bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of Afferent and Efferent Competence of Transplanted GnRH Cells in the Brains of Hypogonadal Female Mice
Authors: Rajendren Gopalan V. , Gibson Marie J.
Published: March 2003

A deletion in the gene encoding gonadotropin‐releasing hormone (GnRH) induces hypogonadism in mice caused by the deficiency of GnRH. Activation of the reproductive axis can be achieved in these hypogonadal (hpg) mice by third cerebro‐ventricular transplantation of preoptic area (POA) containing GnRH neurons, obtained from normal fetal mice. The present study was carried out in female hpg mice with POA grafts (hpg/POA) to investigate anatomical integration of the GnRH cells required for the functional activation of the reproductive system. Ovarian development was present only in mice in which the graft tissue was located close to the median eminence (ME). The total lack of ovarian development in individuals with grafts containing GnRH cells located elsewhere in the brain suggests that the mere presence of GnRH cells does not guarantee ovarian development, but that the location of the graft may be important. Activation of the grafted GnRH cells following mating, as evidenced by the induction of Fos immunoreactivity, was observed in hpg/POA mice in which there was no ovarian development or detectable GnRH immunoreactive fiber innervation of the ME. Although ovarian development was evident in individuals with grafts located close to the ME, release of luteinizing hormone (LH) in response to mating was apparent in only some of these mice. The occurrence of mating and pregnancy only in hpg/POA mice with ovarian development and the reflex release of LH in response to mating suggests that both the efferent and afferent connections of the GnRH system are important for the full functioning of the system.
Stimulatory Effect of Calcitonin Gene‐Related Peptide on Adrenocorticotropin Release from Rat Anterior Pituitary Cells
Authors: Iino Kazumi , Oki Yutaka , Tominaga Tatsumi , Iwabuchi Masayasu , Ozawa Megumi , Watanabe Fumie , Yoshimi Teruya
Published: March 2003

In the present study, we examined the direct regulatory effect of rat calcitonin gene‐related peptide (CGRP) on adrenocorticotropin (ACTH) release from rat cultured anterior pituitary cells. CGRP significantly increased ACTH release at concentrations of 10−8–10−11 M. The ACTH release was gradually increased by CGRP concentrations lower than 10−10 M, and was decreased at concentrations higher than 10−9 M, presenting a bell‐shaped dose‐response curve. As well as having an additive effect on corticotropin‐releasing factor‐induced ACTH release, CGRP stimulated the accumulation of intracellular cAMP. The CGRP‐induced ACTH release was inhibited by a protein kinase A inhibitor, suggesting that its stimulatory effect on the ACTH release was mediated via an adenylate–cyclase–protein kinase system. CGRP‐like immunoreactive nerve fibers have been reported to innervate the anterior pituitary, so that the stimulatory effect of CGRP on the ACTH release suggests that this peptide may be involved in neural regulation of hormone secretion in the anterior pituitary.
Effect of Endotoxin and Interleukin‐1β on Corticotropin‐Releasing‐Factor and Prostaglandin Release by Rat Brainstem Slices
Authors: Molina‐Holgado  , Borrell  , Guaza
Published: March 2003

This study investigated the effects of lipopolysaccharide (LPS) and interleukin‐1β (IL‐1β) on corticotropin releasing factor (CRF) and prostaglandin E2 (PGE2) release by brainstem slices in vitro. First, we characterized our experimental model and demonstrated that high potassium stimulates CRF release from rat brainstem slices in a calcium dependent way. The direct stimulation of brainstem slices with IL‐1β (3–25 pM) did not modify basal or potassium‐stimulated CRF release, although IL‐1β at the dose of 25 pM increased PGE2 production. Peripheral injection (ip) of LPS (1–10 μg/kg) or IL‐1β (1–10 μg/kg) evoked a dose‐related potentiation of the ex‐vivo release of CRF and PGE2 from brainstem slices. However, central (icv) administration of LPS (10–500 ng/rat) potentiated the release of CRF and PGE2 only at the dose of 500 ng/rat, whereas IL‐1β (1–100 ng/rat) failed to modify significantly the ex vivo production of both CRF and PGE2. The results of the present study provide evidence that peripheral, rather than central, endotoxin and IL‐1β administration induce the activation of brainstem CRF and PGE2, supporting the hypothesis that peripheral cytokine signalling to the CNS is mediated by stimulation of peripheral afferents.
Absence of Photoperiodic Modulation of Gonadotrophin Secretion in HPD Rams Following Chronic Pulsatile Infusion of GnRH
Authors: Lincoln  , Clarke
Published: March 2003

Hypothalamo‐pituitary disconnected Soay rams (HPD rams) were treated chronically with a pulsatile infusion of GnRH (250 ng/2 h) for 10 weeks to reactivate the pituitary‐gonadal axis under long days (16L:8D; LD group n=8) and short days (8L:16D, SD group n=8). The aim was to establish whether photoperiod modulates the secretion of gonadotophins (presumably through the daily melatonin signal) by an action at the level of the pituitary gland. The treatment of the HPD rams with GnRH induced a marked, long‐term increase in the peripheral blood concentrations of FSH, LH and testosterone, growth of the testes and reappearance of the androgen‐dependant sexual skin colouration. There were parallel changes in the short‐term, pulsatile fluctuations in the blood concentrations of LH and FSH, the pituitary responsiveness to GnRH and the testicular responsiveness to a standardized injection of oLH, as observed previously in normal intact rams during reactivation of the reproductive axis induced by a switch from long to short days. There were no significant differences in any of the reproductive parameters between the LD and SD HPD groups, although the blood concentrations of prolactin were significantly higher in the LD group, and GnRH provoked a chronic increase in the blood concentrations of prolactin, particularly in the SD group. Overall, the results support the conclusion that (i) melatonin does not act within the pituitary gland to mediate effects of photoperiod on GnRH‐induced gonadotrophin secretion; and (ii) changes in prolactin secretion which result from the local action of melatonin in the pituitary gland do not affect gonadotrophin secretion in the ram.
Axons Containing the Growth Associated Protein GAP‐43 Specifically Target Rat Corticotrophs Following Adrenalectomy
Authors: Paden Charles M. , Babcock Cheyenne , Conner Karrie A. , Duong Dang Khoa T. , Kuhl Jason M.
Published: March 2003

An extensive network of nerve fibers immunoreactive for the neuronal growth associated protein GAP‐43 (GAP‐43‐IR) is present within the anterior pituitary (AP) of the rat, and the density of these fibers has been reported to increase 4 days after adrenalectomy (ADX). In the present study, we employed confocal dual‐label immunofluorescence microscopy to determine whether GAP‐43‐IR fibers are specifically associated with corticotrophs at various intervals after ADX. A dramatic increase in the density of GAP‐43‐IR was apparent 4 days after ADX, and this increase was sustained at 7 and 14 days post‐ADX. The percentage of corticotrophs in apparent contact with GAP‐43‐IR axons was 87% at 4 days after ADX and 92% at 14 days. In addition, fewer than 15% of GAP‐43‐IR terminals were associated with cells other than corticotrophs in either group. This highly specific targeting of corticotrophs during a period in which these cells are undergoing both hypertrophy and hyperplasia indicates that axonal sprouting is occurring in response to ADX. While the less intense GAP‐43‐IR in the AP of intact rats precluded precise quantitative analysis, the majority of corticotrophs also appeared to be selectively innervated in these animals. The observations that GAP‐43‐IR axons selectively contact corticotrophs, and that both the specificity and thoroughness of innervation are maintained by targeted growth of GAP‐43‐IR axons following ADX, strongly suggest that these fibers are of functional significance.
Ultrastuctural Colocalization of Vesicular Cholecystokinin and Corticoliberin in the Periportal Nerve Terminals of the Rat Median Eminence
Authors: Juaneda  , Dubourg  , Ciofi  , Corio  , Tramu
Published: March 2003

Cholecystokinin (CCK) is present in axon terminals distributed around the fenestrated capillary loops of the hypothalamo‐hypophysial portal system. In the hypothalamic paraventricular nucleus, CCK has been shown to coexist with corticoliberin (CRH). However, in the median eminence (ME) nothing is known about the chemical phenotype of the CCK immunoreactive terminals. This study, carried out in the male rat, was designed to examine the possibility of coexistence of CCK immunoreactivity (CCK‐IR) and CRH‐IR in fibres of the ME and to describe, at the electron microscopic level, the vesicular pattern of distribution of CCK‐IR in the pericapillary endings of the ME. The use of the elution‐restaining procedure showed notable similarities between stainings directed against CCK or CRH, respectively, suggesting a colocalization of both peptides in the same terminals. This result was confirmed using a simultaneous double‐staining procedure. At the electron microscope level, double immunogold staining procedure enabled us to observe a consistent localization of CCK‐IR and CRH‐IR over dense‐cored vesicles. Most of the terminals were seen to contain both immunoreactivities which, in addition, were often present together in the same vesicles. However, some rare endings remained exclusively stained either for CCK or for CRH. Our results provide evidence for a concomitant release of CCK and CRH into the portal blood.
Effects of TGF‐β1 on Prolactin Synthesis and Secretion: an In‐Vitro Study
Authors: Coya  , Alvarez  , Perez  , Gianzo  , Diéguez
Published: March 2003

The hypothalamus exerts a predominantly inhibitory influence on prolactin secretion through dopamine. In addition, the expression of anterior pituitary hormone‐gene products are regulated by intrapituitary growth factors. In particular, TGF‐β1 produced in the pituitary regulates lactotroph cell proliferation and prolactin gene‐expression. This study characterized the regulation of in‐vitro prolactin synthesis and secretion by TGF‐β1 using rat anterior pituitary cells in monolayer culture. Furthermore, we studied the interaction of TGF‐β1 with other signals involved in the neuroregulation of prolactin secretion, such as dopamine and TRH, as well as the importance of different signal transduction pathways in this response. TGF‐β1 inhibited prolactin secretion in a time‐ and concentration‐dependent manner, with half‐maximal inhibition occurring at the range of 15–30 pM. The inhibitory effect was observed after 4 h, being maximal after 4 days of exposure of the cells to the peptide. This inhibitory effect was mimicked by TGF‐β2 but not by inhibin, and was not influenced by oestrogens, being similar in male, normal female or oestradiol‐treated rats. Prolonged pretreatment of the cells with TGF‐β1(4 days) did not modify GH or TSH secretion nor dopamine‐induced inhibition of prolactin secretion, and blunted prolactin responses to TRH, Forskolin, But2‐cAMP and to the calcium ionophore A23187. The effect observed after long‐term treatment (24 h to 4 days) is essentially caused by a decrease in prolactin synthesis, since TGF‐β1 inhibited prolactin mRNA levels and de novo prolactin protein synthesis. However, in the short term (up to 12 h) TGF‐β1 inhibition of prolactin secretion was associated with an increase in intracellular prolactin content, dissecting a dual mechanism of action of TGF‐β1. The short‐term TGF‐β1 effect did not modify Erk‐2 phosphorylation, basal or TRH‐induced increase in intracellular calcium concentration, but blunted basal and forskolin stimulated cAMP levels. But2‐cAMP replacement did not revert the inhibition of prolactin secretion. However, pertussis toxin was able to recover a large percentage of TGF‐β1‐induced inhibition of prolactin secretion. This study indicates that TGF‐β1 plays a crucial role as a modulator of lactotroph function, inhibiting prolactin biosynthesis after long‐term treatment, as well as, after short‐term exposure prolactin secretion at the level of the secretory process, through a mechanism pertussis toxin sensitive but independent of Erk‐2 phosphorylation, calcium concentrations or intracellular cAMP.
Radioligand Assays for Oestradiol and Progesterone Conjugated to Protein Reveal Evidence for a Common Membrane Binding Site in the Medial Preoptic Area‐Anterior Hypothalamus and Differential Modulation by Cholera Toxin and GTPγS
Authors: Caldwell  , Walker  , Rivkina  , Pedersen  , Mason
Published: March 2003

In this study membrane oestradiol (E) binding sites in the medial preoptic area‐anterior hypothalamus (MPOA‐AH) of ovariectomized (OVX) rats were characterized using standard radioligand binding techniques employing E conjugated to bovine serum albumin (BSA) at position 6 and radiolabeled with 125I (E‐6‐[125I‐BSA]). In previous studies binding of a radioactive conjugate of progesterone (P) and BSA (P‐3‐[125I‐BSA]) was examined using the same membrane preparation. E‐6‐[125I‐BSA] binding was linear across a tissue concentration range of 0.005–0.02 mg protein/0.1 ml of membrane suspension. An association T1/2 of 9.5 min and a dissociation T1/2 of 52.1 min for E‐6‐[125I‐BSA] were derived from kinetic experiments. Competition binding experiments revealed high (Ki=0.63±(0.50 nM) and low (Ki=161.5(96.5 nM) affinity binding sites for E‐6‐[125I‐BSA], demonstrating different binding parameters than shown in our previous work for P‐3‐[125I‐BSA] binding. Further studies on MPOA‐AH membranes treated with cholera toxin (CTX) and GTPγS suggested that E‐6‐BSA binding sites are associated with G proteins. E‐6‐[125I‐BSA] binding demonstrated both high‐and low‐affinity sites. GTPγS added to the assay reduced both E‐6‐[125I‐BSA] and P‐3‐[125I‐BSA] binding suggesting that G proteins are associated with both binding sites. Extensive analysis of both E‐6‐[125I‐BSA] and P‐3‐[125I‐BSA] binding sites demonstrated a reciprocal relationship such that high‐affinity E‐6‐[125I‐BSA] binding sites exhibit low affinity for P‐3‐[125I‐BSA] and low‐affinity E‐6‐[125I‐BSA] binding sites exhibit high affinity for P‐3‐[125I‐BSA]. Preincubating membranes with CTX or GTPγS reduced high‐affinity E‐6‐[125I‐BSA] binding and enhanced high‐affinity P‐3‐[125I‐BSA] binding. These results suggest that, in the MPOA‐AH, membrane steroid binding sites exist in two interconvertible conformations that preferentially bind either E‐6‐BSA or P‐3‐BSA, depending on their association with a G protein. Additional studies with free steroids revealed that: (1) oestrogens (17β‐oestradiol, diethylstilbestrol) as well as synthetic oestrogen antagonists tamoxifen and ICI 182 780 displaced P‐3‐[125I‐BSA] further suggesting a relationship between membrane binding sites for E and P‐3‐[125I‐BSA] binding sites; and (2) treatment of OVX rats with E decreased displacement by P‐3‐BSA and increased displacement by ICI 182,780 and tamoxifen suggesting these antagonists affect membrane P‐3‐[125I‐BSA] binding sites after in‐vivo E treatment. The membrane binding sites for E and P demonstrate interrelationships not demonstrated by their nuclear receptors.
Effects of Growth Hormone‐Releasing Peptide‐6 on the Nocturnal Secretion of GH, ACTH and Cortisol and on the Sleep EEG in Man: Role of Routes of Administration
Authors: Frieboes  , Murck  , Antonijevic  , Steiger
Published: March 2003

After repeated intravenous (i.v.) boluses of growth hormone‐releasing peptide‐6 (GHRP‐6) we found recently increases of growth hormone (GH), corticotropin (ACTH) and cortisol levels and of the amount of stage 2 sleep. In clinical use, oral (p.o.), intranasal (i.n.) and sublingual (s.l.) routes of administration have advantages over i.v. administration. We compared the sleep‐endocrine effects of 300 μg/kg of body weight (b.w.) GHRP‐6 in enteric‐coated capsules given p.o. at 21.00 h and of 30 μg/kg GHRP‐6 i.n. or 30 μg/kg GHRP‐6 sl. given at 22.45 h in normal young male controls with placebo conditions. After GHRP‐6 p.o. secretion of GH, ACTH and cortisol remained unchanged. The only effect of GHRP‐6 s.l. was a trend toward an increase in GH in the first half of the night. GHRP‐6 i.n. prompted a significant increase in GH concentration during the total night and a trend toward an increase in ACTH secretion during the first half of the night, whereas cortisol secretion remained unchanged. Furthermore, after GHRP‐6 i.n., sleep stage 2 increased in the second half of the night by trend, and spectral analysis of total night non‐rapid eye movement (REM) sleep revealed a decrease of delta power by trend. In contrast sleep stage 2 decreased during the second half of the night after GHRP‐6 p.o. Our data demonstrate that GHRP‐6 is capable of modulating GH and ACTH secretion as well as sleep. However, the effects depend upon dosage, duration and route of administration.
Adrenoceptor Subtype Involvement in Suppression of Prolactin Secretion by Noradrenaline
Authors: Colthorpe  , Nalliah  , Anderson  , Curlewis
Published: March 2003

In sheep, injection of noradrenaline suppresses prolactin secretion by a direct effect at the pituitary gland. The aims of this study were to use primary cultures of ovine pituitary cells to examine the receptor subtypes that mediate the inhibitory effect of noradrenaline on prolactin secretion and, by using receptor antagonists in vivo, determine whether noradrenaline acts as a prolactin release‐inhibiting factor (PIF). Noradrenaline and dopamine suppressed prolactin secretion from ovine pituitary cells with ED50s of 60.9±46.6 and 1.5±1.0×10−9 mol/l, respectively (P<0.05). The in‐vitro prolactin release‐inhibiting effect of noradrenaline (10−7 mol/l) was not blocked by the dopamine antagonists pimozide (D2) or SCH23390 (D1) but was blocked by each of the adrenoceptor antagonists (α1‐adrenoceptor antagonists prazosin and WB4101, the α2‐adrenoceptor antagonist yohimbine and the β‐adrenoceptor antagonist propranolol). The response to adrenoceptor agonists was also tested in vitro. The α1‐adrenoceptor agonists phenylephrine and cirazoline significantly suppressed prolactin. Of the α2‐agonists, clonidine had no effect whereas oxymetazoline and p‐aminoclonidine both suppressed prolactin. The β‐adrenoceptor agonist isoproterenol also suppressed prolactin while the specific β3‐antagonist BRL37344 had no effect. When the adrenoceptor antagonists were tested in vivo in ewes manipulated to be in the luteal phase, only WB4101 significantly (P<0.05) increased plasma prolactin concentrations but this response was small and only observed in one of two experiments. In summary, these experiments suggest that adrenoceptors and not dopamine receptors are responsible for the inhibitory effect of noradrenaline on prolactin secretion in vitro but do not implicate a particular adrenoceptor subtype. The in‐vivo experiments do not provide convincing evidence for a role for noradrenaline as a physiologically important PIF.
Increase in mRNA Concentrations of Pituitary Receptors for Growth Hormone‐Releasing Hormone and Growth Hormone Secretagogues After Neonatal Monosodium Glutamate Treatment
Authors: Kovacs  , Kineman  , Schally  , Flerko  , Frohman
Published: March 2003

Previous studies have demonstrated that neonatal monosodium glutamate (MSG) treatment destroys growth hormone releasing‐hormone (GHRH) neurones within the hypothalamic arcuate nucleus, decreases serum GH and insulin‐like growth factor (IGF‐I) concentrations, and retards linear growth. In the present study we investigated whether expression of pituitary GH, GHRH receptors (GHRH‐R), growth hormone secretagogue receptors (GHS‐R) and liver IGF‐I is altered in this model of GHRH deficiency. In addition, we investigated if treatment of MSG‐lesioned rats with the GHRH agonist, JI‐38, would ‘normalise’ the GH‐axis. Serum GH and IGF‐I concentrations were determined by RIA, GH mRNA levels were evaluated by Northern blotting, and GHRH‐R, GHS‐R and IGF‐I mRNA levels were measured by semiquantitative RT‐PCR. In accord with previous reports, neonatal MSG treatment caused 50% and 76% decreases in serum GH and IGF‐I concentrations, respectively, at 8 weeks of age. The decline in circulating GH was accompanied by a 56% reduction in total pituitary GH content, which was a reflection of the decrease in total pituitary protein. However, GH concentration (per mg protein) was unaltered. Despite the maintenance of a normal GH concentration, GH mRNA concentration (per μg total RNA) was suppressed by 42%, compared to saline‐treated controls (P<0.05). These data indicate that a post‐transcriptional mechanism, such as a reduction in the GH secretory rate, acts to maintain intracellular GH concentrations. The fall in circulating concentrations of GH leads to a 42% decrease in liver IGF‐IB mRNA levels, while liver IGF‐IA transcripts showed only a 27% suppression. In contrast, pituitary GHRH‐R and GHS‐R mRNA levels (per μg total RNA) were increased in MSG‐lesioned rats by 96% and 180% of normal values (P<0.01), respectively. Twice daily treatment of MSG‐lesioned rats (for 2 weeks) with the GHRH agonist, JI‐38, increased serum GH and IGF‐I levels, as measured 20 h after the last agonist injection. However, GH, IGF‐I, GHRH‐R and GHS‐R mRNA levels were not altered at this time. These results demonstrate that intermittent GHRH agonist treatment stimulates pituitary GH secretion and GH in turn stimulates hepatic IGF‐I but that effects on gene expression are not sustained. Collectively, our observations demonstrate a complex interplay between transcriptional, translational and post‐translational mechanisms within each level of the GH‐axis following destruction of GHRH neurones by neonatal MSG treatment.
Sex‐Steroid Induction of Endogenous Opioid Inhibition on Oxytocin Secretory Responses to Stress
Authors: Douglas  , Johnstone  , Brunton  , Russell
Published: March 2003

In pregnancy, endogenous opioids inhibit enhanced basal and stressor‐stimulated oxytocin neurone activity and secretion. By contrast, stress responses of the hypothalamo‐pituitary‐adrenal (HPA) axis are reduced in pregnancy. We investigated whether the high levels of oestradiol and progesterone of pregnancy could induce these changes. Silastic capsules containing oestradiol or progesterone (or control capsules) were implanted s.c. in virgin female rats for 16 or 17 days, with or without progesterone removal on day 15 to mimic the progesterone withdrawal seen at the end of pregnancy. Plasma concentrations of oxytocin, adrenocorticotrophic hormone (ACTH) and corticosterone were measured in jugular vein blood samples from conscious rats. Under basal conditions, naloxone (5 mg/kg) increased oxytocin secretion in all groups, but had no greater effect in sex‐steroid treated rats, and did not induce Fos expression in the supraoptic nucleus. Forced swimming, a stressor, increased oxytocin secretion at 5 min in vehicle‐injected controls, and this response was slightly attenuated in the sex‐steroid treated groups. Pretreatment with naloxone greatly enhanced the response in the sex‐steroid treated rats, and was less effective in the controls. In rats treated with oestradiol alone, naloxone prolonged the response. Thus, the combined sex‐steroid treatment enhanced the responsiveness of oxytocin neurones to the stressor, while simultaneously restraining oxytocin secretion via endogenous opioid inhibition. In the same rats, ACTH and corticosterone secretion was also stimulated by the stressor, but the hypothalamo‐pituitary‐adrenal (HPA) axis response was not attenuated in sex‐steroid treated rats. Naloxone weakly reduced the HPA axis response in controls and was ineffective in the sex‐steroid treated rats. We conclude that oestradiol and progesterone may be responsible for inducing the opioid restraint and enhanced oxytocin neurone responsiveness in pregnancy.
Insulin‐Like Growth Factor I in the Anterior Pituitary of the Clawed Frog Xenopus laevis: Immunocytochemical and Autoradiographic Indication for a Paracrine Action and Corelease with Prolactin
Authors: David  , Bosshard  , Kloas  , Reinecke
Published: March 2003

Insulin‐like growth factor I (IGF‐I) and its receptor are present in human and rat anterior pituitary. However, few data exist on the potential presence of IGF‐I or its receptor in the non‐mammalian pituitary and the cellular sites of IGF‐I production have not been identified in any species. Thus, we investigated the anterior pituitary of the clawed frog Xenopus laevis which is widely used to study growth and differentiation. The study was performed with antisera against mammalian insulin‐like growth factor I (IGF‐I), prolactin (PRL) and growth hormone (GH) using immunohistochemical and immunocytochemical techniques. IGF‐I binding was determined by in‐vitro receptor autoradiography. The PRL‐and GH‐immunoreactive cells exhibited distinct distribution patterns. Neither at the light nor the electron microscopical level any colocalization of PRL‐and GH‐immunoreactivities was apparent. The PRL‐immunoreactive cells exhibited round granules of medium electron density (mean diameter: 312 nm) and the GH‐immunoreactive cells spherical granules of medium electron density (mean diameter: 165 nm). By the use of serial semithin sections IGF‐I‐immunoreactivity was exclusively located in PRL‐immunoreactive cells. At the ultrastructural level, IGF‐I‐immunoreactivity was confined to the secretory granules in coexistence with PRL‐immunoreactivity using the double labelling immunogold technique. Specific IGF‐I binding sites were localized throughout the pituitary. The results provide evidence for a concomitant release of PRL and IGF‐I and suggest autocrine/paracrine actions of IGF‐I in the anterior pituitary.
Calbindin‐D28k Immunoreactivity is a Marker for a Subdivision of the Sexually Dimorphic Nucleus of the Preoptic Area of the Rat: Developmental Profile and Gonadal Steroid Modulation
Authors: Sickel  , McCarthy
Published: March 2003

Calbindin‐D28k (calbindin) is a 28 kilodalton calcium binding protein which potentially plays a role in neuroprotection. We report here the normal development and gonadal steroid modulation of a sexually dimorphic group of calbindin immunoreactive cells within the sexually dimorphic nucleus of the preoptic area (SDN) which we call the calbindin‐immunoreactive SDN or CALB‐SDN. Beginning on PN2, a faintly immunoreactive CALB‐SDN is present, however, the volume is not sexually dimorphic. On PN4, the staining of the CALB‐SDN appears more robust but the volume is still not sexually dimorphic. By PN8 and extending through PN12 and PN26, the latest age analysed, the volume of the CALB‐SDN is larger in males by two‐ to fourfold. Cresyl violet counterstain reveals a similar developmental profile of the SDN as well as clusters of darkly staining calbindin immunonegative cells which lie around the CALB‐SDN. Castration of males on PN0 decreases the volume of the CALB‐SDN by PN12 and administration on the day of birth and PN1 of either testosterone propionate or oestradiol benzoate, but not dihydrotestosterone propionate to females increases the volume of the CALB‐SDN by PN12. By demonstrating the sexual dimorphism and gonadal steroid modulation of the CALB‐SDN, we hereby establish that calbindin is a specific marker of a subdivision of the SDN and can be used as such in future studies.
Cocaine and Amphetamine‐Regulated‐Transcript Peptide Mediation of Leptin Stimulatory Effect on the Rat Gonadotropin‐Releasing Hormone Pulse Generator In Vitro
Authors: Lebrethon  , Vandersmissen  , Gérard  , Parent  , Bourguignon
Published: March 2003

Pulsatile gonadotropin‐releasing hormone (GnRH) secretion was studied in vitro using explants of the retrochiasmatic hypothalamus from prepubertal male and female rats. Leptin caused a dose‐dependent reduction of the GnRH interpulse interval in both sexes. We studied the effects of cocaine‐ and amphetamine‐regulated transcript (CART) since this peptide was shown recently to mediate the anorectic effects of leptin in the hypothalamus. CART caused a reduction of the GnRH interpulse interval. This effect was prevented using an anti‐CART antiserum which could partially overcome leptin stimulatory effects as well. Using hypothalamic explants from Zucker rats homozygous for the leptin receptor mutation ( fa/fa), GnRH pulse frequency was not affected by leptin, while a significant acceleration was caused by the CART‐peptide. In conclusion, leptin involves the hypothalamic CART‐peptide to stimulate the prepubertal GnRH pulse generator in vitro.
Role of Protein Kinases in the Prolactin‐Induced Intracellular Calcium Rise in Chinese Hamster Ovary Cells Expressing the Prolactin Receptor
Authors: Sorin B. , Vacher A. M. , Djiane J. , Vacher P.
Published: March 2003

There is still only limited understanding of the early steps of prolactin signal transduction in target cells. It has been shown that prolactin actions are associated with cell protein phosphorylation, Ca2+ increases, and so on. However, the link between the activation of kinases and calcium influx or intracellular Ca2+ mobilization has not yet been clearly established. Chinese hamster ovary (CHO) cells, stably transfected with the long form of rabbit mammary gland prolactin receptor (PRL‐R) cDNA were used for PRL‐R signal transduction studies. Spectrofluorimetric techniques were used to measure intracellular calcium ([Ca2+]i) in cell populations with Indo1 as a calcium fluorescent probe. We demonstrate that, although protein kinase C activation (PMA or DiC8) caused a calcium influx in CHO cells, prolactin‐induced PKC activation was not responsible for the early effect of prolactin on [Ca2+]i. Activation of protein kinase A (PKA) or protein kinase G did not modify [Ca2+]i and inhibition of PKA pathway did not affect the prolactin response. In the same way, phosphatidylinositol‐3 kinaseinhibition had no effect on the prolactin‐induced Ca2+ increase. On the other hand, tyrosine kinase inhibitors (herbimycin A, lavendustin A, and genistein) completely blocked the effect of prolactin on [Ca2+]i (influx and release). W7, a calmodulin‐antagonist, and a specific inhibitor of calmodulin kinases (KN‐62), only blocked prolactin‐induced Ca2+ influx but had no significant effect on Ca2+ release. Using pharmacological agents, we present new data concerning the involvement of protein phosphorylations in the early effects of prolactin on ionic channels in CHO cells expressing the long form of PRL‐R. Our results suggest that, at least in the very early steps of prolactin signal transduction, serine‐threonine phosphorylation does not participate in the prolactin‐induced calcium increase. On the other hand, tyrosine phosphorylation is a crucial, very early step, since it controls K+ channel activation, calcium influx, and intracellular calcium mobilization. Calmodulin acts later, since its inhibition only blocks the prolactin‐induced Ca2+ influx.
The Effects of Ciliary Neurotrophic Factor on the Hypothalamo‐Pituitary Gonadal Axis In Vitro in Female Rats
Authors: Stanley S. A. , Todd J. F. , Small C. J. , Kim M. S. , Heath M. M. , Anand P. , Ghatei M. A. , Bloom S. R.
Published: March 2003

Ciliary neurotrophic factor (CNTF) is a member of the neuropoietic family of cytokines. CNTF exerts its actions through activation of a receptor complex, which shows similarity of sequence, second messenger systems and distribution to the leptin receptor. Leptin has been demonstrated to exert profound effects on the hypothalamo‐pituitary gonadal axis. This study examines the in vitro effects of CNTF on hypothalamic luteinizing hormone releasing hormone release (LHRH) and pituitary luteinizing hormone (LH) release compared to those of leptin in the female. We report that CNTF stimulates LHRH release from medial basal hypothalamic explants harvested from proestrous female rats and this effect is of similar magnitude to that seen with leptin. In contrast, CNTF suppresses LHRH‐stimulated LH release from dispersed anterior pituitary cells harvested from proestrous female rats but has no effect on basal LH release. Leptin stimulates basal LH release but has no effect on LHRH‐stimulated LH release. The suppressive effect of CNTF on LHRH‐stimulated LH release has been confirmed in perifused anterior hemipituitaries. These results suggest a differential effect of CNTF on the hypothalamo‐pituitary gonadal axis and a possible role in the modulation of pituitary gonadal function.
Effect of Antisense Suppression of Transforming Growth Factor‐β3 Gene on Lactotropic Cell Proliferation
Authors: De A. , Hentges S. , Boyadjieva N. , Sarkar D. K.
Published: March 2003

The mechanism by which oestrogen regulates lactotropic cell proliferation is not well understood. Recently it has been shown that a transforming growth factor beta (TGF‐β) gene‐related peptide, TGF‐β3, is produced in the lactotropes and stimulates lactotropic cell proliferation in vitro. In this study, the role of this growth factor in oestrogen‐induced lactotropic cell proliferation was determined in vitro using oligonucleotide designed to inhibit TGF‐β3 gene expression. We used the oligonucleotide in an antisense orientation, which is complementary to regions in the TGF‐β3 message. Oligonucleotides in sense and missense orientation were also used. We found that the antisense sequence that was effectively incorporated into pituitary cells produced a marked inhibition of the stimulatory action of oestrogen on lactotropic cell proliferation. Sense and missense oligonucleotides produced no significant effects on oestrogen‐stimulated lactotropic cell proliferation. The growth‐inhibitory effect of antisense oligonucleotide was blocked by TGF‐β3 peptide. These results suggest that TGF‐β3 may be involved in the processes that mediate oestrogen‐regulated lactotropic cell proliferation.
Ghrelin Acts on Leptin‐Responsive Neurones in the Rat Arcuate Nucleus
Authors: Traebert M. , Riediger T. , Whitebread S. , Scharrer E. , Schmid H. A.
Published: March 2003

Leptin decreases food intake and increases energy expenditure in rodents by inhibiting neurones in the hypothalamic arcuate nucleus. The growth hormone secretagogue (GHS) ghrelin is known to stimulate food intake and to be the endogenous ligand for the GHS‐receptor, which is strongly expressed in the arcuate nucleus, like the leptin receptor (Ob‐R). In this study, we analysed the effect of systemic ghrelin administration on Fos expression in the arcuate nucleus on neurones expressing Ob‐R. Injection of ghrelin (0.2 mg/kg, i.p) significantly increased the number of neurones expressing Fos protein in the ventromedial arcuate nucleus. Fifty‐seven percent of all Fos‐positive cells in the ventromedial arcuate nucleus were also positive for Ob‐R staining. Furthermore, we investigated electrophysiologically the effect of ghrelin and leptin on the activity of arcuate neurones in an in‐vitro slice preparation. Ghrelin stimulated the electrical activity dose‐dependently in 80% of all cells tested (n=49) with a threshold concentration of 10−11 M; only 8% were inhibited and 12% did not respond. The effect of ghrelin (10−7 M) was weakly antagonized by the peptidic GHS‐receptor antagonist (D‐Lys3)‐GHRP‐6 (10−4 M), which also showed a much weaker affinity (IC50, 0.9 × 10−6 M) to the GHS‐receptor than ghrelin (IC50, 0.3 × 10−9 M). Ghrelin increased the electrical activity in 76% of all cells which were inhibited by leptin (n=17). These data show that ghrelin interacts with the leptin hypothalamic network in the arcuate nucleus. The opposite effect of leptin and ghrelin on neurones in the arcuate nucleus may serve as a neurophysiological correlate of the orexigenic and anorectic effects of ghrelin and leptin.
In‐Vitro Study of the Effect of Adrenaline on the Functional Capacity of Human Neutrophils: Role During Exercise
Authors: Malpica M. I. , Rodríguez A. B. , Sáez M. C. , García J. J. , Barriga C. , Ortega E.
Published: March 2003

We studied the effect of adrenaline on the capacity of human neutrophils to attach, ingest and destroy Candida albicans. The neutrophils were incubated in vitro in the presence of an adrenaline concentration taken as basal (10−10 m), and another that is referred to as being reached following physical exercise (10−9 m). Two higher concentrations (10−7 m and 10−5 m), which are seldom attained in blood but which can occur at specific locations in the organism, were tested as having a possible pharmacological application. At the two high concentrations (10−7 m and 10−5 m), the capacity to attach, ingest and destroy C. albicans was greater than at the physiological concentrations (10−10 m and 10−9 m). Indeed, the capacity to attach and ingest C. albicans was significantly less after incubation with these physiological concentrations than the control values (incubation in the absence of adrenaline). Hence, high concentrations of adrenaline seem to enhance neutrophils' phagocytic capacity compared to physiological plasma concentrations, whereas small variations such as those caused by physical exercise have no effect on this functional capacity.
Nutritional Regulation of Hypothalamic Leptin Receptor Gene Expression is Defective in Diet‐Induced Obesity
Authors: Sahu A. , Nguyen L. , O'Doherty R. M.
Published: March 2003

Leptin action in the hypothalamus plays a critical role in maintaining normal food intake and body weight. Hyperleptinaemia is associated with obesity in humans and animal models, suggesting a state of leptin resistance. Although the mechanism of leptin resistance is not clearly understood, alterations in leptin receptor (Ob‐R) gene expression have been proposed as a potential mechanism mediating modifications in leptin action in obesity and during changes in nutritional status (fed/fasted). The current study examined the effects of diet‐induced obesity (DIO) made by feeding rats a high fat diet for 9 weeks, and nutritional status on levels of long form (Ob‐Rb) and total (Ob‐Rtot) Ob‐R mRNA expression in the hypothalamus. In the fed state, hypothalamic Ob‐Rb mRNA and Ob‐Rtot mRNA levels were similar in DIO and control standard chow fed rats (SC) despite hyperleptinaemia in DIO rats. However, although an overnight fast moderately increased hypothalamic Ob‐Rb mRNA levels in SC rats, fasting did not increase Ob‐Rb mRNA levels in DIO rats. To address the possibility that elevated leptin concentration in DIO rats may mediate an alteration in OB‐R mRNA levels, we examined the effects of adenovirus‐mediated hyperleptinaemia on Ob‐R gene expression in SC rats. Despite substantially elevated plasma and cerebrospinal fluid concentrations of leptin, hypothalamic Ob‐R mRNA levels were similar in both groups. In conclusion, the current study demonstrates that DIO is associated with a loss of nutritional regulation of hypothalamic Ob‐R mRNA levels, and that hyperleptinaemia is not sufficient to alter Ob‐R mRNA expression.
Central Cholecystokinin‐Octapeptide Accelerates the Activity of the Hypothalamic Gonadotropin‐Releasing Hormone Pulse Generator in Goats
Authors: Ichimaru T. , Matsuyama S. , Ohkura S. , Mori Y. , Okamura H.
Published: March 2003

To clarify central actions of cholecystokinin‐octapeptide (CCK‐8) on reproduction, effects of an intracerebroventricular (i.c.v.) administration of CCK‐8 on the activity of the gonadotropin‐releasing hormone (GnRH) pulse generator were examined in ovariectomized (OVX) goats in the absence or presence of oestradiol. Goats were chronically fitted with recording electrodes in the mediobasal hypothalamus, and electrophysiological manifestations of the GnRH pulse generator were monitored as characteristic increases in the multiple‐unit activity (MUA volleys). In OVX goats, a bolus i.c.v. injection of as little as 0.01 nmol of CCK‐8 induced a MUA volley with a short latency, which resulted in a significant decrease in the post‐treatment volley interval compared to that in the saline injected control. Administration of higher doses of CCK‐8 (0.1 and 2 nmol) did not further accelerate the occurrence of the MUA volley, but stimulatory effects were observed for a longer period than that after the 0.01 nmol injection. When goats were treated with oestradiol, while a bolus i.c.v. injection of 0.01 nmol CCK‐8 had no effect, an injection of 0.1 nmol of the peptide significantly decreased the post‐treatment volley interval. On continuous i.c.v. infusion of CCK‐8 at 3 nmol per 200 µl/h for 3 h, MUA volleys with shorter intervals than those in the control were successively induced without any apparent change in basal plasma luteinizing hormone levels in OVX goats. These results demonstrate that central CCK‐8 strongly accelerates the activity of the GnRH pulse generator in goats.
Inducible Nitric Oxide Synthase is Responsible for Nitric Oxide Release from Murine Pituicytes
Authors: Kjeldsen T. H. , Rivier C. , Lee S. , Hansen E. W. , Christensen J. D. , Moesby L.
Published: March 2003

This study investigated whether pituicytes were able to produce and release nitric oxide (NO), and which type of nitric oxide synthase (NOS) would be responsible for this phenomenon. Lipopolysaccharide (LPS) 1 µg/ml was used as inflammatory mediator. Because pituicytes are known to secrete interleukin (IL)‐6 upon stimulation with LPS, this parameter was also investigated. Cultured pituicytes, from 4‐week‐old male mice, were stimulated with LPS for 6 h or 24 h. At 24 h, there was a significant increase in accumulated nitrite indicating NO formation. In contrast, IL‐6 release was already significantly higher 6 h after stimulation and further increased at 24 h. The correlation between accumulated nitrite and secreted IL‐6 was 0.84 after 24 h of incubation with LPS. The expression of inducible NOS (iNOS) mRNA in the pituicytes was significantly higher than the control level after 6 h and 24 h of exposure to LPS, with levels at 6 h being significantly higher than those at 24 h. There was no detected expression of endothelial NOS or neuronal NOS mRNA. Cultured pituicytes were also subjected to immunocytochemistry for iNOS protein at 6, 12, and 24 h after stimulation with LPS. Most cells were positive for iNOS, but there were no observable differences with the time points that we used. Collectively, these results show that pituicytes are able to produce NO, and that the inducible form of NOS is responsible for this production. Furthermore, there is a weak correlation between NO and IL‐6 released from pituicytes after 24 h of stimulation with LPS.
Growth Hormone‐Releasing Hormone‐Producing and Dopaminergic Neurones in the Mouse Arcuate Nucleus Are Independently Regulated Populations
Authors: Phelps C. J. , Romero M. I. , Hurley D. L.
Published: March 2003

Differentiation of hypophysiotropic neurones that regulate the secretion of growth hormone (GH) and prolactin is influenced by GH and prolactin. Genetic GH and prolactin deficiency in mutant rodent models such as the Ames dwarf (df/df) mouse results in an increase in the number of GH‐stimulatory GH‐releasing hormone (GHRH) neurones and a reduction of prolactin‐inhibitory tuberoinfundibular dopaminergic (TIDA) neurones in the arcuate nucleus during postnatal development. The present study tested the hypothesis that these concomitant changes in numbers of tyrosine hydroxylase (TH)‐ and GHRH‐immunoreactive neurones in df/df hypothalamus might represent a neuronal population of fixed number that undergoes a partial change in phenotype during postnatal development. To evaluate this possibility, the postnatal reduction of the df/df TIDA population was prevented by administering prolactin neonatally to preserve TH phenotype; dwarf and normal sibling mice were treated with daily injections of ovine prolactin or vehicle starting at postnatal day 12 and continuing for 30 days. Following this treatment, numbers of arcuate neurones containing GHRH or TH, or both, were quantified using immunocytochemistry. It was hypothesized that prolactin preservation of TH‐immunoreactive cell number would be accompanied by either a decrease in the GHRH‐producing population or an increase in numbers of cells producing both TH and GHRH. In prolactin‐treated normal (DF/df) mice, numbers of arcuate TH‐immunoreactive neurones were similar to those in vehicle‐treated normals. Numbers of TH‐positive neurones in prolactin‐treated dwarfs were higher than in vehicle‐treated dwarfs, and did not differ from numbers in DF/df. Numbers of GHRH‐immunoreactive cells in vehicle‐treated df/df were higher than in vehicle‐treated DF/df, and were not different in prolactin‐treated groups of either dwarf or normal mice. Neurones containing both TH and GHRH constituted 15% of the TH population, and 76% of the GHRH population, in control normal mice; in control dwarfs, double‐labelled cells were 9.3% of TH and 9.9% of GHRH. Numbers of cells immunoreactive for both TH and GHRH were not affected by prolactin treatment in either mouse type. These results demonstrate that the increase in number of GHRH‐expressing neurones in the df/df arcuate nucleus does not occur at the expense of the TH phenotype, and that this increase is not influenced by prolactin feedback. Although coexpression of TH and GHRH in a subpopulation indicates that TIDA and GHRH populations are not exclusive, they appear to be influenced independently by prolactin and GH signals during development.
NFκB Activation in Mouse Pituitary: Comparison of Response to Interleukin‐1β and Lipopolysaccharide
Authors: Parnet P. , Pousset F. , Laye S.
Published: March 2003

The mouse anterior pituitary contains both types of interleukin (IL)‐1 receptors, IL‐1 receptor type I (IL‐1RI) and IL‐1 receptor type II (IL‐1RII). These receptors are expressed mainly on somatotroph cells. In the present study, the ability of the mouse pituitary to respond in vivo to IL‐1 or to lipopolysaccharide (LPS) was demonstrated by measuring, with an electrophoretic mobility shift assay, the presence of an active NFκB complex in cell nuclei from pituitaries of mice injected intraperitoneally with recombinant rat‐IL‐1β or LPS. Using immunohistochemistry with an antibody directed against the p65 NFκB subunit, a rapid and transient NFκB response to LPS was observed. This response was present predominantly in the nuclei of glial fibrillary acidic protein (GFAP)‐positive cells and F4/80‐labelled cells of the posterior and the anterior pituitary 15 min after stimulation and became faint after 2 h. In comparison, the early and strong NFκB response to IL‐1β treatment was localized into somatotroph cells, GFAP positive cells and F4/80‐labelled cells of the posterior and anterior pituitary. Activation of NFκB in response to IL‐1β was no longer apparent in IL‐1RI knockout mice, confirming that this receptor is essential for the transduction of IL‐1 signal in the pituitary, but remained after LPS treatment. In addition, we investigated the effect of IL‐1 on target genes by measuring the mRNA and proteins synthesis of growth hormone (GH), IL‐6 and IL‐1ra in the pituitary and the plasma. IL‐1β was shown to induce a rapid and strong synthesis of IL‐6 and IL‐1ra in the pituitary but failed to regulate GH contents or release. These data suggest that the pituitary is able to respond to a systemic infection via cytokine‐mediated responses transduced by IL‐1.