Riva M A, Creese I
Center for Molecular and Behavioral Neuroscience, Rutgers State University of New Jersey, Newark 07102.
Mol Pharmacol. 1989 Jul;36(1):201-10.
[3H]Dihydroalprenolol ([3H]DHA) has been used extensively in receptor binding studies to measure beta-adrenergic receptors in the central nervous system. Usually, nonspecific binding has been defined by high concentrations of the beta-adrenergic receptor agonist isoproterenol or antagonists such as alprenolol or propranolol. Scatchard plots of such "specific" [3H]DHA saturation data in rat cerebral cortex membranes are linear. However, computer analysis demonstrated that the competition curves of these drugs for 2.0 nM [3H]DHA binding are biphasic, with a continuous inhibition of [3H]DHA binding in the concentration range usually used to determine nonspecific binding. These data indicate that another saturable high affinity site was being labeled by the radioligand and that the definition of nonspecific binding with any of these unlabeled drugs is not satisfactory. We used the nonlinear, least squares, curve-fitting program LIGAND to analyze total [3H]DHA binding, allowing the program to mathematically define nonspecific binding as a function of 3H-ligand concentration. Significantly lower Bmax (-44%) and Kd (-58%) values for beta-adrenergic receptors were found, indicating that under normal experimental procedures (defining [3H]DHA non-specific binding with these nonradioactive drugs) a second binding site was being labeled. We found that [3H]DHA binding to this site could be inhibited by drugs such as RU24969, a 5-hydroxytryptamine1A (5HT1A) and 5HT1B receptor subtype-selective agonist, and CGS12066B, a 5HT1B receptor subtype-selective agonist, which were able to compete for 15-20% of [3H]DHA binding in the nanomolar concentration range, whereas drugs that are selective for other serotonin receptor subtypes inhibited [3H]DHA binding only at much higher concentrations. Another beta-adrenergic receptor antagonist radioligand, [3H]CGP-12177, was found to be more selective for beta-adrenergic receptors. Alprenolol competition curves for [3H]CGP-12177 binding were monophasic and saturation curves, with nonspecific binding defined either by 10 microM alprenolol or by LIGAND, yielded Bmax values close to those obtained with [3H]DHA when its nonspecific binding was defined by LIGAND. [3H]DHA cannot be considered a suitable radioligand to quantify central nervous system beta-adrenergic receptors in the manner in which it has been typically used.
[3H]二氢烯丙洛尔（[3H]DHA）已广泛用于受体结合研究，以测量中枢神经系统中的β-肾上腺素能受体。通常，非特异性结合是通过高浓度的β-肾上腺素能受体激动剂异丙肾上腺素或拮抗剂如烯丙洛尔或普萘洛尔来定义的。大鼠大脑皮层膜中此类“特异性”[3H]DHA饱和数据的Scatchard图呈线性。然而，计算机分析表明，这些药物对2.0 nM [3H]DHA结合的竞争曲线是双相的，在通常用于确定非特异性结合的浓度范围内，[3H]DHA结合受到持续抑制。这些数据表明，放射性配体标记了另一个可饱和的高亲和力位点，并且用这些未标记药物中的任何一种来定义非特异性结合并不令人满意。我们使用非线性最小二乘法曲线拟合程序LIGAND来分析总的[3H]DHA结合，让该程序在数学上把非特异性结合定义为3H-配体浓度的函数。发现β-肾上腺素能受体的Bmax值（-44%）和Kd值（-58%）显著降低，这表明在正常实验程序下（用这些非放射性药物定义[3H]DHA非特异性结合），第二个结合位点被标记了。我们发现，[3H]DHA与该位点的结合可被诸如RU24969（一种5-羟色胺1A（5HT1A）和5HT1B受体亚型选择性激动剂）和CGS12066B（一种5HT1B受体亚型选择性激动剂）等药物抑制，这些药物在纳摩尔浓度范围内能够竞争15 - 20%的[3H]DHA结合，而对其他5-羟色胺受体亚型具有选择性的药物仅在高得多的浓度下才抑制[3H]DHA结合。另一种β-肾上腺素能受体拮抗剂放射性配体[3H]CGP - 12177被发现对β-肾上腺素能受体更具选择性。[3H]CGP - 12177结合的烯丙洛尔竞争曲线是单相的且为饱和曲线，用10 microM烯丙洛尔或LIGAND定义非特异性结合时，得到的Bmax值与用LIGAND定义[3H]DHA非特异性结合时获得的值接近。[3H]DHA不能被认为是一种以其通常使用方式来定量中枢神经系统β-肾上腺素能受体的合适放射性配体。
