Unger J, Ketelbant P, Dumont J E
Endocrinology. 1985 Mar;116(3):958-65. doi: 10.1210/endo-116-3-958.
The lysosomotropic amines are well-known inhibitors of lysosomal protein degradation. These drugs were used in dog thyroid slices to ascertain the role of the lysosome in thyroglobulin hydrolysis and in hormone secretion. NH4Cl (1-20 mM) and chloroquine (5-500 microM) inhibited, in a concentration-dependent manner, the TSH-stimulated secretion. The high concentrations of these compounds also inhibited the basal secretion. The inhibition was not toxic since 1) the nonbutanol extractable iodine, an index of follicle disruption, was not increased, except slightly by 20 mM NH4Cl; 2) the compounds did not inhibit the TSH-induced stimulation of protein iodination; 3) the inhibition of secretion caused by these compounds was reversible; 4) the ultrastructural changes induced by NH4Cl were reversed after its withdrawal. The inhibition of secretion was presumably related to the lysosomal trapping of the drugs because: 1) the time lag for the fall in secretion rate after drug addition was shorter than for the inhibition of secretion at the level of pseudopod formation by carbamylcholine and cytochalasin B; 2) for NH4Cl this delay and the degree of inhibition were modulated by the [H+] gradient between the medium and the lysosome; 3) 20 min after NH4Cl addition, 92% of the lysosomes were vacuolated and swollen (median section area, 126 mu2 vs. 50 mu2 for the controls) and 8% of the lysosomes were swollen and still dense (median, 206 mu2); 20 min after chloroquine addition, 90% of the lysosomes remained dense and had a significantly higher section area (median, 79 mu2) than the controls (P less than 0.001), whereas 10% of the lysosomes were vacuolated and large (median area, 438 mu2). The number of pseudopods measured by scanning electron microscopy significantly decreased only after 1 h (P less than 0.001). This late decrease could not account for the early block of secretion and suggested a lack of membrane recycling. In conclusion, lysosomotropic amines interfere with a post phagocytotic, presumably lysosomal, step in secretion by dog thyroid. These data constitute the first biochemical evidence in the intact cell of the role of lysosomes in TSH-induced thyroglobulin hydrolysis and thyroid hormone secretion.
溶酶体亲和胺是众所周知的溶酶体蛋白降解抑制剂。这些药物被用于犬甲状腺切片，以确定溶酶体在甲状腺球蛋白水解和激素分泌中的作用。氯化铵（1 - 20 mM）和氯喹（5 - 500 microM）以浓度依赖的方式抑制促甲状腺激素（TSH）刺激的分泌。这些化合物的高浓度也抑制基础分泌。这种抑制不是毒性作用，因为：1）作为卵泡破坏指标的非丁醇可提取碘，除了20 mM氯化铵使其略有增加外，并未升高；2）这些化合物不抑制TSH诱导的蛋白碘化刺激；3）这些化合物引起的分泌抑制是可逆的；4）氯化铵撤除后，其诱导的超微结构变化得以逆转。分泌抑制可能与药物在溶酶体中的捕获有关，因为：1）添加药物后分泌速率下降的时间滞后比卡巴胆碱和细胞松弛素B在伪足形成水平抑制分泌的时间滞后短；2）对于氯化铵，这种延迟和抑制程度受培养基与溶酶体之间的[H⁺]梯度调节；3）添加氯化铵20分钟后，92%的溶酶体空泡化并肿胀（中位截面积，126μm²，而对照组为50μm²），8%的溶酶体肿胀且仍致密（中位值，206μm²）；添加氯喹20分钟后，90%的溶酶体仍致密，且截面积显著高于对照组（中位值，79μm²）（P < 0.001），而10%的溶酶体空泡化且较大（中位面积，438μm²）。通过扫描电子显微镜测量的伪足数量仅在1小时后显著减少（P < 0.001）。这种后期减少不能解释分泌的早期阻断，提示缺乏膜再循环。总之，溶酶体亲和胺干扰犬甲状腺分泌过程中吞噬后、可能是溶酶体的一个步骤。这些数据构成了完整细胞中溶酶体在TSH诱导的甲状腺球蛋白水解和甲状腺激素分泌中作用的首个生化证据。
