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Numerous studies have shown that prolactin (PRL) production by GH3 cells grown in serum supplemented media is regulated by several hormones including thyroliberin (TRH). The recent availability of hormonally defined, serum-free media for the growth of GH3 cells has made it possible to determine the effect of TRH in absence of other prolactin regulating hormones. Here we demonstrate that transfer of GH3/B6 cells from serum-supplemented medium to serum-free media results in several important changes: (1) altered growth response to TRH, (2) altered cell attachment and morphology, (3) greatly reduced prolactin production, and (4) greater stimulation of prolactin production by TRH. After 4 days in serum-free medium, TRH stimulates prolactin production by as much as 5-fold instead of approximately 2-fold in serum-supplemented medium. Furthermore, this increased responsiveness to TRH in serum-free medium is accompanied by a 10-fold decrease in the ED50 for TRH (concentration needed for half-maximal response) and paradoxically by a 2-fold reduction in the number of high-affinity TRH binding sites without significant change of their association constant.

The in vivo internalization of thyrotropin-releasing hormone (TRH) was studied by using a semiquantitative immunoelectron microscopic method. Pituitary glands of normal male rats or of rats intravenously injected with 100 ng TRH and sacrificed after 5–60 min were used. Ultrathin sections were obtained by cryoultramicrotomy of fixed pituitary glands. Pituitary cellular types were identified by appropriate antiserums. An antiserum specifically directed against TRH was used. TRH-like immunoreactivity due to endogenous TRH was observed in thyrotropes and prolactin cells, but never in somatotropes, gonadotropes or corticotropes. At the subcellular level, the reaction was detected within the cytoplasmic matrix, the secretory granules, and the nucleus but only occasionally at the plasma membrane. After in vivo injection of TRH, the immunocytochemical reaction (1) was still restricted to thyrotropes and prolactin cells, (2) increased with time elapsed after injection up to 15–30 min and then returned to basal intensity in cytoplasm, secretory granules, and nucleus, and (3) became very frequent at the plasma membrane. These data (1) provide evidence for endogenous TRH within thyrotropes and prolactin cells, i.e., in physiological target cells for TRH, and (2) support the hypothesis that normal TRH target cells can, in vivo, internalize exogenous as well as endogenous TRH into several subcellular compartments including the nucleus.