Pandey K N
Department of Biochemistry and Molecular Biology, Medical College of Georgia, School of Medicine, Augusta 30912-2100.
Biochem J. 1992 Nov 15;288 ( Pt 1)(Pt 1):55-61. doi: 10.1042/bj2880055.
The kinetics of internalization, sequestration and metabolic degradation of atrial natriuretic factor (ANF)-receptor complex were studied in rat thoracic aortic smooth-muscle (RTASM) cells. These parameters were directly determined by measuring 125I-ANF binding to total, intracellular and cell-surface receptors. Pretreatment of cells with the lysosomotropic agent chloroquine and the energy depleter dinitrophenol led to an increase in the intracellular 125I-ANF radioactivity. After 60 min incubation at 37 degrees C, cell-associated 125I-ANF radioactivity fell rapidly in chloroquine-treated cells (> 85%) compared with the controls (< 45%). 125I-ANF radioactivity increased to a peak of 65% of the initial level within 15 min in chloroquine-treated cells compared with only 22% in the control cells. During the initial incubation period at 37 degrees C, chloroquine inhibited the release of both intact and degraded 125I-ANF in a time-dependent manner. However, at later incubation times, the effect of chloroquine was diminished and release of both degraded and intact ligand was resumed. Extracellular unlabelled ANF did not affect the release of degraded 125I-ANF but it accelerated the release of intact ANF by a retroendocytotic mechanism. After the endocytosis, about 30-40% of ANF receptors were restored to the cell surface from the internalized pool of receptors. The restoration was blocked by chloroquine or dinitrophenol but not by cycloheximide. Exposure of RTASM cells to unlabelled ANF resulted in a time- and concentration-dependent loss of ANF receptors. Unlabelled ANF (10 nM) induced a loss of more than 52% of 125I-ANF binding, and a complete loss occurred at micromolar concentrations. It is inferred that ANF-induced down-regulation of its receptor resulted primarily from an increased rate in internalization and metabolic degradation of ligand-receptor complex by receptor-mediated endocytotic mechanisms.
在大鼠胸主动脉平滑肌（RTASM）细胞中研究了心房利钠因子（ANF）-受体复合物的内化、隔离和代谢降解动力学。这些参数通过测量¹²⁵I-ANF与总受体、细胞内受体和细胞表面受体的结合直接确定。用溶酶体促渗剂氯喹和能量消耗剂二硝基苯酚预处理细胞导致细胞内¹²⁵I-ANF放射性增加。在37℃孵育60分钟后，与对照细胞（<45%）相比，氯喹处理的细胞中与细胞相关的¹²⁵I-ANF放射性迅速下降（>85%）。与对照细胞中仅22%相比，氯喹处理的细胞中¹²⁵I-ANF放射性在15分钟内增加到初始水平的65%峰值。在37℃的初始孵育期内，氯喹以时间依赖性方式抑制完整和降解的¹²⁵I-ANF的释放。然而，在后期孵育时间，氯喹的作用减弱，降解和完整配体的释放恢复。细胞外未标记的ANF不影响降解的¹²⁵I-ANF的释放，但通过逆向胞吞机制加速完整ANF的释放。内吞作用后，约30-40%的ANF受体从内化的受体池中恢复到细胞表面。这种恢复被氯喹或二硝基苯酚阻断，但未被环己酰亚胺阻断。将RTASM细胞暴露于未标记的ANF导致ANF受体随时间和浓度依赖性丢失。未标记的ANF（10 nM）导致超过52%的¹²⁵I-ANF结合丢失，在微摩尔浓度下完全丢失。据推测，ANF诱导的其受体下调主要是由于受体介导的胞吞机制导致配体-受体复合物内化和代谢降解速率增加。
