Influence of Cell Density on Desensitization of the Thyroid Cell Cyclic Adenosine 3′5′-Monophosphate Response to Thyrotropin Stimulation*

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Abstract
Stimulation of cultured thyroid cells with TSH produces an acute increase in cAMP content (15–30 min maximum) followed by a decrease over 24 h representing refractoriness to further TSH stimulation. With continued exposure of thyroid cell subcultures to TSH for greater than 4 days, escape from desensitization occurred in that cAMP levels returned to the initial maximum (15–30 min) values. This increase in cAMP levels was unrelated to an increase in cell number. In addition, subculture of thyroid cells for between 1 and 6 days in the absence of TSH followed by acute (30 min) TSH stimulation revealed a progressive decrease in the maximum cAMP levels attained (50% of day 1 values after 4 and 6 days of subculture). The cAMP response to a 24-h period of TSH stimulation was examined in groups of cells after different durations of subculture without TSH. With TSH stimulation on the first day of subculture, as expected, cAMP levels increased markedly after 30 min, declined by 6 h, and remained suppressed for up to 24 h. On the fourth day of subculture, TSH induced the initial rise in cAMP levels and subsequent decline between 30 min and 6 h. However, after 24 h cAMP rebounded to levels higher than those observed after 30 min. Exposure of thyroid cells to culture media used to grow fibroblasts and thyroid cells for up to 6 days did not result in escape from TSH desensitization. In addition, the same phenomenon of escape from TSH desensitization was seen whether or not the medium was changed daily, or not changed at all over a 5-day period. Equal numbers of thyroid cells (5× 104, 10r>, or 4 × 105) were seeded into culture dishes of different surface areas. Only at high cell density (>5 × 104 cells/cm2) was escape from TSH desensitization observed. Subculture for 5 days of a fibroblast-free human thyroid adenoma cell line, whose cells were no longer capable of division, did not result in escape from TSH desensitization. These data indicate that at a critical cell density, corresponding to confluency, desensitization to TSH occurs but is then lost despite continuing TSH stimulation. This escape from TSH desensitization cannot be attributed to contaminating fibroblasts or to the depletion or accumulation of a medium factor(s). The mechanism of desensitization of the cAMP response to TSH stimulation in cultured human thyroid cells is therefore linked to cell density, or contact inhibition.

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