Thyrotropin-releasing hormone inreases prolactin mRNA activity in the cytoplasm of GH-cells as measured by translation in a wheat germ cell-free system

A wheat germ embryo extract was used to translate cytoplasmic RNA isolated from rat pituitary tumor cells (GH-cells). This RNA directed the synthesis of a radioactive product which was precipitated with antiserum specific for rat prolactin. The molecular weight of this immunoprecipitated product was 24,500 as determined by electrophoresis in denaturing gels. Prolactin secreted by intact GH-cells had a molecular weight identical to standard pituitary prolactin, reported to be about 22,500. Our finding that a larger form of prolactin is made by the wheat germ system is similar to results recently described by Maurer, Stone and Gorski (J. Biol. Chem., in press). Thyrotropin-releasing hormone (TRH) stimulates prolactin synthesis in GH-cells, and cytoplasmic RNA isolated from cells treated with TRH directed the synthesis in wheat germ extracts of larger amounts of prolactin than RNA isolated from control cells. The increase in translatable cytoplasmic mRNA for prolactin corresponded to the increase in prolactin synthesis which suggests that the increase in prolactin synthesis in TRH-treated cells is a result of the accumulation of cytoplasmic mRNA for prolactin.     

Cell-free synthesis of a large translation product of prolactin messenger RNA.

RNA prepared from rat anterior pituitaries or from prolactin-secreting pituitary tumors has been shown to direct the synthesis of a large form of prolactin in a cell-free system derived from wheat germ. Immunoprecipitation of cell-free reactions demonstrated the synthesis of a product which was recognized by a specific antiprolactin antisera. Analysis of the immunoprecipitate on sodium dodecyl sulfate containing polyacrylamide gels suggested that the cell-free product has a molecular weight of approximately 28,000 compared to 22,500 for prolactin. RNA prepared by completely different techniques from rat pituitary and a pituitary tumor resulted in identical large translation products. Translation of tumor RNA in a cell-free system from Krebs ascites cells also resulted in a similar large product. The identity of the cell-free product as prolactin was confirmed by comparing peptides derived from the cell-free product and prolactin. The results of these studies suggest that prolactin messenger RNA directs the cell-free synthesis of a product which contains the amino acid sequence of prolactin but which has an addition at one or both ends of the molecule.     

Construction and characterization of a cDNA clone containing a portion of the bovine prolactin sequence.

Poly(A)-containing RNA from the bovine anterior pituitary has been used as a template for the enzymatic synthesis of double-stranded cDNA. The resulting double-stranded cDNA was inserted into the Pst I site of pBR322 with the oligo(dG)-oligo(dC) tailing technique and subsequently cloned in E. coli chi 1776. Clones containing sequences complementary to prolactin mRNA were identified by colony hybridization with partially purified prolactin cDNA. A 250 base pair sequence from one prolactin positive clone was extensively characterized and shown to contain the coding information for amino acids 119-192 of authentic bovine prolactin. The recombinant DNA from this clone was covalently attached to diazotized aminocellulose and used to purify prolactin mRNA from a mixture of mRNAs.     

Comparison of secreted and extracted forms of rat pituitary prolactin

Prolactin secreted by rat anterior pituitary explants into organ culture medium was purified by salt fractionation and gel filtration. A yield of 22 mg/g was obtained, which clearly represented de novo synthesis and secretion of the hormone. Comparative characterization studies were performed on the secreted prolactin and pituitary extracted rat prolactin obtained from the National Institute of Arthritis, Metabolism and Digestive Diseases. The biological and immunological activity estimates of both forms were comparable, although the specific activities of the secreted prolactin were somewhat lower than those of the pituitary prolactin. The secreted and extracted forms of prolactin appeared to be identical in primary structure as evidenced by similar amino acid compositions and identical NH₂-terminal sequences. Circular dichroism spectra suggested that there may be differences in tertiary structure, since the positive tryptophan band at 292 nm, which was observed with extracted hormone, was absent in the secreted prolactin.     

Regulation by calcium of prolactin and growth hormone mRNA sequences in primary cultures of rat pituitary cells

Addition of Ca2+ to primary cultures of female pituitary cells incubated in serum-free medium lacking added Ca2+ yielded no effects on levels of prolactin or growth hormone mRNA, assayed by cytoplasmic dot hybridization. However, incubation of the cells in serum-free medium containing sufficient ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to reduce medium Ca2+ levels below the 10-40 microM present as a trace contaminant yielded a decrease in the levels of both mRNAs. The decrease was dose-dependent at extracellular Ca2+ concentrations below 1.0 microM, had an apparent half-maximum at about 0.3 microM, and did not appear to plateau with increasing incubation times. Following 2-3-day incubations of cells in low Ca2+, a reduction of prolactin mRNA (23-70-fold) consistently greater than the reduction of growth hormone mRNA (9-15-fold) was observed. Similar effects of reduced extracellular Ca2+ were obtained with primary cultures of male pituitary cells. The specificity of these effects of lowered extracellular Ca2+ was demonstrated by the following observations. The decreases in these mRNAs were substantially reversible by readdition of Ca2+ to the incubation medium. Reduction of extracellular Ca2+ led to no detectable changes in cellular ribosomal RNA levels or over-all RNA synthesis. In male pituitary cells, the level of another metal-regulated mRNA, that for metallothionein, was not decreased by a reduction of extracellular Ca2+ that caused a 40-fold decrease in levels of prolactin and growth hormone mRNA. Hence, Ca2+ exhibits specificity in its regulation of pituitary prolactin and growth hormone gene expression.     

Demonstration of prolactin messenger RNA after amplification in the brain in rats]

By means of immunocytochemistry, a central neuronal network containing a prolactin-like substance has been described in the rat. In order to demonstrate the synthesis of this peptide in these cells, we examined the presence of prolactin messenger RNA (PRL mRNA) in several brain samples including the pituitary gland. Amplification of the PRL mRNA was performed by the polymerase chain reaction technique, followed by southern blotting and hybridization with a specific oligonucleotide. Results showed the presence of the expected cDNA (468 bp) in the hypothalamus. Another cDNA with a lower molecular weight was also observed.     

Routing, processing and export of rat pituitary prolactin: identification of a 36 kDa disulphide-bridged oligomeric preprolactin

To gain an insight in the routing, processing and export of rat prolactin, rat pituitary cells were cultured in serum-free medium in the presence of cycloheximide, carbonyl cyanide m-chlorophenylhydrazone, Brefeldin A and monensin. The potential influence of these perturbants, whose well documented effects are the altering of protein synthesis and transport, was studied on rat prolactin molecular size isoforms appearing in cellular extracts and in culture medium. The outcome of the culture experiments as recorded in vertical SDS-PAGE, thiol gradient electrophoresis and sequential SDS-PAGE followed by prolactin specific immunoblotting and densitometry, was as follows: (1) at the cellular level we were able to characterize a novel 36 kDa protein as a disulphide-bridged oligomeric precursor prolactin, which is presumably rapidly transformed in the cis/medial Golgi; to designate monomeric rat prolactin as an early Golgi protein and t o advance evidence that the main processing of the glycosylated rat prolactin is a cis/medial Golgi event; (2) in release none of the perturbants disturbed the relative distribution of monomeric and glycosylated rat prolactin, the main molecular size isoforms currently secreted by untreated pituitary cells, or induced the appearance of transformed molecular size isoforms; (3) the secretion mode indicates that rat prolactin is released via the regulated pathway in the presence of the perturbants used.     

Biosynthesis and degradation of prolactin in the rat anterior pituitary gland. Time course of incorporation of label in vitro and evidence for rapid degradation.

Biosynthesis of prolactin was studied in anterior pituitary glands from female rats, incubated in vitro. In this system [3H]leucine was incorporated into pituitary proteins, including somatotropin (growth hormone) and prolactin. The rate of uptake of label into prolactin (and to a lesser extent into total protein) slowed considerably during the first 2 h of incubation, although the rate of uptake into somatotropin was constant for 8 h. The most probable explanation for this apparent decrease in the rate of prolactin synthesis is degradation of prolactin in the gland. Degradation of this hormone was also demonstrated by incubating prelabelled pituitaries in unlabelled medium and following the content of labelled prolactin, and by studying the hormonal content of pituitary glands (by radioimmunoassay) before and after incubation. Degradation of prolactin appears to be much more rapid than that of somatotropin, and may represent a physiological mechanism whereby over-accumulation of prolactin is prevented when secretion of the hormone has been rapidly switched off.     

The effects of dopamine on prolactin mRNA levels in rat pituitary cells in culture.

Dopamine is known to be the prolactin-release inhibiting factor, but the effects of dopamine itself on regulation of prolactin messenger RNA have been little studied because of the instability of dopamine. We have compared the effects of dopamine and bromocriptine on the levels of prolactin mRNA and on the rates of synthesis, storage, and release of prolactin in primary cultured rat pituitary cells. The cells were incubated for 72 h with no secretagogue (control group) or in the presence of either dopamine (10 mumol/L) plus ascorbic acid (100 mumol/L) or bromocriptine (0.1 mumol/L). Prolactin mRNA was measured in cell extracts by means of slot blots, and newly synthesized prolactin was measured in similar incubations by the addition of [3H]leucine, followed by gel electrophoresis. The levels of total prolactin were measured by radioimmunoassay. Prolactin mRNA was reduced to 78 +/- 9% (mean +/- SEM) of control levels in bromocriptine-treated cells and to 59 +/- 7% in dopamine-treated cells, demonstrating that dopamine stabilized by ascorbic acid was able to reduce the levels of prolactin mRNA in rat pituitary cells in culture. Dopamine may act at sites in addition to the dopaminergic D2 receptor, since the level of prolactin mRNA was reduced more by a supramaximal dose of dopamine than by a supramaximal dose of bromocriptine. The results of the [3H]prolactin and prolactin measurements suggested that availability of mRNA was not a major factor in controlling the rate of prolactin synthesis.     

Hormonal regulation of prolactin storage in a clonal strain of rat pituitary tumor cells

GH4C1 cells (GH cells) are a clonal strain of rat pituitary tumor cells which secrete prolactin. GH cells have been used to study hormone secretion, but they store relatively little prolactin compared to normal prolactin-secreting cells. They are not suitable, therefore, for studying some aspects of pituitary function. We have found that the amount of prolactin GH cells store can be regulated. When GH cells were plated at 10(6) cells/well and treated for six days with 180 nM insulin or 1 nM estradiol, there was a 60 percent increase in prolactin storage compared to control cells. Insulin and estradiol in combination acted synergistically to cause a 190 percent increase in prolactin storage. In contrast, they were additive in increasing extracellular prolactin; there was a 40 percent increase in extracellular prolactin after insulin, a 20 percent increase after estradiol, and a 50 percent increase after insulin plus estradiol. The increases in prolactin storage were always greater than the increases in extracellular prolactin. The increases in prolactin storage were dose-dependent and reached maximal levels after four days of treatment with 180 nM insulin plus 1 nM estradiol. Reducing the plating density to 10(3) cells/well increased the response to insulin and estradiol to nineteenfold. Epidermal growth factor (10 nM) acted synergistically with estradiol and insulin in combination to increase prolactin storage 27-fold. The insulin- and estradiol-induced increase in extracellular prolactin was caused by a specific increase in the rate of prolactin synthesis. The fractional increase in prolactin storage above the increase in prolactin production could not be explained by an increase in prolactin synthesis, an increase in intracellular transit time, or a change in the cell-cycle distribution of the population. Hormone storage can, therefore, be regulated independently from other processes which control hormone production. The prolactin stored in response to insulin and estradiol was releasable by potassium depolarization. Following depletion of intracellular prolactin by depolarization, the cells retained their increased capacity for prolactin storage. The ability to increase prolactin storage will make GH cells a more useful system in which to study pituitary function.     

The effect of ethanol on prolactin secretion in vitro

In order to investigate the action of ethanol on the anterior pituitary gland, the effect of ethanol on prolactin secretion in vitro was studied. Ethanol significantly increased the in vitro incorporation of ³H-leucine into both prolactin contained within the pituitary gland and that released into the medium. The enhancement of ³H labelled-prolactin synthesis induced by ethanol was suppressed by cycloheximide. These results support the hypothesis that ethanol stimulates the in vitro synthesis and release of prolactin by the pituitary gland.     

Combined use of in situ hybridisation and immunocytochemistry for the investigation of prolactin gene expression in immature,pubertal, pregnant,lactating and ovariectomised rats

Summary We have investigated the use of in situ hybridisation together with immunocytochemistry for the study of endocrine cell function, using as an example the expression of prolactin messenger RNA (mRNA) in pituitaries of rats under various endocrinological conditions. In situ hybridisation using a ³²P-labelled cRNA probe for rat prolactin was carried out on sections of 4% paraformaldehyde-fixed pituitaries from prepubertal, pubertal, pregnant, lactating and ovariectomised rats and adjacent sections were immunostained for prolactin. Northern gel analysis was performed on total RNA extracts of pregnant, lactating and control pituitaries. While in ovariectomised rat pituitaries both prolactin immunoreactivity and prolactin mRNA were decreased, no differences in prolactin immunostaining were seen between prepubertal, pubertal, pregnant or lactating rats and controls, even when the supra-optimal dilution technique was used. However, using in situ hybridisation, prolactin mRNA signal was increased in prepubertal rats, and with hybridisation and northern gel analysis the signal was reduced in pregnant rats and markedly increased in lactating rats. The combined use of in situ hybridisation and immunocytochemistry provides morphological information concerning endocrine gene expression and protein synthesis in the pituitary gland.     

Combined use of in situ hybridisation and immunocytochemistry for the investigation of prolactin gene expression in immature, pubertal, pregnant, lactating and ovariectomised rats

We have investigated the use of in situ hybridisation together with immunocytochemistry for the study of endocrine cell function, using as an example the expression of prolactin messenger RNA (mRNA) in pituitaries of rats under various endocrinological conditions. In situ hybridisation using a 32P-labelled cRNA probe for rat prolactin was carried out on sections of 4% paraformaldehyde-fixed pituitaries from prepubertal, pubertal, pregnant, lactating and ovariectomised rats and adjacent sections were immunostained for prolactin. Northern gel analysis was performed on total RNA extracts of pregnant, lactating and control pituitaries. While in ovariectomised rat pituitaries both prolactin immunoreactivity and prolactin mRNA were decreased, no differences in prolactin immunostaining were seen between prepubertal, pubertal, pregnant or lactating rats and controls, even when the supra-optimal dilution technique was used. However, using in situ hybridisation, prolactin mRNA signal was increased in prepubertal rats, and with hybridisation and northern gel analysis the signal was reduced in pregnant rats and markedly increased in lactating rats. The combined use of in situ hybridisation and immunocytochemistry provides morphological information concerning endocrine gene expression and protein synthesis in the pituitary gland.     

Synthesis, cloning and determination of the primary structure of DNA complementary to mRNA of prolactin from the human pituitary gland

The poly(A)-containing mRNA from human pituitary and prolactinoma have been purified and translated in the cell-free system from rabbit reticulocytes. mRNA from prolactinoma was shown to be enriched with specific prolactin mRNA. DNA complementary to the prolactin mRNA from human pituitary was obtained and cloned. Sequencing of the 900 bp insert by the Maxam-Gilbert technique suggested the cDNA cloned to cole for the previously published amino acid sequence, mismatches with mRNA from prolactinoma occurring at the third positions of codons and thus not causing amino acid substitutions.     

Direct and reversible inhibition of estradiol-stimulated prolactin synthesis by antiestrogens in vitro

The antiestrogens tamoxifen and monohydroxytamoxifen inhibited the estradiol-stimulated increase in prolactin synthesis by dispersed cells in culture derived from immature rat pituitary glands. Monohydroxytamoxifen had a relative binding affinity for the estrogen receptor similar to that of estradiol, whereas tamoxifen's relative binding affinity was approximately 3%. This was consistent with the observation that monohydroxytamoxifen was 30 times more potent than tamoxifen as an antiestrogen in vitro. To avoid the possibility that tamoxifen was fractionally metabolized to monohydroxytamoxifen by the pituitary cells, the p-methyl, p-chloro, and p-fluoro derivatives of tamoxifen that are unlikely to be converted to monohydroxytamoxifen were tested for activity. The substitution did not have a detrimental effect on their ability to inhibit the binding of [3H]estradiol to either rat uterine or pituitary gland estrogen receptors. Similarly, the derivatives of tamoxifen inhibited estradiol-stimulated prolactin synthesis at concentrations that were consistent with their relative binding affinities. Although it is clearly an advantage for tamoxifen to be metabolized to the more potent antiestrogen monohydroxytamoxifen, we have shown that this is not a prerequisite for the antiestrogenic actions of tamoxifen. With the direct actions of antiestrogens established, the pituitary cell system was validated for further structure-activity relationship studies. Overall, the inhibition of estradiol-stimulated prolactin synthesis by antiestrogens is competitive and reversible with estradiol, an effect that can be explained by interactions with the estrogen receptor system.     

Quantification of prolactin messenger ribonucleic acid, pituitary content and plasma levels of prolactin, and detection of immunoreactive isoforms of prolactin in pituitaries from turkey embryos during ontogeny.

The content of prolactin mRNA as well as total prolactin content and type of isoforms of prolactin were measured in single pituitary glands from turkey embryos and poults. Levels of mRNA and pituitary content of prolactin remained low until 5 days before hatching, while plasma concentrations remained low until 2 days before hatching. Levels of prolactin mRNA then increased until the day of hatch, stayed stable during the 3 first days of age, and significantly increased until 2 wk of age. Similar changes were observed in pituitary content and plasma levels of prolactin. Two immunoreactive bands of apparent molecular masses of 24 and 27 kDa, corresponding to the nonglycosylated and glycosylated form of prolactin, respectively, were visualized on Western blots. In pituitary glands from embryos at 22 days of incubation, 31.5% of the protein was glycosylated, whereas in embryos at 27 days of incubation and poults at 1 and 7 days of age, 48.6%, 48.0%, and 56. 0% of prolactin was glycosylated, respectively. The results indicate that the increases in the synthesis and the release of prolactin occur mainly around and after the time of hatching in the turkey embryo. Higher percentages of glycosylated isoforms were associated with increasing levels of total prolactin in the pituitary gland. Thus, the synthesis of prolactin and its post-translational modifications may be important factors involved in the physiologic changes occurring around the time of hatching.     

Novel prolactin related mRNAs in rat pituitary cells

From cytoplasm of rat pituitary GH4C1 tumour cells, anti prolactin anti-serum precipitates a polypeptide with apparent molecular weight of 75.000 in addition to prolactin. In vitro translation of size fractionated RNA shows that a 82.000 molecular weight PRL-like polypeptide is encoded by a mRNA larger than the 1 kb prolactin mRNA. Northern blot analysis shows that a rat prolactin cDNA probe hybridize to a 3.2 kb RNA and a 1.5 kb RNA in addition to the 1 kb PRL mRNA. The 82.000 molecular weight translation product and the 3.2 kb mRNA is also detected in rat anterior pituitary cytoplasm. We conclude that at least one high molecular weight mRNA which code for a prolactin-like polypeptide, is present in normal rat anterior pituitary gland and in GH4C1 cells.