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Monolayer cultures of human thyroid cells derived from thyroid adenoma were utilized for the assay of thyroid stimulating substances such as thyrotropin (TSH), cholera toxin and thyroid stimulating immunoglobulin (TSI) in patients with Graves' disease. Adenoma cells were treated with 0.1% collagenase or 2000 unit/ml dispase to thyrocytes. The cells were cultured in MEM containing 10% fetal calf serum under an atmosphere of 5% CO2 in air. Within 24 hours, the cells attached themselves to the plastic surface and formed a monolayer. Cyclic AMP responses to TSH, cholera toxin or Graves' IgG were tested in a medium (PBS) containing 0.5 mM IBMX. The cyclic AMP responses to TSH were generally maximal on the 3rd day of culture and declined thereafter. The response was dose-dependent, and 10 microU/ml of TSH produced a significant increase of cellular cyclic AMP. The response by 1 microU/ml of TSH was 28 approximately 57 fold above the basal. The response was also a function of the incubation period. The maximal response was attained after 1 h incubation. When the cultures were washed after exposure to TSH, the cellular cyclic AMP levels rapidly declined, suggesting that removal of receptor-bound TSH results in a prompt cessation of cyclic AMP production. The thyroid cells in monolayer also responded to cholera toxin. The response was dose-dependent, and cholera toxin as low as 1 ng/ml was able to increase cyclic AMP production. In contrast to the observations in TSH, the cyclic AMP responses induced by cholera were hardly affected by washing the cultures after exposure to cholera toxin. Treatment of the cells with cholera toxin for only 3 min resulted in a continuous stimulation of cyclic AMP production for more than 4 hours. Confirming recent observations by others, most of Graves' IgG stimulated cyclic AMP production in a dose-dependent manner, but some of them inhibited the response at high concentrations. IgG derived from normal subjects did not increase cellular cyclic AMP. The time course in the cyclic AMP responses induced by Graves' IgG was variable among the IgG preparations from different patients. In some patients, the maximal responses were attained after 4 hours of incubation. A significant difference was noted between TSH and Graves' IgG in the stimulation of cyclic AMP production after washing the cultures. When the cultures were treated with Graves' IgG for 30 min, washed and then incubated without Graves' IgG, cellular cyclic AMP levels remained at the levels which were almost equivalent to those observed in the continuous presence of the IgGs.(ABSTRACT TRUNCATED AT 400 WORDS)

The Effects of TSH, Cholera Toxin and Graves' IgG on cAMP Production in Cultured Human Thyroid Adenoma Cells in Monolayer