Turner J T, James-Kracke M R, Camden J M
Department of Pharmacology, School of Medicine, University of Missouri-Columbia.
J Pharmacol Exp Ther. 1990 Jun;253(3):1049-56.
Regulation of the neurotensin receptor-inositol phosphate-intracellular Ca2+ ((Ca2+]i) pathway was studied in HT29 cells. Preincubation with neurotensin or phorbol 12-myristate, 13-acetate decreased the number of cell surface neurotensin receptors and neurotensin-induced increases of inositol trisphosphates and [Ca2+]i. The phorbol 12-myristate, 13-acetate-(43 +/- 1% at 1 microM) but not the neurotensin (65 +/- 9% at 10 nM)-induced decrease in receptors was blocked by staurosporine. The decrease in cell surface receptors was accompanied by a 55 +/- 7% shift of specifically bound 125I-neurotensin from the plasma membrane fraction to the light vesicle fraction of sucrose density gradients if a 37 degrees C incubation step was included. The time course for desensitization of [Ca2+]i mobilization was more rapid (maximal at approximately 1 min) than for loss of receptors (maximal at 45 min). After a 5-min exposure to neurotensin, the cell surface receptor number rapidly returned to control levels in the absence of agonist, but [Ca2+]i sensitivity to neurotensin recovered only partially. Incubation with carbachol, ATP or phorbol 12-myristate, 13-acetate desensitized the subsequent [Ca2+]i response to neurotensin. These results demonstrate a polyphosphoinositide-[Ca2+]i pathway in HT29 cells stimulable by neurotensin and other agents. The results from pre-incubation studies indicate that the neurotensin receptor-signaling pathway is homologously and heterologously regulated. Finally, differences in time courses for loss and recovery of cell surface receptors and desensitization of the [Ca2+]i response, as well as the lack of effect on 125I-neurotensin binding of other agonists that desensitize the neurotensin [Ca2+]i response, suggest that receptor internalization alone does not account for desensitization of the system.
在HT29细胞中研究了神经降压素受体 - 肌醇磷酸 - 细胞内Ca2+（[Ca2+]i）信号通路的调节。用神经降压素或佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯预孵育可减少细胞表面神经降压素受体的数量以及神经降压素诱导的肌醇三磷酸和[Ca2+]i的增加。佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（1μM时为43±1%）而非神经降压素（10nM时为65±9%）诱导的受体减少可被星形孢菌素阻断。如果包含37℃孵育步骤，细胞表面受体的减少伴随着特异性结合的125I - 神经降压素从质膜部分向蔗糖密度梯度的轻囊泡部分的55±7%的转移。[Ca2+]i动员脱敏的时间进程比受体丢失的时间进程更快（约1分钟时达到最大值）（45分钟时达到最大值）。在暴露于神经降压素5分钟后，在无激动剂的情况下，细胞表面受体数量迅速恢复到对照水平，但[Ca2+]i对神经降压素的敏感性仅部分恢复。用卡巴胆碱、ATP或佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯孵育可使随后[Ca2+]i对神经降压素的反应脱敏。这些结果证明了HT29细胞中存在可被神经降压素和其他试剂刺激的多磷酸肌醇 - [Ca2+]i信号通路。预孵育研究的结果表明神经降压素受体信号通路受到同源和异源调节。最后，细胞表面受体丢失和恢复的时间进程以及[Ca2+]i反应脱敏的时间进程的差异，以及其他使神经降压素[Ca2+]i反应脱敏的激动剂对125I - 神经降压素结合无影响，表明仅受体内化不能解释该系统的脱敏。
