McKinney M, Stenstrom S, Richelson E
Mol Pharmacol. 1984 Sep;26(2):156-63.
Cells of the murine neuroblastoma clone N1E-115 possess muscarinic receptors that influence the intracellular level of cyclic nucleotides. The stimulation of [3H]cyclic GMP levels occurs only with intact cells and has an EC50 near the "low-affinity" agonist equilibrium dissociation constant (KL) determined by radioligand binding assays. The inhibition of prostaglandin E1-stimulated [3H]cyclic AMP formation has an EC50 close to the value for the "high-affinity" agonist equilibrium dissociation constant (KH). During sequential subculturing in medium supplemented with newborn bovine serum, the inhibition of [3H]cyclic AMP was maintained, but the [3H]cyclic GMP response declined dramatically, and after 7 subculturings it was essentially absent. The time course for [3H]cyclic GMP formation in a late subculture with an 88% loss of the response was identical with the time course in early subcultures. A normal [3H]cyclic GMP response to bradykinin and histamine was demonstrated to be present in cells that had lost the [3H]cyclic GMP response to carbachol. The EC50 and KD values for the two muscarinic responses and binding sites increased 3- to 4-fold after several subculturings. A 90% loss of low-affinity binding sites was closely correlated with a similar loss of the [3H]cyclic GMP response. High-affinity binding sites did not decline significantly in concentration until the 11th subculture, where the total number of muscarinic sites was only 6% of the earliest subculture. In all subcultures, however, the ability of the muscarinic receptor to decrease [3H]cyclic AMP levels was maintained. These data, which show that the subculturing of N1E-115 cells in medium supplemented with newborn calf serum results in a selective loss of one muscarinic function, strongly support the hypothesis that these cells contain two separate muscarinic receptor-effector systems. One receptor subtype or conformation has a low affinity for the agonist and mediates cyclic GMP formation. The other receptor subtype or conformation has a higher affinity for the agonist and mediates an inhibition of prostaglandin E1-stimulated cyclic AMP formation.
鼠神经母细胞瘤克隆N1E - 115的细胞具有影响环核苷酸细胞内水平的毒蕈碱受体。[3H]环鸟苷酸水平的刺激仅在完整细胞中发生，其半数有效浓度（EC50）接近通过放射性配体结合试验测定的“低亲和力”激动剂平衡解离常数（KL）。前列腺素E1刺激的[3H]环腺苷酸形成的抑制作用的EC50接近“高亲和力”激动剂平衡解离常数（KH）的值。在补充新生牛血清的培养基中连续传代培养期间，[3H]环腺苷酸的抑制作用得以维持，但[3H]环鸟苷酸反应急剧下降，经过7次传代培养后基本消失。在反应丧失88%的后期传代培养物中[3H]环鸟苷酸形成的时间进程与早期传代培养物中的时间进程相同。对卡巴胆碱失去[3H]环鸟苷酸反应的细胞中，对缓激肽和组胺的正常[3H]环鸟苷酸反应被证明是存在的。经过几次传代培养后，两种毒蕈碱反应和结合位点的EC50和KD值增加了3至4倍。低亲和力结合位点90%的丧失与[3H]环鸟苷酸反应的类似丧失密切相关。高亲和力结合位点的浓度直到第11次传代培养时才显著下降，此时毒蕈碱位点总数仅为最早传代培养物的6%。然而，在所有传代培养物中，毒蕈碱受体降低[3H]环腺苷酸水平的能力得以维持。这些数据表明，在补充新生小牛血清培养基中对N1E - 115细胞进行传代培养会导致一种毒蕈碱功能的选择性丧失，有力地支持了这些细胞包含两个独立的毒蕈碱受体 - 效应器系统的假说。一种受体亚型或构象对激动剂具有低亲和力并介导环鸟苷酸的形成。另一种受体亚型或构象对激动剂具有更高的亲和力并介导对前列腺素E1刺激的环腺苷酸形成的抑制作用。
