Physiological role of thiol proteases in thyroid hormone secretion

Abstract

To determine the physiological role of the thiol proteases in T4 and T3 release from thyroglobulin, experiments were performed with 131I-prelabelled rat thyroid lobes incubated in vitro in the presence and absence of leupeptin, an inhibitor of thiol proteases. Basal secretion of [131I]T4 and [131I]T3 from rat thyroid lobes prelabelled in vivo was quite low, but in the presence of 10 mU/ml bovine TSH a marked stimulatory effect was observed. The stimulatory effect of TSH was completely abolished by leupeptin. This was associated with marked inhibition of lysosomal proteolytic activity, suggesting that the inhibitory effect of leupeptin on T4 and T3 secretion could be attributed to its inhibitory action on proteolysis of thyroglobulin. Further evidence for an inhibitory effect of leupeptin on intralysosomal hydrolysis of thyroglobulin was obtained when thyroid lobes were incubated with 131I-in the presence and absence of leupeptin and TSH. The crude lysosomal preparation was fractionated on a Percoll density gradient, which separates 131I-containing particles into a dense peak containing purified lysosomes and a buoyant peak containing pinocytotic vesicles. A marked increase in the 131I-content of the dense peak was observed in the presence of TSH + leupeptin. Analysis of the 131I in the dense fraction by sucrose density gradient centrifugation and by SDS-polyacrylamide gel electrophoresis demonstrated that leupeptin inhibited degradation of 19S thyroglobulin, especially the formation of [131I]peptides of MW < 14K.Ina previous communication from this laboratory we described a procedure for the purification of thyroid lysosomes . We later reported ) that lysosomal digestion of thyroglobulin was markedly inhi¬ bited by pepstatin, an inhibitor of cathespin D, and by leupeptin, an inhibitor of thiol proteases. These results led us to suggest that both cathepsin D and thiol proteases play a role in lysosomal digestion of thyroglobulin (Tg).The present study was undertaken to provide evidence for a physiological role for thiol pro¬ teases in T4 and T3 secretion from the thyroid gland. For this purpose we employed a rat thyroid lobe incubation system, which previously had been shown (Okamura et al. 1979) to display good ability to convert added 131I" to [131I]T4. Leupep¬ tin is known to inhibit protein breakdown in intact cells (Kominami et al. 1980;Furuno et al. 1982), and it semed likely, therefore, that it would prove useful in studies involving the thyroid lobe system. Pepstatin, on the other hand, does not readily cross cell membranes (Dean 1975), and its inhibit¬ ory action was not tested in the present study. Materials and Methods AnimalsSprague-Dawley male rats weighing 175 -250 g were used in all experiments. They were fed the laboratory stock diet.

Acute and chronic leptin effect upon in vivo and in vitro rat thyroid iodide uptake

“…Glands were sliced and incubated in a minimum essential medium (MEM-SIGMA, MO) enriched with 5 mUI/ml of TSH, 0.1 μM of KI, 2 mM of glutamine and 25 mM of NaHCO3, at 37°C, 95% O 2 /5% CO 2 atmosphere in a Dubnoff metabolic shaker (100 cycles/min), as previous reported (Moura et al, 1990). The concentration of TSH and KI, as well as, the 125 I amount were established based on dose-response curve (Okamura et al, 1979, Yoshinari andTaurog, 1986). 125 I (3,700 Bq) was added and after 15, 30, 60, 90 and 120 min, thyroid 125 I content was counted (Cobra Autogamma, Packard Instrument Co., Downers Grove, IL, USA).…”