Higgins L S, Berg D K
Department of Biology, University of California, San Diego, La Jolla 92093.
J Cell Biol. 1988 Sep;107(3):1147-56. doi: 10.1083/jcb.107.3.1147.
Bovine adrenal chromaffin cells have nicotinic acetylcholine receptors (AChRs) that are activated by the splanchnic nerve, resulting in release of catecholamines from the cells. Examination of the AChRs can provide information about the regulation and turnover of synaptic components on neurons and endocrine cells. Previous studies have shown that mAb 35 recognizes the AChR on the cells. Here we show that mAb 35 can remove AChRs from the surface of the cells by antigenic modulation, and that the modulation can be used together with other methods to examine the stability and turnover of the receptors in the plasma membrane. Unexpectedly, the results indicate a disparity between the rate at which AChRs reappear on the cells and the rate at which the ACh response recovers after preexisting AChRs have been removed. Exposure of bovine adrenal chromaffin cultures to mAb 35 results in a parallel decrease in the magnitude of the nicotinic response and the number of AChRs on the cells. The decrease depends on the concentration and divalence of mAb 35, and on the time and temperature of the incubation. The antibody induces receptor aggregation in the plasma membrane under conditions where receptor loss subsequently occurs. After binding to receptor, mAb 35 appears to be internalized, degraded, and released from the cells through a temperature sensitive pathway that requires lysosomal function. These features are characteristic of antigenic modulation. Appearance of new AChRs on the cells either after antigenic modulation or after blockade of existing AChRs with monovalent antibody fragments occurs at a rate equivalent to 3% of the receptors present on control cells per hour. The rate of receptor loss from the cells was measured in the presence of either tunicamycin or puromycin to block appearance of new receptors. Both conditions indicated a receptor half-life of approximately 24 h and a rate of loss of approximately 3%/h. The finding that the rate of receptor loss equaled the rate of receptor appearance was consistent with the observation that the total number of AChRs on untreated cells did not increase with time. In the presence of tunicamycin, loss of receptor-mediated response to nicotine also occurred with a half-time of 24 h. Paradoxically, the rate of recovery of the nicotinic response, determined using two procedures, was more than twice as great as the rate at which new AChRs appeared on the cells.(ABSTRACT TRUNCATED AT 400 WORDS)
牛肾上腺嗜铬细胞具有烟碱型乙酰胆碱受体（AChRs），这些受体可被内脏神经激活，从而导致细胞释放儿茶酚胺。对AChRs的研究可以提供有关神经元和内分泌细胞上突触成分的调节和更新的信息。先前的研究表明，单克隆抗体35能识别细胞上的AChR。在此我们表明，单克隆抗体35可通过抗原调变从细胞表面去除AChRs，并且这种调变可与其他方法一起用于研究质膜中受体的稳定性和更新。出乎意料的是，结果表明AChRs在细胞上重新出现的速率与预先存在的AChRs被去除后ACh反应恢复的速率之间存在差异。将牛肾上腺嗜铬细胞培养物暴露于单克隆抗体35会导致烟碱反应的幅度和细胞上AChRs的数量同时减少。这种减少取决于单克隆抗体35的浓度和二价性，以及孵育的时间和温度。在随后发生受体丢失的条件下，该抗体诱导质膜中的受体聚集。与受体结合后，单克隆抗体35似乎通过需要溶酶体功能的温度敏感途径被内化、降解并从细胞中释放。这些特征是抗原调变的特点。在抗原调变后或用单价抗体片段阻断现有AChRs后，细胞上新AChRs的出现速率相当于对照细胞上每小时存在的受体的3％。在衣霉素或嘌呤霉素存在下测量细胞中受体丢失的速率，以阻止新受体的出现。两种情况均表明受体的半衰期约为24小时，丢失速率约为3％/小时。受体丢失速率等于受体出现速率这一发现与未处理细胞上AChRs总数不随时间增加的观察结果一致。在衣霉素存在下，对尼古丁的受体介导反应的丢失也以24小时的半衰期发生。矛盾的是，使用两种方法确定的烟碱反应恢复速率比细胞上新AChRs出现速率的两倍还多。（摘要截短于400字）
