Donato di Paola E, Cusack B, Yamada M, Richelson E
Department of Psychiatry, Mayo Clinic Jacksonville, Florida.
J Pharmacol Exp Ther. 1993 Jan;264(1):1-5.
Clone N1E-115 cells have specific, high-affinity binding sites for neurotensin (NT). These receptors mediate formation of cyclic GMP and other second messengers. We studied the effects of extended exposure of cells to [D-Lys8]NT(8-13) (NT2), a peptidase-resistant NT analog, on NT's ability to stimulate cyclic GMP synthesis and to bind [3H]NT to its sites on these cells. When intact N1E-115 cells were preincubated with NT2 (1 microM) for various times (15 min to 12 hr) at 37 degrees C, maximal desensitization and binding site (Bmax) loss occurred after only 15 min. Receptor affinity for [3H]NT did not change. At 0 degrees C NT2 for 15 min caused no down-regulation. After 15 min of preincubation with NT2 at 37 degrees C, the recovery of receptor binding and function after a 10 to 20 min lag-time was rapid (T1/2 = 15 min and 19 min, for binding and cyclic GMP response, respectively). After 12 hr of NT2 preincubation, it was slow (T1/2 = 212 min). Incubation of cells with cycloheximide (70 microM) for 5 hr after their exposure to NT2 for 15 min did not change the rate or extent of recovery of NT Bmax. However, cycloheximide did decrease the recovery of NT Bmax after exposure of cells to NT2 for 12 hr. Cycloheximide alone did not change NT Bmax. These data suggest that this decrease of NT receptor binding after short (15 min) and long (12 hr) exposure times to agonist involves two consecutive steps: intracellular sequestration of recyclable NT receptors, followed by receptor degradation causing true down-regulation.
克隆N1E - 115细胞具有针对神经降压素（NT）的特异性、高亲和力结合位点。这些受体介导环磷酸鸟苷（cGMP）和其他第二信使的形成。我们研究了细胞长时间暴露于[D - Lys8]NT(8 - 13)（NT2，一种抗肽酶的NT类似物）对NT刺激cGMP合成以及将[3H]NT与其在这些细胞上的位点结合能力的影响。当完整的N1E - 115细胞在37℃下用NT2（1微摩尔）预孵育不同时间（15分钟至12小时）时，仅15分钟后就出现了最大脱敏和结合位点（Bmax）丧失。受体对[3H]NT的亲和力没有变化。在0℃下，NT2作用15分钟未引起下调。在37℃下用NT2预孵育15分钟后，经过10至20分钟的延迟时间，受体结合和功能的恢复很快（结合和cGMP反应的半衰期分别为15分钟和19分钟）。在NT2预孵育12小时后，恢复很慢（半衰期为212分钟）。细胞在暴露于NT2 15分钟后用放线菌酮（70微摩尔）孵育5小时，并未改变NT Bmax恢复的速率或程度。然而，在细胞暴露于NT2 12小时后，放线菌酮确实降低了NT Bmax的恢复。单独使用放线菌酮不会改变NT Bmax。这些数据表明，在短时间（15分钟）和长时间（12小时）暴露于激动剂后NT受体结合的减少涉及两个连续步骤：可循环利用的NT受体的细胞内隔离，随后是受体降解导致真正的下调。
