Cultures Of Anterior Pituitary Cells Research Articles

Cadmium mimics estrogen-driven cell proliferation and prolactin secretion from anterior pituitary cells.

Cadmium (Cd) is a heavy metal of considerable occupational and environmental concern affecting wildlife and human health. Recent studies indicate that Cd, like other heavy metals, can mimic effects of 17β-estradiol (E2) involving E2 receptor (ER) activation. Lactotrophs, the most abundant cell type in anterior pituitary gland, are the main target of E2, which stimulates cell proliferation and increases prolactin secretion through ERα. The aim of this work was to examine whether Cd at nanomolar concentrations can induce cell proliferation and prolactin release in anterior pituitary cells in culture and whether these effects are mediated through ERs. Here we show that 10 nM Cd was able to stimulate lactotroph proliferation in anterior pituitary cell cultures from female Wistar rats and also in GH3 lactosomatotroph cell line. Proliferation of somatotrophs and gonadotrophs were not affected by Cd exposure. Cd promoted cell cycle progression by increasing cyclins D1, D3 and c-fos expression. Cd enhanced prolactin synthesis and secretion. Cd E2-like effects were blocked by the pure ERs antagonist ICI 182,780 supporting that Cd acts through ERs. Further, both Cd and E2 augmented full-length ERαexpression and its 46 kDa-splicing variant. In addition, when co-incubated Cd was shown to interact with E2 by inducing ERα mRNA expression which indicates an additive effect between them. This study shows for the first time that Cd at nanomolar concentration displays xenoestrogenic activities by inducing cell growth and stimulating prolactin secretion from anterior pituitary cells in an ERs-dependent manner. Cd acting as a potent xenoestrogen can play a key role in the aetiology of different pathologies of the anterior pituitary and in estrogen-responsive tissues which represent considerable risk to human health.

In vitro Suppression of Prolactin During Later Stages of Egg Lay in Domestic Hen (Gallus gallus Domesticus) Anterior Pituitcytes by RNA Interference

2 Abstract: Prolactin (PRL) is a peptide hormone secreted by anterior pituitary gland. PRL increases as per the age and reproductive cycle of birds from normal physiological levels to extremely higher level there by affecting the ovulation, egg formation, oviposition, egg production and hyperprolactinemia. This is mor e pronounced and persistent after 72 weeks of age in birds. Hence a study was conducted in anterior pituitary primary cultured cells obtained from 72 and 82 weeks old white leg horn (WLH) hens to knock down the PRL gene expression by siRNA and observing its effects on PRL, PRL mRNA, protein content of PRL, prolactin receptor (PRLR) and Growth Hormone (GH) mRNA to unravel the functional role of PRL at 72 and 82 week age by siRNA for PRL. Three siRNAs were designed as per standard siRNA protocols and studied the suppression of PRL gene expression in primary cultured cells procured from adult chicken anterior pituitary glands in in vitro conditions. Average percentage reduction of PRL in anterior pituitary primary cell culture following siRNA transfection was 82 and 60% at 72 and 82 weeks respectively. Protein content of PRL was significantly (p<0.01) decreased in siRNA transfected cells compared to controls. Growth Hormone (GH) mRNA and PRL receptor (PRLR) mRNA levels did not change significantly (p<0.01) between control and treated cells. Results clearly suggested that the siRNA designed for PRL specifically decreased PRL gene expression in in vitro conditions. Level of PRLR mRNA and GH mRNA levels expression did not follow the similar pattern of PRL gene expression in anterior pituitary cells. It is concluded that, construction of short specific siRNA for PRL significantly decreased PRL, PRL mRNA and protein content of PRL without showing any effect on PRLR and GH between the two age groups of birds. These results may lead to construction of short specific siRNA for stable and chronic suppression of PRL gene expression during embryogenesis before an increase of PRL occurs for long term knock down of PRL in in vivo conditions. In conclusion , understanding of hyperprolactinemia and the involvement of PRL may provide the basis for the development of therapeutic drugs or methods against hyperprolactinemia by RNA interference.

Localization of Notch signaling molecules and their effect on cellular proliferation in adult rat pituitary

Notch signaling is a cell-to-cell signaling system involved in the maintenance of precursor cells in many tissues. Although Notch signaling has been reported in the pituitary gland, the histological characteristics of Notch receptors and ligands in the gland are unknown. Here, we report the histological gene expression pattern of Notch receptors and ligands and the role of Notch signaling in cellular proliferation in adult rat pituitary gland. In situ hybridization detected transcripts of Notch1 and 2 and Jagged1 and 2. Double-staining with a combination of in situ hybridization and immunohistochemistry revealed that their mRNAs were localized in almost half of the S100-protein-positive cells, which are generally regarded as marginal layer cells and folliculo-stellate cells. In primary culture of anterior pituitary cells, proliferation of S100-protein-positive cells was modulated by Notch signaling inhibitor and solubilized Notch ligand. Furthermore, quantitative analysis revealed that the inhibition of Notch signaling led to the down-regulation of mRNA for the Notch target gene Hes1 and the up-regulation of p57 gene expression. These findings suggest that Notch signaling is involved in the proliferation of S100-protein-positive cells, presumably precursor cells, in adult rat pituitary gland.

Estrogens induce expression of membrane-associated estrogen receptor α isoforms in lactotropes.

Estrogens are key to anterior pituitary function, stimulating hormone release and controlling cell fate to achieve pituitary dynamic adaptation to changing physiological conditions. In addition to their classical mechanism of action through intracellular estrogen receptors (ERs), estrogens exert rapid actions via cell membrane-localized ERs (mERs). We previously showed that E2 exerts a rapid pro-apoptotic action in anterior pituitary cells, especially in lactotropes and somatotropes, through activation of mERs. In the present study, we examined the involvement of mERα in the rapid pro-apoptotic action of estradiol by TUNEL in primary cultures of anterior pituitary cells from ovariectomized rats using a cell-impermeable E2 conjugate (E2-BSA) and an ERα selective antagonist (MPP dihydrochloride). We studied mERα expression during the estrous cycle and its regulation by gonadal steroids in vivo by flow cytometry. We identified ERα variants in the plasma membrane of anterior pituitary cells during the estrous cycle and studied E2 regulation of these mERα variants in vitro by surface biotinylation and Western Blot. E2-BSA-induced apoptosis was abrogated by MPP in total anterior pituitary cells and lactotropes. In cycling rats, we detected a higher number of lactotropes and a lower number of somatotropes expressing mERα at proestrus than at diestrus. Acute E2 treatment increased the percentage of mERα-expressing lactotropes whereas it decreased the percentage of mERα-expressing somatotropes. We detected three mERα isoforms of 66, 39 and 22 kDa. Expression of mERα66 and mERα39 was higher at proestrus than at diestrus, and short-term E2 incubation increased expression of these two mERα variants. Our results indicate that the rapid apoptotic action exerted by E2 in lactotropes depends on mERα, probably full-length ERα and/or a 39 kDa ERα variant. Expression and activation of mERα variants in lactotropes could be one of the mechanisms through which E2 participates in anterior pituitary cell renewal during the estrous cycle.

Utilizing the hair follicle to dissect the regulation and autocrine/paracrine activities of prolactin in humans

to the editor: Ferraris et al. ([4][1]) have recently reported an increase in anterior pituitary cell proliferation in Δ1-9-G129R-hPRL (competitive prolactin receptor antagonist) -expressing transgenic mice compared to wild-type controls. In addition, they reported increased proliferation/decreased

17β-Estradiol modulates the prolactin secretion induced by TRH through membrane estrogen receptors via PI3K/Akt in female rat anterior pituitary cell culture

Considering that estradiol is a major modulator of prolactin (PRL) secretion, the aim of the present study was to analyze the role of membrane estradiol receptor-α (mERα) in the regulatory effect of this hormone on the PRL secretion induced by thyrotropin-releasing hormone (TRH) by focusing on the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway activation. Anterior pituitary cell cultures from female rats were treated with 17β-estradiol (E(2), 10 nM) and its membrane-impermeable conjugated estradiol (E(2)-BSA, 10 nM) alone or coincubated with TRH (10 nM) for 30 min, with PRL levels being determined by RIA. Although E(2), E(2)-BSA, TRH, and E(2)/TRH differentially increased the PRL secretion, the highest levels were achieved with E(2)-BSA/TRH. ICI-182,780 did not modify the TRH-induced PRL release but significantly inhibited the PRL secretion promoted by E(2) or E(2)-BSA alone or in coincubation with TRH. The PI3K inhibitors LY-294002 and wortmannin partially inhibited the PRL release induced by E(2)-BSA, TRH, and E(2)/TRH and totally inhibited the PRL levels stimulated by E(2)-BSA/TRH, suggesting that the mER mediated the cooperative effect of E(2) on TRH-induced PRL release through the PI3K pathway. Also, the involvement of this kinase was supported by the translocation of its regulatory subunit p85α from the cytoplasm to the plasma membrane in the lactotroph cells treated with E(2)-BSA and TRH alone or in coincubation. A significant increase of phosphorylated Akt was induced by E(2)-BSA/TRH. Finally, the changes of ERα expression in the plasmalemma of pituitary cells were examined by confocal microscopy and flow cytometry, which revealed that the mobilization of intracellular ERα to the plasma membrane of lactotroph cells was only induced by E(2). These finding showed that E(2) may act as a modulator of the secretory response of lactotrophs induced by TRH through mER, with the contribution by PI3K/Akt pathway activation providing a new insight into the mechanisms underlying the nongenomic action of E(2) in the pituitary.

Nitric Oxide-Sensitive Guanylyl Cyclase Is Differentially Regulated by Nuclear and Non-Nuclear Estrogen Pathways in Anterior Pituitary Gland

17β-estradiol (E2) regulates hormonal release as well as proliferation and cell death in the pituitary. The main nitric oxide receptor, nitric oxide sensitive- or soluble guanylyl cyclase (sGC), is a heterodimer composed of two subunits, α and β, that catalyses cGMP formation. α1β1 is the most abundant and widely expressed heterodimer, showing the greater activity. Previously we have shown that E2 decreased sGC activity but exerts opposite effects on sGC subunits increasing α1 and decreasing β1 mRNA and protein levels. In the present work we investigate the mechanisms by which E2 differentially regulates sGC subunits' expression on rat anterior pituitary gland. Experiments were performed on primary cultures of anterior pituitary cells from adult female Wistar rats at random stages of estrous cycle. After 6 h of E2 treatment, α1 mRNA and protein expression is increased while β1 levels are down-regulated. E2 effects on sGC expression are partially dependent on de novo transcription while de novo translation is fully required. E2 treatment decreased HuR mRNA stabilization factor and increased AUF1 p37 mRNA destabilization factor. E2-elicited β1 mRNA decrease correlates with a mRNA destabilization environment in the anterior pituitary gland. On the other hand, after 6 h of treatment, E2-BSA (1 nM) and E2-dendrimer conjugate (EDC, 1 nM) were unable to modify α1 or β1 mRNA levels, showing that nuclear receptor is involved in E2 actions. However, at earlier times (3 h), 1 nM EDC causes a transient decrease of α1 in a PI3k-dependent fashion. Our results show for the first time that E2 is able to exert opposite actions in the anterior pituitary gland, depending on the activation of classical or non-classical pathways. Thus, E2 can also modify sGC expression through membrane-initiated signals bringing to light a new point of regulation in NO/sGC pathway.

Prolactin receptor antagonism in mouse anterior pituitary: effects on cell turnover and prolactin receptor expression

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.

Inhibition of Nuclear Factor-Kappa B Sensitises Anterior Pituitary Cells to Tumour Necrosis Factor-α- and Lipopolysaccharide-Induced Apoptosis

Nuclear factor-kappa B (NF-κB), an important pro-inflammatory factor, is a crucial regulator of cell survival. Both lipopolysaccharide (LPS) and tumour necrosis factor (TNF)-α activate NF-κB signalling. Oestrogens were shown to suppress NF-κB activation. Oestrogens exert a sensitising action to pro-apoptotic stimuli such as LPS and TNF-α in anterior pituitary cells. In the present study, we show by western blotting that 17β-oestradiol (E(2)) decreases TNF-α-induced NF-κB/p65 and p50 nuclear translocation in primary cultures of anterior pituitary cells from ovariectomised (OVX) rats. Also, the in vivo administration of E(2) decreases LPS-induced NF-κB/p65 and p50 nuclear translocation. To investigate whether the inhibition of NF-κB pathway sensitises anterior pituitary cells to pro-apoptotic stimuli, we used an inhibitor of NF-κB activity, BAY 11-7082 (BAY). BAY, at a concentration that fails to induce apoptosis, has permissive action on TNF-α-induced apoptosis of lactotrophs and somatotrophs from OVX rats, as assessed by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Pharmacological inhibition of NF-κB signalling enhances E(2)-sensitising effect to TNF-α-induced apoptosis in lactotrophs but not in somatotrophs. In vivo administration of BAY allowed LPS-induced apoptosis in anterior pituitary cells from OVX rats (determined by fluorescence activated cell sorting). Furthermore, LPS-induced expression of Bcl-xL in pituitaries of OVX rats is decreased by E(2) administration. Our results show that inhibition of the NF-κB signalling pathway sensitises anterior pituitary cells to the pro-apoptotic action of LPS and TNF-α. Because E(2) inhibits LPS- and TNF-α-activated NF-κB nuclear translocation, the present study suggests that E(2) sensitises anterior pituitary cells to TNF-α- and LPS-induced apoptosis by inhibiting NF-κB activity.

N-terminal prolactin-derived fragments, vasoinhibins, are proapoptoptic and antiproliferative in the anterior pituitary.

The anterior pituitary is under a constant cell turnover modulated by gonadal steroids. In the rat, an increase in the rate of apoptosis occurs at proestrus whereas a peak of proliferation takes place at estrus. At proestrus, concomitant with the maximum rate of apoptosis, a peak in circulating levels of prolactin is observed. Prolactin can be cleaved to different N-terminal fragments, vasoinhibins, which are proapoptotic and antiproliferative factors for endothelial cells. It was reported that a 16 kDa vasoinhibin is produced in the rat anterior pituitary by cathepsin D. In the present study we investigated the anterior pituitary production of N-terminal prolactin-derived fragments along the estrous cycle and the involvement of estrogens in this process. In addition, we studied the effects of a recombinant vasoinhibin, 16 kDa prolactin, on anterior pituitary apoptosis and proliferation. We observed by Western Blot that N-terminal prolactin-derived fragments production in the anterior pituitary was higher at proestrus with respect to diestrus and that the content and release of these prolactin forms from anterior pituitary cells in culture were increased by estradiol. A recombinant preparation of 16 kDa prolactin induced apoptosis (determined by TUNEL assay and flow cytometry) of cultured anterior pituitary cells and lactotropes from ovariectomized rats only in the presence of estradiol, as previously reported for other proapoptotic factors in the anterior pituitary. In addition, 16 kDa prolactin decreased forskolin-induced proliferation (evaluated by BrdU incorporation) of rat total anterior pituitary cells and lactotropes in culture and decreased the proportion of cells in S-phase of the cell cycle (determined by flow cytometry). In conclusion, our study indicates that the anterior pituitary production of 16 kDa prolactin is variable along the estrous cycle and increased by estrogens. The antiproliferative and estradiol-dependent proapoptotic actions of this vasoinhibin may be involved in the control of anterior pituitary cell renewal.

The systemic inhibition of nitric oxide production rapidly regulates TRH mRNA concentration in the paraventricular nucleus of the hypothalamus and serum TSH concentration. Studies in control and cold-stressed rats

Neurons of the paraventricular nuclei of the hypothalamus (PVN) that synthesize the peptide thyrotropin releasing hormone (TRH) control energy homeostasis. Identifying the circuits which regulate these neurons is critical to fully understand integration of metabolic information and the mechanisms that set thyroid hormone levels. We tested the hypothesis that nitric oxide (NO) acutely controls PVN TRH expression and thyrotropin (TSH) secretion by the anterior pituitary. The subcutaneous treatment of rats with NG-nitro-l-arginine methyl ester (l-NAME), an inhibitor of NO synthases, enhanced PVN TRH mRNA and medio-basal hypothalamic TRH levels, and reduced serum TSH concentration. Analysis of the effect of a NO donor in primary cultures of hypothalamic or anterior pituitary cells suggested that the effect of NO includes a direct action on hypothalamic neurons. The cold stress-induced increase in TSH release was inhibited by sc l-NAME. Therefore, production of NO may control the activity of the hypothalamus–pituitary–thyroid axis.

Kisspeptin Regulates Prolactin Release through Hypothalamic Dopaminergic Neurons

Prolactin (PRL) is tonically inhibited by dopamine (DA) released from neurons in the arcuate and periventricular nuclei. Kisspeptin plays a pivotal role in LH regulation. In rodents, kisspeptin neurons are found mostly in the anteroventral periventricular and arcuate nuclei, but the physiology of arcuate kisspeptin neurons is not completely understood. We investigated the role of kisspeptin in the control of hypothalamic DA and pituitary PRL secretion in adult rats. Intracerebroventricular kisspeptin-10 (Kp-10) elicited PRL release in a dose-dependent manner in estradiol (E2)-treated ovariectomized rats (OVX+E2), whereas no effect was found in oil-treated ovariectomized rats (OVX). Kp-10 increased PRL release in males and proestrous but not diestrous females. Associated with the increase in PRL release, intracerebroventricular Kp-10 reduced Fos-related antigen expression in tyrosine hydroxylase-immunoreactive (ir) neurons of arcuate and periventricular nuclei in OVX+E2 rats, with no effect in OVX rats. Kp-10 also decreased 3,4-dihydroxyphenylacetic acid concentration and 3,4-dihydroxyphenylacetic acid-DA ratio in the median eminence but not striatum in OVX+E2 rats. Double-label immunofluorescence combined with confocal microscopy revealed kisspeptin-ir fibers in close apposition to and in contact with tyrosine hydroxylase-ir perikarya in the arcuate. In addition, Kp-10 was not found to alter PRL release from anterior pituitary cell cultures regardless of E2 treatment. We provide herein evidence that kisspeptin regulates PRL release through inhibition of hypothalamic dopaminergic neurons, and that this mechanism is E2 dependent in females. These findings suggest a new role for central kisspeptin with possible implications for reproductive physiology.

Lack of functional GABAB receptors alters GnRH physiology and sexual dimorphic expression of GnRH and GAD-67 in the brain

GABA, the main inhibitory neurotransmitter, acts through GABA(A/C) and GABA(B) receptors (GABA(B)Rs); it is critical for gonadotropin regulation. We studied whether the lack of functional GABA(B)Rs in GABA(B1) knockout (GABA(B1)KO) mice affected the gonadotropin axis physiology. Adult male and female GABA(B1)KO and wild-type (WT) mice were killed to collect blood and tissue samples. Gonadotropin-releasing hormone (GnRH) content in whole hypothalami (HT), olfactory bulbs (OB), and frontoparietal cortexes (CT) were determined (RIA). GnRH expression by quantitative real-time PCR (qRT-PCR) was evaluated in preoptic area-anterior hypothalamus (POA-AH), medial basal-posterior hypothalamus (MBH-PH), OB, and CT. Pulsatile GnRH secretion from hypothalamic explants was measured by RIA. GABA, glutamate, and taurine contents in HT and CT were determined by HPLC. Glutamic acid decarboxylase-67 (GAD-67) mRNA was measured by qRT-PCR in POA-AH, MBH-PH, and CT. Gonadotropin content, serum levels, and secretion from adenohypophyseal cell cultures (ACC) were measured by RIA. GnRH mRNA expression was increased in POA-AH of WT males compared with females; this pattern of expression was inversed in GABA(B1)KO mice. MBH-PH, OB, and CT did not follow this pattern. In GABA(B1)KO females, GnRH pulse frequency was increased and GABA and glutamate contents were augmented. POA-AH GAD-67 mRNA showed the same expression pattern as GnRH mRNA in this area. Gonadotropin pituitary contents and serum levels showed no differences between genotypes. Increased basal LH secretion and decreased GnRH-stimulated gonadotropin response were observed in GABA(B1)KO female ACCs. These results support the hypothesis that the absence of functional GABA(B)Rs alters GnRH physiology and critically affects sexual dimorphic expression of GnRH and GAD-67 in POA-AH.

Prostaglandin-D synthetase induces transcription of the LH beta subunit in the primary culture of chicken anterior pituitary cells via the PPAR signaling pathway

Downstream effects of prostaglandin-D synthetase (PGDS) in a primary culture of chicken ( Gallus gallus) anterior pituitary cells were investigated to study how PGDS regulated laying in hens. Either PGDS downstream metabolite, PGD 2 or PGJ 2, elevated LHB mRNA and LHB protein levels in dose- and time-dependent manners, and treatment with arachidonic acid (1 μM) alone upregulated 15-deoxy-Δ 12,14-PGJ 2 (15-d-PGJ 2; derived from PGJ 2)/PGJ 2, LHB mRNA, and LHB protein levels (P < 0.05) in the primary culture of chicken pituitary anterior cells. Transfection of the plasmid Enhanced Green Fluorescent Protein-PGDS plasmid into these cells in medium containing 1 μM arachidonic acid additionally increased 15-d-PGJ 2/PGJ 2, LHB mRNA, and LHB protein levels (P < 0.05). In the hypothalamus/pituitary gland of laying hens, there was a high correlation (r = 0.64; P < 0.05) between PGDS and the peroxisome proliferator-activated receptor A ( PPARA) mRNA level in a high egg production strain, the L2 Taiwan country chickens. In commercial Single-Comb White Leghorn layers, there were high correlation coefficients between PGDS and PPARA (r = 0.65; P < 0.05) and between PGDS and PPARG (r = 0.67; P < 0.01) mRNA levels. A broad-range PPARs agonist, GW9578 (5 to 500 nM), enhanced LHB mRNA and LHB protein levels (P < 0.05). The PPARA-specific (GW6471) and PPARG-specific (T0070907) antagonists suppressed endogenous LHB mRNA and LHB protein levels (P < 0.05); in addition, both antagonists attenuated arachidonic acid–induced LHB mRNA levels (P < 0.05) and PGDS-induced (in the presence of 1 μM arachidonic acid) LHB mRNA and LHB protein (P < 0.05) levels in the primary culture of chicken anterior pituitary cells. Higher LHB mRNA/LHB protein ratios in PGD 2-, PGJ 2-, arachidonic acid-, PGDS-, and GW9578-induced as well as GW6471- and T0070907-suppressed anterior pituitary cells suggested that LHB transcription occurred before translation. In conclusion, PGDS induced LHB transcription and subsequent translation via the PPAR signaling pathway.

Effects of differential pulse frequencies of chicken gonadotrophin-releasing hormone-I (cGnRH-I) on laying hen gonadotrope responses in vitro

The aim of this work was to determine the effects of cGnRH I pulse frequencies on FSH and LH release and the changes in features and number of cultured laying hen FSH-cells and LH-cells in vitro. Primary adenohypophyseal cell cultures taken from laying hens were stimulated by four 5 min pulses using 1 or 10 nM cGnRH, administered with interpulses between pulses at 15, 30 or 60 min. Pulse frequencies and dose dependent effects were examined in six separate experiments including two controls. After the last interpulse time, the supernatants were collected and stored at −70° C until the performance of an indirect enzyme-linked immunosorbent assay (ELISA) using chicken LH and chicken FSH antisera at 1:1000 and 1:2000 dilutions, respectively. Supernatants were coated in duplicate on the inner surface of Immulon 2 plates and later blocked with the optimal solutions. They were incubated with each antiserum and subsequently with isotype-specific peroxidase-labeled anti-rabbit antibodies. Hydrogen peroxide/o-phenylenediamine was added as substrate/chromogen and the optical density (OD) was determined at 492 nm. The ABC immunocytochemical method was performed to characterize and re-count the gonadotropes employing anti-chicken FSH and anti-chicken LH as primary antibodies. The number of FSH-LH cells was obtained using stereological analysis and the data were statistically processed. The ODs obtained for each anti-hormone were compared with the control groups and with each other. Significant differences were found in number of aggregated-positive LH cells, which decreased with 1 nM cGnRH-I, 15 vs. 30 min pulses, increased with 30 vs. 60 min pulses, and also with 10 nM cGnRH-I, 30 vs. 60 min pulses. Aggregated positive FSH cells, however, did not show significant differences in percentage at any GnRH dose or pulse frequencies, but did show activity at low pulse frequencies of 15 and 30 min. The results suggest that LH cells varied in percentage in a dose dependent manner at higher pulse frequency (15 min) and were dose independent at low pulse frequency (60 min) and showed inactive features; while FSH cell numbers were unaffected showing features of activity at low pulse frequencies. High and moderate pulse frequencies of cGnRH-I (15-30 min) increased the FSH release in dose independent manner without changes in features or percentage of FSH cells. Low pulse frequency (60 min) of cGnRH-I increased LH release dose independently disminished LH cell percentage and showed changes in cells’ features. These results in avian cells showed differences in responses to GnRH pulse frequencies from those reported earlier in mammals.

Cathepsin D Is the Primary Protease for the Generation of Adenohypophyseal Vasoinhibins: Cleavage Occurs within the Prolactin Secretory Granules

Vasoinhibins are a family of N-terminal prolactin (PRL) fragments that inhibit blood vessel growth, dilation, permeability, and survival. The aspartyl endoprotease cathepsin D is active at acidic pH and can cleave rat PRL to generate vasoinhibins. We investigated whether and where vasoinhibins could be generated by cathepsin D in the adenohypophysis of rats and mice and whether their production could be gender dependent. Vasoinhibins were detected in primary cultures of rat adenohypophyseal cells by Western blot with antibodies directed against the N terminus of PRL but not the C terminus. Ovariectomized, estrogen-treated females show greater levels of adenohypophyseal vasoinhibins than males. Peptide sequencing analysis revealed that the cleaved form of PRL in rat adenohypophyseal extracts contains the PRL N terminus and a second N terminus starting at Ser(149), the reported cleavage site of cathepsin D in rat PRL. In addition, cathepsin D inhibition by pepstatin A reduced vasoinhibin levels in rat adenohypophyseal cell cultures. Confocal and electron microscopy showed the colocalization of cathepsin D and PRL within rat adenohypophyseal cells and secretory granules, and a subcellular fraction of rat adenohypophysis enriched in secretory granules contained cathepsin D activity able to generate vasoinhibins from PRL. Of note, vasoinhibins were absent in the adenohypophysis of mice lacking the cathepsin D gene but not in wild-type mice. These findings show that cathepsin D is the main protease responsible for the generation of adenohypophyseal vasoinhibins and that its action can take place within the secretory granules of lactotrophs.

Effects of isoquinolonesulfonamides on action potential secretion coupling in pituitary cells.

Pituitary lactotrophs fire action potentials spontaneously and the associated voltage-gated calcium influx is sufficient to maintain high and steady prolactin release. Several intracellular proteins can mediate the action of calcium influx on prolactin secretion, including calmodulin-dependent protein kinases. Here, we studied effects of isoquinolonesulfonamides KN-62 and KN-93, calmodulin-dependent protein kinase inhibitors, and KN-92, an inactive analog, on spontaneous electrical activity, voltage-gated calcium influx, cyclic nucleotide production, and basal prolactin release. The effects of these compounds on electrical activity and calcium signaling was measured in single lactotrophs and cyclic nucleotide production and prolactin release were determined in static culture and perifusion experiments of anterior pituitary cells from postpubertal female rats. KN-62 and KN-93 blocked basal prolactin release in a dose- and time-dependent manner, suggesting that calmodulin-dependent protein kinase could mediate the coupling of electrical activity and secretion. However, a similar effect on basal prolactin release was observed on application of KN-92, which does not inhibit this kinase. KN-93 also inhibited cAMP and cGMP production, but inhibition of prolactin release was independent of the status of cyclic nucleotide production. Single cell measurements revealed abolition of spontaneous and depolarization-induced electrical activity and calcium transients in KN-92/93-treated cells, with a time course comparable to that observed in secretory studies. The results suggest that caution should be used when interpreting data from studies using isoquinolonesulfonamides to evaluate the role of calmodulin-dependent protein kinases in excitable endocrine cells, because inactive compounds exhibit comparable effects on action potential secretion coupling to those of active compounds.

Time-resolved fluoroimmunoassay (TR-FIA) of gonadotropins.

Time-resolved fluoroimmunoassay (TR-FIA), which has recently been developed as a non-isotopic immunoassay, was intended for assay of gonadotropins (LH and FSH). Ovine LH or FSH was labeled with N1-(p-isothiocyanatobenzyl)-diethylenetriamine-N1, N2, N3, N3-tetraacetic acid Eu-chelate (DTTA), and competitive binding assay was performed using a 96-weel titer plate previously coated with a second antibody, followed by measurement using time-resolved fluorometry. TR-FIA for standard ovine LH (NIDDK, LH-I-3) or FSH (NIDDK, FSH-I-1) had a sensitivity of about 25 pg/50 microliters sample. The assay system was applied to heterologous assay of porcine gonadotropins. Linearity was obtained by the dilution test using medium from primary culture of porcine anterior pituitary cells. Intra- and inter-assay coefficients of variation of LH and FSH determination in 31 different porcine samples were satisfactorily low, between 3.5 and 8.1% (intra-assay) and between 1.7 and 13.1% (inter-assay). Correlation coefficients between radioimmunoassay and TR-FIA were calculated to be 0.945 for LH and 0.978 for FSH. Stimulation of LH and FSH release with GnRH was observed by TR-FIA. This non-isotopic TR-FIA thus provides as good sensitivity, reproducibility and accuracy as conventional RIAs.

Platelet-derived Growth Factor (PDGF) and PDGF Receptor Expression and Function in Folliculostellate Pituitary Cells

Homo- and heterodimers of platelet-derived growth factor-A (PDGF-A) and PDGF-B chains are involved through PDGF alpha- and beta-receptors in the growth regulation of multiple normal and tumoural cell types as well as in tumour neovascularization. Since little information is available on the impact of PDGF/PDGF receptors in normal and adenomatous pituitary, we studied the expression and action of this growth factor system in a variety of pituitary tumour cell lines and in rat anterior pituitary cell cultures. By RT-PCR, mRNA expression of PDGF-A and -B chains and of both receptors was found in rat pituitary and mouse folliculostellate TtT/GF pituitary tumour cells. Rat somatotroph MtT-S and mouse corticotroph AtT20 tumor cells expressed only a part of the PDGF/PDGF receptor components whereas mouse gonadotroph alphaT3-1 and rat lactosomatotroph GH3 pituitary tumour cells contained neither PDGF nor PDGF receptors. To further characterize the role of PDGF in TtT/GF cells, the effect of PDGF-AB and -BB on growth and vascular endothelial growth factor-A (VEGF-A) release was studied. Proliferation of TtT/GF cells was weakly but significantly stimulated by PDGF. Both in rat pituitary cell cultures and in TtT/GF cells, PDGF-AB and -BB strongly enhanced VEGF-A secretion. The PI3 kinase inhibitor LY 294002 blocked the increase in VEGF-A. Western immunoblotting confirmed the participation of key components of the PI3 kinase/Akt signal pathway (PDK1, Akt-Ser476) in PDGF-stimulated VEGF production. Thus the PDGF/PDGF receptor system is expressed in folliculostellate cells and is involved in VEGF regulation. Its role in endocrine pituitary tumour cell lines and pituitary adenomas need to be clarified in future studies.

Bovine C-terminal octapeptide of RFamide-related peptide-3 suppresses luteinizing hormone (LH) secretion from the pituitary as well as pulsatile LH secretion in bovines

Gonadotropin-inhibiting hormone (GnIH), observed in quail as a member of the RFamide neuropeptide family, suppresses luteinizing hormone (LH) secretion from the avian pituitary. Rats and cattle have an active gene of another member of the RFamide neuropeptide family, termed RFamide-related peptide-3 (RFRP-3), although bovine RFRP-3 is different from that of rats in both length and amino-acid sequence. A single injection of GnIH or RFRP-3 inhibited LH secretion in rodents, which continued for various periods. This study was conducted to evaluate the effects of bovine C-terminal octapeptide of RFRP-3 (RFRP-3-8) on LH secretion from cultured anterior pituitary (AP) cells of cattle, and the effects of RFRP-3-8 injections on pulsatile LH secretion in castrated male calves. The suppressive effect of RFRP-3-8 on LH secretion from AP cells was observed in the presence of gonadotropin-releasing hormone (GnRH), but not in the absence of GnRH in culture media. In another experiment collecting blood samples serially from castrated male calves with repeated intravenous injections of RFRP-3-8 ( n = 6) or saline ( n = 6), the RFRP-3-8 group showed suppressed LH pulse frequency during the injection period ( P < 0.05); however, the RFRP-3-8 group showed no difference from the saline group in all measures of LH secretion in the postinjection period. In conclusion, our results suggested that RFRP-3-8 suppresses LH secretion from cultured AP cells, as well as LH pulse frequency in cattle.