Effect of the GABAA agonist muscimol on prolactin secretion from human prolactin-secreting adenomas and GH3 rat pituitary tumour cells.

The effect of muscimol, a specific potent GABAA receptor agonist, on prolactin release from human prolactin-secreting tissue was investigated using a perifusion system. Perifusion studies on normal rat anterior pituitary tissue, which has identical GABA receptors to those found in normal human pituitary glands, show that muscimol has a specific biphasic effect on prolactin release. This is characterized by an initial transient stimulation (222.3 +/- 21.6% of basal) lasting for 5-10 min followed by a more prolonged inhibitory phase (63.9 +/- 3.1% inhibition of basal). Five human prolactin-secreting adenomas were studied, and in none of the tumours could a biphasic response be demonstrated. One of the prolactin-secreting adenomas had a blunted inhibitory response, but the other 4 showed no inhibitory effect of muscimol on prolactin release. Muscimol had no significant effect on basal or thyrotropin-releasing-hormone (TRH)-stimulated prolactin secretion from GH3 rat pituitary tumour cells. These studies suggest that the GABAergic effect on prolactin secretion is absent or altered in both rat and human prolactin-secreting tumour cells.     

High-level expression of biologically active human prolactin from recombinant baculovirus in insect cells

We examined the feasibility of high-level production of recombinant human prolactin, a multifunctional protein hormone, in insect cells using a baculovirus expression system. The human prolactin cDNA with and without the secretory signal sequence was cloned into pFastBac1 baculovirus vector under the control of polyhedrin promoter. Prolactin was produced upon infection of either Sf9 or High-Five cells with the recombinant baculovirus containing the human prolactin cDNA. The production of recombinant prolactin varied from 20 to 40 mg/L of monolayer culture, depending on the cell types. The prolactin polypeptide with its own secretory signal was secreted into the medium. N-terminal amino acid sequence analysis of the recombinant polypeptide purified from the culture medium indicated that the protein was processed similar to human pituitary prolactin. Carbohydrate analysis of the purified protein indicated that a fraction of the recombinant prolactin made in insect cells appeared to be glycosylated. Also, both secreted and nonsecreted forms of the recombinant prolactin in insect cells were biologically equivalent to the native human prolactin (pituitary derived) in the Nb2 lymphoma cell proliferation assay.     

催乳素及其受体样免疫活性在文昌鱼神经系统、哈氏窝和其它组织的分布

用兔抗人催乳素多克隆抗体和鼠抗人催乳索受体单克隆抗体对文昌鱼神经系统、哈氏窝和其它组织进行免疫组织化学研究。结果显示:催乳素免疫活性细胞及催乳素受体定位在文昌鱼脑泡、神经管、哈氏窝、轮器、内柱、消化管和性腺(卵巢和精巢),表明催乳素在文昌鱼有广泛分布,并且从进化观点来看,证明催乳素是一种高度保守的古老激素。双重免疫染色进一步揭示催乳素及其受体免疫反应阳性物质共存于同一卵母细胞胞膜和胞质以及精巢中精原细胞、初级与次级精母细胞和Sertoli细胞。研究结果首次证明了文昌鱼脑泡和哈氏窝以及其它组织能够合成和分泌催乳素,表明像脊椎动物一样,催乳素可能参与调节文昌鱼体内代谢和对环境的适应以及性腺发育,提示文昌鱼可能出现原始的脑泡-哈氏窝(催乳素)-靶细胞调控轴的雏形。本研究为文昌鱼哈氏窝内分泌学以及催乳素的起源与演化提供新的基础资料。     

Immunohistochemical Localization of Growth Hormone and Prolactin in the Pituitary Gland of Acipenser güldenstaedti Brandt (Chondrostei)

Abstract Prolactin (LTH) and growth hormone (GH) containing cells in A. güldenstaedti have been localized by means of anti-ovine prolactin and anti-bovine growth hormone respectively, coupled indirectly to peroxidase, and localized histochemically with hydrogen peroxide as substrate and 3,3′-diaminobenzidine as capturing agent. The distribution of the anti-prolactin positive cells has been demonstrated and correlated histologically with the acidophilic cells both in the rostral and proximal pars distalis. This cell type is elongated and arranged in follicles in the rostral pars distalis; in the proximal pars distalis they are smaller and oval, without any special orientation. Neither of the other cell types which are scattered among these acidophils contain prolactin. The anti-bovine growth hormone positive cells are evenly distributed in the proximal pars distalis above the hypophysial cleft, and some are also found in the pars intermedia. The anti-GH positive cells have been correlated histologically with the amphiphilic cells in the proximal pars distalis. These cells are arranged in cell cords in close contact with the secondary capillary plexus, near its origin from the primary capillary plexus covering the median eminence.     

Comparative immunoenzymatic localization of prolactin and growth hormone in human and rat pituitaries.

Twelve human and twelve rat pituitaries were stained by an immunohistochemical method using a rabbit anti-ovine prolactin serum, a rabbit anti-human growth hormone serum and a sheep anti-rabbit immunoglobulin serum conjugated with horseradish peroxidase. On the same pituitary section, growth hormone cells were stained brown by using 3-3'-diaminobenzidine as peroxidase substrate, and prolactin cells were stained purplish blue by using 4-chloro-1-naphtol. Growth hormone cells outnumbered prolactin cells, especially in human pituitaries where the proportion is at least 10:1. No cells containing both brown granules stained for growth hormone and blue granules stained for prolactin were found in any of the sections examined. In the fetal pituitaries, there was no apparent hypertrophy of the prolactin cells, although the circulating levels of the hromone are known to be as high in the fetus at term as in the mother and much higher than in nonpregnant women.     

Functional consequences of prolactin signalling in endothelial cells: a potential link with angiogenesis in pathophysiology?

Prolactin is best known as the polypeptide anterior pituitary hormone, which regulates the development of the mammary gland. However, it became clear over the last decade that prolactin contributes to a broad range of pathologies, including breast cancer. Prolactin is also involved in angiogenesis via the release of pro-angiogenic factors by leukocytes and epithelial cells. However, whether prolactin also influences endothelial cells, and whether there are functional consequences of prolactin-induced signalling in the perspective of angiogenesis, remains so far elusive. In the present study, we show that prolactin induces phosphorylation of ERK1/2 and STAT5 and induces tube formation of endothelial cells on Matrigel. These effects are blocked by a specific prolactin receptor antagonist, del1-9-G129R-hPRL. Moreover, in an in vivo model of the chorioallantoic membrane of the chicken embryo, prolactin enhances vessel density and the tortuosity of the vasculature and pillar formation, which are hallmarks of intussusceptive angiogenesis. Interestingly, while prolactin has only little effect on endothelial cell proliferation, it markedly stimulates endothelial cell migration. Again, migration was reverted by del1-9-G129R-hPRL, indicating a direct effect of prolactin on its receptor. Immunohistochemistry and spectral imaging revealed that the prolactin receptor is present in the microvasculature of human breast carcinoma tissue. Altogether, these results suggest that prolactin may directly stimulate angiogenesis, which could be one of the mechanisms by which prolactin contributes to breast cancer progression, thereby providing a potential tool for intervention.     

Prolactin-inducible proteins in human breast cancer cells

The mechanism of action of prolactin in target cells and the role of prolactin in human breast cancer are poorly understood phenomena. The present study examines the effect of human prolactin (hPRL) on the synthesis of unique proteins by a human breast cancer cell line, T-47D, in serum-free medium containing bovine serum albumin. [35S]Methionine-labeled proteins were analysed by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and fluorography. Treatment of cells with hPRL (1-1000 ng/ml) and hydrocortisone (1 microgram/ml) for 36 h or longer resulted in the synthesis and secretion of three proteins having molecular weights of 11,000, 14,000, and 16,000. Neither hPRL nor hydrocortisone alone induced these proteins. Of several other peptide hormones tested, only human growth hormone, a hormone structurally and functionally similar to hPRL, could replace hPRL in causing protein induction. These three proteins were, therefore, referred to as prolactin-inducible proteins (PIP). Each of the three PIPs was purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and specific antibodies were generated to them in rabbits. By immunoprecipitation and immunoblotting (Western blot) of proteins secreted by T-47D cells, it was demonstrated that the three PIPs were immunologically identical to one another. In addition, the 16-kDa and 14-kDa proteins (PIP-16 and PIP-14), and not the 11-kDa protein (PIP-11), incorporated [3H]glycosamine. Furthermore, 2-deoxyglucose (2 mM) and tunicamycin (0.5 micrograms/ml), two compounds known to inhibit glycosylation, blocked the production of PIP-16 and PIP-14, with a concomitant increase in the accumulation of PIP-11. These results indicate PIP-16 and PIP-14 are glycosylated variants of PIP-11. Finally, in vitro translation of poly(A)+ messenger RNA followed by immunoprecipitation revealed a 12.5-kDa protein, possibly the precursor form of PIPs. In addition, T-47D cells treated with hPRL plus hydrocortisone contained 10-fold more mRNA for PIPs than control cells, suggesting that the hormones' action is at the level of gene expression. Our finding represents a first demonstration of prolactin regulation of gene expression in human target cells. The human breast cancer cells, T-47D, appear to be an excellent model to afford future studies on the molecular action of prolactin and on the possible role of prolactin in human breast cancer.     

Studies on prolactin in human serum, urine and milk.

Prolactin activity was measured in serum, urine and milk using a specific human prolactin radioimmunoassay (RIA). Serum, urine and milk were parallel with the human prolactin standard in the RIA. There was no correlation between serum prolactin levels and urinary prolactin activity. Dialysis of urine samples resulted in complete loss of human prolactin activity while the addition of human prolactin to the urine resulted in the recovery of over 50% of the hormone after dialysis. Thus it was concluded that prolactin is not present in urine. In additional experiments it was observed that the RIA prolactin activity in urine was significantly correlated with the osmolality of the urine and that Na+ and K+ were contributory elements. On the other hand, prolactin was found in human milk and correlated well with the expected serum levels of this hormone. This latter finding is interesting because prolactin receptors have been shown to exist on the serosal side of the mammary epithelial cells. The presence of prolactin in milk suggests the possibility of other sites of action for this hormone in addition to the cell membrane.     

Culture of human pituitary prolactin and growth hormone cells

Summary Fragments of pituitary tissue obtained from a total of 37 patients with either breast cancer, diabetic retinopathy, galactorrhea, or acromegaly were dissociated into single cell suspensions prior to cell culture. Release of human growth hormone (hGH) and human prolactin (hPRL) into the culture medium was measured by radioimmunoassay. During a 3-week culture period, prolactin cells released 9–13 times the intracellular levels of hPRL at the time of seeding, whereas hGH release from growth hormone cells was only 1–2 times that of their initial intracellular level during this same time. Both growth hormone and prolactin cells retained distinctive ultrastructural features during culture. The prolactin cells responded to TRH stimulation by elevated release of PRL into the medium. No evidence for mitotic division of prolactin cells in vitro was found.This work was supported by NCI Contract NO 1-CB-23863     

Hydrophobic-interaction chromatography and anion-exchange chromatography in the presence of acetonitrile. A two-step purification method for human prolactin

Described is a two-chromatographic-step preparative-scale technique for the purification of human prolactin from a frozen pituitary homogenate. The method utilizes hydrophobic interaction chromatography on the mildly hydrophobic adsorbent phenyl-Sepharose CL-4B and anion-exchange chromatography on DEAE-cellulose in the presence of acetonitrile. Human prolactin was solubilized at pH10.0 after a prior extraction of pituitaries at pH4.0, the acid pH being ineffective at solubilizing human prolactin but capable of solubilizing large amounts of interfering protein. An 11-fold increase in the potency of the solubilized human prolactin was achieved in this manner. Prolactin could be adsorbed to phenyl-Sepharose at low ionic strengths (I<0.01); few other proteins were adsorbed under these conditions. This is a demonstration of the hydrophobic nature of human prolactin. The amount of phenyl-Sepharose was limited to the minimum (35mg of protein/g of phenyl-Sepharose) necessary to adsorb human prolactin, further reducing the uptake of other pituitary protein. Desorption was achieved by using an acetonitrile gradient (0–30%, v/v), resulting in a purification of human prolactin of 85-fold and recovery of 78%. Acetonitrile (20%, v/v) was also included in all buffers for DEAE-cellulose chromatography, increasing the resolution and recovery of human prolactin, apparently by minimizing non-ionic interactions with the matrix. Prolactin (10mg) was recovered from 63g if pituitaries, an overall recovery of 58%. It was homogeneous by gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, contained less than 0.1% somatotropin (growth hormone), on iodination demonstrated more than 95% binding to excess anti-(human prolactin) serum and could be displaced from anti-(human prolactin) serum in a manner indistinguishable from the serum of a patient with a human prolactin-secreting adenoma.     

Human corpus luteum: Immunocytochemical evidence for presence of prolactin

Summary Six human corpora lutea (day 17–25) of the menstrual cycle and 4 ovarian stromal tissues from 7 cycling women were examined for the presence of the hormone, prolactin, by immunohistochemistry using the indirect peroxidase-antiperoxidase method. After mounting tissue sections of 4 m, endogenous peroxidases were removed with hydrogen peroxide and the sections were incubated for l h at room temperature followed by 16 h at 4° C with a highly specific antisera for human prolactin, nonimmunized normal rabbit serum for a control reaction, or antiserum preadsorbed with excess human prolactin for specificity determination. Following the reaction with the second antibody (goat antirabbit IgG) for l h at room temperature, prolactin was localized using peroxidase anti-peroxidase and 3.3-diaminobenzidine as the chromogen. Prolactin was present and could be localized in the luteal cells of all 6 corpora lutea, but not in any of the ovarian stroma studied. Human adenohypophysis served as a positive tissue control for prolactin immunopositive staining. The localization of immunoreactive prolactin in the corpus luteum demonstrates directly the presence of this hormone in the human ovary, adding further evidence for its role in luteal function.     

Localization of prolactin-like immunoreactivity in grafted human sweat glands

Using a double-antibody immunoperoxidase technique, we demonstrated human prolactin-like material in some cells of the secretory coil and in luminal duct cells of sweat glands of human skin which had been carried as grafts on mice for 32-35 weeks. It therefore seems likely that a population of cells in the secretory coil synthesizes prolactin and can be considered as diffuse peripheral endocrine cells. The prolactin may function locally to regulate sweat electrolyte concentration.     

Haemolytic and bactericidal activity of serum from the ring dove (Streptopelia risoria) after treatment with exogenous prolactin

The purpose of this study was to elucidate the effect of exogenous prolactin on the haemolytic and bactericidal capacity of serum obtained from ring doves (Streptopelia risoria) previously injected with either bovine serum albumin or saline solution. Haemolytic activity was measured in CH-50 units (which represents the capacity of serum complement to lyse 50% of sheep red blood cells in the presence of specific antibody) and the bactericidal activity was estimated from the number of colony-forming units ofStaphylococcus aureus which survived after 24 h of incubation in the presence of serum. The results indicated that: (1) bovine serum albumin stimulated both haemolytic and bactericidal activity, the highest values occurring 24 h and 4 days after administration, respectively. (2) Prolactin induced an increase in the haemolytic activity of complement. (3) The administration of bovine serum albumin to animals previously treated with prolactin produced a greater stimulation than either bovine serum albumin or prolactin alone.Abbreviations BSA bovine serum albumin - CFT complement fixation test - CFU colony-forming units - CH-50 units, the reciprocal of the complement dilution with 50% lysis of the hemolysin-treated erythrocytes - IU international units - PBS phosphate-buffered saline solution - SS saline solution     

转基因小鼠乳腺上皮细胞的体外培养及其对催乳素反应的研究

实现转基因生物乳腺反应器对外源蛋白的高效表达是目前生物制药亟待解决的难题。催乳素对泌乳期乳蛋白的合成与分泌具有重要的调控功能。通过转基因小鼠乳腺上皮细胞模型的建立,研究催乳素如何调控乳蛋白的表达,为提高乳腺反应器高效表达外源蛋白提供技术及理论支撑。应用机械破碎及胶原酶消化法,经差速贴壁纯化,成功培养含人转铁蛋白基因的小鼠乳腺上皮细胞,细胞上清液中检测到人转铁蛋白表达。细胞经牛催乳素诱导后人转铁蛋白的表达水平明显升高。利用转基因小鼠乳腺上皮细胞模型,可以进行催乳素和环境因素等对乳腺上皮细胞合成及分泌蛋白能力影响的研究。     

Microcapillary clonogenic assays for human marrow hematopoietic progenitor cells

The capillary clonogenic cell assay was developed and adapted to culture myeloid and erythroid colonies from human bone marrow cells. The plating efficiencies for femoral bone marrow granulocyte-macrophage progenitors (CFU-gm), erythroid colony-forming units (CFU-e) and erythroid burst-forming units (BFU-e) were 0.143%, 0.229% and 0.141%, respectively. Standard bone marrow progenitor Petri dish assays require a total culture volume of 1 ml per dish, and as such are not suitable for the small numbers of cells often obtained from human bone marrow samples. The microcapillary assay as developed and standardized in our laboratory has the unique advantage of being able to utilize small numbers of cells. This technique is suitable for evaluating the myelotoxicity of investigational new anti-cancer and anti-HIV agents and for further investigation of the mechanisms underlying chemotherapy-induced bone marrow toxicity.     

Prolactin and growth hormone cells in the human hypophysis: A study with immunoenzyme histochemistry and differential staining

Growth hormone and prolactin cells were immunostained in human hypophyses with antibody against rat growth hormone or prolactin and the peroxidase-antiperoxidase complex. Growth hormone cells were round and, in normal pituitaries, arranged in sizable groups. Prolactin cells occurred singly and were less numerous; they were often extensively branched. Only a few prolactin cells stained with carmoisine. Incubation of the antibody with an excess of the appropriate antigen greatly diminished or abolished immunostaining; absorption of anti-prolactin with growth hormone often enhanced it. Prolactin cells were somewhat hypertrophied and hyperplastic in a neonate. Many of them stained with carmoisine. An even greater hypertrophy and hyperplasia of these cells (which pushed apart the growth hormone cells) was found in a lactating woman. Immunostained giant prolactin cells were also observed. Staining of the prolactin cells with carmoisine was extensive. Upon prolonged exposure to anti-growth hormone antibody, ACTH/MSH cells also showed immunostaining which was abolished by absorption of the antiserum with growth hormone but not with synthetic 1-24ACTH. Growth hormone cells evidently correspond to the alpha acidophils of Romeis, prolactin cells in lactation to his eta cells; the relation of his epsilon cells to the pleomorphic "resting" prolactin cells is not clear.     

In vitro studies of prolactin inhibition of luteinizing hormone action on Leydig cells of rats and mice

Previous in vivo studies have shown that in male rabbits prolactin inhibits the testosterone production stimulated by luteinizing hormone (LH) or human chorionic gonadotropin (hCG). This inhibition has now been studied in vitro using both mouse and rat testicular interstitial cells. First, the dose response of human LH (hLH) stimulation of testosterone was studied in detail using testicular interstitial cells from both species. Next, a small but stimulatory dose of hLH was selected and extensive prolactin doses were studied in vitro. NIH B-6 (bovine) prolactin in varying doses was added to the interstitial cells 30 min prior to the addition of a constant dose of hLH. Under these circumstances prolactin inhibited LH action over a wide range of doses. In both species a biphasic dose-response curve existed: large doses of 100 to 1000 ng/ml produced less inhibition or augmented LH action, compared to smaller doses. Next, entire hLH dose-response curves were produced in the presence of three doses of prolactin (0.33, 33, and 1000 ng/ml) as well as in the absence of prolactin. The addition of prolactin shifted the hLH dose-response curve to the right and depressed the maximal response in comparison to the curve without prolactin. Finally, inhibitory doses of prolactin resulted in no detectable change in LH receptor number as estimated from Scatchard plots. It is concluded that prolactin inhibits LH action on interstitial cells as determined by rate of testosterone production except at very large doses of prolactin where LH action is less inhibited or augmented. The inhibitory action of prolactin in this in vitro interstitial cell assay was not accompanied by a decrease in LH receptor number. Thus, a postreceptor action is likely to be involved.