Pecquery R, Giudicelli Y
Biochim Biophys Acta. 1982 Jan 12;714(1):14-25. doi: 10.1016/0304-4165(82)90122-2.
The binding characteristics of the alpha-component of (-)-[3H]norepinephrine to hamster adipocyte membranes were studied. Binding was rapid, equilibrium in 20 min at 25 degrees C. Dissociation of specific binding by 10 microM phentolamine suggested dissociation from two different sites. The time course of dissociation induced by a 50-fold dilution was unchanged by the addition of norepinephrine, suggesting the absence of cooperative binding sites. [3H]norepinephrine binding was saturable, yielding curvilinear Scatchard plots. Computer modeling of these data further supported the existence of two classes of binding sites, one with high affinity (KD = 23 nM) but low binding capacity (96 fmol/mg protein) and one with low affinity (KD = 400 nM) but high binding capacity (1000 fmol/mg protein). Adrenergic ligands competed with [3H]norepinephrine binding in the following order of potency: (-)-norepinephrine greater than (-)-epinephrine much greater than (+)-norepinephrine greater than (-)-isoproterenol. Displacement by the selective alpha-adrenergic drugs prazosin, clonidine, and yohimbine yielded biphasic curves consistent with binding of [3H]norepinephrine to both alpha 1-(14-22%) and alpha 2-(78-86%) receptor subtypes. Although Gpp(NH)p failed to alter the binding of [3H] dihydroergocryptine, it severely reduced the binding affinity of (-)-epinephrine, (-)-norepinephrine and the selective alpha 2-agonist, clonidine. The inhibitory effects of clonidine and of the alpha-component of (-)-epinephrine on the adrenocorticotropin-stimulated cyclic AMP production in the intact adipocyte were closely correlated with their effects on the binding of both [3H]norepinephrine and [3H]dihydroergocryptine. Conversely, yohimbine but not prazosin markedly antagonised the alpha-inhibitory effect of norepinephrine on cyclic AMP production. These data led to conclude that [3H]norepinephrine can be successfully used to study the entire alpha-adrenergic receptor population of hamster fat cells and that the predominant alpha 2-receptor subtype exists in two different affinity states for agonists, the proportions of which are modulated by guanine nucleotides.
研究了（-）-[3H]去甲肾上腺素的α组分与仓鼠脂肪细胞膜的结合特性。结合迅速，在25℃下20分钟达到平衡。10μM酚妥拉明引起的特异性结合解离表明从两个不同位点解离。50倍稀释诱导的解离时间进程不受去甲肾上腺素添加的影响，表明不存在协同结合位点。[3H]去甲肾上腺素结合是可饱和的，产生曲线形的Scatchard图。对这些数据的计算机建模进一步支持存在两类结合位点，一类具有高亲和力（KD = 23 nM）但低结合容量（96 fmol/mg蛋白质），另一类具有低亲和力（KD = 400 nM）但高结合容量（1000 fmol/mg蛋白质）。肾上腺素能配体与[3H]去甲肾上腺素结合的竞争效力顺序如下：（-）-去甲肾上腺素＞（-）-肾上腺素＞＞（+）-去甲肾上腺素＞（-）-异丙肾上腺素。选择性α-肾上腺素能药物哌唑嗪、可乐定和育亨宾的置换产生双相曲线，与[3H]去甲肾上腺素与α1-（14-22%）和α2-（78-86%）受体亚型的结合一致。尽管Gpp（NH）p未能改变[3H]二氢麦角隐亭的结合，但它严重降低了（-）-肾上腺素、（-）-去甲肾上腺素和选择性α2-激动剂可乐定的结合亲和力。可乐定和（-）-肾上腺素的α组分对完整脂肪细胞中促肾上腺皮质激素刺激的环磷酸腺苷产生的抑制作用与其对[3H]去甲肾上腺素和[3H]二氢麦角隐亭结合的影响密切相关。相反，育亨宾而非哌唑嗪显著拮抗去甲肾上腺素对环磷酸腺苷产生的α抑制作用。这些数据得出结论，[3H]去甲肾上腺素可成功用于研究仓鼠脂肪细胞的整个α-肾上腺素能受体群体，并且主要的α2-受体亚型对激动剂存在两种不同的亲和力状态，其比例受鸟嘌呤核苷酸调节。
