Oka J A, Weigel P H
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77550.
J Cell Biochem. 1988 Feb;36(2):169-83. doi: 10.1002/jcb.240360208.
Binding, endocytosis, and degradation of asialo-orosomucoid (ASOR) mediated by the galactosyl (Gal) receptor were examined in isolated rat hepatocytes in complete media supplemented with an osmolite. The specific binding of 125I-ASOR to cells at 4 degrees C was unaffected by up to 0.4 M sucrose or NaCl. Unlike sucrose or NaCl, mannitol stimulated 125I-ASOR binding at low concentrations but inhibited binding at higher concentrations. Continuous internalization at 37 degrees C, which requires receptor recycling, was completely blocked at 0.2 M sucrose or 0.15 M NaCl, corresponding in each case to a total osmolality of about 550 mmol/kg. This effect was reversed and endocytic function was restored by washing the cells, indicating that cell viability was unaffected. The rate of degradation of internalized 125I-ASOR was also inhibited by increasing sucrose concentrations. This inhibition is due to a block in the delivery of ligand to lysosomes and not an effect on degradation per se. In the presence of 0.2 M sucrose, the rate and extent of endocytosis of surface-bound 125I-ASOR were, respectively, 33.0 +/- 8.1% and 69.4 +/- 10.5% (n = 8) of the control without sucrose. Under these conditions, the dissociation of internalized receptor-ASOR complexes was completely inhibited. When sucrose was added, the effect on the endocytosis of surface-bound 125I-ASOR was virtually immediate. Previous studies showed that about 40% of the surface-bound 125I-ASOR which is internalized can return to the cell surface still bound to receptor (Weigel and Oka: J Biol Chem 259:1150, 1984). If 0.2 M sucrose was added after endocytosis occurred, 125I-ASOR still returned to the cell surface, although the rate and extent of return were inhibited by more than 50%. Interestingly, hyperosmolarity is the only treatment we have found which can reversibly inhibit, although only partially, the endocytosis of surface-bound 125I-ASOR.
在添加了渗透压调节剂的完全培养基中，对分离的大鼠肝细胞中由半乳糖基（Gal）受体介导的去唾液酸血清类黏蛋白（ASOR）的结合、内吞作用及降解进行了研究。4℃时，125I-ASOR与细胞的特异性结合不受高达0.4M蔗糖或NaCl的影响。与蔗糖或NaCl不同，甘露醇在低浓度时刺激125I-ASOR的结合，但在高浓度时抑制结合。37℃下需要受体循环的持续内吞作用在0.2M蔗糖或0.15M NaCl时完全被阻断，在每种情况下对应的总渗透压约为550mmol/kg。通过洗涤细胞可逆转这种效应并恢复内吞功能，表明细胞活力未受影响。内化的125I-ASOR的降解速率也因蔗糖浓度增加而受到抑制。这种抑制是由于配体向溶酶体的递送受阻，而非对降解本身的影响。在0.2M蔗糖存在下，表面结合的125I-ASOR的内吞速率和程度分别为无蔗糖对照的33.0±8.1%和69.4±10.5%（n = 8）。在这些条件下，内化的受体-ASOR复合物的解离被完全抑制。添加蔗糖时，对表面结合的125I-ASOR内吞作用的影响几乎是即时的。先前的研究表明，内化的表面结合的125I-ASOR中约40%仍可与受体结合返回细胞表面（Weigel和Oka：《生物化学杂志》259:1150，1984）。如果在内吞作用发生后添加0.2M蔗糖，125I-ASOR仍会返回细胞表面，尽管返回的速率和程度受到超过50%的抑制。有趣的是，高渗是我们发现的唯一一种虽只能部分但可可逆抑制表面结合的125I-ASOR内吞作用的处理方法。
