bluuebunny/crossref_metadata_2025 huggingface dataset:
Cholecystokinin Activates C‐Fos Expression in Hypothalamic Oxytocin and Corticotropin‐Releasing Hormone Neurons
Authors: Verbalis Joseph G. , Stricker Edward M. , Robinson Alan G. , Hoffman Gloria E.
Published: September 2006

The effect of systemically‐administered Cholecystokinin octapeptide (CCK) on hypothalamic oxytocin, vasopressin, and corticotropin‐releasing hormone neurons was studied by analysis of c‐fos antigen expression in immunocytochemically‐characterized neurons in the supraoptic and paraventricular nuclei. CCK (100μg/kg intraperitoneally) caused a marked increase in nuclear c‐fos immunocytochemical staining, which peaked at 60 to 90 min after injection. C‐fos expression was found in most magnocellular oxytocin neurons in the supraoptic nucleus and in all magnocellular subdivisions of the paraventricular nucleus, but in no vasopressin neurons in either area. C‐fos expression was also found in several parvocellular subdivisions of the paraventricular nucleus: in oxytocin neurons within the medial and lateral, but not the dorsal, parvocellular subdivisions, and in corticotropin‐releasing hormone neurons in the medial parvocellular subdivision. Injection of lower doses of CCK showed that c‐fos expression closely paralleled the pattern of pituitary oxytocin secretion in response to CCK, with a threshold for activation at 1 μg/kg, near maximal responses by 10 μg/kg, and maximal responses by 100 μg/kg. These studies demonstrate that the pattern of c‐fos expression in hypothalamic magnocellular neurons following systemic CCK administration mirrors the neurosecretory response of these neurons, both with regard to specificity for the peptides secreted as well as intensity of secretion. They also demonstrate that systemic CCK administration activates c‐fos expression in parvocellular oxytocin and corticotropin‐releasing hormone neurons, and therefore likely causes secretion of oxytocin and corticotropin‐releasing hormone within the brain at the terminal fields of these neurons.
Regulatory Effects of Estrogens on the Pituitary Content of Secretogranin II and Chromogranin A
Authors: Anouar Youssef , de Monti Mireille , Galindo Estelle , Benié Tanon , Aunis Dominique , Duval Jacques
Published: September 2006

Modulation of pituitary content of secretogranin II and chromogranin A has been compared to that of luteinizing hormone (LH) in adult female rats following gonadectomy and subsequent estradiol therapy. Both proteins were quantified using two specific antisera, BS 487 and anti‐CAP‐14, respectively. Ovariectomy promoted an increase in the concentration of secretogranin II and chromogranin A, though to a lesser degree than that of LH. Moreover, intracellular processing of secretogranin II was significantly enhanced. Daily estradiol injection further increased pituitary secretogranin II and LH contents while that of chromogranin A was reduced to sham‐operated control values. These data 1) demonstrate that both granins are differentially regulated through ovarian factors, and 2) confirm that regulation of secretogranin II production may be closely related to that of LH.
Generation and Characterization of an Antiserum Directed Against Neurohypophyseal Prohormones
Authors: Verbalis Joseph G. , Hoffman Gloria E. , Rosenbaum Lawrence C. , Nilaver Gajanan , Loh Y. Peng
Published: September 2006

An antiserum to neurohypophyseal prohormones was generated by immunization of rabbits with a synthetic peptide fragment bridging the prohormone cleavage site between the vasopressin (AVP) and human AVP‐neurophysin sequences of pro‐pressophysin. Polyclonal antibodies directed against this peptide cross‐reacted with intact human pro‐pressophysin (ED50 of 260fmol), but not with either of the final products of enzymatic processing, AVP and human AVP‐neurophysin. Gel electrophoresis and Western immunoblotting of pituitary or hypothalamic extracts from multiple species including mouse, cow and man identified a protein band of molecular weight consistent with intact pro‐pressophysin; in hypothalamic extracts from normally‐hydrated rats no protein bands were stained, but in extracts from Brattleboro rats a faint band in the area of pro‐oxyphysin was identified. Immunohistochemical studies using the antiserum demonstrated the presence of only very small amounts of immunoreactive prohormone in a few widely scattered cells in the hypothalami of normally‐hydrated rats. However, after 5 days of solute loading with 2% NaCl as drinking solution, staining for intact prohormone was prominent in the supraoptic and paraventricular nuclei of the hypothalamus. Combined immunoperoxidase‐immunofluorescence labeling for prohormone and either AVP‐neurophysin or oxytocin‐neurophysin revealed prohormone staining in both types of magnocellular neurons in rat hypothalami. These studies suggest that during states of accelerated synthesis and secretion of neurohypophyseal hormones some accumulation of intact prohormone occurs in both AVP and oxytocin magnocellular neurons.
Acetylcholine Synthesis in a Primary Culture of Porcine Intermediate Lobe Cells
Authors: Tandon A. , Collier B. , Zhang Z. W. , Feltz P.
Published: September 2006

Previous pharmacological studies with the pituitary gland have suggested that acetylcholine (ACh) might be involved in the regulation of intermediate lobe (IL) function. Whether ACh is endogenous to the IL cells or provided from an extrinsic source is unclear. The present experiments tested the possibility that the endocrine cells of the IL may be a source of ACh by measuring certain cholinergic markers in a primary culture of dissociated porcine cells. The endogenous ACh content was readily measurable in both the freshly dissociated IL cells and in 2‐ or 4‐day primary cultures. Choline acetyltransferase activity was also measurable in the freshly dissociated and cultured IL cells and was reduced by 53% in the presence of a specific inhibitor, napthylvinylpyridine (50 μM). IL cells incubated in the presence of [14C]choline (1 μM) were able to synthesize [14C]ACh and the accumulation of the new ACh was inhibited by hemicholinium‐3 (30 μM), a competitive inhibitor of high affinity choline uptake at cholinergic nerve terminals. In conclusion, these results demonstrate that the endocrine cells of the IL are capable of synthesizing and storing ACh.
Effect of Ovariectomy or Oestrogen Implants upon Pituitary Function in Female Hypogonadal Mice Bearing Normal Fontal Preoptic Area Grafts
Authors: Scott I. S. , Goff A. E. Porter , Cox B. S. , Charlton H. M. , Clayton R. N.
Published: September 2006

The effects of ovariectomy and oestrogen feedback for 10 days upon pituitary and serum luteinizing hormone (LH) content, pituitary glycoprotein subunit messenger ribonucleic acid (mRNA) and prolactin mRNA content in normal females, female hypogonadal mice and hypothalamic grafted female hypogonadal mice, bearing a graft of normal mouse preoptic area tissue into the third ventricle, have been investigated. In normal females ovariectomy resulted in a rise in serum LH, LHβ‐subunit and common α‐subunit mRNAs with no significant change in pituitary LH content or follicle‐stimulating hormone (FSH) β‐subunit mRNA. In the hypogonadal females, preoptic area grafting resulted in an elevation in all of the above parameters into the normal range. Ovariectomy in this group resulted in a further elevation of serum LH, LHβ‐subunit and α‐subunit mRNAs with no change in pituitary LH content or FSHβ‐subunit mRNA, which in all cases were comparable to ovariectomized normal animals. Oestrogen treatment caused a fall in pituitary LH content and the serum LH fell below the detection of the assay. LHβ‐subunit and a‐subunit mRNA mirrored this fall but there was no change in FSHβ‐subunit hybridization. These experiments suggest that even though normal neuronal input to the gonadotrophin‐releasing hormone neurons is disrupted, oestrogen‐induced negative feedback can still occur in grafted female hypogonadal animals. Gonadotrophin‐releasing hormone neurons are reported to lack oestrogen receptors but feedback within this graft by co‐transplanting oestrogen‐sensitive neurons remains a possibility, as does feedback at the level of the host median eminence where graft axons extend to the pituitary portal vessels. The similarity of the response in normal and grafted animals indicates that these actions of oestrogen may be effected predominantly at the pituitary level.
Functional Interrelationships Between Adrenergic and Opioid Systems in the Neuroregulation of Growth Hormone Secretion in Infant Rats
Authors: Arce Victor M. , Cella Silvano G. , Locatelli Vittorio , Müller Eugenio E.
Published: September 2006

Functional interrelationships between hypothalamic adrenergic and opioid systems were studied in 10‐day‐old male and female rats. Either clonidine (150 μg/kg, sc), an α2‐adrenoceptor agonist, or FK 33‐824 (1 mg/kg, sc), a synthetic analog of met‐enkephalin, increased plasma growth hormone (GH) levels, the increment being significantly higher with FK 33‐824 than with clonidine. Pharmacologic blockade of opioid receptors with naloxone (5 mg/kg, sc) did not modify either basal GH levels, or the GH response to clonidine, whereas blockade of α2‐adrenoceptors with yohimbine (2.5 mg/kg, sc) reduced basal GH levels and partially counteracted the FK 33‐824‐induced GH rise. Clonidine (150μ/kg, sc, twice daily) administered from postnatal day 5 to 9, increased basal GH levels and pituitary GH content. In these pups, acute administration of clonidine failed to further release GH, but the GH response to acute administration of FK 33‐824 was significantly enhanced. A 5‐day treatment with FK 33‐824 (1 mg/kg, sc, twice daily), neither modified basal GH levels, nor pituitary GH content. Under these conditions, the in vivo GH response to an FK 33‐824 challenge was significantly enhanced, and the response to clonidine was preserved. Pituitaries from FK 33‐824‐pretreated rats were hyperresponsive to GH‐releasing hormone (10−7 M).

In summary, our data indicate that in rat pups: 1) two separate components i.e. one adrenergic, the other extra‐adrenergic, subserve the GH‐releasing effect of opioid peptides; 2) in contrast to short‐term stimulation of α2‐adrenoceptors, stimulation of opioid receptors does not trigger GH synthesis or induce down‐regulation or tolerance; 3) short‐term opioid stimulation does not affect an α2‐adrenergic challenge, but sensitizes to an opioid challenge.
Endogenous Opioids Modulate the Oxytocin Response to Insulin‐Induced Hypoglycaemia and Partially Mediate the Inhibitory Effect of Ethanol in Man
Authors: Coiro V. , Capretti L. , Davoli C. , SperoniJ G. , Bianconi L. , Cavazzini U. , Marcato A. , Papadia C. , Volpi R. , Chioderat P.
Published: September 2006

The possible inhibition exerted by ethanol on the oxytocin (OT) response to insulin‐induced hypoglycaemia was tested in man. Furthermore, the possibilities that endogenous opioids play a role in the control of hypoglycaemia and/or ethanol action on OT were examined. Insulin tolerance tests were performed in three groups of eight age‐ and weight‐matched normal men treated with: 1) naloxone, group 1 1 mg bolus naloxone + 2.5 mg over 105 min, group 2 2 mg bolus naloxone + 5 mg over 105 min, group 3 4 mg bolus naloxone + 10 mg over 105 min; 2) ethanol (50 ml in 110ml of whiskey) to all the groups; 3) a combination of ethanol + naloxone; 4) normal saline. Furthermore, the effect of ethanol + naloxone (4+10mg) in the absence of insulin‐induced hypoglycaemia was evaluated in seven additional subjects. During this latter test, the plasma levels of OT remained unchanged. Insulin‐induced hypoglycaemia produced a 2.2‐fold increment in plasma OT levels in the control experiments. This response was not changed by the treatment with the lowest dose of naloxone (1+2.5mg) in group 1, but it was significantly enhanced by administration of naloxone at higher doses (mean peak OT levels rose 2.8‐fold in both group 2 and group 3). In all subjects the OT response to hypoglycaemia was completely abolished by ethanol. However, when ethanol was given together with naloxone, the hypoglycaemia‐induced OT rise was only partially inhibited by ethanol. At all doses naloxone produced similar effects; in fact, in all groups OT rose 1.5‐fold in response to hypoglycaemia during insulin tolerance test + ethanol + naloxone. Neither naloxone nor ethanol altered the basal secretion of OT, as tested during 45 min before the insulin tolerance test.

These data demonstrate that the OT response to insulin‐induced hypoglycaemia is inhibited by ethanol. Furthermore, the data indicate that endogenous opioids are involved in the control of hypoglycaemia‐stimulated OT secretion and partially modulate ethanol inhibitory action.
Glutamate Peripherally Administered Exerts Somatostatin‐Releasing Action in the Conscious Rat
Authors: Benyassi Ahmed , Tapia‐Arancibia Lucia , Arancibia Sandor
Published: September 2006

This study was designed to determine whether glutamate is able to stimulate somatostatin release from in vivo conscious animals when somatostatin release is monitored in unanaesthetized rats stereotaxically implanted with a push‐pull cannula in the median eminence. One week after implantation, the median eminence was perfused with artificial cerebrospinal fluid alone or with the addition of either CGS 19755, an N‐methyl‐D‐aspartate (NMDA) receptor antagonist (1(10−6 M), or glutamate (10−5 M). The latter (which is able to cross the brain‐blood barrier at large doses) was also peripherally administered (1 g/kg ip). Median eminence perfusate samples were collected every 15 min and somatostatin was measured by a sensitive radioimmunoassay.

In rats receiving ip glutamate injection, somatostatin release from the median eminence was significantly increased (73.3±10.4 versus 24.8 ± 6.2; P < 0.01; n = 5) when compared to baseline levels measured in the same animals, but no effect was observed when local perfusion of the median eminence with glutamate (10−4 to 10−5 M; n = 6) was performed. Glutamate‐induced somatostatin release was completely blunted (n = 5) by prior local administration of CGS 19755 (10−5 M), a potent NMDA‐type receptor antagonist able to cross the blood‐brain barrier. In contrast, administration of glutamic acid diethylester, a competitive antagonist of non‐NMDA receptors, at doses of 10−4 M (n = 4), was not able to alter this response.

Our results are the first in vivo evidence in favour of a neuroendocrine role for glutamate on somatostatin release whose site of action seems to exclude the median eminence.
The Influence of Photoperiod on the Hypothalamic Content of Beta‐Endorphin and the Luteinizing Hormone Responses to Naloxone and to Steroid Withdrawal in the Male Syrian Hamster
Authors: Juss T. S. , Maywood E. , Walker A. P. , Herbert J. , Hastings M. H.
Published: September 2006

The aim of this study was to investigate the influence of inhibitory photoperiods upon opioidergic function, as determined by changes in the hypothalamic content of β‐endorphin and the luteinizing hormone response to opioidergic receptor blockade, in the male Syrian hamster over the course of gonadal involution and spontaneous gonadal recrudescence. Animals exposed to an 8 h light: 16 h dark cycle (8L: 16D) for 14 weeks underwent gonadal regression. Regression was also observed in animals held for 7 weeks on one of a range of short daylengths of between 11.5 h and 13.5 h, the degree of atrophy being greatest in those animals on the shortest daylength. The tissue concentration of β‐endorphin within the mediobasal hypothalamus was significantly higher in animals exposed to 8L: 16D for 14 weeks than in gonadally active controls held on long days (16L: 8D). Exposure to photoperiods of less than 13.5 h for 7 weeks also caused a significant increase in the β‐endorphin content of the mediobasal hypothalamus and there was a positive correlation between the concentration of β‐endorphin, the degree of gonadal atrophy and the shortness of the photoperiod. Endorphin levels within the preoptic area were not affected by photoperiodic treatments.

Exposure of intact animals to 8L: 16D for 12 weeks caused gonadal atrophy and an associated loss of the luteinizing hormone responses to both naloxone and castration. Castrated animals receiving testosterone replacement (cast + T) also exhibited photoinhibition, in the form of reduced serum levels of luteinizing hormone, and this was similarly accompanied by a loss of sensitivity to naloxone and to withdrawal of steroid. Prolonged exposure to 8L:16D led to spontaneous reactivation of the gonadotrophic axis as a consequence of the development of scotorefractoriness. In both gondally intact animals and in cast + T groups, this was associated with a restoration, in parallel, of the luteinizing hormone responses to naloxone and to castration/ steroid withdrawal. The time‐course of the restoration of the response to steroid withdrawal in castrates was not significantly different to that observed in intact animals. The luteinizing hormone response to naloxone took significantly longer to redevelop in cast + T groups than it did in gonadally intact animals.

The data demonstrate that central opioid systems are sensitive to photoperiod and are consistent with the hypothesis that opioids are involved in the neuroendocrine regulation of reproductive responses to daylength.
Involvement of Dopamine in Inhibition of Food Intake by Cholecystokinin Octapeptide in Male Rats
Authors: Bednar I. , Forsberg G. , Lindén A. , Qureshi G. A. , Södersten P.
Published: September 2006

Deprivation of food reduced the level of dopamine in the cerebrospinal fluid of male rats and subsequent ingestion of food or intraperitoneal injection of Cholecystokinin octapeptide restored the level. Injection of a dopamine receptor agonist (apomorphine) or Cholecystokinin octapeptide inhibited food intake and these effects were reversed by pretreatment with a dopamine receptor antagonist (cis‐flupentixol). Blockade of cholecystokinin‐A receptors, by treatment with L‐364,718, but not cholecystokinin‐B receptors, by treatment with L‐365,260, blocked the inhibitory effect of Cholecystokinin octapeptide on food intake but did not affect the inhibitory effect of apomorphine. It is suggested that Cholecystokinin interacts with dopamine in the control of food intake.
Mapping of the Bovine Oxytocin Gene Control Region: Identification of Binding Sites for Luteal Nuclear Proteins in the 5′ Non‐Coding Region of the Gene
Authors: Walther Norbert , Wehrenberg Uwe , Brackmann Beate , Ivell Richard
Published: September 2006

In view of the small number of hormone‐producing cells, the factors regulating oxytocin gene expression in the classic site of synthesis, in the magnocellular neurons of the hypothalamus, have not yet been characterized. In the early bovine corpus luteum there is a tissue‐specific oxytocin expression involving many more cells. This tissue therefore was chosen as a experimental system to identify deoxyribonucleic acid elements and nuclear proteins involved in the regulation of oxytocin gene expression. 3.2 kb from the 5’non‐coding region of the bovine oxytocin gene have been sequenced and subcloned fragments used as probes for gel retardation and footprinting experiments. Binding sites for luteal as well as more ubiquitous proteins were detected in the oxytocin promoter region and in an artiodactyl‐specific dispersed repeated deoxyribonucleic acid element. A binding site in the promoter region with a superficial similarity to an estrogen‐responsive element (‐159 to ‐152) was shown not to bind this steroid hormone receptor but to bind two nuclear proteins alternatively. One is a luteal protein, the other a more general transcription factor belonging to the steroid hormone receptor superfamily and similar, if not identical to the COUP protein. This alternative binding of a tissue‐ and phase‐specifically expressed protein or an ubiquitous factor to the same site in the oxytocin promoter suggests a role for these two proteins in the transient up‐regulation and subsequent down‐regulation of the oxytocin gene during the differentiation of the bovine corpus luteum.
Long‐Term Dynamic in vitro System for Investigating Rat Pineal Melatonin Secretion
Authors: Rékési Z. , Csernus V. , Horvéth J. , Vigh S. , Mess B.
Published: September 2006

Several details regarding the control of melatonin (MT) secretion from the pinealocytes are still to be clarified. To obtain more data on the mechanism and kinetics of MT secretion and on the interactions between bioactive materials affecting MT production, a perifusion system has been developed in our laboratory. In this dynamic in vitro system the surviving pinealocytes maintain their full responsiveness for at least 5 days. In order to determine the MT contents of large numbers of samples collected in the perifusion system, a sensitive MT radioimmunoassay was set up utilizing our specific MT antibody.

In our perifusion system the basal MT release does not change significantly during the 5‐day experiments. Norepinephrine (NE) was used at 1 μM concentration for 30 min as a marker of the responsiveness of pinealocytes, given at the beginning and at the end of the same experiments. No significant difference was found in the MT responses to NE stimulation over 5 days.

The kinetics of MT response and the dose‐response relationship were investigated after NE exposure at various concentrations (100 nM to 10 mM). NE at 100 nM was found to be ineffective. Between 1 μM and 1 mM concentrations NE increased the MT release in a dose‐dependent manner. No significant difference was found between the responses above 1 mM concentration. NE seems to be a specific stimulator of pineal MT production.

The MT production reached the maximum value after a relatively long lag (2 to 3 h) when NE application had been stopped, and returned to basal values after 5 to 6 h. This prolonged time‐course of MT secretion, in contrast with the fast responses of pituitary cells to releasing hormones, suggests that NE stimulated the synthesis of MT rather than the release of stored hormone.

The modulatory effects of light‐dark cycle on basal and stimulated MT release of perifused pineals was also investigated: Neither basal nor NE‐stimulated (100 nM to 10μM) MT release was influenced significantly by light‐dark conditions showing that the light‐dark cycle does not have a direct modulatory effect on MT secretion under in vitro circumstances.

Based on our observations, this perifusion system should be a useful tool for investigating: 1) hormone interactions on the regulation of pineal MT release, and 2) accurate kinetics of MT response.
Angiotensin Peptides in Brain and Pituitary of Rat and Sheep
Authors: Lawrence Anne C. , Clarke Lain J. , Campbell Duncan J.
Published: September 2006

Several lines of evidence indicate that angiotensin peptides may be formed in the brain, where angiotensin II (Ang II) and angiotensin‐(1–7) (Ang‐(1–7)) may function as neurotransmitters. However, there is considerable controversy concerning the identity and levels of angiotensin peptides in the brain. We have used a novel high performance liquid chromatography‐based radioimmunoassay to measure Ang‐(1–7), Ang II, Ang‐(1–9) and Ang I in various brain regions and in the pituitary of the rat and sheep. We also studied the effect of different methods of tissue extraction, and the effect of the converting enzyme inhibitor ramipril, on angiotensin peptide levels in the rat hypothalamus. The levels of Ang‐(1–7), Ang II, Ang‐(1–9) and Ang I were low (<25 fmol/g) in all brain regions examined, except for the sheep median eminence and cerebellar cortex where Ang II levels were 385±116 and 193±37 fmol/g (mean ± SEM, n = 6), respectively. Pituitary Ang II levels were 103±13 fmol/g in the rat and 63±18 fmol/g in the sheep. The levels of Ang‐(1–7), Ang‐(1–9) and Ang I were much lower than those of Ang II in brain and pituitary. Ang‐(1–7) levels in the rat hypothalamus were low (<6 fmol/g) but methods of extraction which involved freezing and thawing of the tissue resulted in substantially higher levels of this peptide. Ang II levels in the rat hypothalamus (18±3 fmol/g) were reduced to undetectable levels (<6 fmol/g) by ramipril administration. The low levels of angiotensin peptides in the hypothalamus and brainstem indicate that if these peptides function as neurotransmitters in these regions, then they are of particularly low abundance. Moreover, our results indicate that the high levels of Ang‐(1–7) reported previously for rat hypothalamus may be artefactual, due to the method of tissue extraction.
Progesterone Receptor‐Containing Neurons in the Guinea‐Pig Mediobasal Hypothalamus have Axonal Projections to the Medial Preoptic Area
Authors: Warembourg Maryvonne , Poulain Pierre , Jolivet André
Published: September 2006

The location and number of progesterone receptor‐containing neurons in the mediobasal hypothalamus that project to the medial preoptic area were determined by combining retrograde fluorescent tract tracing with progesterone receptor immunocytochemistry. Injections of the retrograde tract tracer Fluoro‐gold were made in the preoptic area of female guinea‐pigs ovariectomized and primed with estradiol. After 5 days survival to allow for retrograde transport, tissue sections were incubated with monoclonal antibodies to the progesterone receptor to detect the presence of progesterone receptor‐immunoreactive neurons.

Cell bodies were labelled with Fluoro‐gold throughout the arcuate nucleus. These neurons were not concentrated in any particular area of the nucleus but were diffusely distributed bilaterally. Retrogradely‐labelled neurons were also observed in the ventrolateral and ventromedial nuclei mainly contralateral to the injection site. Progesterone receptor immunofluorescence labelled a subpopulation (7% to 10%) of these retrogradely‐labelled cells particularly in the arcuate nucleus, including the median eminence. The double‐labelled cells were more numerous in the anterior two‐thirds of the arcuate nucleus. Although our estimates of the proportion of hypothalamic progesterone receptor‐immunoreactive neurons that sent axons directly to the medial preoptic area were low, (about 0.35%), these neurons may be part of a neural circuit involved in the regulation of reproductive processes.
Development of Oxytocinergic Neurons in Monolayer Cultures Derived from Embryonic, Fetal and Postnatal Rat Hypothalami
Authors: Madarász E. , Környei Z. , Poulain D. A. , Theodosis D T.
Published: September 2006

Monolayer cultures from hypothalami of embryonic, fetal and postnatal rats were established to study the development of oxytocin‐secreting neurons in vitro. Particular culture conditions, known to enhance the survival and growth of different types of neural cells in vitro, were used to investigate the conditions necessary for the appearance and survival of these peptide‐producing cells in culture. They included increasing the concentration of potassium in the culture milieu and/or the addition of triiodothyronine (T3). The use of immunocytochemical procedures with a monoclonal antibody that recognizes oxytocin‐associated neurophysin (NP‐OT) and polyclonal antibodies specific for oxytocin permitted us to identify the neurons. In cultures derived from embryonic (E16‐E17) hypothalami, no NP‐OT‐ or oxytocin‐positive neurons were detected and their appearance could not be provoked by increasing extracellular potassium concentration or by administration of T3. On the other hand, in cultures obtained from fetal (E18‐E19) rat hypothalami, a few neurons showed immunoreactivity for NP‐OT (but not for oxytocin); the immunoreactivity was localized essentially in somata and proximal portions of neurites. When 108 M T3 was included in the culture medium, there were cells showing immunoreactivity not only for NP—OT, but also for oxytocin, visible in somata and in dendritic‐ and axonal‐like processes. In comparison, T3 did not influence the total number of neurons developing in these cultures. Lastly, in cultures derived from young postnatal (PO—P2) rat hypothalami, NP—OT‐ and oxytocin‐immunopositive neurons were found regularly; their appearance did not require any special pretreatment of the cultures. In all cultures, high extracellular potassium concentration (25 mM) resulted in a general improvement in the survival of neurons but did not induce the appearance of more oxytocin‐immunoreactive cells. Our observations support in vivo results showing that although presumptive oxytocin‐producing cells appear early in the development of the hypothalamus, their maturation, and in particular, their ability to produce oxytocin, occurs late at the time of birth. A factor that selectively enhances their differentiation, is the thyroid hormone, T3.
Spontaneous Intracellular Calcium Oscillations and Gsα Subunit Expression are Inversely Correlated with Secretory Granule Content in Pituitary Cells
Authors: Chiavaroli Carlo , Cooper Dermot M. F. , Boyajian Christine L. , Murray‐Whelan Rosemary , Demaurex Nicolas , Spiegel Allen M. , Schlegel Werner
Published: September 2006

Cells of the pituitary tumour cell line GH4C1 were exposed to epidermal growth factor, estradiol and insulin for 5 days, a treatment which resulted in 1) increased prolactin storage in secretory granules, 2) the loss of spontaneous [Ca2+]1 oscillations, and 3) a selective reduction of the protein Gsα, seen in immunoblots, cholera toxin labelling, and vasoactive intestinal peptide stimulation of adenylyl cyclase. In contrast, the glucocorticoid dexamethasone, which increases the expression of Gsα, partially restored spontaneous [Ca2+]1 oscillations and decreased prolactin storage. It is concluded that Gsα levels in tumoral cells result in spontaneous electrical activity which may empty prolactin stores by the continuous activation of exocytosis.
Melatonin Regulates the Synthesis and Secretion of Several Proteins by Pars Tuberalis Cells of the Ovine Pituitary
Authors: Morgan Peter J. , Barrett Perry , Davidson Gary , Lawson Wilfred
Published: September 2006

The pars tuberalis (PT) of the pituitary may be an important target for melatonin action, but the secretory output of the melatonin‐responsive cells is unknown. Using [35S]methionine, protein synthesis and secretion have been studied in primary cultures of ovine PT cells, and analysed by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. Only 4% of the labelled proteins appeared in the medium with the majority retained in the cells. Stimulation of the cells with 10μM forskolin increased the accumulation of several labelled proteins in the medium without corresponding changes in the cell (72, 62, 44, 39, 29, 24, 23, 18 and 14 kd). Two‐dimensional gel electrophoresis showed the proteins to have mildly acidic isoelectric points. Melatonin (1 μM) counteracted the stimulatory effect of forskolin on all but one (23 kd) of these secreted proteins. Immunoprecipitation showed this to be prolactin. Furthermore, melatonin alone appeared to have an inhibitory effect on the synthesis and release of proteins into the medium. The synthesis and secretion of the melatonin‐responsive proteins was not inhibited by actinomycin D (1 μg/ml), indicating control at the translational level. This contrasts with the regulation of prolactin which is actinomycin D‐sensitive. Pulse‐chase experiments demonstrated that it requires 30 min for the secretory proteins to appear in the medium, consistent with intracellular processing and packaging prior to secretion. The secretory proteins labelled in the ovine PT, and responsive to melatonin, did not appear to be specific to the PT, as a similar profile of labelled secretory proteins was produced in primary cultures of pars distalis cells. However, melatonin had no effect on the synthesis and secretion of proteins by the pars distalis. These results demonstrate that in the ovine PT melatonin regulates the synthesis and export of several secretory proteins. These are possibly packaging proteins of secretory granules, similar to the granin family of proteins. Thus, the results confirm that melatonin‐responsive cells are secretory cells and further imply that the PT‐specific product is not a protein.
Glucocorticoids are Required for Food Deprivation‐Induced Increases in Hypothalamic Neuropeptide Y Expression
Authors: Ponsalle Patricia , Srivastava Laxmi S. , Uht Rosalie M. , White Jeffrey D.
Published: September 2006

Neuropeptide Y (NPY), a 36 amino‐acid peptide found within the hypothalamus, is thought to be an important regulator of food intake. Hypothalamic NPY gene expression, synthesis and secretion are all known to be increased in models of increased metabolic demand in which serum glucocorticoids are also elevated. The present studies were designed to test the hypothesis that glucocorticoids are required for increased hypothalamic preproNPY mRNA levels induced by food deprivation (FD). First, animals underwent bilateral sham‐adrenalectomy (sham) or not (control), and were subjected to 72 h FD, or not. Total RNA was isolated from hypothalamic tissue blocks and the content of preproNPY mRNA was measured by solution hybridization/RNase protection analysis. This study revealed that there was no significant difference in hypothalamic preproNPY mRNA content between shamfed and control‐fed groups, or between sham‐FD and control‐FD groups. In the second experiment, animals underwent bilateral adrenalectomy (ADX), were allowed to feed ad libitum and were sacrificed 1 day, 4 days and 7 days after ADX. Nuclease protection analysis revealed no significant effect of ADX on hypothalamic preproNPY mRNA levels over this time‐course. Finally, we examined the role of glucocorticoids in regulating NPY gene expression following FD. Animals underwent bilateral ADX, or not. At the time of surgery, ADX animals received placebo, or corticosterone (B) replacement in the form of constant release pellets, at one of two doses. Food was removed from half of the animals in each group 24 h after surgery; all animals were sacrificed 72 h thereafter. There was no difference in preproNPY mRNA content between the ADX‐FD and ADX‐fed groups, relative to the fed controls. Replacement with corticosterone [ADX(B)] did not alter preproNPY mRNA content in fed animals, however preproNPY mRNA content in FD animals was increased 2.5‐fold. These studies demonstrate that glucocorticoids are necessary and serve a stimulatory role in the increase in hypothalamic preproNPY mRNA levels observed under conditions of FD, and suggest that hypothalamic NPY gene expression may be directly responsive to peripheral metabolic and hormonal signals.
Oestrogen Positive Feedback Reduces Arcuate Proopiomelanocortin Messenger Ribonucleic Acid
Authors: Rosie R. , Thomson E. , Blum M. , Roberts J. L. , Fink G.
Published: September 2006

Oestradiol‐17ß, in its positive feedback mode, as well as stimulating the release of luteinizing hormone‐releasing hormone. (LHRH), also stimulates LHRH mRNA synthesis in medial preoptic neurons. Our aim was to determine whether the action of oestradiol might be mediated in part by inhibiting arcuate proopiomelanocortin (POMC) neurons which terminate on LHRH neurons. Female rats were ovariectomized at about 1000 h of dioestrus, given a subcutaneous injection of oil or oestradiol benzoate and killed at 1600 h the same day (presumptive dioestrus), presumptive pro‐oestrus and oestrus and at 1000 h of presumptive pro‐oestrus. In situ hybridization of brain sections was carried out with a [35S]UTP‐labelled riboprobe containing 538 bases of a cDNA identical to exon 3 of the POMC gene, which includes the ß‐endorphin coding domain. The amount of POMC mRNA in cells of the anterior arcuate nucleus, as assessed by grain counts, was significantly decreased in oestradiol benzoate‐ compared with oil‐treated animals at 1600 h of pro‐oestrus, but at none of the other times studied. This finding, together with other data, suggests that the positive feedback stimulation of LHRH mRNA synthesis by oestradiol‐17ß may be due, in part, to the disinhibition of LHRH neurons consequent on inhibition of POMC mRNA biosynthesis in arcuate neurons.
The Role of the Mu1 Opioid Receptor Subtype in the Regulation of Prolactin and Growth Hormone Secretion by Beta‐Endorphin in Female Rats: Studies with Naloxonazine
Authors: Janik James , Callahan Phyllis , Rabii Jamshid
Published: September 2006

The μ opioid receptor subtype has been reported to mediate the prolactin secretory response to opioids. This receptor subtype has been implicated in the morphine‐induced prolactin increase, as well as the prolactin response to μ‐specific opioid peptides. Subtypes of the μ receptor have been proposed and the μ1, site has been postulated as the receptor subtype involved in the morphine‐induced prolactin secretory response. However, the role of this receptor subtype in mediating the endocrine effects of the endogenous opioid peptides has not been characterized. In order to determine the physiological significance of this receptor subtype, animals were pretreated with saline, WIN 44,441–3 (a μ, δ and κ antagonist) or naloxonazine (a μ1 antagonist) followed by a stimulatory dose of morphine or β‐endorphin. A dose response study for β‐endorphin was conducted to determine the minimal stimulatory dose of β‐endorphin on the prolactin and growth hormone (GH) secretory response. The dose response study indicated that β‐endorphin is a more potent stimulus for prolactin release than for GH. A dose as low as 25 ng increased prolactin levels as much as 100‐fold in both lactating and diestrous female rats. In contrast, 2.5 μg β‐endorphin was required in order to consistently and significantly increase circulating levels of GH by 2‐ to 3‐fold. WIN 44,441–3 antagonized the stimulatory effects of β‐endorphin on both prolactin and GH secretion. Naloxonazine pretreatment abolished the morphine‐induced prolactin secretory response, without affecting the GH increase in diestrous females. Naloxonazine also antagonized the prolactin response to β‐endorphin in both lactating and diestrous females. In addition, it attenuated the GH secretory response but did not totally abolish it. These data indicate that β‐endorphin elicits an increase in prolactin release through an opioid specific receptor which appears to be the μ1 opioid receptor subtype. They further suggest that β‐endorphin may increase GH levels, at least partially, via its action at this μ1 site.
Novel Hypothalamic and Preoptic Sites of Prepro‐Melanin‐Concentrating Hormone Messenger Ribonucleic Acid and Peptide Expression in Lactating Rats
Authors: Knollema S. , Brown E. R. , Vale W. , Sawchenko P. E.
Published: September 2006

The deduced structure of the rat melanin‐concentrating hormone (MCH) precursor predicted the existence of at least two peptides that may be processed from it, one similar to teleost MCH and a second novel neuropeptide, NEI. Cellular localization studies confirmed that prepro‐MCH (ppMCH) mRNA and the MCH and NEI peptides are expressed predominantly in cells in the zona incerta and caudal lateral hypothalamic area with minor contingents seen in the olfactory tubercle and pons. A moderate MCH‐and NEI‐immunoreactive axonal projection to the median eminence and, particularly, to oxytocin‐rich regions of the posterior pituitary suggested some anatomical heterogeneity of ppMCH‐expressing neurons in the hypothalamus, and an involvement in neuroendocrine function. In the present study, immunohistochemical and hybridization histochemical methods were used to follow MCH gene and peptide expression as a function of reproductive status in female rats. Nursing dams sacrificed after 8 to 21 days of lactation consistently displayed ppMCH mRNA and MCH and NEI immunoreactivity in a discrete and contiguous band, encompassing the ventral aspect of the medial part of the medial preoptic nucleus, the periventricular preoptic nucleus, and the most rostral aspects of the paraventricular nucleus of the hypothalamus (PVH). Combined immunohistochemical (for oxytocin) and hybridization histochemical (for ppMCH mRNA) staining failed to reveal a significant degree of congruence in the two chemically‐specified cell populations in the PVH of lactating dams. The apparent induction of ppMCH‐derived peptides and mRNA in the preoptic area and PVH was not apparent in animals sacrificed 4 to 8 days after weaning, during late pregnancy, or at any point in the estrous cycle. Moreover, no frank alterations in ppMCH mRNA were evident in the principal sites at which ppMCH is expressed at constitutively high levels, i.e. in the lateral hypothalamic area and zona incerta, as a function of reproductive status. The loci and apparent state‐dependency of the induction of ppMCH mRNA and peptide expression suggests a role for these gene products in the control of lactation.
Evidence that Release of Dopamine in the Brain is Involved in the Inhibitory Effect of Cholecystokinin Octapeptide on Ingestion of Intraorally Administered Sucrose in Male Rats
Authors: Bednar I. , Qureshi G. A. , Södersten P.
Published: September 2006

To study the possibility that release of dopamine in the brain mediates the inhibitory effect of Cholecystokinin octapeptide on ingestive behaviour, the effect of amphetamine on intake of pellets or an intraorally administered sucrose solution was compared with that of Cholecystokinin octapeptide. Additionally, comparisons were made between the effect of Cholecystokinin octapeptide and pargyline, a monoamine oxidase inhibitor, and α‐methyl‐ρ‐tyrosine, a tyrosine hydroxylase inhibitor. While amphetamine dose‐dependently inhibited pellet intake it failed to inhibit sucrose intake in doses which caused behavioural stereotypies (<800 μg). Cholecystokinin octapeptide (5 μg) inhibited ingestive behaviour in both tests. A very high dose of amphetamine (2 mg) was required to inhibit sucrose intake to a level comparable to that of Cholecystokinin octapeptide. Pargyline (5 to 25 mg) or α‐methyl‐p‐tyrosine (25 to 100 mg) dose‐dependently inhibited pellet intake but had only weak effects on the intake of sucrose. Pargyline increased the concentration of dopamine and 3‐methoxytyramine in the dorsal striatum and decreased the concentration of 3,4‐dihydroxyphenylacetic acid. α‐Methyl‐ρ‐tyrosine decreased the concentration of dopamine and 3,4‐dihydroxyphenylacetic acid, but not that of 3‐methoxytyramine. Injection of amphetamine (2 mg), but not Cholecystokinin octapeptide, in rats pretreated with pargyline increased the concentration of 3‐methoxytyramine in the dorsal striatum and this effect was blocked by pretreatment with α‐methyl‐ρ‐tyrosine. Pretreatment with α‐methyl‐ρ‐tyrosine partially reversed the inhibitory effect of Cholecystokinin octapeptide on sucrose ingestion, enhanced the effect of amphetamine but did not affect that of apomorphine, a dopamine agonist. The results support the possibility that the inhibitory effect of Cholecystokinin octapeptide on consummatory ingestive behaviour, in part, is mediated via release of dopamine in the brain.
Stress Affects the Activated Form of the Corticosteroid‐Receptor Complex in the Rat Brain
Authors: Maroulakou Ioanna G. , Kitraki Efthimia , Stylianopoulou Fotini
Published: September 2006

Glucocorticoid actions in the brain, particularly in the hippocampus and the hypothalamus, are critically involved in the response of the organism to stress. The key molecules in this process are the corticosteroid receptors, which upon activation, migrate and act in the nucleus. We have investigated the effect of stress on the activated form of the cytosolic glucocorticoid receptor from the above brain areas, using anion exchange chromatography. Exposure of rats to chronic stress resulted in the disappearance of the chromatographic peak, which corresponds to the activated form (DE II) of the hormone‐receptor complex. For this phenomenon to occur, 1) the animal must be exposed to chronic, and not to acute stress, and 2) the adrenals of the animal must be intact. The disappearance of the activated form of the hormone‐receptor complex (DE II) following chronic stress is most probably due to proteolysis of the receptor molecule, since it is specifically inhibited by the protease inhibitor leupeptin. This phenomenon may represent an adaptive mechanism which helps the organism cope with a repeated stressor.
Immunocytochemical Localization of 11 Beta‐Hydroxysteroid Dehydrogenase in Hippocampus and Other Brain Regions of the Rat
Authors: Sakai Randall R. , Lakshmi Vijaya , Monder Carl , McEwen Bruce S.
Published: September 2006

The dehydrogenase form of 11 β‐hydroxysteroid dehydrogenase (11‐DH) which catalyzes the oxidation of the biologically active steroid, corticosterone, to its inactive metabolite, 11‐dehydrocorticosterone, is found in rat brain. The distribution and localization of 11‐DH‐like labeling in the rat brain was examined by immunocytochemistry. 11‐DH‐like immunostaining was found in all subfields of the hippocampus and in many other parts of the brain, including the preoptic area (POA), central nucleus of the amygdala, bed nucleus of the stria terminalis (NIST) and the cerebral cortex. Percentages of 11‐DH‐positive cells ranged from 10% in the POA and NIST to 50% to 60% in the hippocampus. When combined with neuronal or glial markers, 11‐DH‐like immunostaining was found to be predominantly localized within neurons, ranging from 10% or less glial labeling in hippocampus, amgydala and cortex to 22% glial labeling in the POA and NIST. Immunostaining was present in both the cytoplasmic and nuclear components of some cells in addition to their projections. In the kidney, 11‐DH has been postulated to be a key component in a mechanism by which aldosterone gains access to renal Type I receptors despite the presence of much higher concentrations of glucocorticoids. The present data is consistent with a similar mechanism occurring in at least some parts of the brain, although the hippocampus appears to be an important exception because it does not appear to be differentially responsive to aldosterone in spite of its high 11‐DH activity and immunoreactivity. However, the hippocampus is not implicated in neural control of salt appetite and fluid balance, whereas some of the other brain regions like the POA, NIST and amygdala are believed to be involved. Other aspects of 11‐DH localization must therefore be examined in future studies, including the co‐presence of mineraiocorticoid receptors and 11‐DH in the same or adjacent cells and the possible significance of the relatively high glial localization of 11‐DH immunoreactivity in the POA and NIST.