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Basal and thyroliberin-stimulated prolactin synthesis in single-cell cultures and in populations of rat pituitary cells.

ABSTRACT: 1. Newly synthesized prolactin was obtained from cultures of rat pituitary tumour cells (GH4C1 cells) after incubation with [35S]methionine. 2. Radioactive synthesized and secreted prolactin was quantified by an immunoprecipitation method by using disc-gel electrophoresis of the dissolved immunoprecipitate in the presence of sodium dodecyl sulphate. By using a microanalytical modification, hormone synthesis and secretion could also be studied in single-cell cultures. This technique was combined with a cytoimmunofluorescence method in which rhodamine-conjugated antibodies were used for studying intracellular prolactin. 3. The presence of radioactive synthesized and secreted prolactin was demonstrated in nine out of 13 single-cell cultures. Cell cultures containing 10 cells or more and clonal populations originating from one cell always secreted radioactive prolactin. 4. Thyroliberin treatment (2 muM) for 24h increased the extracellular accumulation of radioactive prolactin in five out of seven single-cell cultures and always in populations of cells. 5. The number of cells showing prolactin specific fluorescence increased from 20 to 50% and the intensity of this fluorescence became greater after thyroliberin treatment. 6. Studies of [35S]prolactin secretion from single cells and immunochemical detection of intracellular prolactin showed that some cells in an unsynchronized population did not secret radioactive prolactin or show prolactin specific fluorescence. 7. The quantitative effect of thyroliberin as studied in single-cell cultures suggested that the main if not the only effect was to increase prolactin synthesis in cells already producing hormone.

SUBMITTER: Gautvik KM

PROVIDER: S-EPMC1163944 | biostudies-other | 1976 Jul

REPOSITORIES: biostudies-other

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Project description:1. The effects of prostaglandins E2 and F2alpha on prolactin synthesis were examined in a clonal strain of rat pituitary tumour cells, and compared with those of thyroliberin. 2. The prostaglandins and thyroliberin gave a dose-related and time-dependent stimulation of prolactin synthesis. The maximal effects (about twofold increases) were observed after 54h of treatment with 25nM-prostaglandin E2 and 2.5nM-prostaglandin F2alpha. A similar stimulation of prolactin synthesis was observed after 250nM-thyroliberin. The combined treatment with prostaglandins and thyroliberin did not increase prolactin synthesis over and above that obtained with each compound alone. 3. After removal of prostaglandins E2 and F2alpha there was a complete reversal of prolactin synthesis to pre-stimulation values 18h later (t1/2less than or equal to 9h). The rapid reversible effect of prostaglandins was in contrast with that of thyroliberin, where prolactin synthesis returned to control values with a t1/2 of about 42 h. 4. Prostaglandin E2 (5mum) and thyroliberin (5mum) increased cellular concentrations of cyclic AMP eight- and four-fold respectively. Maximal effects were observed after 2-5min of incubation. The increases in cyclic AMP were biphasic; normal values were obtained 60 min after the start of incubation with prostaglandin E2 or thyroliberin. 5. The dose/response curve showed that prostaglandin E2 caused maximal increase of cyclic AMP at 50nM. Concentrations of prostagland in E2 that caused half-maximal stimulation of cyclic AMP accumulation and of prolactin synthesis were 4 and 5nM respectively. 6. Combined treatment with prostaglandin E2 and thyroliberin in concentrations that separately caused maximal cyclic AMP increases did not result in a further increase in this cyclic nucleotide. 7. These results are consistent with a role of cyclic AMP in mediating the effects or prostaglandins and thyroliberin on prolactin synthesis. However, if cyclic AMP is involved as a common intracellular mediator of prolactin synthesis, it cannot alone explain all the effects of prostaglandins and thyroliberin in this cell system.

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Basal and thyroliberin-stimulated prolactin synthesis in single-cell cultures and in populations of rat pituitary cells.

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