bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of GH3 Cells Overexpressing Basic Fibroblast Growth Factor (FGF‐2)
Authors: Wada Satoko , Kaji Hidesuke , Yoshimura Koji , Chihara Kazuo
Published: November 2003

Basic fibroblast growth factor (FGF‐2) is not only a potent mitogen for various cells but also a multifunctional factor with angiogenic and chemotactic activity, and the capacity to induce the synthesis of various proteinases and to modulate endocrine function. To clarify the role played by FGF‐2 in the progression of pituitary tumor, we fused rat FGF‐2 cDNA to the promoter SRα, consisting of the early promoter of SV40 and HTLV(I)‐LTR, and we cotransfected GH3 cells with pSV2‐neo by an electroporation method. After selection by G418, we obtained 7 neomycin‐resistant clones. Southern blot analysis of genomic DNA revealed the presence of transfected rat FGF‐2 cDNA in 4 of the 7 clones. To measure FGF‐2 molecules, we established a new immuno‐fluorometric assay system, using 3 monoclonal antibodies against different portions of human FGF‐2. This assay had a minimum sensitivity of 10 pg/ml and cross‐reacted neither with acidic fibroblast growth factor (FGF‐1) nor insulin‐like growth factor 1 (IGF‐1), even at a concentration of 100 ng/ml. Although FGF‐2 was undetectable in the culture medium of any of the clones, the cell homogenate contained a significant amount of FGF‐2 (7.2 ng/mg protein) in 1 of the 4 FGF‐2‐transfected clones (GH3FGF(+)), whereas FGF‐2 was not detected (<5.2 pg/mg protein) in the cell homogenates of either the parent GH3 cells or the control cells transfected with pSV2‐neo alone (GH3FGF(−)). GH3FGF(+) grew as adherent cells and formed epithelial sheets with a growth rate similar to that of control cells. The amount of prolactin(PRL) released by TRH was greater in GH3FGF(+) than that in GH3 or GH3FGF(−). On the other hand, the sensitivity to SRIF was increased in GH3FGF(+) compared with that in other clones. The findings of these in vitro studies indicate that FGF‐2, if it is expressed in pituitary tumor cells, plays little if any role in cell growth but may modulate certain cell functions such as responsiveness to hormones.
Differential LHRH Secretion, Dye Coupling, and Protein Expression in Two Morphologically Distinct Cell Types Identified in GT1–7 Cultures
Authors: Matesic Diane , Attardi Barbara , Dellovade Tammy , Pfaff Donald , Germak John
Published: November 2003

The immortalized neuronal cell line, GT1–7, has been shown to secrete LHRH in a pulsatile manner and to possess many other characteristics of hypothalamic LHRH neurons in vivo, and thus provides a potential model system for studying biochemical and physiological mechanisms regulating LHRH secretion. In the present study, two morphologically and functionally distinct types of cells have been identified in GT1–7 cultures and each type purified to over 95% homogeneity. One type (N cells) appeared more neuronal with extended neurites and somewhat rounded cell perikarya, while the other type (G cells) had flatter cell perikarya that contained filopodia but no neurites. Growth properties of the two cell types also differed. The doubling time for proliferation of N cells was nearly two‐fold shorter than that for G cells and N cells displayed ‘piling up’ whereas G cells exhibited contact inhibition. Functionally, N cells, but not G cells, were dye‐coupled as measured by a fluorescence photobleaching assay. While both cell types expressed LHRH, N cells released significantly higher levels of LHRH into the culture media and exhibited more intense LHRH immunostaining. The two cell types also showed differences in immunostaining for other proteins. N cells, unlike G cells, immunostained positive for neuron‐specific enolase (NSE), whereas G cells, unlike N cells, stained immunopositive for vimentin. Both cell types expressed SV‐40 T antigen protein, indicating that they were derived from the same transgenic mouse hypothalamic tumour. The physiological significance of these two cell types in GT1–7 cultures remains to be determined, but elucidation of their morphological and biochemical properties is intended to contribute to better understanding and application of this experimentally important neuroendocrine cell line.
The Role of Aromatization in the Restoration of Male Rat Reproductive Behavior
Authors: Vagell Michael E. , McGinnis Marilyn Y.
Published: November 2003

This study assessed the role of aromatization in the expression of male reproductive behavior by testing the effects of the aromatase inhibitor, fadrozole, on the restoration of male sexual behavior and partner preference in testosterone‐treated gonadectomized rats. We measured nuclear estrogen receptor occupation to determine whether fadrozole blocked brain aromatase. In addition, nuclear androgen receptor assays were used to verify that fadrozole does not block androgen receptors. Mini‐osmotic pumps fitted to brain infusion cannulas were used to deliver fadrozole (20 μg/day) into the right lateral ventricle. The majority of animals receiving fadrozole treatment with two, 10 mm testosterone filled Silastic capsules (T/F group) failed to display any sexual behavior 7 and 13 days following implant surgery. In contrast, animals receiving fadrozole treatment which were implanted with two, 10 mm testosterone capsules and one, 5 mm 1% estradiol capsule (T/F/E group) copulated normally, indicating that fadrozole’s inhibition of male sex behavior was specifically due to blocking aromatase activity. Moreover, the animals which received only one, 5 mm 1% estradiol capsule (E group) also failed to exhibit male sexual behavior. Partner preference for either a sexually receptive female or a non‐receptive female was measured in a three chambered apparatus for an index of sexual motivation. Repeated measures contrasts on the group x test interaction indicated that the T/F group was not significantly different from the T group. In addition, the E group did not show a preference for the receptive females and was significantly different from the T group. Fadrozole treatment resulted in a 59% decrease in brain nuclear estrogen receptor occupation relative to the T group. Fadrozole had no significant effect on brain nuclear androgen receptor occupation. Our results lend support to the hypothesis that both androgen receptor activation and aromatization are necessary for the restoration of male sexual behavior in rats. However, we found that estradiol is neither necessary nor sufficient for the restoration of partner preference.
Slow Wave Sleep Drives Inhibition of Pituitary‐AdrenalSecretion in Humans
Authors: Bierwolf Claudia , Struve Kai , Marshall Lisa , Born Jan , Fehm Horst L.
Published: November 2003

During the first half of nocturnal sleep, the secretory response of the pituitary‐adrenal axis to either CRH or vasopressin (VP) administration is reduced. Two experiments were performed aiming (i) to investigate the impact of sleep on the response to a combined CRH/VP administration and (ii) to specify the onset of sleep associated pituitary‐adrenal suppression and its relation to specific sleep stages. In experiment I, we compared the effect of simultaneous administration of VP (0.5 IU i.v., within 6 min) and CRH (50 μg bolus i.v., in the third min of VP infusion) on the secretion of ACTH, cortisol and GH in healthy men during the first nocturnal epoch of slow wave sleep (SWS) and during nocturnal wakefulness. The increase of ACTH and cortisol concentrations after combined VP/CRH administration was distinctly higher during wakefulness than sleep (P<0.01). In experiment II, CRH (30 μg/h, after an initial bolus of 30 μg) was continuously infused in 7 healthy men on 2 nights. On one of the nights, the men were allowed to sleep (between 23.00 h and 05.00 h) after a 3‐h period of wakefulness, on the other night they stayed awake throughout the experiment. In both conditions, CRH enhanced ACTH/cortisol plasma levels. Compared with concentrations during continuous wakefulness, sleep and in particular SWS was associated with a suppression of ACTH/cortisol levels (P<0.05). The findings further support an inhibitory influence of early nocturnal sleep on pituitary‐adrenal activity. The effect appears to be strongest during SWS and is probably mediated via hypothalamic secretion of a release inhibiting factor of ACTH.
NO and Angiotensin II Effects on Blood Pressure andFluid Homeostasis
Authors: Liu Hanwu , Terrell Mary Lee , Bui Vuong , Summy‐Long Joan Y. , Kadekaro Massako
Published: November 2003

Angiotensin II (50 ng/5 μl) and L‐NAME (250 μg/5 μl), an inhibitor of NO synthase (NOS), were administered intracerebroventricularly alone or in combination to conscious rats. Mean arterial blood pressure (MABP) increased reaching a peak within 5 min in all groups compared to controls treated with the vehicle, artificial CSF (5 μl). MABP returned to basal levels at 30 min after angiotensin II and remained stable for the following 90 min. In animals treated with L‐NAME alone, after the initial pressor response, MABP declined but began to increase progressively from 30 min until the end of the experiment at 120 min. When administered with angiotensin II, however, the initial pressor response was prolonged. Angiotensin II‐induced drinking was significantly attenuated by L‐NAME. In control rats, inhibiting NOS elevated plasma levels of oxytocin and vasopressin but in angiotensin II‐stimulated animals, only oxytocin was further elevated after L‐NAME. Thus, NO formed centrally inhibits basal secretion of oxytocin and vasopressin as well as the resting blood pressure. During stimulation with angiotensin II, NO facilitates drinking, limits the pressor response and selectively inhibits oxytocin release.
Production of an Oxytocin like Substance by the Subcommissural Organ (SCO), Related to the Reproductive Cycle in Oviparous and Viviparous Reptiles
Authors: D’Uva Ermelinda Limatola , Maria Gaia Monti  , Di Montefiano Rosita
Published: November 2003

The subcommissural organ (SCO) is a circumventricular organ of glial origin typical of all vertebrates. The SCO releases its secretion into the third ventricle to constitute Reissner’s fibre (RF). Reportedly, in reptiles, SCO has cyclic secretory activity related to the reproductive cycle.

In this immunocytochemical study we show that, in females of oviparous reptiles (Lacertidae: Podarcis sicula) and in a viviparous species (Scincidae: Chalcides chalcides), SCO secretion consists of hormones, in part of the oxytocin‐like (OXY‐like) type. The amount of OXY‐like material in the cells and in the third ventricle varies according to the different stages of the reproductive cycle. In the oviparous species, OXY‐like hormone secretion can be induced by FSH administration at 28 °C, in the period of winter reproductive stasis as well. In the viviparous skink, showing an annual single ovulatory cycle, OXY‐like secretion is present in the basal region of the cells, and is released into the third ventricle only at delivery.

 The role of an OXY‐like hormone in the SCO is here discussed in relation to the different stages of the reproductive cycle. Its influence on the hypothalamus‐hypophysis‐gonad axis and its role in the transport of eggs into the ducts in the oviparous species, and at delivery in the viviparous one, are also suggested.
Pharmacological Characterization of Oxytocin‐Binding Sites in Rat Spinal Cord Membranes: Comparison with Embryonic Cultured Spinal Cord Neurones and Astrocytes
Authors: Evrard M. E. , Strosser M. T. , Di Scala‐Guenot D.
Published: November 2003

Detection and pharmacological characterization of OT‐binding sites were performed on 12‐day‐old rat spinal cord membranes and on embryonic cultured spinal neurones and astrocytes after 12 days in culture. In neurone‐enriched cultures, OT‐binding sites were detected by autoradiography on cells morphologically comparable to neurone‐specific enolase immunoreactive cells. In astrocyte cultures, as shown by combination of autoradiography and immunocytochemistry, OT‐binding sites were detected on cells expressing the glial fibrillary acidic protein (a specific astrocytic marker).
Microinjection of the Tachykinin Neuropeptide K into the Ventromedial Hypothalamus Disrupts the Hormonal Onset of Maternal Behavior in Female Rats
Authors: Sheehan Teige P. , Numan Michael
Published: November 2003

The medial amygdala exerts an inhibition of maternal behavior in virgin rats, but neither the site to which it projects to exert this effect nor the neurotransmitter used in such a pathway is known. There is also evidence that the ventromedial nucleus of the hypothalamus exerts an inhibition of maternal behavior, and the medial amygdala projects to this nucleus, suggesting that it may receive a projection from the medial amygdala which is inhibitory for maternal behavior. Tachykinin injection into the hypothalamus inhibits reproductive behavior in male rats, and there is a tachykininergic projection from the medial amygdala to the ventromedial hypothalamus. Consequently, the present study was conducted to evaluate the hypothesis that the tachykinin neuropeptide K can inhibit maternal behavior after injection into the ventromedial hypothalamus. Female rats were primed to be maternal by pregnancy termination and estrogen injection. Four doses of the peptide (279 pmol, 186 pmol, 116 pmol, and 66 pmol) were bilaterally injected into the ventromedial hypothalamus, and all were effective in delaying the onset of maternal behavior. Evidence that neuropeptide K disrupts maternal behavior in animals that have already begun to be maternal is also presented. Site specificity of neuropeptide K’s effect to within the region of the ventromedial hypothalamus was supported, as injection into the mediodorsal thalamus was without effect. Some possibly relevant neuroendocrine effects are addressed, as well as the possibility that tachykinins may act within the ventromedial hypothalamus to promote virgin female rats’ fear of pup odors.
Melatonin Receptors in the Brain and Pituitary Gland of Hypothalamo‐Pituitary Disconnected Soay Rams
Authors: Williams L. M. , Lincoln G. A. , Mercer J. G. , Barrett P. , Morgan P. J. , Clarke I. J.
Published: November 2003

In Soay rams in which the pituitary gland has been surgically separated from the hypothalamus, blood prolactin concentrations vary in response to changes in photoperiod and the administration of melatonin, as in intact animals, providing evidence that melatonin acts within the pituitary gland to control prolactin secretion. In this study the presence of potentially functional melatonin receptors in the pars tuberalis and zona tuberalis (PT/ZT) of hypothalamo‐pituitary disconnected (HPD) Soay rams is confirmed using both in vitro autoradiography with the ligand 2‐(125I)‐ iodomelatonin and in situ hybridization for the melatonin receptor. There was no effect of the HPD operation on the pattern and quantity of 2‐(125I)iodomelatonin binding in the brain demonstrating that this binding is independent of hypothalamic regulation. The possibility that melatonin may control prolactin secretion directly via specific receptors on lactotrophs was investigated using dual in situ hybridization with a (35S) labelled probe for the ovine melatonin receptor (Mel1ab) and a Digoxigenin labelled probe for ovine prolactin. Melatonin receptor gene expression was observed in the PT/ZT in both intact and HPD rams, however, there was no colocalization with prolactin gene expression; only in the ZT was there a close association between cells expressing the melatonin receptor and lactotrophs. The results provide strong support for the view that melatonin acts via the PT/ZT to mediate the effects of photoperiod on the seasonal cycle in prolactin secretion.
Endopeptidase EC 3.4.24.15 Presence in the Rat Median Eminence and Hypophysial Portal Blood and its Modulation of the Luteinizing Hormone Surge
Authors: Wu T. J. , Pierotti† Adrian R. , Jakubowski‡ Moshe , Sheward W. John , Glucksman Marc J. , Smith A. Ian , King Joan C. , Fink George , Roberts James L.
Published: November 2003

The endopeptidase EC 3.4.24.15 (EP24.15) is a zinc metalloendopeptidase that is widely distributed in a variety of tissues, including the testes, pituitary and the central nervous system. Among its numerous roles in metabolizing and processing biologically‐active peptides, the enzyme degrades gonadotropin‐releasing hormone (GnRH) by cleaving the central Tyr5‐Gly6 bond. The aim of the present studies was to determine whether EP24.15 can modulate the concentrations of GnRH within the hypothalamo‐hypophysial portal blood and thereby play a physiological role in reproduction. Our data suggest the presence of immunoreactive EP24.15 in the perivascular space of the median eminence and that this enzyme is secreted into portal blood. We have also shown a physiological role for this enzyme in that an inhibition of its activity with a specific inhibitor augmented the steroid‐induced LH increase in ovariectomized rats. The present results suggest that secretory and post‐secretory mechanisms are important in shaping the GnRH signal from the central nervous system; GnRH metabolism by EP24.15 may be one such mechanism.
Implication of Nitric Oxide in NGF‐Induced Cell Differentiation: Differences Between Neuronal and Beta Cells
Authors: Fayad Wissam , Czernichow Paul , Scharfmann Raphaël
Published: November 2003

Both pancreatic beta cells (insulin‐secreting cells) and neuronal cells express functional receptors for nerve growth factor. However, while the effect of nerve growth factor on neuronal differentiation is well known, its role on pancreatic beta cells is not established. It has been demonstrated that in PC12 cells, a well characterized NGF‐responsive cell line, NGF increases the production of nitric oxide by inducing the expression of nitric oxide synthase. Nitric oxide is subsequently responsible for growth arrest, a step necessary for neuronal differentiation, visualized by the extension of neuronal‐like processes. In the present study, we studied the effect of nerve growth factor on nitric oxide synthesis in INS‐1 cells, an insulin‐producing cell line which possesses the machinery necessary to respond to nerve growth factor. It was demonstrated that the expression of none of the three isoforms of nitric oxide was induced by nerve growth factor in INS‐1 cells, strongly suggesting that nerve growth factor does not induce an increase in nitric oxide production in this cell line. Finally, we demonstrated that whereas growth arrest occurred in INS‐1 cells cultured in the presence of a donor of nitric oxide (SNP), the simultaneous addition of SNP and nerve growth factor is not sufficient to induce the extension of neuronal‐like processes in INS‐1 cells. These dissimilarities strongly suggest that NGF plays a different role in neuronal and pancreatic beta cells.
Gonadectomy Alters Tyrosine Hydroxylase and Norepinephrine Transporter mRNA Levels in the Locus Coeruleus in Rabbits
Authors: Yang Shu‐Ping , Francis Pau K.‐Y. , Spies Harold G.
Published: November 2003

The central noradrenergic system has a major regulatory role on gonadotropin‐releasing hormone/luteinizing hormone (GnRH/LH) secretion in rabbits. Exogenous administration of norepinephrine (NE) alters GnRH/LH release in a sex steroid‐dependent manner, i.e. NE stimulates GnRH/LH release in oestrogen‐primed ovariectomized (OVX) animals but not in non‐primed individuals. To investigate how gonadal steroids influence noradrenergic neuronal activities in the locus coeruleus (LC), mRNA levels of tyrosine hydroxylase (TH) and NE transporter (NET), two key factors regulating NE synthesis and uptake, were compared 3 weeks after gonadectomy (GDX). Intact male (n=5) and female (n=6) New Zealand White rabbits were sacrificed along with castrated males (n=4) and OVX females (n=5). The brainstem from each individual was sectioned and the LC was punched for detection of TH and NET mRNA levels using the ribonuclease protection assay (RPA). Trunk blood was collected to determine immunoactive serum LH values. Levels of LH were elevated in both males and females after GDX. Luteinizing hormone concentrations averaged 0.10±0.05 ng/ml in intact males vs 1.64±0.31 ng/ml in castrated males (P<0.01) and 0.30±0.08 ng/ml in intact vs 9.80±3.50 ng/ml in OVX females (P<0.05), respectively. Removal of the gonads also increased TH mRNA levels in the LC in both males and females. In intact males, TH mRNA levels were 0.796±0.181 pg/&mgr;g total RNA, whereas in castrates mRNA levels averaged 1.667±0.345 pg/&mgr;g total RNA (P<0.05). In intact females, TH mRNA levels were 0.617±0.054 pg/&mgr;g total RNA while the OVX group averaged 1.084±0.202 pg/&mgr;g total RNA (P<0.05). Similar increases in NET mRNA were noted after GDX in both sexes. In males, NET mRNA levels were 1.461±0.401 pg/&mgr;g total RNA in intacts vs 3.666±0.649 pg/&mgr;g total RNA in castrates (P<0.05). In females, NET mRNA levels averaged 1.336±0.212 pg/&mgr;g total RNA and 3.297±0.835 pg/&mgr;g total RNA in the intact and OVX groups, respectively (P<0.05). The data indicate that GDX enhances gene expression of both TH and NET. The results support the hypothesis that the feedback regulation of sex steroids on LH secretion in rabbits of both sexes involves transcriptional/translational processes of at least TH and NET in brainstem NE cells.
Secretory and Morphological Heterogeneity of Porcine Somatotropes during Postnatal Development
Authors: Castaño Justo P. , Ruiz‐Navarro Antonio , Malagón María M. , Hidalgo‐Díaz Carmen , Gracia‐Navarro Francisco
Published: November 2003

Previous studies from our laboratory have demonstrated that porcine somatotropes can be separated into two subpopulations of low (LD) and high density (HD) by centrifugation in a Percoll gradient. The two subsets are present throughout porcine postnatal growth, although their relative proportions vary with age. In prepubertal animals, HD cells exhibit higher secretory granule content and release more GH than LD cells under basal culture conditions. In the present study, we analysed the ultrastructure of separated LD and HD cells from neonate and mature female pigs, and quantified cell size as well as the relative abundance of several subcellular organelles on immunoidentified somatotropes. Subsequently, GH release under basal conditions was assessed for cultures of unseparated cells and also for LD and HD somatotropes obtained at different stages of postnatal development. Results from the morphometric study demonstrated that LD somatotropes were significantly smaller in size, contained less secretory granules and displayed a more developed endoplasmic reticulum than their HD counterparts, regardless of the age of the pituitary donors. In terms of secretory ability, a significant age‐associated decrease in GH release was observed in monolayer cultures of unseparated cells from prepubertal and mature pigs compared to neonates. A similar decline in GH‐releasing ability was detected for cultures of HD cells. For LD cells, GH secretion only decreased significantly in mature animals. In spite of the divergent pattern followed by both subpopulations during growth, HD somatotropes released significantly more GH than LD somatotropes at the three ages studied. Taken together, our findings demonstrate that the population of porcine somatotropes is mainly composed of two subtypes, LD and HD, which differ in density, morphology and basal secretory activity. These differences are essentially maintained during porcine postnatal development. The progressive reduction in the secretory capacity of HD and LD somatotropes, coupled to the decrease in the relative abundance reported for the HD subpopulation, provides the cytological basis for a better understanding of the decline in GH release associated with age in pigs.
Neuronal Synchronization and Ionic Mechanisms for Propagation of Excitation in the Functional Network of Immortalized GT1–7 Neurons: Optical Imaging with a Voltage‐Sensitive Dye
Authors: Hiruma Hiromi , Uemura Tsuguo , Kimura Fukuko
Published: November 2003

Immortalized gonadotropin releasing hormone (GnRH) neurons (GT1 cell line) in culture release GnRH in a pulsatile manner, suggesting that GT1 cells form a functional neuronal network. Optical imaging techniques and a voltage‐sensitive fluorescent dye (RH795) were used to study the mechanism of neuronal synchronization and intercellular communication in cultured GT1–7 cells (one of the subclones of the GT1 cell line). The majority (79%) of GT1–7 cells in contact with one another revealed synchronized fluctuations in spontaneous neuronal activity. When a cell in contact with other cells was electrically stimulated, the evoked excitation was propagated to neighbouring cells. The ionic mechanisms involved in the propagation of electrical signals between interconnected GT1–7 cells were investigated using various blockers of Na+, Ca2+ and K+ channels. The propagation of stimulus‐evoked excitation was prevented by the voltage‐dependent Na+ channel blocker tetrodotoxin. It was also prevented by the voltage‐dependent Ca2+ channel blockers, Ni+ (nonselective), nimodipine (L‐type) and flunarizine (T‐type>L‐type), but not apparently affected by &ohgr;‐agatoxin IVA (P‐ and Q‐type) and &ohgr;‐conotoxin MVIIA (N‐type). The propagation was not influenced by the K+ channel blockers, quinine, tetraethylammonium and Ba2+, but in some cases, it was enhanced by 4‐aminopyridine (4‐AP) and prevented by apamin. These results suggest that voltage‐dependent Na+ channels and L‐ and T‐type Ca2+ channels are involved in the propagation of electrical signals in the GT1–7 neuronal network. Ionic mechanisms, through 4‐AP‐ or apamin‐sensitive K+ channels, also seem to be involved in the regulation of signal propagation. These mechanisms may underlie the functioning of the neuronal network formed by immortalized GnRH neurons.
GnRH Perikarya in Medial Basal Hypothalamus of Pubertal Female Rhesus Macaque are Ensheathed With Glia
Authors: Witkin Joan W. , O’Sullivan Honor , Miller Reginald , Ferin Michel
Published: November 2003

A previous study in our laboratory revealed that the cell bodies of gonadotropin releasing hormone (GnRH) neurons in the preoptic area (POA) of early to midpubertal female rhesus monkeys were extensively invested with thick glial processes. Because the medial basal hypothalamus (MBH) plays a critical role in the control of pulsatile and cyclic gonadotropin release in the primate, we have now focused on the ultrastructural milieu of GnRH neurons of this region in the same sample of monkeys. The ensheathment of the perikarya of GnRH neurons in the MBH with such glial processes was more pronounced than in the POA. Whereas the mean proportion of the cell membrane covered by these glia was 57% in the POA, it was 72% in the MBH. In addition, the cell bodies of GnRH neurons in the MBH of the pubertal monkey (unlike those in the POA) were less well innervated than were those in the adult cycling monkey, further highlighting differences between these brain regions. Differences in the anatomical milieu of the MBH between immature monkeys, in which GnRH release is still relatively quiescent, and adult cycling monkeys are consistent with the hypothesis that GnRH neurons within the MBH are under particular constraint in the immature animals. The functional significance of these observations must, however, await further studies.
Somatostatin Plays a Dual, Stimulatory/Inhibitory Role in the Control of Growth Hormone Secretion by Two Somatotrope Subpopulations from Porcine Pituitary
Authors: Ramírez José L. , Torronteras Rafael , Castaño Justo P. , Sánchez‐Hormigo Angela , Garrido José C. , García‐Navarro Socorro , Gracia‐Navarro Francisco
Published: November 2003

Previous results from our laboratory demonstrated the existence of two subpopulations of porcine somatotropes of low‐ (LD) and high density (HD) that exhibit differences in ultrastructure and respond in an opposite manner to somatostatin (SRIF) in vitro. In LD cells, SRIF did not affect basal growth hormone (GH) release but partially blocked the stimulatory effect induced by GH‐releasing factor (GRF). Conversely, SRIF paradoxically stimulated the secretory activity of HD somatotropes. Here, we have analysed in detail the basic parameters that characterize this differential response. To this end, the time‐ and dose‐dependent effects of SRIF‐14 were evaluated on separate monolayer cultures of both subpopulations. Likewise, the direct effect of the peptide on individual somatotropes from each subset was assessed by cell immunoblot assay. Finally, we compared the effects of SRIF‐14 and SRIF‐28 on cultures of LD and HD cells. SRIF‐14 (10−7 M) induced a rapid (30 min) and sustained (4 h) 2‐fold increase in GH release from HD cells, whereas it did not affect GH secretion from LD somatotropes. Surprisingly, a low dose of SRIF (10−15 M) stimulated GH release from both LD (154.1±8.2% of basal, P<0.05) and HD (337.2±55.5% of basal, P<0.05) subpopulations, even more effectively than higher doses of the peptide. Results from cell blotting showed that SRIF stimulatory effects were exerted directly upon individual somatotropes. Finally, SRIF‐28 elicited similar responses to those observed for SRIF‐14 in both somatotrope subpopulations, yet 10−15 M SRIF‐28 was less potent than the same dose of SRIF‐14 in stimulating GH release from HD cells. Our present findings demonstrate that SRIF can function as a true GH‐releasing factor in cultures of porcine pituitary cells by acting specifically and directly upon somatotropes. Furthermore, together with previous observations, these results strongly suggest that SRIF is not merely an inhibitor of GH release in pigs, but might play a dual modulatory role. Heterogeneity of the somatotrope population contributes greatly to this divergent effect of SRIF.
Melatonin Inhibits Naloxone‐Induced Luteinizing Hormone Release in Ovariectomized Estrogen‐Primed Rats
Authors: Akema Tatsuo , Chiba Atsuhiko , Ikeda Tomoyuki , Nagami Yuta , Kimura Fukuko , Toyoda Jun‐ichi
Published: November 2003

The present study aimed to examine the effect of melatonin on naloxone‐induced luteinizing hormone (LH) secretion in ovariectomized estrogen‐primed rats. A single intracerebroventricular (ICV) injection of naloxone (&mgr; opioid receptor blocker, 15 &mgr;g) or an intravenous (IV) injection of LH‐releasing hormone (LHRH, 50 ng/kg) elicited a transient and significant increase in the serum LH concentration within 10 min. While an ICV injection of 100 ng melatonin by itself did not change the basal LH release, it almost completely inhibited the naloxone‐induced LH release. Melatonin (10 ng) also significantly reduced the effect of naloxone. However, an ICV injection of 100 ng melatonin did not affect the LHRH‐induced LH release. In separate experiments, the effect of melatonin on naloxone‐induced pulsatile LH secretion was studied in estrogen‐treated rats. A continuous IV infusion of naloxone (20 mg/kg/h) induced LH pulses in rats treated ICV with saline. An ICV administration of 100 ng melatonin, which by itself did not affect basal LH secretion, significantly reduced the frequency, but not the amplitude, of LH pulses induced by the naloxone infusion. These results show that melatonin has a suprapituitary site of action to inhibit naloxone‐induced LH release, and suggest that melatonin has an effect in inhibiting the activity of the hypothalamic LHRH pulse generator, either directly or indirectly, in female rats.
Age‐Dependent Accumulation of Hybrid Vasopressin‐Oxytocin Gene Products but not Hybrid Oxytocin‐Vasopressin Products in the Endoplasmic Reticulum of Brattleboro Rats
Authors: Budd Timothy C. , Morris John F.
Published: November 2003

The age‐dependence of the incidence of magnocellular neurosecretory neurons containing abnormal accumulations of peptide in the rough endoplasmic reticulum was examined in homozygous Brattleboro rats and in their wild‐type Long Evans counterparts. Neurons in which the immunophenotype of the peptide aggregates indicate that somatic cross‐over mutations involving the 5′ end of the vasopressin gene and the 3′ end of the oxytocin gene have occurred, increased with age in homozygous Brattleboro rats, reaching a maximum of 24 cells per hypothalamus (approximately 0.6% of the vasopressin neurons). The increase occurred in both male and female animals but was significantly greater in females. The average incidence of such cells was 6 times greater in the supraoptic than in the paraventricular nucleus. No such cells could be detected in either nucleus of Long Evans rats despite the evidence for hybrid mRNA in these animals. Moreover, no accumulation of peptide translated from the hybrid mRNAs derived from the 5′ end of the oxytocin gene and the 3′ end of the vasopressin gene could be detected in either Brattleboro or Long Evans animals. These results strongly suggest that the accumulation of peptide in the rough endoplasmic reticulum of vasopressin neurons in homozygous Brattleboro rats is due to an abnormality other than the somatic crossing‐over mutation. A second type of abnormal magnocellular neuron with accumulations of peptide in the rough endoplasmic reticulum, in which the immunophenotype of the peptide reveals products derived only from the oxytocin precursor, was present in both Long Evans and Brattleboro rats, but did not increase with age in Brattleboro rats. The incidence of these cells was similar in the supraoptic and paraventricular nuclei.
Inhibition of Brain Oestrogen Receptors by RU 58668
Authors: Vagell Michael E. , McGinnis Marilyn Y.
Published: November 2003

The purpose of this study was to determine if injections of the 11ß‐substituted steroidal antioestrogen, RU 58668, would block two measures of oestrogen receptor action in ovariectomized adult female rats. Using an in vitro nuclear exchange assay, it was found that RU 58668 reduced cell nuclear []>
3H]‐oestradiol binding in brain tissue 24 h after it was injected. However, pituitary cell nuclear [3H]‐oestradiol binding was significantly reduced just 2 h after the antioestrogen was injected. Our results demonstrate that RU 58668 can reach the brain following a subcutaneous injection, but that it needs more time to reach the brain than it does to reach the pituitary. Since the levels of hypothalamic and pituitary progestin receptors are known to be regulated by oestradiol, cytosolic [3H]‐R5020 binding was used as an in vitro assay of endogenous oestrogen receptor action. RU 58668 blocked induction by oestradiol of cytosolic [3H]‐R5020 binding in both the brain and pituitary 48 h after it was injected. In the absence of oestradiol, RU 58668 did not stimulate cell nuclear [3H]‐oestradiol binding or cytosolic [3H]‐R5020 binding in either brain or pituitary. In conclusion, injections of RU 58668 blocked two measures of oestrogen receptor action in the brain and pituitary without showing oestrogenic activity itself.
Effect of POMC1–76, its C‐terminal Fragment γ3‐MSH and Anti‐POMC1–76 Antibodies on DNA Replication in Lactotrophs in Aggregate Cell Cultures of Immature Rat Pituitary
Authors: Tilemans Diane , Ramaekers Dirk , Maria Andries  , Denef Carl
Published: November 2003

Treatment of aggregate cell cultures of 14‐day‐old rat pituitary for 40 h with purified human (h) POMC1–76 dose‐dependently augmented the number of DNA replicating lactotrophs as estimated by autoradiography of []>
3H]‐thymidine (3H‐T) incorporation in cells immunostained for prolactin (PRL). No such effect was seen on the total number of 3H‐T labelled cells (the majority of which did not contain any pituitary hormone in a detectable amount) or on the total number of lactotrophs. The effect of hPOMC1–76 on 3H‐T incorporation in lactotrophs was blocked by concomitant treatment with anti‐hPOMC1–76 monoclonal and polyclonal antibodies cross‐reactive with rat POMC1–74. The latter anti‐hPOMC1–76 antibodies also decreased the number of 3H‐T incorporating lactotrophs in the absence of hPOMC1–76. γ3‐MSH, which is the C‐terminal domain of hPOMC1–76, mimicked the effect of hPOMC1–76 on 3H‐T incorporation in lactotrophs but its potency was lower than that of hPOMC1–76. Other melanocortin (MC) peptides such as α‐ and β‐MSH were also effective but were less potent than γ3‐MSH. The difference in potency was not due to partial degradation of the peptides. hPOMC1–76 did not affect 3H‐T incorporation in other pituitary cell types. In contrast γ3‐MSH also augmented the number of 3H‐T labelled somatotrophs and thyrotrophs. In the embryonic kidney 293 cell line stably transfected with the MC‐3 receptor, γ3‐MSH (10 nM) augmented cAMP formation up to 30 times. In contrast, hPOMC1–76 (100 nM) was inactive in this test system, indicating this peptide is not an agonist at the MC‐3 receptor. The present investigation further supports the role of rat POMC1–74 as a paracrine growth factor in the development of lactotrophs. The active core of POMC1–76 does not seem to be restricted to its C‐terminal domain γ3‐MSH as the latter peptide displays a growth promoting effect that is different from that of POMC1–76: it is less potent, it is not specific for lactotrophs and whereas the effect of γ3‐MSH may be mediated by the MC‐3 receptor that of POMC1–76 is not.
Changes in Distribution of Cystatin C, Apolipoprotein E and Ferritin in Rat Hypothalamus after Hypophysectomy
Authors: Katakai Kenji , Shinoda Motoo , Kabeya Kenji , Watanabe Masatomo , Ohe Yoshihide , Mori Masatomo , Ishikawa Koichi
Published: November 2003

To clarify the mechanism underlying the process of degeneration of injured CNS neurons, we have immunohistochemically examined the distribution of cystatin C, apolipoprotein E, IgG, transferrin and ferritin in the hypophysectomized rat hypothalamus. Stainings for ferritin revealed that reactive microglial cells massed in the paraventricular and supraoptic nuclei 14 days after hypophysectomy, when the degeneration of vasopressin neuronal cell bodies was apparent. Cystatin C‐positive magnocellular neurons first appeared at 4 days and the number of intensely‐stained cells increased rapidly up to the 7th day of hypophysectomy, followed by a decrease thereafter. Most of such cystatin C‐positive neurons were simultaneously stained with anti‐vasopressin serum. Accumulation of apolipoprotein E in extra‐cellular spaces was obvious in the both hypothalamic magnocellular nuclei at 7 days. Several apolipoprotein E‐positive cells were localized in the supraoptic nucleus, although the number of apolipoprotein E‐positive cells was much smaller than that of cystatin C‐positive cells. The experiments performed with the transferrin and IgG antibodies showed undetectable levels of such molecules in and around the degenerating magnocellular neurons during whole experimental periods. These findings suggest the importance of cystatin C and apolipoprotein E in the process of degeneration and/or regeneration of magnocellular neurons after hypophysectomy.
Nitric Oxide Is Involved in the Genesis of Pulsatile LHRH Secretion from Immortalized LHRH Neurons
Authors: López Francisco José , Moretto Marcelo , Merchenthaler István , Negro‐Vilar Andrés
Published: November 2003

Neuronal networks controlling endocrine events present synchronous activity which is required for maintaining physiological functions, including reproduction. Although pulsatile hormone secretion is of paramount importance, the mechanism(s) by which secretory episodes are generated remain largely unknown. Nitric oxide (NO) has become the prototype of a new family of signaling molecules in the body. Nitric oxide diffuses from the source cell and controls activity of neighboring neurons as well as itself as a retrograde messenger. Cells of the luteinizing hormone‐releasing hormone (LHRH) neuronal network, the key component in the control of reproduction, are scattered and loosely arranged in the anterior hypothalamus. A diffusible neurotransmitter could provide a means for establishing synchronous activation of the LHRH neuronal network leading to physiologically‐relevant pulsatile LHRH secretion. In this study, we demonstrate that immortalized LHRH‐producing neurons (GT1–7 cells) express NO synthase (NOS) mRNA and protein. Furthermore, GT1–7 cells are NADPH‐diaphorase‐positive (a marker of NOS activity) and the histochemical reaction can be abolished by treatment with a competitive NOS blocker. The presence of citrulline in these cells provides further evidence for the biological activity of NOS. These observations indicate that an active NO synthesizing machinery is present in immortalized LHRH neurons. In addition, we show that LHRH secretion is enhanced by NO in a cGMP‐dependent manner. Since pulsatile LHRH secretion from immortalized LHRH neurons in vitro is abolished by NOS blockers and NO scavengers, it appears that NO is a unique neurotransmitter that is necessary to set LHRH neurons in phase to establish synchronized pulsatile LHRH secretion.
The value of simulations in the management education of nurses: students’ perceptions
Authors: Wildman S. , Reeves M.
Published: November 2003

This paper examines the use of simulations in the education of student nurses as part of their preparation for management responsibility. A review of the literature indicates that there is little published work on this topic and that those evaluations that have been reported invariably occur at the end of the exercise, thus giving an incomplete picture of the simulation. The nature and format of the simulation, used with 128 students undertaking a pre‐registration/diploma in higher education course, is described. Results from student evaluations immediately following the exercise and after 13 weeks of clinical experience are presented. The findings indicate that this is a realistic and enjoyable way of learning which adequately prepares students for management experience in subsequent clinical allocations. Issues concerned with internal and external validity of simulations and areas for further research are explored.
Symptoms of the stomatognathic system in temporomandibular and cervical spine disorders
Authors: DE WIJER A. , STEENKS M.H. , BOSMAN F. , HELDERS P.J.M. , FABER J.
Published: November 2003

This study was performed to assess the prevalance of signs and symptoms of temporomandibular disorders (TMD) in patients with cervical spine disorders (CSD) and to compare patients with CSD and subgroups of patients with TMD with regard to the results of orthopaedic tests of the stomatognathic system. A group of 103 consecutive patients with signs and symptoms of CSD and a group of 111 consecutive patients with TMD were examined. All subgroups of TMD patients showed a significantly smaller range of motion than the CSD patients. Patients with TMD had limited mouth opening (<40 mm) on active and passive mouth opening more often than CSD patients. TMD patients with myogenous problems reported oral habits more often than CSD patients, although no objective differences between CSD and TMD patients were found. Subgroups of TMD patients reported joint sounds, and pain on palpation and joint play tests of the temporomandibular joint (TMJ) more frequently than CSD patients. Joint sounds on active movements, pain on palpation of the TMJ, and pain on joint play tests correctly classified 82% of the patients with TMD and 72% of the patients with CSD. In spite of the biomechanical and anatomical relationship between the neck and the stomatognathic system, the results of the study show that CSD patients have signs and symptoms of TMD comparable with those of the adult Dutch population. It was concluded that the function of the masticatory system should be evaluated in patients with neck complaints in order to rule out a possible involvement of the masticatory system.
Overexpression of nitric oxide synthases in tendon overuse
Authors: Szomor Z. L. , Appleyard R. C. , Murrell G. A. C.
Published: November 2003

Tendon disorders with a chronic nature, including the rotator cuff, are extremely common, and represent a major clinical problem. Mechanical overload has been proposed as an important etiologic factor in tendinopathies. Nitric oxide (NO), a free radical produced by nitric oxide synthases (NOSs), is a potent regulator and stimulator of biological processes including tendon degeneration and healing. It is also involved in response to mechanical stimuli in different tissues. In an animal model of acutely injured tendon healing temporal and differential expression of NOS isoforms has been demonstrated, suggesting that different patterns of NOSs expression may have different biological functions. Therefore, we hypothesized that tendon overuse may result in a differential upregulation of NOSs, particularly iNOS. An animal model of supraspinatus tendon overuse was utilized, which consisted of treadmill running. A group of animals of the same strain and age subjected to normal cage activity were used as controls. Following a 4‐week exercise protocol supraspinatus tendons were harvested, RNA was extracted, and subjected to competitive reverse transcription and polymerase chain reaction (RT‐PCR) to determine the expression levels of inducible‐, endothelial‐, and neuronal‐NOS isoforms (i‐, e‐, and nNOS). The mRNA expression of all three NOS isoforms increased in the supraspinatus tendons as a result of overuse exercise. iNOS and eNOS mRNA expression increased fourfold (p < 0.01), and there was an increase, but statistically not significant, in nNOS mRNA expression in the overused tendons when compared with the controls. This study is the first to show that NOS isoforms are upregulated in rotator cuff tendon as a result of chronic overuse, and suggests the involvement of nitric oxide in the response of tendon tissue to increased mechanical stress. © 2005 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res
Collagen degradation products modulate matrix metalloproteinase expression in cultured articular chondrocytes
Authors: Fichter M. , Körner U. , Schömburg J. , Jennings L. , Cole A. A. , Mollenhauer J.
Published: November 2003

Destruction of collagen within osteoarthritic cartilage depends in part on collagen‐degrading matrix metalloproteases (MMP). Degradative fragments of type II collagen (Col II) occur in normal and in osteoarthritic cartilage, and may contribute to regulation of matrix turnover by interfering with normal cell–matrix communication pathways. Therefore, the effects of different types of collagen fragments on mRNA and protein levels of MMP‐2, MMP‐3, MMP‐9, and MMP‐13 in cultured bovine articular knee chondrocytes and explants were examined. Primary chondrocytes and explants were incubated with fragments from whole cartilage collagen matrix (Colf) and from purified type II collagen (Col2f), or with a synthetic 29‐mer peptide representing the amino‐terminal domain of type II collagen (Ntelo). Gelatin zymography revealed increases of proMMP‐2, a shift towards active MMP‐2 and increases in proMMP‐9, depending on the type of fragment. In situ hybridization of cartilage sections displayed MMP‐3 mRNA in virtually all cells. Moderate to strong increases in MMP‐2, MMP‐3, MMP‐9, and MMP‐13 mRNA levels were detected by quantitative PCR. The results demonstrate stimulating effects of collagen fragments on both mRNA and/or protein from MMP ‐2, ‐3, ‐9, and ‐13, and suggest a novel mechanism of MMP induction and activation that includes a particular role for N‐telo in controlling catabolic pathways of matrix turnover. © 2005 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res
Liaison psychiatric nursing in an inner city accident and emergency department
Authors: Ryrie I. , Roberts M. , Taylor R.
Published: November 2003

The Mental Health Nursing Review Team recommend research to examine the potential of liaison mental health nursing. This paper describes a study into the work of two emergency psychiatric nurses (EPN) attached to a central London accident and emergency (A&E) department. A semi‐structured interview schedule was employed to generate data on role development and purpose, and a retrospective survey of clinical activity was carried out over a 94‐day period. Specific attributes and working practices, which promoted integration within the A&E department, are identified as are the reasons for referring patients to the EPNs, patients' primary problems on assessment, assessment outcome and the staff involved in each assessment. Findings are considered in relation to the various models of psychiatric consultation and liaison found in the literature. The scope and quality of the available data is also assessed and recommendations are made to enable a more robust prospective study to be undertaken.