Relationship between agonist-induced muscarinic receptor loss and desensitization of stimulated phosphoinositide turnover in two neuroblastomas: methodological considerations.
Relationship between agonist-induced muscarinic receptor loss and desensitization of stimulated phosphoinositide turnover in two neuroblastomas: methodological considerations.
复制标题
两种神经母细胞瘤中激动剂诱导的毒蕈碱受体丧失与刺激的磷酸肌醇周转脱敏之间的关系：方法学考虑。
DOI：
--
复制
发表时间：
1990
影响因子：
3.5
通讯作者：
S. Fisher
中科院分区：
文献类型：
--
作者：
A. K. Thompson;S. Fisher
在人SK-N-SH神经母细胞瘤细胞中加入全部和部分M受体激动剂后，持续的磷脂酰肌醇(PPI)水解率可达2小时，通过在Li+存在的情况下[~3H]肌醇磷酸盐(IP)的累积，或通过从~(32)PI增加磷脂酸盐和磷脂酰肌醇(PI)的标记来监测。即使40-50%的细胞表面M胆碱能受体(MAChRs)被隔离，这种增强的PPI水解率仍保持不变。只有在细胞暴露于激动剂2-4小时后，才能检测到卡巴胆碱刺激的PPI水解物的脱敏，并伴随着类似的mAChR总数的减少。SK-N-SH细胞与部分激动剂Bethan ecol的长时间孵育也可导致刺激的PPI翻转的脱敏，但速度慢于卡巴胆碱，并丢失更少的mAChR位点。在小鼠NIE-115神经母细胞瘤中，mAChR刺激的[~3H]IP形成的脱敏作用似乎发生得更快。然而，Li+不能完全阻止这些细胞中累积的[3 H]IP的降解。当增加[~(32)P]磷脂酰肌醇标记来评估PPI在NIE-115细胞中的周转时，在长达2小时的时间内没有明显的脱敏现象。我们得出的结论是：1)在SK-N-SH细胞中，脱敏比mAChRs的滞留更类似于下调，2)在两个神经母细胞瘤中，刺激的PPI水解脱敏相对较慢，3)脱敏的外观随检测方法的不同而不同。
The addition of both full and partial muscarinic agonists to human SK-N-SH neuroblastoma cells resulted in a sustained phosphoinositide (PPI) hydrolysis for up to 2 hr, as monitored either by the accumulation of [3H]inositol phosphates (IP) in the presence of Li+, or alternatively, by an increased labeling of phosphatidate and phosphatidylinositol (PI) from 32Pi. This enhanced PPI hydrolysis was maintained even though 40-50% of cell-surface muscarinic cholinergic receptors (mAChRs) became sequestered. Desensitization of carbachol-stimulated PPI hydrolysis was only detected after a 2-4 hr prior exposure of the cells to the agonist and was accompanied by a comparable reduction in total mAChR number. Prolonged incubation of SK-N-SH cells with the partial agonist bethanechol also resulted in a desensitization of stimulated PPI turnover but at a slower rate than that observed for carbachol and with a loss of fewer mAChR sites. Desensitization of mAChR-stimulated [3H]IP formation appeared to occur more rapidly in mouse NIE-115 neuroblastoma. However, Li+ could not fully prevent the degradation of accumulated [3H]IP in these cells. When an increase in [32P]phosphatidylinositol labeling was used to assess PPI turnover in NIE-115 cells, no desensitization was evident for up to 2 hr. We conclude that 1) in SK-N-SH cells, desensitization more closely parallels the down-regulation than the sequestration of mAChRs, 2) in both neuroblastomas, stimulated PPI hydrolysis desensitizes relatively slowly and 3) the appearance of desensitization can vary as a function of the assay method employed.