Monolayer Cultures Of Pituitary Cells Research Articles

Relationship between Estradiol, Ergocryptine, and Thyroid Hormone: Effects on Prolactin Synthesis and Prolactin Messenger Ribonucleic Acid Levels*

The effects of combinations of estradiol, ergocryptine, and T3 on PRL synthesis and PRL mRNA levels in monolayer cultures of pituitary cells have been investigated. Incubation of pituitary cells with [35S]methionine, followed by analysis of the newly synthesized proteins on a polyacrylamide gel, suggested that estradiol blocked the ability of ergocryptine to inhibit PRL synthesis, but that estradiol did not affect the ability of T3 to inhibit PRL synthesis. Quantitative studies utilizing immunoprecipitation of newly synthesized PRL demonstrated that PRL synthesis was reduced to 52% of control in ergocryptine-treated cells and returned to 83% of control in cells treated with ergocryptine plus estradiol. In the same experiment, T3 treatment reduced PRL synthesis to 50% of control, and PRL synthesis remained at 50% of control values after treatment with T3 plus estradiol. A concentration of 0.1 nM T3, which had little effect by itself, augmented the ability of ergocryptine to inhibit PRL synthesis, even in t...

STIMULATION OF RELEASE OF LUTEINIZING HORMONE FROM CULTURED PITUITARY CELLS BY 2- AND 4-HYDROXYLATED OESTROGENS

We have examined the effect of the catechol oestrogens 2-hydroxyoestradiol (2-OHE2), 4-hydroxyoestradiol (4-OHE2) and 2-hydroxyoestrone (2-OHE1) and their corresponding primary oestrogens on secretion of LH and FSH by enzymatically dispersed rat anterior pituitary cells in monolayer culture. Basal LH levels in the medium were significantly higher than in control wells when cells were exposed to 10(-8) M-oestradiol-17 beta for 40 h: oestrone and all three catechol oestrogens (in the same doses) also stimulated basal LH concentrations to levels quantitatively similar to those seem after oestradiol treatment. The same effects were observed when steroids were given at 10(-9) mol/l. Oestradiol, 2-OHE2, and 4-OHE2 but not 2-OHE1 increased pituitary responsiveness to LH releasing hormone (LH-RH) (given in a range of doses from 10(-11) to 10(-6) mol/l). The responses of cells treated with 2-OHE2 and 4-OHE2 were similar, though less than the response seen after treatment with oestradiol. This contrasts with the very different oestrogenic effects of 2- and 4-OHE2 previously observed in vivo. Neither oestradiol nor the catechol oestrogens had any effect on basal or LH-RH-stimulated FSH release.

Thyrotoxicosis and a Thyrotropin-Secreting Pituitary Tumor Causing Unilateral Exophthalmos

Hyperthyroidism due to a TSH-secreting pituitary tumor has been noted by a number of investigators. We describe a unique case in which a 17-yr-old female presented with clinical hyperthyroidism, a goiter, and unilateral exophthalmos. Serum T4, free T4, and T3 (RIA) were consistently elevated along with elevated TSH levels (range, 10-100 microunits/ml). Skull x-rays and computed tomography scan revealed a tumor invading the right orbit. Other pituitary function studies were normal and LATs was undetectable. Surgery performed resulted in 70% removal of the pituitary tumor and confirmed the presence of tumor infiltration into the right orbit. TRH tests done pre- and postoperatively (patient still clinically hyperthyroid with elevated T4 and TSH levels) showed TSH and PRL responsiveness. Electron microscopy of the tumor demonstrated features typical of pituitary thyrotrophs. Monolayer cultures of pituitary cells released TSH over time into the media but did not respond to TRH stimulation. Pituitary adenoma tissue content of immunoreactive TSH was 65 microunits/g wet tissue and demonstrated immunosimilarity with human standard. We conclude that the patient had a TSH-secreting pituitary tumor responsive to TRH stimulation.

Effects of colchicine on the morphology and prolactin secretion of rat anterior pituitary cells in monolayer culture.

The effects of incubation of rat anterior pituitary cells in monolayer culture with 10(-6) M colchicine have been investigated during time-intervals extending from 1 to 96 hours. Prolactin release, as measured by radioimmunoassay, was rapidly inhibited by colchicine, this inhibition being accompanied by increased cellular prolactin content for up to 24 hours of treatment and followed by decreased values of cellular prolactin concentration at later time-intervals. Immunocytochemical localization showed an increased positive reaction for prolactin up to 24 hours after colchicine treatment, whereas transmission electron microscopy demonstrated, in parallel, an increased number of intracellular prolactin secretory granules during the same interval. Longer periods of treatment (24-96 hours) resulted in the appearance of more lysosomes, autophagic vacuoles and microfilaments in the cells, whereas the number of Golgi elements was decreased. Following four hours of colchicine treatment and at later stages, microtubules could no longer be observed in the sections. Scanning electron microscopic data showed that colchicine treatment induced dramatic changes in the cell surface morphology: at short time intervals (4 and 8 hours), the number of microvilli decreased and the cell surface became folded, whereas, later, "bleb"-like protrusions of variable dimensions partially covered the cell surface and seemed to be released from it. These data show a good correlation between secretory activity of prolactin-producing cells and morphological changes induced by colchicine treatment.

Molecular Weight Forms of Adrenocorticotropic Hormone Secreted by Primary Cultures of Mouse Anterior Pituitary*

Extracts of mouse anterior pituitary cells in monolayer culture were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis to separate the molecular weight forms of ACTH. The gels were sliced and each segment was eluted. The eluates were assayed for ACTH immunoactivity. Approximately 10% of the immunoactivity in the extracts was found to be present as 20,000--32,000 mol wt ACTH. The remainder of the immunoactivity was equally distributed between two forms of ACTH with apparent molecular weights of 11,800 and 4,500. This distribution is very similar to that found in extracts of mouse anterior pituitary. Mouse anterior pituitary cultures were incubated for 2 h in serum-free tissue culture medium. ACTH was concentrated from the medium and fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The medium was found to contain predominantly the 4,500 and 11,800 forms of ACTH. When vasopressin was added to these cultures (100 ng/ml), the rate of secretion of ACTH was more than doubled in serum free medium. Analysis of the medium from vasopressin-stimulated cultures showed that the 4,500 and 11,800 mol wt forms of ACTH were again the predominant forms present.

Variations in the response of enzyme-dissociated rat pituitary cells to thyrotropin releasing hormone

While exploring the interaction between thyrotropin releasing hormone (TRH) and normal rat anterior pituitary cells in monolayer culture we observed that cells dissociated with the use of trypsin did not respond to TRH with an increase in either TSH or prolactin (PRL) release. The dissociated cells were cultured for 3 days, then washed to remove serum proteins and exposed to 10 −6M TRH for 3 hours. TSH and PRL secretion from stimulated and unstimulated cultures was determined by radio-immunoassay and normalized using cell protein. When such trypsin-dissociated cells were exposed to 0.5 mM dibutyryl cyclic AMP the release of both TSH and PRL doubled indicating that the intracellular secretory machinery was functional and that the block to TRH was proximal to the formation of cyclic AMP and presumably at the level of a TRH surface receptor. Previous studies have shown that such trypsin-dissociated cells respond to LHRH and a crude hypothalamic extract with a dose dependent increase in LH, FSH and ACTH release. This rules out a non-specific effect of trypsin. When pituitary cells were dissociated with a non-trypsin technique, the unstimulated release of both TSH and PRL was comparable to that found with the trypsin-dissociated cultures. However, these cultures did respond to TRH with an increase in TSH release although again no effect was seen with PRL. The susceptibility of the cells to trypsin suggests the possibility that a protein moiety may be closely associated with the function of the receptor.

Synthesis of preprolactin and conversion to prolactin in intact cells and a cell-free system.

Monolayer cultures of pituitary cells were pulse-labeled with [3H]leucine for several minutes and the incorporated radioactivity was analyzed by immunoprecipitation and electrophoresis on sodium dodecyl sulfate containing polyacrylamide gels. Following a 3-min labeling period, a peak of radioactivity with a mobility similar to that of preprolactin was observed, as well as radioactivity co-migrating with prolactin. Competition with unlabeled prolactin demonstrated the specificity of the immunoprecipitation reaction. After 5 min of pulse-labeling followed by 5-min chase in medium with unlabeled leucine, only a product with the mobility of prolactin remained. Addition of a membrane fraction from dog pancreas to a wheat germ cell-free translation system containing pituitary mRNA resulted in the conversion of preprolactin to prolactin. Partial sequence analysis demonstrated that the processed product contained the correct NH2 terminus of prolactin. Thus, both intact pituitary cells and a cell-free heterologous system are able to synthesize preprolactin and cleave it to prolactin offering strong evidence that preprolactin is the biosynthetic precursor to prolactin.

Surface morphology and secretory activity of rat anterior pituitary cells in primary culture

So far, there has been no report concerning the surface morphology of cultured secretory cells in different states of activity, as observed by scanning electron microscopy. The anterior pituitary cells in monolayer culture offer an unique system to study the modifications of cell conformation in relation with changes of activity since these cells can be specifically modulated by stimulating or inhibiting factors. Scanning electron microscopy of anterior pituitary cells in primary culture was thus performed in different states of secretory activity. Adult female Sprague-Dawley rats at random stage of the estrous cycle, were used for the preparation of the primary cultures of anterior pituitary cells as previously described (Endocrinology 98: 1528, 1976). The cells 7. 5 x 105 in 1. 5 ml of Dulbecco's modified Eagle's medium containing 10% horse serum and 0. 25% foetal calf serum were plated in 3. 5 x 10 mm Petri dishes and were used six days after plating.

Isolation, structural elucidation and synthesis of a tetradecapeptide with in vitro ACTH-releasing activity corresponding to residues 33–46 of the α-chain of porcine hemoglobin

A peptide found in acetic acid extracts of porcine hypothalami and capable of stimulating the release of ACTH in vitro was isolated in pure state, structurally identified as Phe-Leu-Gly-Phe-Pro-Thr-Thr-Lys-Thr-Tyr-Pre-Pro-His-Phe and synthesized. This tetradecapeptide, which corresponds to amino acid residues no. 33–46 in the sequence of the α-chain of porcine hemoglobin, probably represents an artefact of extraction or isolation procedures. Since this peptide stimulates ACTH release from rat pituitary fragments and from monolayer cultures of pituitary cells, but not in vivo , caution must be exercised in interpreting the results of in vitro assays for corticotropin releasing factor.

Inhibition of growth hormone and thyrotropin release by growth hormone-release inhibiting hormone

Addition of increasing doses of synthetic growth hormone-release inhibiting hormone (GH-RIH) leads to a progressive decrease of the basal and N 6monobutyryl cyclic AMP-,theophylline- and prostaglandin E 2-induced release of immunoreactive growth hormone (GH) and thyrotropin (TSH) release from rat anterior pituitary cells in monolayer culture. A halfmaximal effect is measured at 3 × 10 −9 M GH-RIH while a maximal inhibition to 10–20% of the control level is found at 1 × 10 −7 M. Using rat hemipituitaries and measurement of GH release by both polyacrylamide gel electrophoresis and radioimmunoassay, a maximal effect of GH-RIH was found in the first 5 min of incubation. The inhibitory effect of GH-RIH on GH release remained constant for at least 3 h. GH-RIH does not affect the basal or induced release of prolactin and luteinizing hormone nor the high K +-induced release of GH and TSH.