RESUMO
BACKGROUND: Glycosylated prolactin (G-PRL) is considered as the major post-translational modification of prolactin (PRL) showing reduced lactotropic and mitogenic activities compared to non-glycosylated prolactin (NG-PRL). AIM: To evaluate the evolution of G-PRL in normoprolactinemic children and adolescents and to analyze possible variations in glycosylated/total prolactin (T-PRL) ratios. METHODS: T-PRL, G-PRL and NG-PRL were evaluated in 111 healthy female and male children and adolescents (4.1-18 years), classified as group 1 (Tanner I), group 2 (Tanner II-III) and group 3 (Tanner IV-V). G-PRL and NG-PRL were identified by chromatography on concanavalin-A-Sepharose. RESULTS: G-PRL/T-PRL (median-range): females, group 1: 0.59 (0.17-0.77), group 2: 0.56 (0.31-0.78), group 3: 0.60 (0.38-0.79); males, group 1: 0.64 (0.39-0.80), group 2: 0.61 (0.24-0.79), group 3: 0.62 (0.35-0.90); the p value is not significant among the different groups in both genders. G-PRL/T-PRL ratios do not change when comparing low (first quartile) versus high (third quartile) T-PRL levels in the different groups. CONCLUSION: Our study would appear to support cosecretion of G-PRL and NG-PRL from childhood to the end of puberty. Such cosecretion would not be dependent on sex steroid levels. It is important to point out that puberty does not change the proportions of G-PRL and NG-PRL.
Assuntos
Desenvolvimento do Adolescente , Desenvolvimento Infantil , Prolactina/análogos & derivados , Prolactina/sangue , Puberdade/sangue , Adolescente , Algoritmos , Argentina , Criança , Pré-Escolar , Cromatografia de Afinidade , Feminino , Glicosilação , Hormônios Esteroides Gonadais/sangue , Humanos , Masculino , Adeno-Hipófise/crescimento & desenvolvimento , Adeno-Hipófise/metabolismo , Prolactina/metabolismo , Puberdade/metabolismo , Radioimunoensaio , Sefarose/análogos & derivadosRESUMO
Since anterior pituitary expresses prolactin receptors, prolactin secreted by lactotropes could exert autocrine or paracrine actions on anterior pituitary cells. In fact, it has been observed that prolactin inhibits its own expression by lactotropes. Our hypothesis is that prolactin participates in the control of anterior pituitary cell turnover. In the present study, we explored the action of prolactin on proliferation and apoptosis of anterior pituitary cells and its effect on the expression of the prolactin receptor. To determine the activity of endogenous prolactin, we evaluated the effect of the competitive prolactin receptor antagonist Δ1-9-G129R-hPRL in vivo, using transgenic mice that constitutively and systemically express this antagonist. The weight of the pituitary gland and the anterior pituitary proliferation index, determined by BrdU incorporation, were higher in transgenic mice expressing the antagonist than in wild-type littermates. In addition, blockade of prolactin receptor in vitro by Δ1-9-G129R-hPRL increased proliferation and inhibited apoptosis of somatolactotrope GH3 cells and of primary cultures of male rat anterior pituitary cells, including lactotropes. These results suggest that prolactin acts as an autocrine/paracrine antiproliferative and proapoptotic factor in the anterior pituitary gland. In addition, anterior pituitary expression of the long isoform of the prolactin receptor, measured by real-time PCR, increased about 10-fold in transgenic mice expressing the prolactin receptor antagonist, whereas only a modest increase in the S3 short-isoform expression was observed. These results suggest that endogenous prolactin may regulate its own biological actions in the anterior pituitary by inhibiting the expression of the long isoform of the prolactin receptor. In conclusion, our observations suggest that prolactin is involved in the maintenance of physiological cell renewal in the anterior pituitary. Alterations in this physiological role of prolactin could contribute to pituitary tumor development.
Assuntos
Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica , Antagonistas de Hormônios/farmacologia , Adeno-Hipófise/metabolismo , Prolactina/análogos & derivados , Prolactina/fisiologia , Receptores da Prolactina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Antagonistas de Hormônios/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Tamanho do Órgão , Adeno-Hipófise/citologia , Adeno-Hipófise/efeitos dos fármacos , Adeno-Hipófise/patologia , Prolactina/antagonistas & inibidores , Prolactina/genética , Prolactina/metabolismo , Prolactina/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores da Prolactina/antagonistas & inibidores , Receptores da Prolactina/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Human prolactin (hPRL) is a 199 aminoacid protein hormone with a wide spectrum of biological activities which is best known for its stimulation of lactation and development of mammary gland. This protein contains only one potential asparagine-linked glycosylation site, which is partially (10-30%) occupied when the protein is synthesized in eukaryotic cells. Although the biological activity of glycosylated hPRL (G-hPRL) has been found to be approximately 4-fold lower than that of hPRL, its physiological function is not yet well defined. In order to better characterize and study this hormone variant, we carried out its laboratory scale purification from conditioned medium of genetically modified CHO cells that had been supplemented with cycloheximide. Addition of cycloheximide increased the absolute concentration of G-hPRL approximately 4-fold and the glycosylated versus non-glycosylated hPRL concentration ratio by approximately 7-fold. G-hPRL purification was carried out via a two-step process based on a cationic exchanger and a size-exclusion HPLC (HPSEC) column. Characterization was carried out by HPSEC, Western blotting, MALDI-TOF-MS and in vitro bioassay based on Nb2 and Ba/F3-LLP cells, the biological activity being of the same order (11-15 IU mg(-1)) in the two assays. Our results show that addition of cycloheximide can be an important strategy for increasing glycosylated protein production, facilitating the purification and characterization of these isoforms.
Assuntos
Cicloeximida/farmacologia , Prolactina/análogos & derivados , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Animais , Bioensaio , Western Blotting , Células CHO , Cromatografia em Gel , Cricetinae , Cricetulus , Meios de Cultivo Condicionados/farmacologia , Glicosilação/efeitos dos fármacos , Humanos , Camundongos , Prolactina/biossíntese , Prolactina/química , Prolactina/isolamento & purificação , Prolactina/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
PROBLEM: Identification of the cell types responsible for the synthesis of decidual prolactin-related protein (dPRP) in the pregnant mouse endometrium. METHOD OF STUDY: Histochemistry and immunocytochemistry were used to determine peri-implantation dPRP and perlecan distribution in the mouse uterus. RESULTS: We identified dPRP in pre-decidual and mature decidual cells from days 5 to 12 of pregnancy. On day 8, dPRP immunoreactivity was detected within cytoplasmic granules of a specific population of granulated decidual cells (GDCs). In mesometrial decidual cells, weak immunoreactivity was seen from days 7 to 14. Between days 11 and 14, dPRP was found in cytoplasm and in the extracellular matrix surrounding islands of spongiotrophoblast. Perlecan, a heparan sulfate proteoglycan, was co-localized with dPRP. CONCLUSION: GDCs are a putative source of dPRP in pregnant mice. Co-localization of perlecan with dPRP suggests that the former acts as a dPRP reservoir and facilitates its paracrine effect in developing placental tissues.
Assuntos
Endométrio/citologia , Endométrio/metabolismo , Prolactina/análogos & derivados , Animais , Grânulos Citoplasmáticos/metabolismo , Decídua/citologia , Decídua/imunologia , Decídua/metabolismo , Endométrio/imunologia , Feminino , Proteoglicanas de Heparan Sulfato/metabolismo , Imuno-Histoquímica , Células Matadoras Naturais/citologia , Camundongos , Microscopia Eletrônica , Gravidez , Prolactina/biossíntese , Prolactina/metabolismo , Vacúolos/metabolismoRESUMO
The synthesis, purification and characterization of G129R-hPRL and S179D-hPRL, the two better-studied antagonists of human prolactin (hPRL), is described. Both of these have been expressed for the first time, in their authentic form, by a stable CHO cell line, at secretion levels of 7.7 and 4.3 microg/10(6) cells/day, respectively. Previous studies had shown that these hPRL analogs, when produced in bacterial cytoplasm, consistently contained misfolded forms and multimers according to the specific denaturation, refolding and purification conditions. These versions also have an N-terminal extra methionine. An extensive physico-chemical characterization was carried out after a practical two-step purification process and included SDS-PAGE and Western blotting analysis, matrix-assisted laser-desorption ionization time-of-flight mass spectral (MALDI-TOF-MS) analysis, high-performance size-exclusion chromatography (HPSEC) and reversed-phase high-performance liquid chromatography (RP-HPLC). This last technique revealed a considerable difference in hydrophobicity due to a single amino acid substitution, with S179D-hPRL less (t(RR) = 0.85 +/- 0.010) and G129R-hPRL more (t(RR) = 1.10 +/- 0.013) hydrophobic than hPRL, where t(RR) is the relative retention time. The biological characterization was based on further refinement of a sensitive proliferation assay using the pro-B murine cell line (Ba/F3) transfected with the long form hPRL receptor cDNA such that the minimal detectable dose was 0.04 ng of hPRL/mL, the Ba/F3-LLP assay. On the basis of this assay, the relative residual agonistic activity of these two products, determined against a hPRL international standard in four independent assays, was 53 x 10(-3) for S179D-hPRL and 70 x 10(-5) for G129R-hPRL. We believe that the present synthesis and characterization could be extremely helpful for studies of these two proteins, which have been reported to antagonize tumor growth-promoting effects of hPRL in vivo in animal models of breast and prostate cancer.
Assuntos
Prolactina/análogos & derivados , Prolactina/farmacologia , Animais , Western Blotting , Células CHO , Cromatografia Líquida de Alta Pressão , Cricetinae , Cricetulus , Meios de Cultura , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Humanos , Prolactina/química , Proteínas Recombinantes/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Hyperprolactinemia without clinical manifestations has been reported in some patients with systemic lupus erythematosus (SLE) because an increase of prolactin (PRL) is produced due to the BIG/BIG molecular variant (molecular variant < 150 kD). This research project aimed to determine levels of PRL: its bioactive form, the little nonglycosylated form (NGPRL) and variants with decreased bioactivity such as the BIG/BIG and the little glycosylated (GPRL), in 29 women and five men with SLE. PRL was assayed by IRMA with a kit from Immunotech Laboratory, the BIG/BIG form by precipitation with polyethyleneglycol 6000, and the NGPRL and GPRL by chromatography on Concanavalin-A- Sepharose. Increased PRL was detected in seven patients (20.6%) of whom three had increased BIG/BIG, six had increased GPRL and only four had increased NGPRL. The three cases with increased BIG/BIG were contrasted by chromatography on Sephadex G-100. No increased PRL or any of the other variants assayed were found in men. Results were similar when PRL was evaluated in the same blood samples by a different IRMA (DPC Laboratory). The etiology of the hyperprolactinemia in some of these patients is unknown, but their lack of symptoms (galactorrhea or amenorrhea) could be due to the BIG/BIG forms and basically to the glycosylation of the hormone. As for the relation between PRL and SLE activity, we found that hyperprolactinemic patients were younger, had a shorter history of illness, although it was not statistically significant, and a higher SLEDAI score. This would indicate a relation between hyperprolactinemia and lupus activity. The patients with increased BIG/BIG form also had a very active illness at the time of the study.
Assuntos
Lúpus Eritematoso Sistêmico/metabolismo , Prolactina/análogos & derivados , Prolactina/sangue , Adulto , Feminino , Humanos , Hiperprolactinemia/sangue , Hiperprolactinemia/complicações , Lúpus Eritematoso Sistêmico/complicações , Masculino , Pessoa de Meia-Idade , Peso Molecular , Prolactina/químicaRESUMO
Decidual cells are endometrial fibroblasts that redifferentiate during pregnancy in several species of mammals. In this work, we describe a subpopulation of resident decidual cells in the mouse endometrium that are joined by intercellular junctions and have cytoplasmic granules. Decidualization was induced in pseudopregnant mice on the 4th day of pseudopregnancy by injection of 30 microl of arachis oil into the uterine lumen. The uteri were collected on day 8 of pseudopregnancy (at 4 p.m., 8 p.m. and 11 p.m.) and on day 9 (at 8 a.m.). The tissues were fixed for light and electron microscopy. During day 8 of pseudopregnancy, granulated cells were present at the antimesometrial pole of the endometrium; they were concentrated at the periphery of the antimesometrial decidua and disappeared on day 9 of pseudopregnancy. The cytoplasm of the granulated decidual cells had acidophilic granules that stained also with periodic acid-Schiff (PAS). These granules stained with anti-rat prolactin antibody in both light and electron microscope immunocytochemical preparations. Vacuoles of various sizes were always present in the granulated cells. A PAS-positive and prolactin-stained material was often deposited at the periphery of the vacuoles. Our results indicate that the granulated decidual cells are the source of decidual prolactin which accumulates in cytoplasmic granules. These granulated cells therefore form a transient gland in the mouse antimesometrial endometrium (granulated decidual gland).
Assuntos
Grânulos Citoplasmáticos/metabolismo , Decídua/citologia , Decídua/metabolismo , Prolactina/metabolismo , Animais , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/ultraestrutura , Feminino , Imuno-Histoquímica/métodos , Camundongos , Microscopia Eletrônica/instrumentação , Microscopia Eletrônica/métodos , Gravidez , Prolactina/análogos & derivadosRESUMO
Se investigaron los efectos de la prolactina porcina glicosilada (gpPrl) y la no glicosilada (ngpPrl) aislada y junto a la gonadotropina coriónica humana (HCG) en la acumulación de progesterona (P) y estradiol (E2). Se obtuvieron células de granulosa a partir de folículos preovulatorios de ratas inmaduras tratadas con gonadotropina obtenida de suero de yegua preñada (PMSG). Cuando se adicionaron concentraciones de 1, 10 y 100 ng/mL de gpPrl o ngPrl a cultivos celulares no estimulados con HCG aumentó la acumulación de P a las 72 h de incubación, aunque la gpPrl mostró un efecto estimulatorio más marcado. La acumulación de E2 se incrementó con la administración de concentraciones de 1 y 10 ng/mL de gpPrl junto con 0,1 UI/mL de HCG, la concentración de 100 ng/mL no mostró un efecto estimulatorio significativo. Los resultados obtenidos sugieren que la producción de P y E2 por células de granulosa de rata en cultivo depende de la forma de Prl usada en los experimentos así como de la presencia de HCG como agente estimulatorio de la esteroidogénesis en el medio de cultivo
Assuntos
Animais , Feminino , Ratos , Técnicas de Cultura de Células , Células da Granulosa , Gonadotropina Coriônica , Estradiol , Progesterona , Prolactina/análogos & derivados , Prolactina/isolamento & purificação , Ratos Sprague-DawleyRESUMO
Reaction of ovine prolactin (oPRL) with a 150-fold molar excess of N-acetylimidazole over protein content resulted in the modification of 2.5 tyrosine residues and 1.2 lysine residues. Acetylation greatly decreased the in vitro binding capacity to lactogenic sites. This binding capacity was partially restored by ammonium bicarbonate treatment, which removes O-acetyl groups from tyrosine residues but not N-acetyl groups from lysine residues. The modification extent of the tyrosine residues was determined. The results suggest that acetylation of tyrosine 44 or of tyrosine 96 is likely to be responsible for the decrease in binding activity of acetylated oPRL, and that one of these residues may play a role in the interaction of oPRL with lactogenic receptors.
Assuntos
Prolactina/análogos & derivados , Acetilação , Aminoácidos/análise , Animais , Dicroísmo Circular , Imidazóis , Técnicas In Vitro , Lisina/química , Microssomos Hepáticos/metabolismo , Fragmentos de Peptídeos/química , Prolactina/química , Prolactina/metabolismo , Ratos , Receptores da Prolactina/metabolismo , Ovinos , Espectrofotometria Ultravioleta , Tirosina/químicaRESUMO
The alpha-amino group of ovine prolactin (oPRL) and human growth hormone (hGH) was selectively modified by transamination with glyoxylic acid. No difference was found in the binding capacity of transaminated oPRL to rat liver lactogenic receptors with respect to its control, although both samples showed a decrease in its binding capacity with reference to the native hormone. This decrease was due to conformational changes caused by the reaction conditions and not by the transamination itself, as shown by the circular dichroism spectra. Transaminated hGH retained the full binding capacity of the hormone. These results suggest that the alpha-amino group is not relevant for the binding to lactogenic liver receptors in both lactogenic hormones.