Dispersed Anterior Pituitary Cells Research Articles

Identification of neuromedin U precursor-related peptide and its possible role in the regulation of prolactin release

The discovery of neuropeptides provides insights into the regulation of physiological processes. The precursor for the neuropeptide neuromedin U contains multiple consensus sequences for proteolytic processing, suggesting that this precursor might generate additional peptides. We performed immunoaffinity chromatography of rat brain extracts and consequently identified such a product, which we designated neuromedin U precursor-related peptide (NURP). In rat brain, NURP was present as two mature peptides of 33 and 36 residues. Radioimmunoassays revealed NURP immunoreactivity in the pituitary, small intestine, and brain of rats, with the most intense reactivity in the pituitary. Intracerebroventricular administration of NURP to both male and female rats robustly increased plasma concentrations of prolactin but not of other anterior pituitary hormones. In contrast, NURP failed to stimulate prolactin release from dispersed anterior pituitary cells. Pretreatment of rats with bromocriptine, a dopamine receptor agonist, blocked the prolactin-releasing activity of NURP. In rats pretreated with the antagonist sulpiride, intracerebroventricular administration of NURP did not increase plasma prolactin concentrations more than administration of saline. These data suggest that NURP induces prolactin release by acting indirectly on the pituitary; dopamine from the hypothalamus, which inhibits prolactin release, may be involved in this activity of NURP.

Arginine vasotocin is the major adrenocorticotropic hormone-releasing factor in the bullfrog Rana catesbeiana

In a previous study, we showed that corticotropin-releasing factor (CRF) is the major thyroid-stimulating hormone (TSH)-releasing factor in the bullfrog (Rana catesbeiana) hypothalamus. Our findings prompted us to ascertain whether CRF or arginine vasotocin (AVT), a known adrenocorticotropic hormone (ACTH) secretagogue in several vertebrates, is the main stimulator of the release of ACTH from the bullfrog pituitary. Both the frog CRF and AVT stimulated the release of immunoassayable ACTH from dispersed anterior pituitary cells in vitro in a concentration-dependent manner. AVT, however, exhibited far more potent ACTH-releasing activity than CRF. Although CRF by itself weakly stimulated ACTH release, it acted synergistically with AVT to enhance the release of ACTH markedly. Mesotocin and AVT-related peptides such as hydrin 1 and hydrin 2 showed relatively weak ACTH-releasing activity. Subsequently, cDNAs encoding the bullfrog AVT V1a-type and V1b-type receptors were molecularly cloned. Reverse transcriptase-PCR using specific primers revealed that the anterior lobe of the pituitary predominantly expressed AVT V1b-type receptor mRNA but scarcely expressed AVT V1a-type receptor mRNA. Abundant signals for V1b-type receptor mRNA in the corticotropes were also detected by in situ hybridization. The results obtained by the experiments with the bullfrog pituitary indicate that AVT acts as the main ACTH-releasing factor through the AVT V1b-type receptor and that CRF acts synergistically with AVT to enhance the release of ACTH.

A possible role of central serotonin in L‐tryptophan‐induced GH secretion in cattle

To clarify the role of serotonin (5-HT) in the regulatory mechanism of L-tryptophan (TRP)--induced growth hormone (GH) secretion in cattle, changes in 5-HT concentrations in the cerebrospinal fluid (CSF) in the third ventricle (3V) and GH in plasma before and after the peripheral infusion of TRP were determined simultaneously. The direct effect of TRP on GH release from the dispersed anterior pituitary cells was also assessed. A chronic cannula was placed in 3V by stereotaxic surgery, then CSF and blood were withdrawn under physiological conditions. TRP (38.5 mg/kg BW) was infused through an intravenous catheter from 12.00 to 14.00 hours and CSF and blood sampling were performed from 11.00 to 18.00 hours at 1-h intervals. The concentration of 5-HT in CSF was determined by high-performance liquid chromatography with electrochemical detection. GH, melatonin (MEL), and cortisol (CORT) concentrations were measured by radio-immunoassay and enzyme-immunoassay. Concentrations of 5-HT were increased by TRP infusion. The TRP infusion significantly increased GH release. On the other hand, TRP did not stimulate GH release from the bovine pituitary cells. MEL and CORT concentrations were not altered by TRP infusion. These results suggest that TRP induced GH release via the activation of serotonergic neurons in cattle.

Living-cell imaging of transgenic rat anterior pituitary cells in primary culture reveals novel characteristics of folliculo-stellate cells

Folliculo-stellate (FS) cells in the anterior pituitary gland appear to possess multifunctional properties. Recently, the development of transgenic rats (S100b-green fluorescent protein (GFP) rats) that express GFP specifically in FS cells in the anterior pituitary gland has allowed us to distinguish and observe living FS cells in other kinds of pituitary cells. We used S100b-GFP rats to investigate the topographic affinity of FS cells for other pituitary cells. We observed living FS cells in enzymatically dispersed anterior pituitary cells of S100b-GFP rats under a fluorescent microscope, and noted that FS cells markedly extended and contracted cytoplasmic processes and formed interconnections with neighboring FS cells. In addition, FS cells adhered to small clusters of GFP-negative cells, which were primarily hormone-producing cells, and these clusters further aggregated during the course of cytoplasmic contraction. In the presence of laminin, fibronectin, and varying types of collagen, FS cells showed marked changes in shape and specific proliferative activity; however, GFP-negative cells did not. On reverse transcription-PCR analysis and immunohistochemistry, FS cells were shown to express integrin subunits, which are the cell surface receptors for extracellular matrix (ECM). In the anterior pituitary gland, FS cells and the various types of hormone-producing cells generate a unique topography in the presence of basement membrane components and interstitial collagens. The novel characteristics of FS cells observed in the present study suggest that in the anterior pituitary gland, FS cells play important roles in determining and/or maintaining local cellular arrangement in the presence of ECM components.

Genetic Labeling: New Approaches to Creating a Gonadotroph “ID”

Genetic Labeling: New Approaches to Creating a Gonadotroph “ID”

Intermedin 17–47 does not function as a full intermedin antagonist within the central nervous system or pituitary

A fragment of intermedin (IMD), IMD17–47, has been shown to antagonize the hypotensive effects of intravenous IMD administration; however, the effects of IMD17–47 have not been studied in other systems such as brain and pituitary gland. IMD17–47 was administered intracerebroventricularly (i.c.v.) into male rats alone or prior to administration of IMD; and blood pressure and food and water intakes measured. Multiple doses of IMD17–47 failed to alter basal blood pressure and heart rate, but did partially reverse the stimulatory effects of IMD given i.c.v. on blood pressure and heart rate. A low dose of IMD17–47 by itself significantly increased basal food and water intake. However, a higher dose of the antagonist did not alter food or water intake compared to control treated rats. No dose of IMD17–47 was able to reverse the inhibitory effects of IMD administered i.c.v. on food and water intake. Furthermore, IMD17–47 failed to significantly alter the inhibitory effects of IMD on growth hormone releasing hormone-stimulated growth hormone release from dispersed anterior pituitary cells in culture. A siRNA molecule designed to compromise IMD production was able to reduce brain IMD levels and did, upon i.c.v. administration, cause increased water drinking in male rats. This tool may provide a better method than the use of the IMD17–47 compound to study the role of endogenous IMD within the CNS and pituitary.

Cocaine- and Amphetamine-Regulated Transcript Is Localized in Pituitary Lactotropes and Is Regulated during Lactation

Cocaine- and amphetamine-regulated transcript (CART) is a highly expressed peptide implicated in the regulation of feeding, reward and reinforcement, and stress-related behaviors. CART has been localized to discrete cell populations in the brain, gut, adrenal gland, and pancreas. In contrast, CART-producing cell types in the pituitary gland remain ill defined. In the present study, double-label immunohistochemistry, employing a high-affinity antiserum we generated against CART-(62-102), was used to identify CART-producing cells in the pituitary gland. In the anterior pituitary, the majority of CART immunoreactivity (-ir) was localized in lactotropes; minor populations of CART-ir cells were identified as somatotropes and corticotropes. In the posterior pituitary, CART-ir extensively colocalized with oxytocin-containing fibers; in contrast, only a few vasopressin fibers contained CART-ir. As expected, CART colocalized with oxytocin in magnocellular neurons of the supraoptic nucleus. The effects of bromocriptine, a potent dopamine receptor agonist, were examined to determine whether CART mRNA expression and protein release are regulated in a similar fashion as prolactin. Similar to prolactin, CART mRNA expression and protein release were significantly decreased after bromocriptine treatment of dispersed rat anterior pituitary cells in culture. To explore the putative physiological role of pituitary CART, we compared levels of CART mRNA expression in lactating and nonlactating female rats. CART mRNA levels were significantly increased in the anterior pituitary and supraoptic nucleus of lactating rats. Furthermore, levels of CART in the systemic circulation were significantly elevated at the onset of lactation, peaked on d 10 of lactation and returned to baseline values 10 d after pups were weaned. The current study describes the cellular localization and regulation of CART expression and protein release from the rat pituitary gland. These findings suggest a putative role for CART in lactation.

Intermedin/Adrenomedullin-2 Inhibits Growth Hormone Release from Cultured, Primary Anterior Pituitary Cells

Intermedin (IMD), a novel member of the adrenomedullin (AM), calcitonin gene-related peptide (CGRP), amylin (AMY) peptide family, has been reported to act promiscuously at all the known receptors for these peptides. Like AM and CGRP, IMD acts in the circulation to decrease blood pressure and in the brain to inhibit food intake, effects that could be explained by activation of the known CGRP, AM, or AMY receptors. Because AM, CGRP, and AMY have been reported to affect hormone secretion from the anterior pituitary gland, we examined the effects of IMD on GH, ACTH, and prolactin secretion from dispersed anterior pituitary cells harvested from adult male rats. IMD, in log molar concentrations ranging from 1.0 pm to 100 nm, failed to significantly alter basal release of the three hormones. Similarly, IMD failed to significantly alter CRH-stimulated ACTH or TRH-stimulated prolactin secretion in vitro. However, IMD concentration-dependently inhibited GHRH-stimulated GH release from these cell cultures. The effects of IMD, although requiring higher concentrations, were as efficacious as those of somatostatin and, like somatostatin, may be mediated, at least in part, by decreasing cAMP accumulation. These actions of IMD were not shared by other members of the AM-CGRP-AMY family of peptides, suggesting the presence of a novel, unique IMD receptor in the anterior pituitary gland and a potential neuroendocrine action of IMD to interact with the hypothalamic mechanisms controlling growth and metabolism.

Dehydroepiandrosterone Restoration of Growth Hormone Gene Expression in Aging Female Rats, in Vivo and in Vitro: Evidence for Actions via Estrogen Receptors

A decline in dehydroepiandrosterone (DHEA) and GH levels with aging may be associated with frailty and morbidity. Little is known about the direct effects of DHEA on somatotropes. We recently reported that 17beta-estradiol (E2), a DHEA metabolite, stimulates the expression of GH in vitro in young female rats. To test the hypothesis that DHEA restores function in aging somatotropes, dispersed anterior pituitary (AP) cells from middle-aged (12-14 months) or young (3-4 months) female rats were cultured in vitro with or without DHEA or E2 and fixed for immunolabeling or in situ hybridization. E2 increased the percentage of AP cells with GH protein or mRNA in the aged rats to young levels. DHEA increased the percentages of somatotropes (detected by GH protein or mRNA) from 14-16 +/- 2% to 29-31 +/- 3% (P < or = 0.05) and of GH mRNA (detected by quantitative RT-PCR) only in aging rats. To test DHEA's in vivo effects, 18-month-old female rats were injected with DHEA or vehicle for 2.5 d, followed by a bolus of GHRH 1 h before death. DHEA treatment increased serum GH 1.8-fold (7 +/- 0.5 to 12 +/- 1.3 ng/ml; P = 0.02, by RIA) along with a similar increase (P = 0.02) in GH immunolabel. GHRH target cells also increased from 11 +/- 1% to 19 +/- 2% (P = 0.03). Neither GH nor GHRH receptor mRNAs levels were changed. To test the mechanisms behind DHEA's actions, AP cells from aging rats were treated with DHEA with or without inhibitors of DHEA metabolism. Trilostane, aminogluthemide, or ICI 182,780 completely blocked the stimulatory effects of DHEA, suggesting that DHEA metabolites may stimulate aging somatotropes via estrogen receptors.

Changes in Expression of Hypothalamic Releasing Hormone Receptors in Individual Rat Anterior Pituitary Cells during Maturation, Puberty and Senescence

Anterior pituitary (AP) is formed by five different cell types, each one producing a different AP hormone whose secretion is regulated by a specific hypothalamic-releasing hormone (HRH). On the other hand, a significant number of AP cells express multiple HRH receptors (multiresponsive cells). Plastic changes in expression of HRH receptors in individual AP cells are involved in critical endocrine events. Here we have characterized the changes in functional responses to CRH, LHRH, TRH, and GHRH in individual AP cells throughout the whole life span of the rat. To this end, calcium responses to the HRHs were followed by single-cell imaging in freshly dispersed AP cells prepared from rats of different ages (0-540 postnatal days). Three different cell pools were identified: 1) monoresponsive cells, holding a single class of HRH receptor; 2) multiresponsive cells; and 3) nonresponsive cells. The relative abundance of each pool changed with age. Nonresponsive cells were abundant at birth, multiresponsive cells were abundant at puberty, and monoresponsive cells dominated at senescence. The relative abundance of each HRH receptor changed largely with age but not gender. In addition, the contribution of monoresponsive and multiresponsive cells to responses to each HRH changed very much with age. Thus, the anterior pituitary shows large changes in cell populations typed by functional responses to HRHs during maturation, puberty, and senescence.

Reduced Hypothalamic Vasopressin Secretion Underlies Attenuated Adrenocorticotropin Stress Responses in Pregnant Rats

We sought to explain decreased ACTH secretory responses to stress in pregnant rats by investigating hypothalamic CRH and vasopressin secretion and actions on anterior pituitary corticotrophs. In late pregnancy median eminence, CRH content was reduced (by 12%). Anterior pituitary proopiomelanocortin mRNA expression, measured by in situ hybridization but not radioimmunoassayed ACTH content, was also reduced (by 45% on d 21); CRH receptor (CRHR)1 mRNA expression was unaltered in pregnancy, but V1b receptor mRNA expression was reduced (by 19%). ACTH secretory responses, measured in jugular blood, to CRH (200 ng/kg iv) or vasopressin (1.7 microg/kg, iv) were reduced on d 21 vs. virgins (49% and 44%), but the response to combined CRH and vasopressin injection was intact. Either antalarmin (CRHR1 antagonist; 20 mg/kg ip) or dP(Tyr(Me)2),Arg-NH2(9))AVP (V1a/b antagonist; 10 microg/kg, iv) pretreatment reduced the ACTH secretory response to forced swimming (90 sec) in virgin rats (by 57% and 40%), but only antalarmin was effective in pregnant rats (53% decrease). In vitro, measuring ACTH secretion from acutely dispersed anterior pituitary cells showed increased corticotroph sensitivity in pregnancy to CRH and to CRH augmentation by vasopressin, attributable to increased intracellular cAMP action. Hence, in late pregnancy, reduced anterior pituitary CRHR1 or V1b receptor expression did not impair corticotroph responses to CRH or vasopressin. Rather, diminished secretagogue secretion in vivo accounts for reduced action of stress levels of exogenous CRH or vasopressin alone; the late pregnancy attenuated ACTH secretory response to swim stress is deduced to be due to reduced vasopressin release by parvocellular paraventricular nuclei neurones.

Tumor Necrosis Factor-Alpha-Induced Nitric Oxide Restrains the Apoptotic Response of Anterior Pituitary Cells

We previously reported that tumor necrosis factor-α (TNF-α) inhibits cell proliferation whereas it stimulates apoptosis of anterior pituitary cells in an estrogen-dependent manner. Also, we showed that nitric oxide (NO) mediates the inhibitory effect of TNF-α on prolactin release. Here, we studied the effect of TNF-α on nitric oxide synthase (NOS) activity and expression in anterior pituitary cells from cycling and ovariectomized (OVX) rats, and the role of NO in TNF-α induced apoptosis of anterior pituitary cells. NOS activity was higher in anterior pituitary cells from rats in proestrus than in diestrus and was stimulated by 17β-estradiol (10^–9 M, E2). TNF-α (50 ng/ml) stimulated NOS activity in anterior pituitary cells from rats at both stages of the estrous cycle and in cells from OVX rats cultured either with or without E2. Inducible NOS (iNOS) gene expression was higher in anterior pituitary cells from rats in proestrus than in diestrus and its expression was enhanced by TNF-α. Acute administration of E2 to OVX rats increased endothelial NOS (eNOS) expression in the anterior pituitary gland. Also, E2 increased eNOS mRNA in dispersed anterior pituitary cells from OVX rats, and this effect was blocked by TNF-α. nNOS expression in the anterior pituitary gland was higer at proestrus than at diestrus but eNOS expression was similar at both stages. TNF-α decreased eNOS mRNA in anterior pituitary cells from rats at proestrus or diestrus. In anterior pituitary cells from OVX rats, TNF-α failed to induce apoptosis but was able to induce it when cells were incubated with NAME or NMMA, NOS inhibitors that did not affect cell viability per se. In the presence of E2, NAME induced apoptosis and enhanced the proapoptotic effect of TNF-α. In conclusion, our study shows that TNF-α upregulates iNOS gene expression whereas it downregulates estrogen-induced eNOS expression in anterior pituitary cells. Endogenous NO may restrain rather than mediate the proapoptotic effect of TNF-α in anterior pituitary cells.

Central Neuropeptide B Administration Activates Stress Hormone Secretion and Stimulates Feeding in Male Rats

Neuropeptide B (NPB) was identified to be an endogenous, peptide ligand for the orphan receptors GPR7 and GPR8. Because GPR7 is expressed in rat brain and, in particular, in the hypothalamus, we hypothesized that NPB might interact with neuroendocrine systems that control hormone release from the anterior pituitary gland. No significant effects of NPB were observed on the in vitro releases of prolactin, adrenocorticotropic hormone (ACTH) or growth hormone (GH) when log molar concentrations ranging from 1 pM to 100 nM NPB were incubated with dispersed anterior pituitary cells harvested from male rats. In addition NPB (100 nM) did not alter the concentration response stimulation of prolactin secretion by thyrotropin-releasing hormone, ACTH secretion by corticotropin-releasing factor (CRF) and GH secretion by GH-releasing hormone. However, NPB, when injected into the lateral cerebroventricle (i.c.v.) of conscious, unrestrained male rats, elevated prolactin and corticosterone, and lowered GH levels in circulation. The threshold dose for the effect on corticosterone and prolactin levels was 1.0 nmol, while that for the effect on GH release was 3.0 nmol NPB. Pretreatment with a polyclonal anti-CRF antiserum completely blocked the ability of NPB to stimulate ACTH release and significantly inhibited the effect of NPB on plasma corticosterone levels. NPB administration i.c.v. did not significantly alter plasma vasopressin and oxytocin levels in conscious rats. It did stimulate feeding (minimum effective dose 1.0 nmol) in sated animals in a manner similar to that of the other endogenous ligand for GPR7, neuropeptide W. We conclude that NPB can act in the brain to modulate neuroendocrine signals accessing the anterior pituitary gland, but does not itself act as a releasing or inhibiting factor in the gland, at least with regard to prolactin, ACTH and GH secretion.

Cocaine- and amphetamine-regulated transcript peptide in the rat anterior pituitary gland is localized in gonadotrophs and suppresses prolactin secretion.

Cocaine- and amphetamine-regulated transcript (CART) mRNA and CART peptide are abundant in the hypothalamic nuclei that control anterior pituitary function. CART peptide has also been localized in the anterior pituitary gland itself, although its role in pituitary function has not as yet been elucidated. In the present study, we investigated the localization and function of CART peptide in the anterior pituitary gland. Immunohistochemical observations revealed that CART peptide colocalized with FSH and LH in gonadotroph cells but that it was absent from the other hormone-producing cells. Immunoelectronmicroscopy suggested that CART peptide and gonadotropin were colocalized in the same secretory granules. CART peptide suppressed prolactin release from dispersed anterior pituitary cells 15 min after its addition into the media [basal production, 234.9 +/- 14.6 ng/ml vs. CART 55-102 peptide 100 nm, 125.0 +/- 18.2 ng/ml (P < 0.01, n = 5)]. Prolactin release was suppressed by CART in a dose-related manner; on the other hand, CART peptide did not affect the secretion of other anterior pituitary hormones. CART peptide synthesis by these cells was elevated 15 min after the addition of leptin to the media (100 nm), as determined by immunoblotting, but LHRH (10 nm) did not significantly affect CART peptide expression. These findings suggest that CART synthesis in the anterior pituitary may be stimulated by leptin and that CART peptide may play a role in the regulation of anterior pituitary hormone secretion in the rat.

Development of radioimmunoassay for bullfrog thyroid-stimulating hormone (TSH): effects of hypothalamic releasing hormones on the release of TSH from the pituitary in vitro

A bullfrog ( Rana catesbeiana) thyroid-stimulating hormone (TSH) β-subunit (TSHβ) antiserum was produced by employing a C-terminal peptide synthesized on the basis of the amino acid sequence deduced from bullfrog TSHβ cDNA. Immunohistochemical studies revealed that the bullfrog adenohypophyseal cells that immunologically reacted with the anti-bullfrog TSHβ corresponded to those positively stained with an antiserum against human (h) TSHβ. The antiserum was used for the development of a specific and sensitive radioimmunoassay (RIA) for the measurement of bullfrog TSH. The sensitivity of the RIA was 0.75 ± 0.07 ng TSH/100 μl assay buffer. The interassay and intraassay coefficients of variation were 7.6 and 5.3%, respectively. Several dilutions of pituitary homogenates of larval and adult bullfrogs, or medium in which bullfrog pituitary cells were cultured, yielded dose–response curves that were parallel to the standard curve. Bullfrog prolactin, growth hormone, luteinizing hormone, follicle-stimulating hormone, and α-subunit derived from glycoprotein hormones did not react in this assay. Immunoassayable TSH in the pituitary culture medium was confirmed to exist in the form of TSHβ coupled with the α-subunit by an immunoprecipitation experiment using the TSHβ antiserum and an α-subunit antiserum. TSH released from pituitary cells into the medium was also confirmed to possess a considerable activity in stimulating the release of thyroxine from the thyroid glands of larval bullfrogs in vitro. The effects of hypothalamic hormones such as mammalian gonadotropin-releasing hormone (mGnRH), ovine corticotropin-releasing hormone (oCRH), and thyrotropin-releasing hormone (TRH) on the release of TSH by dispersed anterior pituitary cells of the bullfrog larvae and adults were also studied. CRH markedly stimulated the release of TSH from both adult and larval pituitary cells. Both TRH and GnRH moderately stimulated the release of TSH from adult pituitary cells but not from the larval cells. This is the first report on the development of an RIA for amphibian TSH, which has provided the direct evidence that the release of TSH from the amphibian pituitary is enhanced by the hypothalamic releasing hormones such as CRH, TRH, and GnRH.

Neuropeptide W acts in brain to control prolactin, corticosterone, and growth hormone release.

The endogenous, peptide ligand for the orphan receptors GPR7 and GPR8 was identified to be neuropeptide W (NPW). Because these receptors are expressed in brain and in particular in hypothalamus, we hypothesized that NPW might interact with neuroendocrine systems that control hormone release from the anterior pituitary gland. No significant effects of NPW were observed on the in vitro releases of prolactin (PRL), ACTH, or GH when log molar concentrations ranging from 1 pM to 100 nM NPW were incubated with dispersed anterior pituitary cells. However, NPW, when injected into the lateral cerebroventricle of conscious, unrestrained male rats, in a dose-related fashion elevated PRL and corticosterone and lowered GH levels in circulation. The threshold dose for all three effects was 1.0 nmol. We conclude that endogenous NPW may play a regulatory role in the organization of neuroendocrine signals accessing the anterior pituitary gland but does not itself act as a true releasing or inhibiting factor in the gland. Central administration of NPW23 also stimulated water drinking and food intake. The ability of exogenous peptide to decrease GH but stimulate PRL secretion and activate the hypothalamo-pituitary adrenal axis, together with the observed behavioral effects, suggests that endogenous NPW may play a role in the hypothalamic response to stress.

Prolactin-releasing peptide and its homolog RFRP-1 act in hypothalamus but not in anterior pituitary gland to stimulate stress hormone secretion.

The RF-amide peptides (RFRPs), including prolactin (PRL)-releasing peptide-31 (PrRP-31) and RFRP-1, have been reported to stimulate stress hormone secretion by either direct pituitary or indirect hypothalamic actions. We examined the possible direct effects of these peptides on PRL and adrenocorticotropin (adrenocorticotropic hormone [ACTH]) release from dispersed anterior pituitary cells in culture and on PRL and ACTH secretion following intracerebroventricular (i.c.v.) administration in vivo. Neither peptide significantly altered PRL or ACTH release from cultured pituitary cells (male rat donors). Central administration of 1.0 and 3.0 nmol of PrRP-31, but only the higher dose of RFRP-1, significantly elevated serum corticosterone levels in conscious male rats. The effect of PrRP-31 was not blocked by pretreatment (i.v.) with the corticotropin-releasing hormone (CRH) antagonist, alpha-helical CRH 9-41; however, pretreatment of the animals (i.v.) with an antiserum to CRH significantly lowered the hypothalamic-pituitary- adrenal axis response to central administration of PrRP-31. On the other hand, the release of PRL was significantly elevated by 3.0 nmol of RFRP-1, but not PrRP-31, in similarly treated, conscious male rats. Pretreatment with the catecholamine synthesis inhibitor, alpha-methyl-para-tyrosine, prevented the stimulation of PRL secretion observed following central administration of RFRP-1. RFRP-1 similarly did not alter PRL secretion in rats pretreated with the dopamine, D(2) receptor blocker, domperidone. These results suggest that the RF-amide peptides are not true neuroendocrine regulators of stress hormone secretion in the rat but, instead, act centrally to alter the release of neuroendocrine factors that do act in the pituitary gland to control PRL and ACTH release. In the case of RFRP-1, stimulation of PRL secretion is potentially owing to an action of the peptide to inhibit dopamine release into the median eminence. The corticosterone secretion observed following central administration of PrRP-31 does not appear, based on our current results, to be solely owing to an action of the peptide on CRH-producing neurons but, instead, may be a result of the ability of PrRP-31 to increase as well the exposure of the corticotrophs in vivo to other ACTH secretagogues, such as oxytocin or vasopressin.

Prolactin-Releasing Peptide Releases Corticotropin-Releasing Hormone and Increases Plasma Adrenocorticotropin via the Paraventricular Nucleus of the Hypothalamus

Intracerebroventricular (ICV) injection of prolactin-releasing peptide (PrRP) is known to increase plasma adrenocorticotropin (ACTH) and cause c-fos expression in the hypothalamic paraventricular nucleus (PVN). We hypothesize that this is the site at which PrRP acts to increase plasma ACTH. We have used ICV injection and direct intranuclear injection of PrRP into the PVN to investigate the sites important in the stimulation of ACTH release in vivo. To investigate the mechanism of action by which PrRP increases ACTH, we have used primary culture of pituitary cells and measured neuropeptide release from in vitro hypothalamic incubations. ICV administration of PrRP increased plasma ACTH 10 min post-injection (PrRP 5 nmol 81.0 ± 23.5 pg/ml vs. saline 16.8 ± 14.1 pg/ml, p < 0.05). Intra-PVN injection of PrRP increased ACTH 5 min post-injection (PrRP 1 nmol 22.9 ± 5.0 pg/ml vs. saline 10.3 ± 1.4 pg/ml, p < 0.05). This effect continued until 40 min post-injection (PrRP 1 nmol 9.9 ± 1.5 pg/ml vs. saline 6.2 ± 0.5 pg/ml, p < 0.05). In vitro PrRP (1–100 nmol/l) did not effect basal or corticotropin-releasing hormone (CRH)-stimulated ACTH release from dispersed anterior pituitary cells. PrRP increased hypothalamic release of CRH (PrRP 100 nmol/l 1.4 ± 0.2 nmol/explant vs. the basal 1.1 ± 0.2 nmol/explant, p < 0.05) but not arginine vasopressin. PrRP also stimulated neuropeptide Y release (PrRP 100 nmol/l 56.5 ± 11.8 pmol/explant vs. basal 24.0 ± 1.9 pmol/explant, p < 0.01), a neuropeptide known to stimulate the hypothalamo-pituitary-adrenal axis. Our data suggest that in vitro PrRP does not have a direct action on the corticotrope but increases plasma ACTH via the PVN and this effect involves the release of hypothalamic neuropeptides including CRH and neuropeptide Y.

The influence of GnRH, oxytocin and vasoactive intestinal peptide on the secretion of β-endorphin and production of cAMP and cGMP by porcine pituitary cells in vitro

The objective of this study was to determine whether gonadotrophin-releasing hormone (GnRH), oxytocin (OT) and vasoactive intestinal polypeptide (VIP) modulate β-endorphin-like immunoreactivity (β-END-LI) secretion by dispersed anterior pituitary cells of pigs and in vivo priming with steroid hormones, estradiol benzoate (EB) and progesterone (P 4), influences the cell reactivity to peptide hormones tested. Additionally, the aim of this research was to examine the involvement of cyclic nucleotides (cAMP and cGMP) in transduction of signals induced by GnRH, OT and VIP in porcine pituitary cells. Pituitaries were collected from ovariectomized (OVX) gilts that were divided into four experimental groups. Animals of group 1 (OVX) received 1 ml corn oil (placebo)/100 kg body weight (b.w.), group 2 (OVX+EB I) and group 3 (OVX+EB II) were treated with EB at the dose 2.5 mg/100 kg b.w., 30–36 and 60–66 h before slaughter, respectively. Animals of group 4 (OVX+P 4) were injected with P 4 at the dose 120 mg/100 kg b.w. for 5 subsequent days before slaughter. Anterior pituitaries were dispersed with trypsin and then pituitary cells were cultured (10 6 per well) in McCoy’s 5A medium containing horse serum (10%) and fetal calf serum (2.5%) for 3 days at 37 °C under an atmosphere of 95% air and 5% CO 2. Subsequently, plates were rinsed with fresh McCoy’s 5A medium and pituitary cells were treated with one of the following agents: GnRH (100 ng/ml), OT (10 −6 M) or VIP (10 −7 M) and incubated for 3.5 h at 37 °C. GnRH did not affect β-END-LI secretion by pituitary cells of OVX (group 1) and OVX+P 4 (group 4) gilts. When the pituitary cells were incubated in the presence of OT and VIP, significant increases were observed. After priming of OVX gilts with EB, 30–36 h before slaughter (group 2), we noted a significant increase in β-END-LI release from pituitary cells only in the presence of VIP. Pituitary cells from gilts treated with EB, 60–66 h before slaughter (group 3), produced markedly elevated amounts of β-END-LI after GnRH, OT or VIP addition. GnRH markedly stimulated cGMP release from cultured pituitary cells in all experimental groups and significantly increased cAMP production by the cells from OVX, OVX+EB II and OVX+P 4 animals. The addition of OT enhanced both cAMP and cGMP output in all experimental groups of pigs. VIP stimulated cAMP release from pituitary cells derived from OVX, OVX+EB I and OVX+EB II animals. cGMP output was markedly elevated under the influence of VIP from pituitary cells of OVX, OVX+EB II and OVX+P 4 gilts. In conclusion, our results suggest that GnRH, OT and VIP can modulate β-endorphin release from porcine pituitary cells and imply the involvement of cAMP and cGMP in transduction of signals induced by studied peptides in the cells.

In vitro and in vivo herpetic vector-mediated gene transfer in the pituitary gland: impact on hormone secretion.

Herpes simplex virus type 1 (HSV-1)-derived vectors are known to be effective tools to deliver transgenes into normal and neoplastic anterior pituitary (AP) cells in vitro. Our objective was to assess the in vitro and in vivo effects of tsK/beta-gal, a temperature-sensitive HSV-1-derived vector harbouring the E. coli beta-galactosidase gene, on AP hormone secretion as well as on transgene expression in rat AP tumours (hyperplastic prolactinomas). The impact of vector infection on prolactin (PRL) and GH release was determined in vitro in normal and hyperplastic (lactotrophic) dispersed AP cells exposed for 24 h to tsK/beta-gal as well as in vivo in ectopic AP grafts. In some oestrogen-induced prolactinoma-carrying rats, vector suspension was stereotaxically injected into the glands to assess transgene expression in vivo. GH and PRL release was measured by specific RIAs. In vivo transgene expression was assessed by immunohistochemistry for beta-galactosidase and enzymohistochemistry (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside). Ectopic pituitary grafts and stereotaxic surgery were performed following standard procedures. At a multiplicity of infection of 0.5, the vector induced a 30 and 22% fall in PRL and GH release respectively in normal AP cells, whereas the corresponding hormone release inhibition for hyperplastic AP cells was 41 and 33% for PRL and GH respectively. In ectopic pituitary grafts, the effect of vector infection on hormone secretion was assessed by measuring serum PRL levels in the host rats every 5 days for 4 weeks post-grafting. In the pituitary-grafted rats that received the viral vector, serum PRL failed to increase to the levels achieved in control-grafted animals. Finally, pituitary tumours stereotaxically injected with tsK/beta-gal showed widespread expression of the beta-galactosidase transgene around the injection areas. The results reported here have implications for basic studies using gene transfer to pituitary gland as well as potential gene therapy approaches to pituitary diseases.