Schloos J, Wellstein A, Palm D
Naunyn Schmiedebergs Arch Pharmacol. 1987 Jul;336(1):48-59. doi: 10.1007/BF00177750.
The agonist/alpha 2-adrenoceptor interactions at human platelet membranes have been examined in radioligand binding studies with the full agonist ligand 3H-UK-14,304 [5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline] and the antagonist ligand 3H-yohimbine. From association kinetics of different concentrations of 3H-UK-14,304 (0.75-8.1 nmol/l) a KD-value of 2.37 nmol/l in agreement with the high-affinity KD-value (KDH = 1.60 +/- 0.15 nmol/l) obtained from equilibrium binding studies was derived. In the presence of Gpp(NH)p about 6% of specific radioligand binding was observed in the association reaction. Addition of Gpp(NH)p at equilibrium resulted in a rapid loss (t 1/2 less than 1 min) of approximately 80% of bound radioligand. Dissociation after addition of an excess of phentolamine (10 mumol/l) showed a biphasic time course independent of the radioligand concentration with the proportions of 1/5 of rapidly (t 1/2 less than 2 min) and 4/5 of slowly dissociating ligand (k-1 = 0.033 +/- 0.004 min-1). Application of a sequential binding model resulted in KD-values from this approach also in agreement with KDH from equilibrium binding studies. The rank order of potency for different agonists and antagonists to compete for binding with 3H-UK-14,304 indicated an alpha 2-adrenoceptor interaction: (-)adrenaline greater than or equal to clonidine greater than (-)noradrenaline greater than (-)isoprenaline and yohimbine = rauwolscine greater than phentolamine greater than prazosin greater than or equal to corynanthine greater than timolol respectively. The analysis of competition isotherms of UK-14,304 versus 3H-yohimbine (Hill-coefficient = 0.59 +/- 0.03) showed that the agonist binds to two affinity states of the alpha 2-adrenoceptor, with high (KDH = 1.77 +/- 0.50 nmol/l) and low affinity (KDL = 71.2 +/- 11.6 nmol/l) respectively. From these experiments a fraction of 56.9% +/- 2.1% of the total number of alpha 2-adrenoceptors (Bmax = 198.4 +/- 8.0 fmol/mg of protein) in the high-affinity state was calculated. Similar results were obtained from 3H-UK-14,304 saturation isotherms according to a two-state binding model (KDH = 1.60 +/- 0.15 nmol/l; KDL = 66.2 +/- 10.7 nmol/l; BmaxH = 57.6% +/- 2.3%). Adrenoceptor agonists competed for specific binding of 3H-UK-14,304 and 3H-yohimbine in a manner that suggests that the 3H-UK-14,304 (approximately 3.5 nmol/l) labeled sites represent predominantly the agonist induced or stabilized high-affinity state of the alpha 2-adrenoceptor.(ABSTRACT TRUNCATED AT 400 WORDS)
在放射性配体结合研究中，使用完全激动剂配体3H-UK-14,304 [5-溴-6-(2-咪唑啉-2-基氨基)-喹喔啉]和拮抗剂配体3H-育亨宾，检测了人血小板膜上激动剂/α2-肾上腺素能受体的相互作用。根据不同浓度3H-UK-14,304（0.75 - 8.1 nmol/l）的结合动力学，得出KD值为2.37 nmol/l，这与从平衡结合研究中获得的高亲和力KD值（KDH = 1.60 ± 0.15 nmol/l）一致。在Gpp(NH)p存在下，结合反应中观察到约6%的特异性放射性配体结合。在平衡时加入Gpp(NH)p导致约80%的结合放射性配体迅速丢失（t1/2小于1分钟）。加入过量酚妥拉明（10 μmol/l）后的解离显示出双相时间进程，与放射性配体浓度无关，快速解离（t1/2小于2分钟）的配体占1/5，缓慢解离的配体占4/5（k-1 = 0.033 ± 0.004 min-1）。应用序贯结合模型从该方法得出的KD值也与平衡结合研究中的KDH一致。不同激动剂和拮抗剂与3H-UK-14,304竞争结合的效价顺序表明存在α2-肾上腺素能受体相互作用：（-）肾上腺素≥可乐定＞（-）去甲肾上腺素＞（-）异丙肾上腺素，育亨宾 = 萝芙木碱＞酚妥拉明＞哌唑嗪≥育亨宾碱＞噻吗洛尔。UK-14,304与3H-育亨宾竞争等温线分析（希尔系数 = 0.59 ± 0.03）表明，激动剂与α2-肾上腺素能受体的两种亲和力状态结合，分别具有高亲和力（KDH = 1.77 ± 0.50 nmol/l）和低亲和力（KDL = 71.2 ± 11.6 nmol/l）。根据这些实验计算出处于高亲和力状态的α2-肾上腺素能受体总数（Bmax = 198.4 ± 8.0 fmol/mg蛋白质）的比例为56.9% ± 2.1%。根据双态结合模型从3H-UK-14,304饱和等温线获得了类似结果（KDH = 1.60 ± 0.15 nmol/l；KDL = 66.2 ± 10.7 nmol/l；BmaxH = 57.6% ±
