Rab proteins are small GTPases that control distinct vesicular transport steps. Along the endocytic pathway, Rab5a is a ratelimiting catalyst of internalization, and Rab7 controls trafficking through late endosomes to lysosomes. The dependence of thyroid hormone production by thyrocytes on thyroglobulin endocytosis and intracellular processing in late endosomes͞lysosomes suggests that its rate can be regulated by the expression or function of these endocytic catalysts. We compared the expression level and membrane recruitment of Rab5a and Rab7 in autonomous thyroid adenomas (where the cAMP cascade is constitutively activated) and surrounding quiescent tissues. The concentrations of Rab5a and Rab7, but not of Rab8, were coordinately increased up to 6-fold in adenomas, and correlated with a proportionate decrease in soluble thyroglobulin content (reflecting colloid depletion by accelerated endocytic uptake in hyperactive tissue). In adenomas, a higher proportion of Rab5a and Rab7 was membrane associated, and the equilibrium density of particulate Rab7 and iodine shifted toward lysosomal fractions, indicating that progression along the degradation pathway also was promoted. In cultures of polarized human thyrocytes from normal patients, thyroid-stimulating hormone or forskolin increased, to a similar extent, Rab5a and Rab7 but not Rab8 expression, apical endocytosis of thyroglobulin and lucifer yellow, and basolateral secretion of T 3 and T4. Taken together, these in vivo and in vitro observations demonstrate that thyroid-stimulating hormone, via cAMP, coordinately enhances the expression of Rab5a and Rab7, which promote Tg endocytosis and transfer to lysosomes, respectively, resulting in accelerated thyroid hormone production.

Nitric oxide (NO) is a well-known mediator of autoimmune processes. In the thyroid gland, it is produced in response to interleukin 1 (IL-1) and may mediate cytokine action at an early stage of autoimmune thyroiditis. In this study, we have investigated whether NO is involved in cytokine-induced cytotoxic effects and epithelial barrier alterations in thyrocytes. Human thyroid epithelial cells were cultured as tight polarised monolayers on a permeable support and exposed or not to IL-1 (100 U/ml), alone or in combination with interferon-(IFN-; 100 U/ml) added to the basal compartment. NO production was not detected in control thyrocytes, but was significantly induced by the combination of IL-1 with IFN-, in a time-dependent fashion. Similarly, expression of the inducible isoform of nitric oxide synthase (NOSII), determined by immunoblot and immunofluorescence confocal microscopy, was not detected in control cells, but was markedly induced after 48-h exposure to both cytokines. This treatment significantly increased the release of cytosolic lactate dehydrogenase (LDH) in the apical and basolateral media and decreased transepithelial electrical resistance. Although IFN-was not sufficient to induce NO production, it could by itself decrease transepithelial resistance and synergised the IL-1 effect on LDH release. The NOS inhibitor, -nitro-argininemethyl ester, suppressed the cytokine-induced NO production and decreased the LDH release, but failed to prevent the loss of transepithelial resistance. These results indicated that human thyrocytes express NOSII and produce NO in response to IL-1 +IFN-and suggest that NO acts as a mediator of cytokine-induced cytotoxicity in the thyroid gland and may promote the exposure of autoantigens to the immune system. In contrast, NO does not appear to mediate the cytokine-induced disruption of the thyroid epithelial barrier.

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We have shown that large lysosomes appear in thyroids of aging male cream hamsters. To investigate the role of this lysosomal change in the age-dependent reduction in hormone secretion, thyroids of young (<4 months of age) and old (>22 months of age) male and female hamsters were labeled with 125I at near isotopic equilibrium. Changes in thyroid morphology were analyzed by light- and electron-microscopic morphometry. Changes in thyroglobulin processing were analyzed by subcellular fractionation and identification of 125I-compounds by sucrose gradients and reverse-phase high-pressure liquid chromatography (HPLC). Sexual dimorphism present in thyroids of young animals became more marked upon aging. The parallel increase in thyroid weight and thyroglobulin content was more conspicuous in old females than in old males. Two morphological observations were specific to old females: (1) large follicles with flat epithelium and evenly labeled colloid and (2) deposits of amyloid material (possibly immunoglobulin light chain-related) between follicles. Although lysosomes were enlarged in female and male aged thyroids, they did not accumulate iodine. However, after isopycnic centrifugation of crude lysosomal fractions in Percoll gradients, 125I in old thyroids was not distributed mainly in the dense fraction L1 (lysosomes) as in young thyroids, but partly in particles of lower density (light L2 and buoyant fractions). 125I in the lighter particles was mostly found in intact thyroglobulin and in large iodopeptides. This 125I shift towards less dense particles was more marked in females than in males. These results indicate that age delays thyroglobulin progression towards dense lysosomes and suggest that the slower traffic of thyroglobulin in the endocytic pathway contributes to the reduction in thyroid hormone secretion in the aged cream hamster.