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ABSTRACT
The 21-amino steroid U74006F is a potent inhibitor of lipid peroxidation and has been shown to affect beneficially the acutely injured central nervous system. Therapeutically, it is desirable for this compound to be devoid of steroid side-effects. We have demonstrated a significant (P < 0·001) inhibition of basal ACTH secretion from cultured rat pituitary cells during a 24-h incubation at concentrations (10–100 μmol/l) previously demonstrated to inhibit lipid peroxidation in vitro. U74006F also inhibited corticotrophin-releasing factor (CRF)-stimulated ACTH secretion significantly and the combination of dexamethasone and U74006F completely blocked CRF-41-stimulated ACTH secretion. Administration of U74006F in vivo (30 mg/kg, orally, every 6 h for 30 h) had no effect on ACTH levels in normal rats (84±38 vs 45±6 ng/l in control animals) but increased ACTH levels in adrenalectomized rats (1330±295 vs 464±79 ng/l in control animals, P < 0·02). This increase in ACTH was not observed when adrenalectomized animals were maintained on the same regime of U74006F for 5 days. Our data suggest that U74006F is capable of exerting inhibitory effects on ACTH secretion in vitro. In vivo, effects on ACTH secretion were stimulatory rather than inhibitory and only occurred short-term in adrenalectomized animals or chronically in adrenalectomized rats maintained on dexamethasone. No effects on the pituitary-adrenocortical axis were seen following short-term or chronic administration of U74006F in normal rats.
Journal of Endocrinology (1990) 126, 203–209
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ABSTRACT
The mechanism responsible for the suppression of GH secretion in hyperglycaemia and hypoglyceamia in rats has been investigated using perifusion of anterior pituitary cells. When perifused with Krebs-Ringer bicarbonate containing normal (5 mmol/l), high (20 mmol/l) and low (1 mmol/l) concentrations of glucose, the GH responses to GH-releasing factor (GRF) were 85 ± 5, 85·5 ± 5·4 and 89 ± 3·0 (s.e.m.)% respectively compared with the initial response to GRF at 5 mmol/l in each column. The mean GH response to GRF from anterior pituitary cells of normal rats was 6·58 ± 0·88 μg/three pituitaries, which was not statistically different from that of cells from rats with streptozotocin-induced diabetes (5·40 ± 0·68 μg/three pituitaries). It is concluded that GH suppression in diabetic rats and during hypoglycaemia is not mediated by changes in the GH response to GRF.
Journal of Endocrinology (1989) 122, 657–660
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ABSTRACT
We have previously shown that a heat-stable component of Russell's viper venom (RVV) releases GH in a dose-dependent manner from cultured rat anterior pituitary cells. We have now investigated the intracellular mechanisms involved in RVV-stimulated GH release by concomitant administration of RVV with known intracellular mediators in rat pituitary cells.
3-Isobutyl-1-methylxanthine (IBMX; 0·5 mmol/l), added to cultured rat anterior pituitary cells simultaneously with RVV, at concentrations up to a maximally effective dose of 10 μg/ml, increased GH release (3·7-fold, 4·0-fold and 2·0-fold; P < 0·001) compared with the effect of venom alone. These effects were additive, indicating that RVV and IBMX stimulate through different intracellular messengers. RVV failed to increase the formation of basal or IBMX-stimulated intracellular cyclic AMP (cAMP), confirming that RVV affects GH release through a cAMP-independent pathway. 12-0-Tetradecanoylphorbol-13-acetate (TPA; 0·1 μmol/l), added simultaneously with various doses of RVV (0·1–10 μg/ml), did not increase GH release beyond the maximal effect of RVV. This result indicates that RVV might be stimulating GH release through a similar mechanism to that of TPA (by activating protein kinase C).
When pituitary cells were perifused with Ca2+-free medium or verapamil (50 μmol/l), RVV-stimulated GH release was inhibited by 65 and 42% respectively. This reflects the recognized requirement of Ca2+ for secretory processes. However, RVV (10 μg/ml) had no significant effect on intracellular free Ca2+ concentrations as measured using the fluorescent Ca2+ probe quin-2.
These findings indicate that the mechanism of action of RVV on GH release is independent of a change in both cAMP levels and intracellular free Ca2+ concentrations, and is dependent upon protein kinase C.
Journal of Endocrinology (1990) 127, 111–117
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ABSTRACT
We have demonstrated that the novel hypothalamic peptide pituitary adenylate cyclase-activating poly-peptide (PACAP-38; 0·1–100 nmol/l) caused an increase in the release of GH, ACTH, LH and α-subunit and accumulation of intracellular cyclic AMP from dispersed rat anterior pituitary cells in static culture for 24 h. There were no significant effects on TSH or prolactin release over the same time-period. PACAP-38 (10 nmol/l) increased the release of GH by 1·3-fold (P<0·05), ACTH by 1·9-fold (P<0·05), LH by 3·5-fold (P<0·001) and α-subunit by 2·0-fold (P< 0·005) and the accumulation of intracellular cyclic AMP by >2-fold (P<0·001) after 24 h. However, the time-course for the effect of PACAP-38 (1 mmol/l) on hormone release and intracellular cyclic AMP levels showed a temporal dissociation. The effect of PACAP-38 on GH and ACTH levels did not reach significance until 24 h whereas the effect of PACAP-38 on LH and α-subunit release reached significance after 4 h implying a different mechanism of action for their release.
To investigate the PACAP-induced secretion of LH and α-subunit further, we examined the effects of PACAP after down-regulation of protein kinase C (PKC). PACAP-38 at a dose maximal for the stimulation of LH and α-subunit release (10 nmol/l) added together with the PKC activator, 12-0-tetradecanoyl-phorbol-13-acetate (TPA; 0·1 μmol/l) had no greater effect on LH and α-subunit release than TPA alone over a 4 h incubation period. Increasing the pretreatment time with TPA (0–5 h) at a dose (0·1 μmol/l) known to deplete PKC activity substantially, reduced the ability of PACAP-38 to stimulate LH and α-subunit release and intracellular cyclic AMP levels significantly. We conclude that the stimulatory actions of PACAP on LH and α-subunit relies in part on PKC activity.
Journal of Endocrinology (1992) 134, 33–41
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ABSTRACT
The direct effects of ketoconazole on ACTH secretion have been investigated using a rat pituitary cell culture system. Ketoconazole had no significant effects on basal ACTH secretion, corticotrophin-releasing factor- or arginine vasopressin-stimulated ACTH secretion, nor did it affect dexamethasone inhibition of ACTH secretion. In-vivo studies demonstrated an increased ACTH level (168 vs 76 ng/l) accompanied by a fall in plasma corticosterone (193 vs 307 μg/l) in normal rats given ketoconazole (24 mg/kg, five oral doses given 8 hourly). No effects were seen in adrenalectomized rats or at lower doses (6 mg/kg) in normal or adrenalectomized rats. A high dose of ketoconazole (24 mg/kg, twice daily oral doses) also caused increased ACTH levels in normal rats (129 vs 86 ng/l) when given for 7 days. No effects were seen in adrenalectomized rats or on plasma corticosterone levels in normal rats. Our data suggest that ketoconazole at these doses has no direct effects on pituitary ACTH secretion but causes an increase in plasma ACTH due to its inhibition of adrenal steroid synthesis.
J. Endocr. (1986) 108, 37–41
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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
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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
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ABSTRACT
In five healthy normal male volunteers, pretreatment with the cholinergic muscarinic antagonist pirenzepine (30 mg i.v.) almost abolished the growth hormone (GH) response to a maximal dose (120 μg i.v.) of growth hormone-releasing hormone (GHRH) (GH response at 40 min 5.6 ± 1.3 mU/l with GHRH and pirenzepine vs 40.8 ± 5.3 mU/l with GHRH alone, P <0.02). Concomitant i.v. infusion of galanin (40 pmol/kg/min) with pirenzepine not only restored but significantly potentiated the GH response to GHRH (GH at 40 min 72.2 ± 10.5 mU/l, P <0.001 vs GHRH and pirenzepine, P <0.02 vs GHRH alone). Previous studies have proposed that cholinergic pathways control GH release via samatostatin and this study suggests that galanin may act by modulating hypothalamic somatostatinergic tone either directly or, possibly, by facilitating cholinergic neurotransmission.
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ABSTRACT
In this study we have examined the effects of TRH, thyroid hormones and dopamine on the rat anterior pituitary content of neuromedin U-like immunoreactivity. Oral administration of TRH (20 mg/100 g per day) to euthyroid animals evoked a fivefold increase in peptide content after 12 days of treatment. This effect was found to be dependent upon circulating levels of thyroid hormone, since administration of TRH to thyroidectomized animals failed to show a similar effect without simultaneous treatment with tri-iodothyronine. The possibility that the TRH-induced increase in anterior lobe neuromedin U content reflected alterations in prolactin secretion or synthetic rate was also examined. Treatment of euthyroid animals with a dopamine agonist and antagonist was, however, without effect. These results demonstrate a unique relationship between TRH and thyroid hormone levels in increasing the anterior lobe content of neuromedin U immunoreactivity.
Journal of Endocrinology (1989) 122, 471–476
Veterinary Basic Sciences, Royal Veterinary College, Royal College Street, London NW1 0TU, UK
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Veterinary Basic Sciences, Royal Veterinary College, Royal College Street, London NW1 0TU, UK
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Veterinary Basic Sciences, Royal Veterinary College, Royal College Street, London NW1 0TU, UK
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Veterinary Basic Sciences, Royal Veterinary College, Royal College Street, London NW1 0TU, UK
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The insulinotrophic effects of glucagon-like peptide 1 (GLP-1) are mediated by its seven-transmembrane receptor (GLP-1R) in pancreatic β-cells. We have transiently transfected the GLP-1R and a proopiomelanocortin (POMC) promoter-driven human preproinsulin gene vector (pIRES) into the AtT-20 pituitary corticotrophic cell line, to investigate the possibility of creating a regulated, insulin-expressing cell line. Receptor expression was confirmed by RT-PCR and functionality was demonstrated by measuring changes in cAMP levels in response to GLP-1. Rapid (5 min) stimulation of cAMP production was observed with 100 nM GLP-1, 24 h after transfection of 2 μg GLP-1R DNA. AtT-20 cells co-transfected with GLP-1R and human glycoprotein hormone α-subunit or rat POMC promoters revealed GLP-1-stimulated cAMP activation of transcription. Co-transfection of the pIRES vector with the GLP-1R resulted in GLP-1-stimulated activation of POMC promoter-driven preproinsulin gene transcription but insulin secretion was not detected. However, using an adenoviral expression system to infect AtT-20 cells with GLP-1R and the preproinsulin gene (including 120 bp of its own promoter) resulted in a 6.4 ± 0.6-fold increase in cAMP and a 4.9 ± 0.8-fold increase in insulin secretion in response to 100 nM GLP-1. These results demonstrate, for the first time, functional GLP-1R-mediated preproinsulin gene transcription and secretion in a transplantable cell line.
