Immunocytochemical analysis of prolactin production by monolayer cultures of GH3 rat anterior pituitary tumor cells: II. Variation in prolactin content of individual cell colonies, and dynamics of stimulation with thyrotropin-releasing hormone (TRH)

Abstract

The preceeding report (Hoyt and Tashjian, '80) correlates immunocytochemical localizations and mean prolactin concentrations in GH₃ monolayers maximally stimulated with TRH; the present does so over the duration of TRH treatment. Low density seeding produced numerous discrete GH₃ cell colonies. Cultures were harvested ½, 4, 12, 24, 72, and 144 hr after administration of TRH (50 ng/ml) or saline (control). All cells (42,658 total) in at least 10 microscopic fields/monolayer, 1 cell colony/field, were classed as unstained, heavily (H), moderately (M), or weakly (W) stained for prolactin. In controls, colonies contained 51‐91 prolactin‐positive cells/100 of population. Colonies with few positive cells had many more W than M cells, and the reverse was true in those with many positive cells. In all colonies, the effect of TRH was biphasic. Initial (0‐4 hr) release of prolactin was overlapped, beginning at 3‐4 hr, by a progressive increase of intracellular hormone. After 144 hr, the prolactin content of treated cultures had increased to 190% of control, and prolactin‐positive cells were more numerous (114% of control). These increases were lower than those reported in the preceeding paper after 48 hr of TRH treatment, when intracellular prolactin equalled 450% of control and positive cells equalled 129% of control. These inconsistencies reflect differences in the control level of prolactin production rather than in the absolute effects of TRH, which were virtually identical in the successive experiments. We conclude that: (1) TRH acts to alter hormone production in cells already making prolactin; (2) TRH increases somewhat the number of prolactin‐containing cells; (3) the relative contribution of such “new” cells to increased hormone output depends on the basal level of prolactin production, which differs among individual GH₃ cell colonies and varies over time in culture. This diversity does not diminish the usefulness of GH₃ cells as biochemical models of hormone biosynthesis. It does hinder their valid morphological evaluation, which apparently must be controlled as carefully as biochemical experiments and should include immunocytochemical localizations, at least for the hormone at issue.