Evidence for an internal and functional circadian clock in rat pituitary cells.

In primary cultures of rat pituitary cells and in a pituitary sommatolactotroph cell line (GH4C1), endogenous core-clock- as well as hormone-genes such as prolactin displayed a rhythmic expression pattern, fitted by a sinusoidal equation in which the period value was close to the circadian one. This is consistent with the presence of a functional circadian oscillator in pituitary cells whose importance was ascertained in GH4C1 cell lines stably expressing a dominant negative mutant of BMAL1. In these cells, both endogenous core-clock- and prolactin-genes no more displayed a circadian pattern.

Inactivation of transcription factor pit-1 to target tumoral somatolactotroph cells.

The treatment of growth hormone (GH)- and prolactin (PRL)-secreting tumors resistant to current therapeutic molecules (somatostatin and dopamine analogues) remains challenging. To target these tumors specifically, we chose to inactivate a gene coding for a crucial factor in cell proliferation and hormonal regulation, specifically expressed in pituitary, by using a dominant-negative form of this gene involved in human pituitary deficiencies: transcription factor Pit-1 (POU1F1) mutated on arginine 271 to tryptophan (R271W). After lentiviral transfer, the effect of R271W was studied in vitro on human tumoral somatotroph and lactotroph cells and on the murine mammosomatotroph cell line GH4C1 and in vivo on GH4C1 subcutaneous xenografts in nude mice.

Conditional overexpression of the wild-type Gs alpha as the gsp oncogene initiates chronic extracellularly regulated kinase 1/2 activation and hormone hypersecretion in pituitary cell lines.

In pituitary cells, activation of the cAMP pathway by specific G protein-coupled receptors controls differentiative functions and proliferation. Constitutively active forms of the alpha subunit of the heterotrimeric G(s) protein resulting from mutations at codon 201 or 227 (gsp oncogene) were first identified in 30-40% of human GH-secreting pituitary adenomas. This rate of occurrence suggests that the gsp oncogene is not responsible for initiating the majority of these tumors.

Regulation of the RAP1/RAF-1/extracellularly regulated kinase-1/2 cascade and prolactin release by the phosphoinositide 3-kinase/AKT pathway in pituitary cells.

In pituitary cells, prolactin (PRL) synthesis and release are controlled by multiple transduction pathways. In the GH4C1 somatolactotroph cell line, we previously reported that MAPK ERK-1/2 are a point of convergence between the pathways involved in the PRL gene regulation. In the present study, we focused on the involvement of the phosphoinositide 3-kinase (PI3K)/Akt pathway in the MAPK ERK-1/2 regulation and PRL secretion in pituitary cells.

Lentiviral vectors efficiently transduce human gonadotroph and somatotroph adenomas in vitro. Targeted expression of transgene by pituitary hormone promoters.

Despite important advances in human therapeutics, no specific treatment for both non-functioning gonadotroph and resistant somatotroph adenomas is available. Gene transfer by viral vectors can be considered as a promising way to achieve a specific and efficient treatment. Here we show the possibility of efficient gene transfer in human pituitary adenoma cells in vitro using a human immunodeficiency virus (HIV)-type 1-derived vector.

Epidermal growth factor triggers an original, caspase-independent pituitary cell death with heterogeneous phenotype.

Programmed cell death (PCD) is physiologically involved in the regulation of cell division and differentiation. It encompasses caspase-dependent mitochondrial and nonmitochondrial pathways. Additional caspase-independent pathways have been characterized in mitochondrial PCDs but remain hypothetical in nonmitochondrial PCDs.

Evidence for a direct negative coupling between dopamine-D2 receptors and PLC by heterotrimeric Gi1/2 proteins in rat anterior pituitary cell membranes.

Dopamine (DA) is known to inhibit basal and hormone TRH- or angiotensin II (AngII)-stimulated PRL secretion and inositol phosphate accumulation in rat pituitary cells in primary culture. This inhibition persists when cells are incubated in a calcium-free medium (a condition in which DA could not inhibit PLC activities by blocking calcium influx) and is abolished by a Pertussis toxin treatment. These data suggest that DA receptor could be negatively coupled to PLC by a direct mechanism involving a Pertussis toxin-sensitive G protein.

Vasoactive intestinal polypeptide and pituitary adenylate cyclase-activating polypeptides stimulate mitogen-activated protein kinase in the pituitary cell line GH4C1 by a 3',5'-cyclic adenosine monophosphate pathway.

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP38) regulate anterior pituitary cell secretion and proliferation. In the somatolactotrope GH4C1 cell line, these effects are mediated through the type-II-like PACAP receptor (VPAC2) coupled to the cAMP pathway. In this study, the control of the extracellularly responsive kinases (ERKs) by VIP and PACAP38 was investigated in GH4C1 cells.

Psycholeine, a natural alkaloid extracted from Psychotria oleoides, acts as a weak antagonist of somatostatin.

The effects of psycholeine, a plant alkaloid, were investigated on binding of radiolabelled somatostatin ([125I]N-Tyr-SRIF) and on somatostatin (SRIF)-induced inhibition of adenylate cyclase activity and growth hormone (GH) secretion by rat anterior pituitary cells. Psycholeine was shown to displace specific binding of [125I]N-Tyr-SRIF to pituitary membrane preparations, with an IC50 of 10(-5) M. At this concentration, psycholeine was also effective in significantly reducing the SRIF-induced inhibition of adenylate cyclase activity previously stimulated by growth hormone releasing factor (GRF).

Angiotensin II effects on second messengers involved in prolactin secretion are mediated by AT1 receptor in anterior pituitary cells.

The two forms of angiotensin II (Ang II) receptors, AT1 and AT2 subtypes, have been demonstrated in many other cells beside the anterior pituitary cells. Attempting to investigate the subtype(s) of Ang II receptors implicated in the multiple transduction mechanisms involved in Ang II stimulation of prolactin (PRL) release by lactotropes, we studied the effect of selective nonpeptidergic Ang II antagonists on the PRL release, adenylate cyclase (AC), and phospholipase C activities. In intact cells, the AT1 antagonist DuP753 blocked Ang II-induced PRL release, reversed in a dose dependent manner Ang II-evoked inositol phosphates production, and inhibited completely the PLC and protein kinase C (PKC) dependent cAMP accumulation induced by Ang II.

Selective effect of a diet-induced decrease in the arachidonic acid membrane-phospholipid content on in vitro phospholipase C and adenylate cyclase-mediated pituitary response to angiotensin II.

Young rats were fed on an essential fatty acid (EFA)-deprived diet for 6 weeks after weaning. Their pituitary was removed and adenohypophyseal cells dispersed and maintained in culture. Membrane lipids were analyzed and basal and stimulated levels of hormone secretion were measured after 4-day incubation in a culture medium containing or not 160 microM arachidonic acid 20:4n-6 (AA) in order to obtain EFA-deficient or EFA-restored pituitary cells, respectively.

Neuropeptide Y enhances LHRH binding to rat gonadotrophs in primary culture.

Incubation of dispersed adenohypophyseal cells from intact male rats with Neuropeptide Y (NPY) or Peptide YY (YY) at 21 degrees C increased maximal 125I LHRHa binding (Bmax) by about 50%. In presence of 10(-7) M NPY, Bmax calculated from saturation isotherm curves was 15.3 +/- 1.

Epidermal growth factor treatment induces D2 dopamine receptors functionally coupled to delayed outward potassium current (IK) in GH4C1 clonal anterior pituitary cells.

GH4C1 cells, a clonal cell line from a rat pituitary tumor, have been widely used as a model to study the regulation of prolactin secretion. These cells, however, do not express dopamine D2 receptors and are therefore not suitable for exploring mechanisms involved in dopamine inhibition of prolactin secretion. The recent demonstration that epidermal growth factor (EGF) is able to induce functional expression of D2 receptors in GH3 cells, a parental clonal cell line, overcomes this difficulty.

Somatostatin receptors on thyrotropin-secreting pituitary adenomas: comparison with the inhibitory effects of octreotide upon in vivo and in vitro hormonal secretions.

The in vivo and in vitro inhibitory effects of a somatostatin (SRIH) analog, octreotide, upon TSH, alpha-subunit, GH, and PRL have been studied, as well as SRIH receptors and their coupling to adenylate cyclase, in nine TSH-secreting pituitary adenomas. From in vivo and cell culture studies, the TSH- and alpha-subunit-secreting adenomas appeared heterogeneous, with four out of the nine tumors cosecreting GH and/or PRL. A single sc injection of octreotide (100 micrograms) lowered plasma concentration of TSH by 40 +/- 5% (mean +/- SE of 5), of alpha-subunit by 27 +/- 9% (n = 5), of GH by 60 +/- 5% (n = 4), and of PRL by 27 +/- 9% (n = 4).

Alkaloids from Psychotria oleoides with activity on growth hormone release.

Bioactivity-guided purification of a crude alkaloid extract of Psychotria oleoides has afforded a new alkaloid, psycholeine [1], together with quadrigemine C [2], a tetrameric pyrrolidinoindoline compound of unknown stereochemistry. A comparison study of nmr and cd spectra of quadrigemine C and hodgkinsine [3], a trimeric pyrrolidinoindoline substance, led us to suggest the stereochemistry of quadrigemine C. The structure and configuration of psycholeine was determined by spectroscopic means and chemical correlation with quadrigemine C.

A second endogenous molecular form of mammalian hypothalamic luteinizing hormone-releasing hormone (LHRH), (hydroxyproline9)LHRH, releases luteinizing hormone and follicle-stimulating hormone in vitro and in vivo.

In vitro and in vivo release of pituitary hormones were studied in the presence of (hydroxyproline9)LHRH ((Hyp)LHRH), a newly characterized endogenous molecular form of LHRH. Results were compared to those obtained with LHRH itself. (Hyp)LHRH, as LHRH, stimulated both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release in a homothetic manner.

PKC modulation of inhibitory coupling of angiotensin II receptors with adenylate cyclase in lactotroph cells.

Angiotensin II (AII) and thyreoliberin (TRH) have recently been shown to stimulate intracellular cAMP formation in rat lactotroph cells, in addition to their already documented coupling to phospholipase C. The effect on intracellular cAMP is unaffected by pertussis toxin (PTX) and is not due to a direct coupling to adenylate cyclase (AC); it results instead from a protein kinase C (PKC)-dependent process. In contrast, when tested in membrane preparations, AII, but not TRH, induces a PTX-sensitive inhibition of AC.

Involvement of protein kinase-C in the effect of angiotensin-II on adenosine 3',5'-monophosphate production in lactotroph cells.

When applied to rat anterior pituitary cells, angiotensin-II (AII) exerted two opposite effects on adenylate cyclase (AC) activity: a pertussis toxin (PTX)-sensitive inhibition of the enzyme with a maximal effect of -42 +/- 2% in crude cell membrane preparations, and, in contrast, a non-PTX-sensitive stimulation of cAMP production (maximal effect = 38 +/- 3%) in intact cells. The apparent affinity of both effects was equal to 1.8 nM.

Dual mechanisms of inhibition by dopamine of basal and thyrotropin-releasing hormone-stimulated inositol phosphate production in anterior pituitary cells. Evidence for an inhibition not mediated by voltage-dependent Ca2+ channels.

In primary cultures of anterior pituitary cells, dopamine inhibited basal and thyrotropin-releasing hormone (TRH)-stimulated inositol monophosphate, bisphosphate, and trisphosphate production. This inhibition by dopamine can be resolved into two distinct components. One of the components was rapid and already present after 10 s.

Arachidonate Metabolism in the Anterior Pituitary: Effect of Arachidonate Inhibitors on Basal and Stimulated Secretion of Prolactin, Growth Hormone and Luteinizing Hormone. II. Hormone Release from Dispersed Pituitary Cells.

Abstract In the accompanying study, we reported the effects of inhibitors of arachidonic acid metabolism on the regulation of prolactin, growth hormone (GH) and luteinizing hormone secretion by male hemipituitaries. The present work extends these investigations to primary cell cultures of the same origin. Arachidonic acid metabolism was inhibited by either 5, 8, 11, 14-eicosatetraynoic acid (ETYA), a blocker of cyclooxygenase- and lipoxygenase-catalysed pathways, or the cyclooxygenase inhibitors, indomethacin and aspirin.

Resistance to bromocriptine in prolactinomas.

Bromocriptine therapy normalizes PRL secretion in most, but not all, patients with prolactinomas. This study was undertaken to determine the mechanism(s) responsible for bromocriptine resistance in patients with a PRL-secreting macroadenomas (n = 5) or microadenomas (n = 3). Their mean basal plasma PRL value was 807 +/- 220 (+/- SE) micrograms/L before treatment, and their nadir mean value was 354 +/- 129 micrograms/L during chronic therapy with 15-30 mg bromocriptine daily; four of the eight patients had an increase in tumor size during therapy.

Differential modulation of D1 and D2 dopamine-sensitive adenylate cyclases by 17 beta-estradiol in cultured striatal neurons and anterior pituitary cells.

Primary cultures of anterior pituitary cells from female rats and of mouse embryonic striatal neurons were used to study the effects of 17 beta-estradiol on D1- and D2-dopamine (DA)-sensitive adenylate cyclase. 17 beta-Estradiol pretreatment (10(-9) M, 72 h) suppressed the D2-DA-induced inhibition of adenylate cyclase activity in anterior pituitary cells. The steroid (10(-9) M, 24 h) also blocked the D2-DA-evoked response in striatal neurons whereas it enhanced by twofold the D1-DA-induced stimulation of the enzyme activity in these neurons.

Involvement of central somatostatin in the alteration of GH secretion in starved rats.

In order to determine the central or peripheral origin of the starvation-induced modifications of growth hormone (GH) and thyroid-stimulating hormone (TSH) secretions, the effects of starvation were studied in freely moving male rats with hypothalamo-hypophyseal disconnection. Five days after the disconnection GH secretion exhibited lower maximal values and higher trough levels and ultradian pulsatile secretion was lost as compared to controls. TSH levels were also decreased.

Dihydropyridine-sensitive calcium channel activity related to prolactin, growth hormone, and luteinizing hormone release from anterior pituitary cells in culture: interactions with somatostatin, dopamine, and estrogens.

In the present work, we determined the activity of voltage-dependent dihydropyridine (DHP)-sensitive Ca2+ channels related to PRL, GH, and LH secretion in primary cultures of pituitary cells from male or female rats. We investigated their modulation by 17 beta-estradiol (E2) and their involvement in dopamine (DA) and somatostatin (SRIF) inhibition of PRL and GH release. BAY-K-8644 (BAYK), a DHP agonist which increases the opening time of already activated channels, stimulated PRL and GH secretion in a dose-dependent manner.

Somatostatin receptors on pituitary somatotrophs, thyrotrophs, and lactotrophs: pharmacological evidence for loose coupling to adenylate cyclase.

Pharmacological characterization of somatostatin (SRIF) receptors located on somatotrophs, thyrotrophs, and lactotrophs was attempted by measuring the effects of 14 structural agonists of somatostatin (SRIF) on the inhibition of basal and GRF-stimulated GH and basal and TRH-stimulated PRL and TSH secretion. We also checked the abilities of the analogs to displace [125I]N-Tyr-SRIF binding to pituitary cell membranes and their potency to inhibit adenylate cyclase activity. There was a very good correlation (r = 0.