Thompson A K, Fisher S K
Neuroscience Laboratory, University of Michigan, Ann Arbor 48104.
J Biol Chem. 1991 Mar 15;266(8):5004-10.
The ability of muscarinic receptors, present in either the cell surface or sequestered compartments of intact human SK-N-SH neuroblastoma cells, to stimulate phosphoinositide hydrolysis has been examined. When cells were first exposed to carbachol for 1 h at 37 degrees C, approximately 50% of the cell surface receptors became sequestered, and this was accompanied by a comparable reduction in the subsequent ability of muscarinic agonists to stimulate phosphoinositide turnover, as monitored by the release of labeled inositol phosphates at 10 degrees C. At this temperature, muscarinic receptor cycling between the two cell compartments is prevented. Upon warming the carbachol-pretreated cells to 37 degrees C, receptor cycling is reinitiated and stimulated phosphoinositide turnover is fully restored within 5-8 min. When measured at 10 degrees C, the reduction of stimulated phosphoinositide turnover observed following carbachol pretreatment was similar in magnitude for both hydrophilic (carbachol, oxotremorine-M) and lipophilic (arecoline, oxotremorine-2, and L-670,548) agonists. The loss of response for both groups of agonists could be prevented if the incubation temperature was maintained at 37 degrees C, rather than at 10 degrees C. At the latter temperature carbachol pretreatment of SK-N-SH cells reduced the maximum release of inositol phosphates elicited by either carbachol or L-670,548 but not the agonist concentrations required for half-maximal stimulation. Radioligand binding studies, carried out at 10 degrees C, indicate that following receptor sequestration, significantly higher concentrations of carbachol were required to occupy the available muscarinic receptor sites. In contrast the lipophilic full agonist L-670,548 recognized receptors present in control and carbachol-pretreated cells with comparable affinities. Analysis of the inositol lipids present after carbachol pretreatment indicate that only a minimal depletion of the substrates necessary for phospholipase C activation had occurred. The results indicate that the agonist-induced sequestration of muscarinic receptors from the cell surface results in a loss of stimulated phosphoinositide hydrolysis when measured under conditions in which the return of the sequestered receptors to the cell surface is prevented. Thus, only those receptors present at the cell surface are linked to phospholipase C activation.
已对完整的人SK-N-SH神经母细胞瘤细胞的细胞表面或隔离区室中存在的毒蕈碱受体刺激磷酸肌醇水解的能力进行了研究。当细胞首先在37℃下暴露于卡巴胆碱1小时时，约50%的细胞表面受体被隔离，同时毒蕈碱激动剂刺激磷酸肌醇周转的后续能力也出现类似程度的降低，这通过在10℃下标记的肌醇磷酸的释放来监测。在此温度下，毒蕈碱受体在两个细胞区室之间的循环被阻止。将经卡巴胆碱预处理的细胞升温至37℃时，受体循环重新启动，刺激的磷酸肌醇周转在5-8分钟内完全恢复。在10℃下测量时，卡巴胆碱预处理后观察到的刺激的磷酸肌醇周转的降低对于亲水性（卡巴胆碱、氧震颤素-M）和亲脂性（槟榔碱、氧震颤素-2和L-670,548）激动剂在程度上相似。如果孵育温度维持在37℃而不是10℃，两组激动剂的反应丧失均可被阻止。在后者温度下，SK-N-SH细胞的卡巴胆碱预处理降低了由卡巴胆碱或L-670,548引起的肌醇磷酸的最大释放，但未降低半数最大刺激所需的激动剂浓度。在10℃下进行的放射性配体结合研究表明，受体隔离后，需要显著更高浓度的卡巴胆碱来占据可用的毒蕈碱受体位点。相比之下，亲脂性完全激动剂L-670,548以相当的亲和力识别对照细胞和经卡巴胆碱预处理的细胞中存在的受体。对卡巴胆碱预处理后存在的肌醇脂质的分析表明，磷脂酶C激活所需底物仅发生了最小程度的消耗。结果表明，在阻止隔离的受体返回细胞表面的条件下测量时，激动剂诱导的毒蕈碱受体从细胞表面隔离导致刺激的磷酸肌醇水解丧失。因此，只有存在于细胞表面的那些受体与磷脂酶C激活相关联。
