2 value of 6.77 at concentrations between 10-7 and 10-6 M. At 10-5 M, clonidine inhibited phenylephrine-induced contractions and dramatically reduced maximum contractions. 4) In contrast, clonidine did not inhibit contractions produced by high KCl or prostaglandin F2α. 5) Inhibition of phenylephrine-induced sustained contractions by clonidine was also produced in the presence of yohimbine. However, guanfacine did not inhibit phenylephrine-induced sustained contractions. These findings suggest that clonidine inhibits phenylephrine-induced contraction of rat thoracic aortae by competitive antagonism of α1-ARs, which is mediated through a mechanism independent of α2-AR stimulation.
