Claeysen S, Sebben M, Bécamel C, Eglen R M, Clark R D, Bockaert J, Dumuis A
Centre National de la Recherche Scientifique Unité Propre de Recherche, National de la Santé et de la Recherche Médicale de Pharmacologie-Endocrinologie, Montpellier, France.
Mol Pharmacol. 2000 Jul;58(1):136-44. doi: 10.1124/mol.58.1.136.
We studied the pharmacological properties of twenty-four 5-hydroxytryptamine (5-HT)(4) receptor ligands known to act as antagonists on 5-HT(4) receptors positively coupled to adenylyl cyclase endogenously expressed in mouse colliculi neurons. In COS-7 cells expressing human or mouse 5-HT(4(a)) receptors (100-8000 fmol/mg of protein), we found neutral antagonists, partial agonists, and inverse agonists. The majority of neutral antagonists belong to the benzodioxanyl ketone class, whereas partial agonists belong to different chemical classes. We found only two inverse agonists, GR 125487 and SB 207266, which are both indoles. Analysis of pharmacological characteristics of the constitutively active wild-type and constitutively active mutated receptors revealed that 1) the ratio between the efficiencies of the full agonist 5-HT and the partial agonist RS 23597 was invariable when the receptor density increased, but was dependent on receptor structure; 2) similarly, the efficacy of the inverse agonist SB 207266 was not dependent on receptor density but was dependent on receptor structure; 3) when the receptor concentration increased, the EC(50) values of the full agonist 5-HT were not modified and the increase in basal constitutive activity, as well as its stimulation by 5-HT, followed a parallel evolution; and 4) the stimulation of basal constitutive activity by 5-HT was not modified by the overexpression of Galphas. All these results indicate that in COS-7 cells, the coupling of the 5-HT(4) receptor to adenylyl cyclase was linear with no indication of spare receptors even at high receptor density (8 pmol/mg). These results are also in accordance with a precoupling between the activated receptor (f(R*)) and adenylyl cyclase. Such observations allowed us to use the two-state model to calculate the constant J, i.e., the equilibrium allosteric constant denoting the ratio of the receptor in the inactive versus active state (J = [R]/[R*]). We found that J was a receptor structural characteristic, independent of receptor density.
我们研究了24种5-羟色胺（5-HT）（4）受体配体的药理学特性，这些配体已知可作为5-HT（4）受体的拮抗剂，该受体与内源性表达于小鼠丘脑中的腺苷酸环化酶呈正偶联。在表达人或小鼠5-HT（4（a））受体（100 - 8000 fmol/mg蛋白质）的COS - 7细胞中，我们发现了中性拮抗剂、部分激动剂和反向激动剂。大多数中性拮抗剂属于苯并二氧杂环戊酮类，而部分激动剂则属于不同的化学类别。我们仅发现两种反向激动剂，GR 125487和SB 207266，它们均为吲哚类。对组成型活性野生型和组成型活性突变受体的药理学特性分析表明：1）当受体密度增加时，完全激动剂5-HT与部分激动剂RS 23597的效率之比不变，但取决于受体结构；2）同样，反向激动剂SB 207266的效力不依赖于受体密度，但依赖于受体结构；3）当受体浓度增加时，完全激动剂5-HT的EC（50）值未改变，基础组成型活性的增加及其受5-HT的刺激呈平行变化；4）5-HT对基础组成型活性的刺激不受Gαs过表达的影响。所有这些结果表明，在COS - 7细胞中，5-HT（4）受体与腺苷酸环化酶的偶联是线性的，即使在高受体密度（8 pmol/mg）下也没有备用受体的迹象。这些结果也与活化受体（f（R*））和腺苷酸环化酶之间的预偶联一致。这些观察结果使我们能够使用双态模型来计算常数J，即表示受体处于非活性状态与活性状态之比的平衡变构常数（J = [R]/[R*]）。我们发现J是受体的结构特征，与受体密度无关。
