Sénard J M, Mauriège P, Daviaud D, Paris H
INSERM U-317, Institut de Physiologie, Université Paul Sabatier, Toulouse, France.
Eur J Pharmacol. 1989 Mar 21;162(2):225-36. doi: 10.1016/0014-2999(89)90285-9.
The HT29 human adenocarcinoma cell-line was used to investigate the binding of 3H-adrenaline at alpha 2-adrenoceptors. All aspects of the study indicated that alpha 2-adrenoceptors were specifically labeled. 3H-adrenaline bound with high affinity (KD = 2.28 +/- 0.41 nM) to a single population of non-interacting sites. The rank order of adrenoceptor antagonists (yohimbine greater than phentolamine much greater than prazosin) and agonists (UK-14,304 greater than clonidine greater than (-)-adrenaline greater than (-)-noradrenaline greater than (+)-adrenaline greater than (+)-noradrenaline greater than amidephrine) to compete with 3H-adrenaline binding showed that the labeled sites were alpha 2-selective and stereospecific. Comparison of the binding parameters of 3H-adrenaline with those of [3H]clonidine (partial-agonist) and [3H]yohimbine (antagonist) indicated that 3H-adrenaline and [3H]clonidine labeled a similar number of sites (156 +/- 13 versus 175 +/- 21 fmol/mg protein) and that [3H]yohimbine (Bmax = 246 +/- 22 fmol/mg protein) labeled more sites than the 3H-agonists. Data on the inhibition of [3H]yohimbine binding by (-)-adrenaline was better fitted to a two-site model and revealed (1) that the KiL/KiH ratio was higher for (-)-adrenaline than for clonidine (2) that both agonists recognized the same percentage of high-affinity receptors. The results from a kinetic study of 3H-adrenaline binding were apparently inconsistent with the equilibrium data. Both the association and dissociation were bi-exponential, suggesting a relative heterogeneity of the labeled sites. The tritiated physiological full-agonist was moreover able to induce tight-binding. The practical consequences of this property are discussed.
使用HT29人腺癌细胞系研究3H-肾上腺素与α2-肾上腺素能受体的结合。该研究的所有方面均表明α2-肾上腺素能受体被特异性标记。3H-肾上腺素以高亲和力（KD = 2.28 +/- 0.41 nM）与单一群体的非相互作用位点结合。肾上腺素能拮抗剂（育亨宾大于酚妥拉明远大于哌唑嗪）和激动剂（UK-14,304大于可乐定大于(-)-肾上腺素大于(-)-去甲肾上腺素大于(+)-肾上腺素大于(+)-去甲肾上腺素大于酰胺福林）与3H-肾上腺素结合竞争的排序表明，标记位点具有α2选择性和立体特异性。3H-肾上腺素与[3H]可乐定（部分激动剂）和[3H]育亨宾（拮抗剂）的结合参数比较表明，3H-肾上腺素和[3H]可乐定标记的位点数量相似（156 +/- 13对175 +/- 21 fmol/mg蛋白质），且[3H]育亨宾（Bmax = 246 +/- 22 fmol/mg蛋白质）标记的位点比3H-激动剂更多。(-)-肾上腺素对[3H]育亨宾结合的抑制数据更符合双位点模型，并显示(1)(-)-肾上腺素的KiL/KiH比值高于可乐定；(2)两种激动剂识别的高亲和力受体百分比相同。3H-肾上腺素结合动力学研究的结果显然与平衡数据不一致。结合和解离均为双指数形式，表明标记位点存在相对异质性。此外，氚标记的生理性全激动剂能够诱导紧密结合。讨论了该特性的实际影响。
