Flow cytometric analysis of functional anterior pituitary cells from female rats

1990 ◽  
Vol 126 (2) ◽  
pp. 261-NP ◽  
Author(s):  
D. Wynick ◽  
M. S. Venetikou ◽  
R. Critchley ◽  
J. M. Burrin ◽  
S. R. Bloom
Keyword(s):  
Cell Surface ◽  
Cell Size ◽  
Anterior Pituitary ◽  
Laser Light ◽  
Female Rats ◽  
Pituitary Cells ◽  
Light Scatter ◽  
Cell Sorter ◽  
Anterior Pituitary Cells ◽  
Surface Labelling

ABSTRACT Laser-light scatter signals generated from living cells provide useful information with regard to both cell size (forward-angle light scatter) and granularity (ninety-degree or perpendicular light scatter). By measuring angles of light scatter and fluorescence, a fluorescence-activated cell sorter is capable of analysing and sorting cells on the basis of their size, granularity and cell-surface fluorescence. Using an electronically programmable individual cell sorter we were able to analyse single, viable, dispersed anterior pituitary cells of the female rat on the basis of their laser light scatter characteristics. Two distinct populations of differing granularity were defined: 26±2·2% (mean ± s.e.m.) were more granular and 74±3·5% less granular. Acutely dispersed anterior pituitary cells were labelled with antibodies against four of the anterior pituitary hormones, and cell size and granularity were compared amongst the different hormonal cell types. Somatotrophs were the most granular cell type, gonadotrophs were the largest and corticotrophs the smallest, whilst lactotrophs were of intermediate size. Labelling was demonstrated to be dependent upon the secretory state of the cell. Hypothalamic stimulating factors increased cell-surface labelling, whilst dopamine and somatostatin decreased labelling. These changes compare favourably with published data obtained by immunocytochemistry. Using dual-colour fluorescence cell surface labelling we were unable to define a population of cells secreting both prolactin and growth hormone (mammosomatotrophs). Journal of Endocrinology (1990) 126, 261–268

Purification of functional lactotrophs and somatotrophs from female rats using fluorescence-activated cell sorting

1990 ◽  
Vol 126 (2) ◽  
pp. 269-274 ◽  
Author(s):  
D. Wynick ◽  
R. Critchley ◽  
M. S. Venetikou ◽  
J. M. Burrin ◽  
S. R. Bloom
Keyword(s):  
Cell Surface ◽  
Cell Sorting ◽  
Anterior Pituitary ◽  
Cell Types ◽  
Female Rats ◽  
Normal Female ◽  
Pituitary Cells ◽  
Female Rat ◽  
Fluorescence Activated Cell Sorting ◽  
Surface Labelling

ABSTRACT As the secretory granules of anterior pituitary cells fuse with the cell surface, there would appear to be sufficient hormone present on the cell surface to be labelled by polyclonal hormone antibodies and thus analysed by flow cytometry. We have therefore applied fluorescence-activated cell sorting to these labelled pituitary cells. Percentage purity and depletion of other cell types was assessed by immunocytochemistry and the reverse haemolytic plaque assay (RHPA). Results demonstrate that fluorescence-activated cell sorting allows almost complete purification of functional lactotrophs and somatotrophs to 96·7 ±1·7 (s.e.m.)% and 98±1·0% respectively by immunocytochemistry, and to 95·8 ±1·1% and 97±0·8% respectively by RHPA. Depletion of other anterior pituitary cell types to less than 2% was demonstrated by both immunocytochemistry and RHPA. Fluorescence-activated cell sorting to this degree of purity was routinely possible with cell yields of 91 ±3·4%. To obtain such purity/depletion, it was necessary to use specific antisera of high titre, at concentrations which ensured maximal cell-surface labelling associated with maximal stimulation of hormonal secretion by the appropriate hypothalamic stimulatory factor. Separating cells on the basis of the intensity of prolactin cell-surface labelling demonstrated a low level of binding of the prolactin antibody to gonadotrophs (but not of sufficient fluorescence intensity to be sorted into the prolactin enriched population), raising the possibility of prolactin receptors on gonadotrophs. We were unable to demonstrate the presence of mammosomatotrophs in the normal female rat, since purified lactotrophs did not contain or secrete GH nor did purified somatotrophs contain or secrete prolactin. Journal of Endocrinology (1990) 126, 269–274

Identification of epidermal growth factor-secreting cells in the anterior pituitary of lactating female rats

1996 ◽  
Vol 148 (2) ◽  
pp. 319-324 ◽  
Author(s):  
A Mouihate ◽  
D Verrier ◽  
J Lestage
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Anterior Pituitary ◽  
Plaque Assay ◽  
Female Rats ◽  
Pituitary Cells ◽  
Cellular Origin ◽  
Anterior Pituitary Cells ◽  
Epidermal Growth ◽  
Reverse Haemolytic Plaque Assay

Abstract Epidermal growth factor (EGF) is synthesized and secreted by mammalian anterior pituitary cells. It stimulates GH and prolactin (PRL) secretion, but the cellular origin of EGF is relatively unexplored. The objective of this study was to characterize the cells that secrete EGF in the anterior pituitary of lactating rats. An EGF reverse haemolytic plaque assay (RHPA) was used to identify EGF-secreting cells and this RHPA was combined with immunofluorescence using antibodies to the six major adenohypophysial hormones (i.e. PRL, GH, LH, FSH, TSH and ACTH). Approximately 20% (20·33 ± 2·96%) of the cells in the pituitary of lactating rats secrete EGF. The EGF-secreting cell population was composed of the following labelled cells: PRL (27%), GH (20%), LH (18%), FSH (14%), TSH (14%) and ACTH (5%). The present study showed that EGF is released by a subpopulation of anterior pituitary cells composed of all the classic hormone-containing cells. Journal of Endocrinology (1996) 148, 319–324

scholarly journals Pathogenesis of Anti–PIT-1 Antibody Syndrome: PIT-1 Presentation by HLA Class I on Anterior Pituitary Cells

2019 ◽  
Vol 3 (11) ◽  
pp. 1969-1978 ◽  
Author(s):  
Keitaro Kanie ◽  
Hironori Bando ◽  
Genzo Iguchi ◽  
Keiko Muguruma ◽  
Ryusaku Matsumoto ◽  
...  
Keyword(s):  
Cell Surface ◽  
Anterior Pituitary ◽  
Hla Class I ◽  
Class I ◽  
Gh3 Cells ◽  
Proximity Ligation Assay ◽  
Pituitary Cells ◽  
Anterior Pituitary Cells ◽  
Hla Class I Molecules ◽  
Presentation Pathway

Abstract Context Anti–pituitary-specific transcriptional factor-1 (anti–PIT-1) antibody syndrome is characterized by acquired and specific deficiencies in growth hormone, prolactin, and thyroid-stimulating hormone. Although PIT-1–reactive cytotoxic T lymphocytes (CTLs) have been speculated to recognize anterior pituitary cells and to cause the injury in the pathogenesis of the syndrome, it remains unclear whether endogenous PIT-1 protein is processed through the proteolytic pathway and presented as an antigen on anterior pituitary cells. Objective To examine how PIT-1 protein is processed and whether its epitope is presented by major histocompatibility complex (MHC)/HLA class I on anterior pituitary cells. Materials and Methods Immunofluorescence staining and proximity ligation assay (PLA) were performed using anti–PIT-1 antibody and patients’ sera on PIT-1–expressing cell line GH3 cells and human induced pluripotent stem cell (iPSC)-derived pituitary tissues. Results PIT-1 was colocalized with MHC class I molecules, calnexin, and GM130 in the cytosol. PLA results showed that PIT-1 epitope was presented by MHC/HLA class I molecules on the cell surface of GH3 cells and iPSC-derived pituitary cells. The number of PIT-1/HLA complexes on the cell surface of pituitary cells in the patient was comparable with that in the control subject. Conclusions Our data indicate that PIT-1 protein is processed in the antigen presentation pathway and that its epitopes are presented by in MHC/HLA class I on anterior pituitary cells, supporting the hypothesis that PIT-1–reactive CTLs caused the cell-specific damage. It is also suggested that number of epitope presentation was not associated with the pathogenesis of anti–PIT-1 antibody syndrome.

PACAP receptor mediated cAMP/PKA activity in Cu-GnRH stimulated anterior pituitary cells of female rats

2013 ◽  
Vol 13 ◽  
pp. 31
Author(s):  
Alina Gajewska ◽  
Marlena Zielińska ◽  
Ewa Wolińska-Witort ◽  
Gabriela Siawrys
Keyword(s):  
Anterior Pituitary ◽  
Female Rats ◽  
Pituitary Cells ◽  
Anterior Pituitary Cells

Characterization of relaxin-stimulated cyclic AMP in cultured rat anterior pituitary cells: influence of dopamine, somatostatin and gender

1989 ◽  
Vol 3 (3) ◽  
pp. 175-182 ◽  
Author(s):  
M. J. Cronin ◽  
T. Malaska
Keyword(s):  
Cyclic Amp ◽  
Anterior Pituitary ◽  
Physiological Role ◽  
Female Rats ◽  
Continuous Exposure ◽  
Pituitary Cells ◽  
Maximal Effect ◽  
Adult Males ◽  
Anterior Pituitary Cells ◽  
Stimulation Of

ABSTRACT Relaxin is a hormone associated with pregnancy that relaxes uterine smooth muscle and softens the connective tissues of the cervix and pelvis. In spite of these well-characterized tissue responses, the second messenger system linked to the relaxin receptor and the range of target tissues are only modestly understood. We found that relaxin enhanced the cyclic AMP levels in anterior pituitary cells from adult female rats. Relaxin induced a maximal 5·7±0·5-fold (mean±s.e.m.) stimulation of cyclic AMP accumulation and had an excitatory concentration for half maximal effect (EC50) of 0·4±0·1 nm, while human relaxin A and B chains had no such activity (EC50> 1 μm). Pertussis toxin amplified the efficacy of relaxin by 1·5±0·1-fold, indicating the intervention of a G coupling protein. The response to relaxin was reversible with washing, and desensitized slowly with continuous exposure to relaxin. In an attempt to define the physiological role for relaxin at the anterior pituitary, we found that two of the major hypophysiotrophic hormones of the brain (dopamine and somatostatin) markedly inhibited the relaxin stimulation of cyclic AMP. There was also a significant correlation of the response magnitude with the gender of the donor rat. Anterior pituitary cells from adult males exhibited a mean twofold maximal stimulation after relaxin, compared with the sixfold increase measured in cells from female rats. We hypothesize a novel physiological function of relaxin, that of signalling the feminine anterior pituitary.

scholarly journals The hormonal status modulates the effect of neurokinin A on prolactin secretion in female rats

1998 ◽  
Vol 159 (3) ◽  
pp. 389-395 ◽  
Author(s):  
D Pisera ◽  
S Theas ◽  
A De Laurentiis ◽  
M Lasaga ◽  
B Duvilanski ◽  
...  
Keyword(s):  
Pituitary Gland ◽  
Anterior Pituitary ◽  
In Vitro Release ◽  
Anterior Pituitary Gland ◽  
Female Rats ◽  
Male Rats ◽  
Neurokinin A ◽  
Pituitary Cells ◽  
Anterior Pituitary Cells ◽  
Ovx Rats

We have previously reported that neurokinin A (NKA), a tachykinin closely related to substance P, increases the release of prolactin (PRL) from the anterior pituitary gland of male rats, but not from pituitaries of ovariectomized (OVX) female rats. In this study, we evaluated the influence of estrogens in the action of NKA on PRL secretion in female rats. NKA stimulated the in vitro release of PRL from pituitary glands of OVX-chronically estrogenized rats, and of proestrus and estrus rats, but had no effect in anterior pituitaries of diestrus rats. In addition, we observed that cultured anterior pituitary cells of OVX rats responded to NKA only when they were incubated for 3 days in the presence of estradiol 10(-9) M. This effect was blocked by L-659,877, an NK-2 receptor antagonist. We also studied the action of NKA on PRL release during lactation. The response of anterior pituitary cells to NKA was variable over this period. The maximal sensitivity to NKA was observed at day 10 of lactation. Furthermore, the blockade of endogenous NKA by the administration of an anti-NKA serum to lactating rats reduced the PRL surge induced by the suckling stimulus. These results show that the responsiveness of the anterior pituitary gland of female rats to NKA is modulated by the endocrine environment, and suggest that NKA may participate in the control of PRL secretion during the estrus cycle and lactation.

Estrogen increases the release of epidermal growth factor from individual pituitary cells in female rats

1995 ◽  
Vol 146 (3) ◽  
pp. 495-500 ◽  
Author(s):  
A Mouihate ◽  
J Lestage
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Anterior Pituitary ◽  
Plaque Assay ◽  
Female Rats ◽  
Pituitary Cells ◽  
Anterior Pituitary Cells ◽  
Epidermal Growth ◽  
The Mean ◽  
Estradiol 17Β

Abstract Rat mixed anterior pituitary cells from cycling females were microscopically demonstrated to produce epidermal growth factor (EGF), in culture, using a reverse hemolytic plaque assay. Anterior pituitary cells of proestrous female rats secrete more EGF than those of metestrous rats, as evaluated by the mean area and the percentage of EGF plaque-forming cells. Estradiol-17β (10 nm) treatment of metestrous culture for 24 h increased significantly the size of EGF plaque-forming cells (from 1290±58 to 2566 ± 57 μm2, P<0·01) and the percentage of EGF plaque-forming cells (from 20·57 to 28·19%; P<0·01) to a level as high as observed in proestrous cultures (basal mean area 2171 ± 157 μm2, and basal percentage of EGF plaque-forming cells 22·71%). These results suggest that the hormonal status of female rats influences EGF secretion in the anterior pituitary gland and that estradiol is implicated in this phenomenon. Journal of Endocrinology (1995) 146, 495–500

Further studies on the regulation, localization and function of the TRH-like peptide pyroglutamyl-glutamyl-prolineamide in the rat anterior pituitary gland

1995 ◽  
Vol 146 (2) ◽  
pp. 293-300 ◽  
Author(s):  
J M M Rondeel ◽  
W Klootwijk ◽  
E Linkels ◽  
P H M Jeucken, W ◽  
L J Hofland ◽  
...  
Keyword(s):  
Body Weight ◽  
Luteinizing Hormone ◽  
Pituitary Gland ◽  
Anterior Pituitary ◽  
Anterior Pituitary Gland ◽  
Female Rats ◽  
Pituitary Cells ◽  
Anterior Pituitary Cells ◽  
Lhrh Antagonist

Abstract Recent evidence shows that thyrotrophin-releasing hormone (TRH) immunoreactivity in the rat anterior pituitary gland is accounted for by the TRH-like tripeptide prolineamide-glutamyl-prolineamide (pGlu-Glu-ProNH2, <EEP-NH2). The present study was undertaken to investigate further the regulation, localization and possible intrapituitary function of <EEP-NH2. Anterior pituitary levels of <EEP-NH2 were determined between days 5 and 35 of life, during the oestrous cycle and after treatment with the luteinizing hormone-releasing hormone (LHRH) antagonist Org 30276. Treatment of adult males with the LHRH antagonist either for 1 day (500 μg/100 g body weight) or for 5 days (50 μg/100 g body weight) reduced anterior pituitary <EEP-NH2 levels by 25–30% (P<0·05 versus saline-treated controls). Anterior pituitary <EEP-NH2 increased between days 5 and 35 of life. In females, these levels were 2- to 3-fold higher (P<0·05) than in males between days 15 and 25 after birth; these changes corresponded with the higher plasma follicle-stimulating hormone (FSH) levels in the female rats. After day 25, <EEP-NH2 levels in female rats decreased in parallel with a decrease in plasma FSH. Injections with the LHRH antagonist (500 μg/100 g body weight), starting on day 22 of life, led to reduced contents of <EEP-NH2 in the anterior pituitary gland of female rats on days 26 and 30 (55 and 35% decrease respectively). Levels of <EEP-NH2 in the anterior pituitary gland did not change significantly during the oestrous cycle. Fractionation of anterior pituitary cells by unit gravity sedimentation was found to be compatible with the localization of <EEP-NH2 in gonadotrophs. In vitro, <EEP-NH2 dose-dependently inhibited TRH-stimulated growth hormone (GH) release from anterior pituitary cells obtained from neonatal rats, but no consistent effects were seen on the in vitro release of luteinizing hormone (LH), FSH, prolactin (PRL) or thyroid-stimulating hormone (TSH) under basal or TRH/LHRH-stimulated conditions. Furthermore, <EEP-NH2 did not affect the in vitro hormone release by anterior pituitary cells obtained from adult rats. In vivo, <EEP-NH2 (0·3–1·0 μg intravenously) did not affect plasma PRL, TSH, LH, FSH and GH in adult male rats. We conclude that <EEP-NH2 in the anterior pituitary gland is regulated by LHRH, is probably localized in gonadotrophs and may play a (paracrine) role in neonatal GH release. Journal of Endocrinology (1995) 146, 293–300

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