Landi M, Bianchetti A, Croci T, Manara L
Research Center Sanofi-Midy S.p.A., Milan, Italy.
Biochem Pharmacol. 1992 Aug 18;44(4):665-72. doi: 10.1016/0006-2952(92)90401-4.
[3H]Dihydroalprenolol ([3H]DHA) specific binding (determined by the difference in the presence and absence of 20 microM (-)isoprenaline) to rat colon membranes was saturable (Bmax = 39.6 fmol/mg protein), of high affinity (Kd = 0.87 nM) and stereospecific (IC50 330 and 3510 nM for (-)- and (+)isoprenaline, respectively); the Hill coefficient was close to one, indicating binding homogeneity. [3H]DHA (0.6 nM) specific binding was potently inhibited (Ki range 1.9-3.3 nM) by the non-selective beta-adrenoceptor antagonists pindolol, alprenolol, but not by the non-adrenergic compounds 5-hydroxytryptamine, 8-hydroxydipropylaminotetraline, methysergide, dopamine and verapamil (Ki greater than 10,000 nM). The selective beta 1- and beta 2-adrenoceptor antagonists CGP 20,712A and ICI 118,551 resulted in biphasic competition binding curves, whose low and high affinity components were compatible with two populations of binding sites accounting for about 75 (beta 2) and 25% (beta 1) of total sites. The relative competing potencies of reference adrenergic agonists also suggested a prevalence of beta 2-adrenergic sites. The new agonists phenylethanolaminotetralines (PEATs), highly selective for the atypical beta-adrenoceptors whose abundance in rat colon has been confirmed by comprehensive functional studies, had variable affinity for the [3H]DHA-labelled sites depending on chirality, but with no substantial correlation with their pharmacological potency. Only 40% of [3H]DHA binding, at a concentration about 10 times its Kd for high affinity sites (beta 1 and beta 2), was prevented by saturating concentrations of isoprenaline. Under this condition, the representative PEAT, SR 58611A, highly potent and selective for atypical beta-adrenoceptors in functional tests, and its pharmacologically inactive enantiomer, both inhibited the residual binding equipotently. In conclusion, [3H]DHA binding did not detect atypical beta-adrenoceptor sites in rat colon membranes, most probably because of its weaker affinity for them than for the coexisting beta 1 and beta 2 sites. PEAT stereoisomers proved essential for assessing both the stereospecificity and the functional significance of this atypical binding and to compare their affinity for [3H]DHA-labelled sites and pharmacological potency.
[3H]二氢阿普洛尔（[3H]DHA）与大鼠结肠膜的特异性结合（通过存在和不存在20微摩尔（-）异丙肾上腺素时的差异来确定）具有饱和性（Bmax = 39.6飞摩尔/毫克蛋白质）、高亲和力（Kd = 0.87纳摩尔）且具有立体特异性（（-）-和（+）-异丙肾上腺素的IC50分别为330和3510纳摩尔）；希尔系数接近1，表明结合具有同质性。非选择性β-肾上腺素能受体拮抗剂吲哚洛尔、阿普洛尔能有效抑制（Ki范围为1.9 - 3.3纳摩尔）[3H]DHA（0.6纳摩尔）的特异性结合，而非肾上腺素能化合物5-羟色胺、8-羟基二丙基氨基四氢萘、甲基麦角新碱、多巴胺和维拉帕米则无此作用（Ki大于10,000纳摩尔）。选择性β1-和β2-肾上腺素能受体拮抗剂CGP 20,712A和ICI 118,551产生双相竞争结合曲线，其低亲和力和高亲和力成分与两类结合位点相符，分别约占总位点的75%（β2）和25%（β1）。参考肾上腺素能激动剂的相对竞争效力也表明β2-肾上腺素能位点占优势。新型激动剂苯乙醇胺四氢萘（PEATs）对非典型β-肾上腺素能受体具有高度选择性，全面的功能研究已证实其在大鼠结肠中大量存在，根据手性不同，它们对[3H]DHA标记位点的亲和力各异，但与它们的药理效力无显著相关性。在浓度约为其对高亲和力位点（β1和β2）的Kd值10倍时，饱和浓度的异丙肾上腺素仅能阻止40%的[3H]DHA结合。在此条件下，代表性的PEAT，SR 58611A，在功能测试中对非典型β-肾上腺素能受体具有高效力和选择性，及其药理惰性对映体，均能等效抑制剩余结合。总之，[3H]DHA结合未检测到大鼠结肠膜中的非典型β-肾上腺素能受体位点，很可能是因为它对这些位点的亲和力比对共存的β1和β2位点的亲和力弱。PEAT立体异构体对于评估这种非典型结合的立体特异性和功能意义以及比较它们对[3H]DHA标记位点的亲和力和药理效力至关重要。
