Lactotroph Cells Research Articles

Dopamine D4 receptor-mediated inhibition of cyclic adenosine 3',5'-monophosphate production does not affect prolactin regulation.

Under physiological conditions, PRL synthesis and secretion are predominantly under negative control by dopamine acting through dopamine D2 receptors present in the pituitary lactotroph cells. To investigate the role of D4 receptors in the regulation of PRL synthesis and secretion, we stably transfected the human D4 receptor complementary DNA into the somatomammotrophic cell line GH4C1. The pharmacological characteristics of D4 expressed in GH4C1 were in close agreement with previous D4 receptor studies in Chinese hamster ovary and COS-7 cells. In GH4C1 cells, activation of D4 receptor variants (D4.2, D4.4, and D4.7) resulted in a similar level of reduction in forskolin- and vasoactive intestinal peptide (VIP)-stimulated cAMP levels (33% and 50%, respectively). In addition, the forskolin-stimulated activity of cAMP response elements fused to the VIP promoter driving the lacZ reporter gene could be blocked by D4 activation. However, quinpirole treatment had a minimal effect on transiently expressed luciferase reporter gene driven by a proximal PRL promoter in one of the D4-expressing cell lines. In contrast, the dopamine D2short receptor expressing GH4ZR7 cells treated with quinpirole displayed a significant decrease (51.3 +/- 4.1%) in PRL promoter activity. VIP-stimulated PRL release was not affected by D4 receptor activation, whereas in GH4ZR7 cells, a significant decrease in VIP-stimulated PRL levels was observed. Neither PRL promoter activity nor PRL secretion levels were affected in control untransfected GH4C1 cells. From this study it appears that although the D4 receptor may be expressed in the anterior pituitary, it does not have a major effect on PRL promoter activity or PRL secretion in GH4C1 cells despite its ability to reduce cAMP production. This might explain why D4- over D2-preferring antipsychotics such as clozapine do not cause hyperprolactinemia.

Dopamine D4 Receptor-Mediated Inhibition of Cyclic Adenosine 3',5'-Monophosphate Production Does Not Affect Prolactin Regulation

Under physiological conditions, PRL synthesis and secretion are predominantly under negative control by dopamine acting through dopamine D2 receptors present in the pituitary lactotroph cells. To investigate the role of D4 receptors in the regulation of PRL synthesis and secretion, we stably transfected the human D4 receptor complementary DNA into the somatomammotrophic cell line GH4C1. The pharmacological characteristics of D4 expressed in GH4C1 were in close agreement with previous D4 receptor studies in Chinese hamster ovary and COS-7 cells. In GH4C1 cells, activation of D4 receptor variants (D4.2, D4.4, and D4.7) resulted in a similar level of reduction in forskolin- and vasoactive intestinal peptide (VIP)-stimulated cAMP levels (33% and 50%, respectively). In addition, the forskolin-stimulated activity of cAMP response elements fused to the VIP promoter driving the lacZ reporter gene could be blocked by D4 activation. However, quinpirole treatment had a minimal effect on transiently expressed luciferase reporter gene driven by a proximal PRL promoter in one of the D4-expressing cell lines. In contrast, the dopamine D2short receptor expressing GH4ZR7 cells treated with quinpirole displayed a significant decrease (51.3 ± 4.1%) in PRL promoter activity. VIP-stimulated PRL release was not affected by D4 receptor activation, whereas in GH4ZR7 cells, a significant decrease in VIP-stimulated PRL levels was observed. Neither PRL promoter activity nor PRL secretion levels were affected in control untransfected GH4C1 cells. From this study it appears that although the D4 receptor may be expressed in the anterior pituitary, it does not have a major effect on PRL promoter activity or PRL secretion in GH4C1 cells despite its ability to reduce cAMP production. This might explain why D4- over D2-preferring antipsychotics such as clozapine do not cause hyperprolactinemia.

Prolactin Signaling in Mammary Gland Development

Prolactin Signaling in Mammary Gland Development

Commentary on the use of immortalized neuroendocrine cell lines for physiological research.

A great deal of work has been done recently using the GTI_ 7 immortalized cells (1) as a model of a "highly differentiated" GnRH neuron (reviewed in refs. 2-5). GT 1 cells, maintained in cell culture, exhibit several morphological (1,6), biochemical (1, 7), and physiological (1,3) functions characteristic of adult, differentiated GnRH neurons. In addition, though never demonstrated for GnRH neurons in situ, G T 1 cells in culture exhibit intrinsic, pulsatile secretory activity (8-10). They have been found to "differentiate" in situ and restore reproductive function in adult hpg mice following their direct introduction into the brain (11). Whereas several advantages of using GTt cells to model the GnRH neuron have been enumerated (2-4), potential disadvantages have received less attention. The in vitro study of such transformed cell lines has several limitations. The extent to which observed electrophysiological, receptor, second messenger, gene expression, and secretory properties ofGT i cells are subsequently found accurately to characterize in situ GnRH neurons in adult brain is unknown. Potential limitations include the following problems, several of which are common to the study of all cell lines. First, it is unclear whether the level of differentiation of postmitotic, immortalized cells can be maintained over cell generations. For instance, increased expression ofT-antigen in transformed pituitary lactotroph cell lines is correlated with progressive loss of the lactotroph phenotype (12). If this occurs with increasing numbers of cell passages, to what extent is the fully differentiated phenotype compromised by the lesser concentrations of T-antigen found in GT 1 founder cells? Second, significant effects ofT-antigen expression in these cells on normal physiological cell functions may exist, but are unrecognized. Third, when stereotaxically placed in the hypothalamus of hpg mice, axons

Regulation of intracellular chloride concentration in rat lactotrophs: possible role of mitochondria

Increasing evidence is accumulating for the involvement of chloride ions in the stimulus-secretion coupling of pituitary cells. We show that the mean intracellular chloride concentration [Cl −]i of rat lactotroph cells maintained in culture is high, close to 60 mM (59.4 mM), using the Cl − sensitive fluorescent probe 6-methoxy- N-(3-sulfopropyl) quinolinium (SPQ), coupled with whole-cell patch-clamp recordings. We demonstrate that this high level is correlated with the presence of mitochondrial stores of Cl − as shown by the release of Cl − in response to various metabolic inhibitors. We determine that CCP (50 μM) induces a mean [Cl −]i increase of 15.8±5.8 mM, using combined electrophysiology and microspectrofluorimetry methods. These data strongly suggest that cell metabolism, including the mitochondrial function, modulate [Cl −]i.

Different behavior of lactotroph cell subpopulations in response to angiotensin II and thyrotrophin-releasing hormone.

1. In the present investigation we have extended the study of lactotroph subpopulations in primary pituitary cell cultures. Male rats with or without previous estrogenization followed by A-II or TRH treatments were selected as experimental models. 2. The TRH increased up to 50% the PRL released in both whole and OROX+EB rats (P < 0.05). In contrast, A-II treatment introduced no changes in PRL secretion from cell cultures derived from whole male rats but attained a significant augmentation (about 75%) of PRL secreted by ORQX+EB pituitary cells. 3. The addition of TRH and A-II to cultures of ORQX+EB-derived lactotrophs induced cytological changes compatible with a high secretory activity. In estrogen-treated rats the prevailing lactotroph subpopulation is type I. In cell cultures from control and A-II treated whole male pituitaries, the majority of lactotrophs consists of atypical subpopulations of II and III cells, with smaller secretory granules (between 150 and 300 nm in diameter). 4. Morphometry of immunostained lactotrophs performed on light microscopic preparations revealed that about 30-36% of the total cell count were lactotrophs. This percentage was fixed and did not change significantly after TRH and A-II treatments. 5. The present results confirm the presence of morphological and functional subtypes of lactotroph cells in rat pituitary. Typical PRL cell population shows the highest responsiveness to angiotensin II and TRH action. This functional heterogeneity of lactotroph subtypes may reflect an important and scarcely explored factor in the regulatory process of prolactin secretion.

Dominant dwarfism in transgenic rats by targeting human growth hormone (GH) expression to hypothalamic GH-releasing factor neurons.

Expression of human growth hormone (hGH) was targeted to growth hormone-releasing (GRF) neurons in the hypothalamus of transgenic rats. This induced dominant dwarfism by local feedback inhibition of GRF. One line, bearing a single copy of a GRF-hGH transgene, has been characterized in detail, and has been termed Tgr (for Transgenic growth-retarded). hGH was detected by immunocytochemistry in the brain, restricted to the median eminence of the hypothalamus. Low levels were also detected in the anterior pituitary gland by radioimmunoassay. Transgene expression in these sites was confirmed by RT-PCR. Tgr rats had reduced hypothalamic GRF and mRNA, in contrast to the increased GRF expression which accompanies GH deficiency in other dwarf rats. Endogenous GH mRNA, GH content, pituitary size and somatotroph cell number were also reduced significantly in Tgr rats. Pituitary adrenocorticotrophic hormone (ACTH) and thyroid-stimulating hormone (TSH) levels were normal, but prolactin content, mRNA levels and lactotroph cell numbers were also slightly reduced, probably due to feedback inhibition of prolactin by the lactogenic properties of the hGH transgene. This is the first dominant dwarf rat strain to be reported and will provide a valuable model for evaluating the effects of transgene expression on endogenous GH secretion, as well as the use of GH secretagogues for the treatment of dwarfism.

Signalling pathways mediating secretory and mitogenic responses to galanin and pituitary adenylate cyclase-activating polypeptide in the 235-1 clonal rat lactotroph cell line.

The neuropeptides galanin and pituitary adenylate cyclase-activating peptide (PACAP) have been implicated in the physiological regulation of lactotroph function. Using the 235-1 clonal lactotroph rat cell line we have studied the signalling pathways mediating the secretory and mitogenic responses to galanin and PACAP. Both peptides stimulated prolactin release to a similar maximal extent. PACAP (100 nM) stimulated an increase in the proliferation rate of 235-1 cells, but was significantly less effective than 100 nM galanin (161.8 +/- 2.3% vs 296.1 +/- 9.1% of control). PACAP stimulated cAMP accumulation with an ED50 of 3.2 nM, and a maximal effect of almost two-fold at a concentration of 100 nM. Galanin depleted cAMP, by 30% at a concentration of 100 nM. The aminosteroid phospholipase C (PLC) inhibitor U-73122 virtually abolished maximal peptide stimulated prolactin release. Depletion of inositol phosphates or downregulation of protein kinase C reduced maximal peptide stimulated prolactin release from about 260% to about 160% of unstimulated release. Both peptides at a concentration of 100 nM caused a sustained increase in intracellular calcium when incubated with cells for 30 min. These results demonstrate that both peptides stimulate prolactin release and the proliferation rate of 235-1 cells. The most important signalling pathway for prolactin release activated by both peptides is via PLC, although they also regulate cAMP levels, which are increased by PACAP and decreased by galanin. Despite maximal peptide stimulated prolactin release being equal, galanin has a greater mitogenic effect on 235-1 cells than PACAP, raising the possibility that it activates an additional mitogenic signalling pathway.

Effects of tamoxifen on serum prolactin levels, pituitary immunoreactive prolactin cells and uterine growth in estradiol-treated ovariectomized rats.

Pituitary effects of the antiestrogen tamoxifen are not well established, although estrogen is known to have a stimulatory role in prolactin secretion. Effects of tamoxifen on serum prolactin levels, pituitary wet weight and number of prolactin cells were studied. Ovariectomized female Wistar rats were injected, subcutaneously, with estradiol valerate, 50 or 300 micrograms/rat per week for 2 or 10 weeks. Tamoxifen was injected during the last days of estrogen treatment. Data were compared with two other groups, treated with estradiol valerate alone or estradiol valerate plus the dopamine agonist bromocriptine. Serum prolactin levels were increased by estrogen treatment with all doses used. Furthermore, rats treated with 300 micrograms of estradiol valerate, for 2 and 10 weeks, showed a clear increase in pituitary weight and number of prolactin cells (p < 0.05). Bromocriptine decreased prolactin levels, pituitary weight and the number of prolactin cells (p < 0.05). Tamoxifen associated to subacute period of estrogen administration resulted in a significant reduction of serum prolactin levels and pituitary weight (p < 0.05). No effects on prolactin levels or number of prolatin cells were observed with tamoxifen associated to chronic estrogen treatment. Tamoxifen also presented a dose-related inhibitory effect upon estrogen-stimulated rises in uterine weight and DNA content. In conclusion, the results of the present paper showed that tamoxifen reduced estrogen-stimulated prolactin levels in some, but not in other hormonal conditions and that these effects were not mediated by an inhibition of lactotroph cell growth. Further studies are needed to define the exact role of antiestrogens at molecular level in hyperprolactinemic states and their eventual connection with dopamine and its agonists.

Pituitary PRL secretion induced by tetraethylammonium is inhibited by dopamine through D 2 receptors

We have evaluated the inhibitory effect of dopamine on PRL secretion induced by blocking K + channels. Tumor-derived GH 4C 1 cells and collagenase-dispersed normal anterior pituitary (AP) cells from young adult male rats were perifused with Krebs-Ringer Hepes medium. In both cell types blocking K + channels with tetraethylammonium (TEA) induced PRL secretion but did not stimulate cyclic AMP generation. Blocking Na + channels with 1 μM tetrodotoxin had no effect on basal or TEA-induced PRL secretion. Dopamine inhibited the TEA-induced rise in [Ca 2+] i in GH 4C 1 cells expressing dopamine D 2 short receptors. In normal AP cells, 1–100 nM dopamine blocked PRL secretion induced by 20 mM TEA in a log-linear concentration-dependent fashion, with a plateau at > 100 nM dopamine (IC 50 30 nM). The D 2 dopaminergic receptor agonist, quinpirole, at 100 nM completely blocked PRL secretion induced by 20 mM TEA. The D 2 dopaminergic receptor antagonist, sulpiride, at 10 μM reversed the inhibitory effect of 10 μM dopamine on PRL secretion induced by 20 mM TEA. Pretreatment of cells with 100 ng/ml pertussis toxin (PTX) for 24 h prevented 100 nM dopamine inhibition of PRL secretion induced by 20 mM TEA. The data indicate that in both normal lactotroph cells and in tumor-derived cells expressing D 2 receptors, PRL secretion stimulated by blocking K + channels is inhibited by dopamine binding to D 2 receptors on the plasma membrane. This inhibition involves interaction with PTX-sensitive G i protein.

Reconstitution of protein kinase A regulation of the rat prolactin promoter in HeLa nonpituitary cells: identification of both GHF-1/Pit-1-dependent and -independent mechanisms.

Expression of the rat PRL (rPRL) gene is highly restricted to pituitary lactotroph cells and is induced by the cAMP-dependent protein kinase A (PKA) pathway. Current data indicate that this PKA effect requires at least one of the redundant pituitary-specific elements of the proximal rPRL promoter, suggesting the involvement of the pituitary-specific transcription factor, GHF-1/Pit-1. To directly determine whether GHF-1 is necessary and sufficient to mediate the PKA activation of the rPRL promoter, we established a cotransfection reconstitution assay whereby the activity of an intact and site-specific mutants of the (-425 to +73) rPRL promoter-luciferase reporter gene was reconstituted by cotransfecting expression vectors encoding for either the PKA beta catalytic subunit, GHF-1, or both, into HeLa nonpituitary cells. Cotransfection of PKA beta alone significantly stimulated rPRL promoter activity in HeLa cells in a GHF-1-independent manner, and this PKA beta effect was mapped to the most proximal GHF-1 site [footprint (FP) I; -67/-36]. Site-specific alterations of either FP II (-130/-120), or of the basal transcription element (BTE; -112/-80), did not significantly affect the PKA beta response. As expected, the transactivation effect of cotransfected GHF-1 mapped to the GHF-1/Pit-1 binding sites, FP I and/or FP III, of the rPRL promoter. Finally, cotransfection of PKA beta and GHF-1 resulted in a marked synergistic response of the rPRL promoter, and this response also localized to the FP I site. These data confirm not only that GHF-1/Pit-1 and the FP I site are involved in mediating the PKA response, but also imply that a distinct and possibly ubiquitous factor is involved by binding to FP I and functionally interacting with GHF-1 to modulate PKA beta regulation of the rPRL promoter.

Expression of plurihormonal mRNAs in somatotrophic adenomas detected using a nonisotopic in situ hybridization method: Comparison with lactotrophic adenomas

We used a nonisotopic in situ hybridization (ISH) method to investigate the expression of pituitary hormone, including glycoprotein hormone mRNAs in 17 somatotrophic and four lactotrophic adenomas. Our ISH studies of lactotrophic adenomas showed that their hormonal gene expression was confined to prolactin, whereas those of somatotrophic adenomas showed that some of them expressed plurihormonal genes. In some somatotrophic adenomas that were immunohistochemically negative for pituitary hormones, positive reactions, mainly for adrenocorticotropic hormone (ACTH), follicle-stimulating hormone β subunit (FSHβ), and luteinizing hormone β subunit (LHβ) mRNAs, were observed in our ISH studies. These results suggest that some somatotrophic adenomas may originate from plurihormonal primordial stem cells, which we have presumed serve as precursors for various hormone-expressing cells. It is unclear why some somatotrophic adenomas derived from plurihormonal primordial stem cells manifest clinically only as the acromegalic hyperfunction syndrome or gigantism. Additional translational factors or some other somatic mutations may play important roles in the clinical manifestations of such adenomas. In conclusion, some somatotrophic adenomas appear to be derived from plurihormonal primordial stem cells, whereas lactotrophic adenomas are well differentiated tumors that originate from lactotrophic cells, which represent the final stage of acidophilic cell line differentiation.

P1-304 - Effects of the novel dopaminergic agent ΟΡΟ-14597 on dopamine D2 receptors in lactotroph cells in rat anterior pituitary gland

P1-304 - Effects of the novel dopaminergic agent ΟΡΟ-14597 on dopamine D2 receptors in lactotroph cells in rat anterior pituitary gland

Secretion of 23 kDa and glycosylated prolactin by rat pituitary cell culture in serum-free media: a comparative morphological, cyto- and immunochemical study

The secretion of 23 kDa prolactin by rat pituitary cells has been thoroughly investigated, but secretion of glycosylated rat prolactin is not currently known. This is mainly due to the lack of an antiserum which is solely specific for glycosylated rat prolactin and therefore we studied the basal secretion of this variant by an indirect method. Rat pituitary cells were cultured in total culture medium and three different serum-free media (DMEM, keratinocyte-serum-free medium, protein-free hybridoma medium) and secretion of 23 kDa and glycosylated rat prolactin was recorded by radioactive techniques and immunoblotting. The pituitary cell quality was monitored by electron microscopy, cell activation-and cell death assessment. In short-range culture (2 days) the pituitary cell quality and behaviour was very good and comparable in total culture medium, DMEM and keratinocyteserum-free medium, i.e. numerous secretory granules, moderate amount of ER, cristae well in place in the mitochondriae. In medium-range culture (8 days) only cells cultured in total culture medium and DMEM presented a parallel behaviour: migration of cells toward each other, marked degranulation, massive array of ER. The inner membrane of the mitochondria was no longer folded into cristae leaving an unoccupied central space. At day 2 of the culture span secretion of 23 kDa rat prolactin was very comparable in all media used; hereafter, secretion of 23 kDa rat prolactin in total culture medium and DMEM assumed the well known pattern of peaking and slowing down, whereas in the other serumfree media it steadily decreased over the culture span. Pertaining to the important novel point of glycosylated rat prolactin secretion, it was low in comparison to the one of 23 kDa rat prolactin and it assumed a near steady pattern in all media used. 26 kDa rat prolactin was identified as the preferentially secreted glycoform, and the 23 kDa isoform as the major secretory product of rat pituitary lactotroph cells.

Immunohistochemical Demonstration of Transforming Growth Factor-beta-1 in Human Pituitary Adenomas.

The expression of transforming growth factor-beta-1 (TGF-beta-1) was investigated in five normal human pituitary glands and 48 human pituitary adenomas using immunoreactivity to polyclonal antibodies against human TGF-beta-1. The relationship between hormone production and the expression of TGF-beta-1 was also investigated. TGF-beta-1 was detected in both normal cells of pituitary gland and in tumor cells of pituitary adenoma. Fifteen of 48 pituitary adenomas showed positive immunoreactivity for TGF-beta-1. Immunohistochemical staining of serial sections revealed coexpression of TGF-beta-1 and pituitary hormones in pituitary adenomas (PRL, ACTH, GH) and in normal pituitary glands (PRL, ACTH, LH). Intense and widespread positive immunoreactivity was observed in lactotroph cell adenomas. These results suggest TGF-beta-1 may be important in the differentiation and proliferation of the cells of normal adenohypophysis and adenoma.

Beta-Mercaptoethanol differentially acts on the rat somatotrophs and lactotrophs in primary culture to suppress the secretion of immunoreactive hormones.

We previously reported that beta-mercaptoethanol (ME) reduced the content of immunoreactive prolactin (iPRL) within the adenohypophyseal cells, primarily through its direct effect on the disulfide bonding of the PRL molecule. Because of the structural similarities between PRL and growth hormone (GH), the effects of ME on the hormonal dynamics of iGH were compared to those of iPRL. ME reduced secretion and intracellular content of both iGH and iPRL in the cultured rat adenohypophyseal cells. However, iGH was more resistant to the suppressive effects of ME than iPRL. This was particularly so with regard to the intracellular hormonal contents. While 0.01% ME caused approximately 90% reduction in the iPRL content of the lactotrophic cells, the highest tested dose of ME, i.e. 0.1%, showed only 20% reduction in the iGH content of the somatotrophic cells. Also, the minimum effective dose of ME to suppress iGH secretion was 10-fold higher than that required for the suppression of iPRL secretion. Significant suppression of iGH secretion was not observed until 120 min after the onset of ME incubation as opposed to 2-9 min for the suppression of iPRL. These findings, together with the lack of any direct effects of ME on the iGH molecule itself, strongly suggested that ME acted via different mechanisms and/or pathways in the somatotrophs and lactotrophs to suppress respective hormonal dynamics.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of activin/inhibin subunit messenger ribonucleic acids by gonadotroph adenomas.

As an initial step in determining whether activin could play a role in the development of gonadotroph adenomas, we attempted to determine if activin/inhibin subunit messenger ribonucleic acids (mRNAs) are expressed by these pituitary tumors. We selected 19 pituitary adenomas that had been excised by transsphenoidal surgery and determined by immunocytochemical criteria to be gonadotroph adenomas. Total RNA was extracted from these adenomas and reverse transcribed. The resulting pit-1 complementary DNA, expected to be present only in somatotroph, lactotroph, and thyrotroph cells, was amplified by the polymerase chain reaction to test the possibility that the adenoma tissue was contaminated by normal pituitary tissue. Only the 10 adenomas that did not express pit-1 mRNA were subjected to further analysis by polymerase chain reaction amplification of the adenoma reverse transcriptase products for the activin/inhibin beta B-, beta A-. and alpha-subunits. All 10 adenomas expressed beta B-subunit, none of the 10 expressed the beta A-subunit, and 6 of the 10 expressed the alpha-subunit. We conclude that gonadotroph adenomas express mRNAs for activin/inhibin beta B- and alpha-subunits. The relationship of beta B expression to the pathogenesis of gonadotroph adenomas remains to be determined.

Cloning of a cDNA encoding an ectoenzyme that degrades thyrotropin-releasing hormone.

Thyrotropin-releasing hormone (TRH) is an important extracellular signal substance that acts as a hypothalamic-releasing factor, which stimulates the release of adenohypophyseal hormones and functions as a neurotransmitter/neuromodulator in the central and peripheral nervous system. The inactivation of TRH after its release is catalyzed by an ectoenzyme localized preferentially on neuronal cells in the brain and on lactotrophic pituitary cells. This enzyme exhibits a very high degree of substrate specificity as well as other unusual properties. The activity of the adenohypophyseal enzyme is stringently controlled by estradiol and thyroid hormones, indicating that this enzyme itself may serve regulatory functions. Fragments of the enzyme isolated from rat or pig brain were generated by enzymatic digestion or cyanogen bromide cleavage, purified by reverse-phase HPLC, and sequenced. PCR amplification and screening of cDNA libraries from rat brain and pituitary led to the identification and isolation of a cDNA that encodes a protein of 1025 amino acids. The analysis of the deduced amino acid sequence was consistent with the identification of the enzyme as a glycosylated, membrane-anchored Zn metallopeptidase. Furthermore, Northern blot analysis demonstrated that the mRNA levels paralleled the tissue distribution of the enzyme and that in pituitary tissue the transcript levels rapidly increased when the animals were treated with triiodothyronine. Finally, transient transfection of COS-7 cells with this cDNA led to the expression of an active ectopeptidase that displayed the characteristics of the TRH-degrading ectoenzyme.

Immunohistochemical Demonstration of Estrogen Receptor in Human Adenohypophysial Cells and Pituitary Adenomas.

The localization of estrogen receptor in normal human pituitary glands and in human pituitary adenomas was investigated using immunoreactivity to polyclonal antibodies against human estrogen receptor in five normal pituitary samples and 45 specimens of pituitary adenoma. The relationship between the expression of estrogen receptor and the production of pituitary hormones was clarified using double immunohistochemical staining. Estrogen receptor was present in both cells of normal pituitary glands and pituitary adenomas. In normal pituitary glands, adenohypophysial cells were immunoreactive for estrogen receptor irrespective of hormone production. In pituitary adenomas, 35 of 45 specimens showed positive immunoreactivity to estrogen receptor. All cases of lactotroph cell adenoma, corticotroph cell adenoma, gonadotroph cell adenoma, and plurihormonal adenoma with clinical signs of acromegaly showed positive immunoreactivity to estrogen receptor. The present study demonstrates the intracellular localization of estrogen receptor in normal adenohypophysial cells of human pituitary glands and in human pituitary adenomas.

Increase in Pit-1 mRNA is not required for the estrogen-induced expression of prolactin gene and lactotroph proliferation.

Estrogen has been shown to stimulate lactotroph proliferation and expression of the prolactin (PRL) gene. Recently it has been established that Pit-1, a pituitary-specific transcription factor, is required for lactotroph proliferation. Furthermore, in vitro studies showed that an increase in the PRL promoter activity caused by estrogen was dependent of the amount of cotransfected Pit-1-expressing plasmid. These findings led us to examine whether the induction of Pit-1 mRNA is required for the estrogen-increased PRL gene expression in the rat anterior pituitary in vivo. Short term estrogen treatment was achieved by means of a single intramuscular injection of estradiol dipropionate. DNA synthesis, the levels of PRL and Pit-1 messenger RNAs in the anterior pituitary were determined. Estradiol dipropionate resulted in a significant increase in DNA synthesis 24 h after administration and in PRL mRNA after 48 h. In contrast, the Pit-1 mRNA level was not altered. Since Pit-1 is expressed not only in lactotroph but also in somatotroph and thyrotroph, and the lactotroph cell population has been reported to be less than 10% in the pituitary, the change in the Pit-1 mRNA level in lactotrophs was not seen following only short term estrogen treatment. An increase in the lactotroph cell population was therefore achieved by chronic estrogen treatment (subcutaneous implantation of a silastic tube containing 17 beta-estradiol powder for 30 days). This treatment resulted in the marked proliferation of lactotrophs and a 3-fold increase in PRL mRNA. However, no alteration in Pit-1 mRNA was observed. These results suggest that the increase in Pit-1 mRNA is not required for the estrogen-induced lactotroph proliferation or PRL gene expression.