Inhibition by lysosomotropic amines of dog thyroid secretion in vitro

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.