Production and Release of Thyrotropin and its Subunits by Monolayer Cultures Containing Bovine Anterior Pituitary Cells*

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Abstract
We have developed a dispersed cell monolayer system derived from bovine anterior pituitary glands. Fresh 1-to 6-week-old calf anterior pituitaries were mechanically and enzymatically dispersed and incubated with Dulbecco's Modified Minimal Essential Medium containing 10% hypothyroid goat serum. The media and cell extracts from confluent monolayers were analyzed for bovine TSH and free α and TSHβsubunits by specific homologous RIAs. Basal levels of TSH, free α and free TSHβsubunits in the media were 6.2±0.3, 8.0±0.3, and 0.95±0.05 ng/106 cells-24 h, respectively. Hence, an 8- to 10-fold excess of free α over free TSHβsubunits was released into the medium. Intracellular basal levels of TSH, free α and free TSHβsubunits were 27.6±1.7, 10.7±0.2, and 2.6 ±0.3 ng/106 cells. 24 h, respectively, and indicated a 3- to 4-fold excess of free α over free TSHβsubunits within the cells. The total α-subunit to totalβ-subunit ratio was 2:1. TRH stimulated release of TSH and its subunits in a dose-dependent fashion, with a half-maximal dose of 2 nM and a maximal response dose of 10 nM. Stimulation with 100 nM TRH increased the levels of TSH, free α and free TSHβsubunits (450-900%, 180-200%, and 300-400%, respectively) in medium, with concomitant decreases within cells. Treatment with thyroid hormones decreased basal and blunted TRH-stimulated levels of TSH and its subunits in medium but had no effect on intracellular stores. However, large doses of T4 (25 nM) or T3 (1 nM) did not completely abolish the TRH (100 nM)-stimulated hormone response. TRH and thyroid hormones affect the release of TSH and TSHβto a greater extent than they do the α-subunit. Finally, total α and TSHβ subunit production was increased with TRH stimulation and decreased with thyroid hormone exposure. Thus, an in vitro system to study the net production and secretion f TSH and its subunits in the normal pituitary thyrotrope has been established.

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