bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hypothalamic Gonadotropin‐Releasing Hormone Secretion: Methodological Aspects of in vitro Systems
Authors: Wibullaksanakul Sunee , Handelsman David J. 
Published: September 2006

AbstractThe regulation of gonadotropin‐releasing hormone (GnRH) secretion from the medial basal hypothalamus (MBH) of adult male rats has been studied using two types of in vitro methods, the dynamic superfusion and static incubation systems. We compared both systems for methodological features including stability of baseline GnRH release and technical limitations or artifacts as well as for response to norepinephrine and naloxone, an opiate antagonist, which represent the two major physiological neuromodulators regulating the GnRH neuron. Baseline GnRH release rate per MBH was similar in both systems for at least 6 h, although owing to dilution of effluent samples by flow‐through medium, the dynamic superfusion system required six MBH per chamber compared with the static incubation system which required only one MBH per chamber. Procedural losses of GnRH were low with either system without the need for proteolytic enzyme inhibitors and were mainly due to adhesion to plastic surfaces of chambers and tubings in the dynamic superfusion system. Mechanical agitation of chambers by either manual swinging or continuous shaking increased GnRH release rate. Addition of hypertonic KCl (final 50 mM) or equivalent NaCl or mannitol solution in a minimal volume directly into the tissue chambers, induced a non‐specific GnRH release due to osmotic effects in the dynamic superfusion system. In contrast, the static incubation system, where hypothalamic tissue is exposed to the exact final concentration of solute by complete change of media, was more resistant to GnRH release by hypertonic stimulus. Thus, only in the static incubation system did 50 mM KCl concentrations cause GnRH release through depolarization alone. Using systems optimized to avoid technical artifacts, stimulation with naloxone demonstrated dose‐dependent GnRH release in both systems whereas norepinephrine gave a clear dose‐dependent GnRH release only in the static system.Thus both incubation systems produce stable and similar baseline GnRH release for at least 6 h. The static incubation system proved superior requiring fewer hypothalami, a shorter preincubation stabilization period, allowing more accurate concentration of additives and was less prone to mechanical and osmotic artifacts while preserving responsiveness to physiological stimuli. The static incubation system, being technically simpler, more robust and accurate, provided a more valid approach to the in vitro study of GnRH release and its regulation by neurochemicals.
Distribution, Isolation and Sequence Analysis of the C‐Terminal Heptapeptide of Pro‐Enkephalin A (YGGFMRF) from the Ovine Median Eminence
Authors: Giraud A. S. , Clarke I. J. , Rundle S. E. , Parker L. M. , Funder J. W. , Simpson R. J. , Smith A. I. 
Published: September 2006

AbstractUsing a polyclonal antiserum raised against the C‐terminal heptapeptide of pro‐enkephalin A, we have isolated the opioid heptapeptide Tyr‐Gly‐Gly‐Phe‐Met‐Arg‐Phe (MERF) from ovine median eminence and mapped its distribution in that structure. MERF‐immunoreactivity was confined to the pars externa (neurosecretory zone) where it colocalized with corticotrophin‐releasing factor in the majority of terminals. No larger, N‐terminally extended forms of MERF were detected in median eminence extracts suggesting that pro‐enkephalin is fully processed to its constituent enkephalin congeners, and that the bioactive products, including MERF, act at the level of the hypothalamus in regulating anterior pituitary function.
Binding Sites for Vasopressin in the Human Pituitary are Associated with Corticotrophs and may Differ from Other Known Vasopressin Receptors
Authors: Du Pasquier D. , Loup F. , Dubois‐Dauphin M. , Dreifuss J. J. , Tribollet E. 
Published: September 2006

AbstractIn view of the fact that Vasopressin can induce pituitary adrenocorticotrophin release, we performed an autoradiographical study of [3H]arginine vasopressin binding in human pituitary tissue obtained post‐mortem from adults and foetuses. Sites of specific, high affinity binding (IC50 3 to 5 nM) were detected as patches in the anterior lobe and at the junction between the anterior and neural lobes. The neural lobe was not labelled. Immunocytochemical studies performed on human pituitary tissue showed that [3H]arginine vasopressin only marked zones which correspond to areas rich in cells immunoreactive to adrenocorticotrophin. We conclude that in the human pituitary, corticotrophs bear vasopressin binding sites. Since non‐radioactive synthetic structural analogues of vasopressin acting as V1 and V2 agonists or antagonists failed to compete for binding of radioligand in the human pituitary, while a V1‐type agonist displaced [3H]arginine vasopressin binding in the rat pituitary, we postulate that binding sites in the human pituitary may differ from the previously known vasopressin receptors.
Reduced Oxytocin Release from the Neural Lobe of Lactating Rats is Associated with Reduced Pituitary Content and does not Reflect Reduced Excitability of Oxytocin Neurons
Authors: Higuchi T. , Bicknell R. J. , Leng G. 
Published: September 2006

AbstractLactating rats show reduced oxytocin release compared with virgin female rats in response to a variety of stimuli, including stress and osmotic stimulation. We sought to establish whether this is a consequence of a reduced response in the oxytocin cells, or of a change in stimulus‐secretion coupling at the level of the neurosecretory terminals in the neural lobe. Blood sampling experiments in anaesthetized rats showed that systemic administration of cholecystokinin resulted in significantly less oxytocin release in lactating rats than in virgin female rats. Electrophysiological recordings of single cells in the supraoptic nucleus, however, showed no difference in the responsiveness of oxytocin cells to this stimulus. Oxytocin release evoked by electrical stimulation or by depolarization with high potassium solutions was lower in isolated neural lobes from lactating rats than in glands from non‐lactating rats, whereas evoked vasopressin release was similar in the two groups. The lactating rat neural lobes had a reduced oxytocin content: to study the consequences of depletion we compared hormone release evoked by electrical stimulation in vitro in neural lobes from normal male rats, and from male rats given 2% NaCI to drink for 2 or 4 days. Saline drinking resulted in a reduction in gland content of both oxytocin and vasopressin, and the evoked release of both hormones was also significantly reduced when expressed as a percentage of the gland content, as was also seen for oxytocin release for glands from lactating rats. Finally, measurement of the extracellular potassium response to stimulation of the isolated neural lobe as an index of the excitability of neural lobe neurosecretory axons was unchanged in lactating rats compared with virgin female rats. Together, the data indicate that reduced oxytocin release observed in lactating rats is a simple consequence of reduced oxytocin content in the neural lobe rather than of a reduced excitability of the oxytocin neurons.
Calcium‐Activated Potassium Channels in Rat Dissociated Ventromedial Hypothalamic Neurons
Authors: Treherne J. M. , Ashford M. L. J. 
Published: September 2006

AbstractA potassium‐selective channel, characterized by a single channel conductance of 160 pS was demonstrated to be present in rat freshly dispersed ventromedial hypothalamic nucleus neurons. The single channel activity was shown to be dependent, using inside‐out membrane patches, upon the presence of intracellular calcium ions, with maximal sensitivity between 10−6 and 10−6 M[Ca2+], and to be modulated by membrane voltage, depolarization causing an increase in open‐state probability in the presence of an activating concentration of calcium. Therefore these properties place this channel into the category of a large conductance (maxi‐K+) calcium‐activated potassium (Ca2+‐K+) channel. This channel is active in cell‐attached recordings from glucoreceptive cells when depolarized by glucose or tolbutamide with openings often associated with action current repolarization. These openings were shown to be abolished in the presence of extracellular Cd2+ and La3+ ions, which block calcium channels, suggesting that extracellular calcium entry upon cell depolarization is responsible for their activation.On a few occasions, a larger conductance (250 pS) Ca2+‐K+ channel was observed in inside‐out membrane patches isolated from ventromedial hypothalamic nucleus neurons. In contrast to the 160 pS channel, the presence of intracellularly‐applied ATP caused a concentration‐dependent, reversible inhibition of its open‐state probability.
Occlusion of the Melatonin‐Free Interval Blocks the Short Day Gonadal Response of the Male Syrian Hamster to Programmed Melatonin Infusions of Necessary Duration and Amplitude
Authors: Maywood E. S. , Lindsay J. O. , Karp J. , Powers J. B. , Williams L. M. , Titchener L. , Ebling F. J. P. , Herbert J. , Hastings M. H. 
Published: September 2006

AbstractPhotoperiodic control of the neuroendocrine axis is mediated by changes in the duration of the nocturnal melatonin signal. This study tested the hypothesis that reading of the signal depends upon the presence of a period free of melatonin between successive signals. Adult male Syrian hamsters were pinealectomized and received chronic subcutaneous infusions of melatonin or saline for 6 weeks. Animals which received saline had large testes. Those which received a single daily infusion which lasted for 10 h (50 ng/h) followed by 14 h without infusion underwent gonadal atrophy. Other animals received a compound melatonin signal in which the melatonin‐free interval was occluded by a continuous infusion (25 ng/h). Superimposed upon this was a 10 h phasic increase in infusion rate such that the maximum rate of infusion was equivalent to that observed in controls (25 ng/h increase, 50 ng/h peak rate), or the increase in rate over the baseline was the same as in controls (50 ng/h increase, 75 ng/h peak rate). In neither group did the animals undergo gonadal regression. Analysis of iodomelatonin binding sites by in vitro autoradiography failed to reveal any systematic difference between animals which did and did not respond to melatonin and so the absence of a response could not be attributed to loss of receptors. These data demonstrate that the photoperiodic system cannot identify the melatonin signal solely upon the features of nocturnal peak height or amplitude of the peak over baseline. They are consistent with the hypothesis that the melatonin‐free interval plays a significant role in photoperiodic time measurement.
Further Characterization of Rat 26,000 Prolactin as a Glycoprotein with Essentially O‐Linked Carbohydrate Chains
Authors: Bollengier F. , Hooghe R. , Velkeniers B. , Mahler A. , Vanhaelst L. , Hooghe‐Peters E. 
Published: September 2006

AbstractIn the rat two major molecular variants of prolactin are recorded i.e. 23,000 Mr and glycosylated 26,000 Mr. In order to further characterize the glycosylated 26,000 rat prolactin molecular variant, rat pituitary cell lysates were digested with several glycoen‐zymes and the digestion products submitted to sodium dodecyl sulphate polyacrylamide gel electrophoresis and subsequent immunoblotting. The results were as follows: treatment with 1) neuraminidase, specific for sialic acid, yielded an Mr decrease of the glycosidic variant from 26,000 to 24,500, 23,800, 23,000 and 22,000; 2) endo‐α‐N‐acetylgalactosaminidase, which releases the disaccharide Gal (β 1–3) GalNac from O‐glycans, split 26,000 rat prolactin into a doublet of Mr 26,000 to 25,500; and 3) mixed exoglycosidases from Turbo cornutus caused a gradual Mr shift from 26,000 to 23,000.Affinity chromatography on wheat germ agglutinin Sepharose 6MB and soybean agglutinin agarose of rat pituitary homogenates and competitive inhibition tests showed that glycosylated rat prolactin has distinct affinity for these lectins.From the experimental data it is proposed that glycosylated rat prolactin is O‐linked through threonine by the disaccharide Gal (β 1–3) GalNac and possesses at least GalNac, and/or Gal and sialyl residues.
Evaluation of a Possible Role for the Dopamine D1 and D2 Receptors in the Steroid‐Dependent Suppression of Luteinizing Hormone Secretion in the Seasonally Anoestrous Ewe
Authors: Curlewis J. D. , Naylor A. M. , McNeilly A. S. 
Published: September 2006

AbstractThis study was undertaken to determine whether dopaminergic suppression of pulsatile luteinizing hormone (LH) secretion during seasonal anoestrus in the ewe is mediated via the dopamine D1 or D2 receptor. This was tested by 1) assessing the response to dopamine D1 and D2 antagonists during seasonal anoestrus, and 2) determining the ability of D1 and D2 agonists to suppress pulsatile LH secretion during the breeding season. In seasonally anoestrous ewes the D2 antagonist pimozide increased LH pulse frequency although this effect did not reach significance (P = 0.07). The D1 antagonist SCH 23390 had no effect on LH pulse frequency. LH pulse amplitude and mean LH were not affected by either treatment. During the breeding season, ovariectomized oestradiol‐implanted ewes were injected intracerebroventricularly with vehicle, LY 171555 (dopamine D2 agonist) and SKF 38393 (D1 agonist) with each drug tested at 50 μg and 200 μg. At the higher dose, LY 171555 significantly (P<0.05) reduced LH pulse frequency in the 2 h period immediately after treatment. Mean LH declined at both doses but only in the first hour after treatment. SKF 38393 did not affect LH pulse frequency, pulse amplitude or mean LH. These results suggest that the D1 receptor is not involved in the suppression of pulsatile LH secretion during seasonal anoestrus. Dopaminergic suppression of pulsatile LH secretion is mediated via the D2 receptor but the significance of this neurotransmitter in the seasonal suppression of LH remains to be elucidated.
Effects of Naloxone on the Preparturient Increase in Adrenocorticotrophin and Cortisol in Foetal Sheep
Authors: Brooks A. Nigel , Challis John R. G. 
Published: September 2006

AbstractParturition in sheep is preceded by a rapid increase in foetal pituitary‐adrenal activity. Administration of exogenous opioids increases foetal plasma adrenocorticotrophin (ACTH) and cortisol concentrations after Day 125 of pregnancy (term = 145 days). In order to test the hypothesis that endogenous opioids regulate the increase in pituitary‐adrenal activity prior to parturition in foetal sheep, we measured changes in plasma ACTH and cortisol in response to administration of either naloxone (1.2 mg intravenous bolus followed by a 2.5 mg/h continuous infusion for 2 h; foetal body weight approximately 2.5 kg) or saline to chronically catheterized foetal sheep at 2‐day intervals between Day 140 and the onset of parturition. In addition, we utilized a computerized algorithm (Munro) to examine the precise characteristics of ACTH and cortisol secretion in control and naloxone‐treated foetuses.The pattern of ACTH and cortisol secretion in saline‐infused control foetuses was highly pulsatile but varied considerably between individual foetuses. ACTH pulses occurred on average every 40 min but were not always coincident with pulses of cortisol. There were no significant changes in average ACTH concentrations, pulse frequency or amplitude between foetuses sampled at 3 to 4 and 1 to 2 days before parturition. In contrast, average cortisol concentrations and pulse amplitude were significantly (P < 0.05) increased, whereas pulse frequency was reduced, in foetuses at 1 to 2 days before parturition. Naloxone caused a significant (P < 0.05) inhibition of ACTH concentrations when compared with pretreatment values and with saline‐treated controls, an effect which persisted for at least 2 h after the end of naloxone infusion. However, there were no significant differences during naloxone treatment in the frequency or amplitude of ACTH or cortisol pulses nor in the mean cortisol concentration.We conclude that ACTH and cortisol are secreted in a pulsatile fashion in the late gestation foetal sheep and that these pulses are not always coincident with each other. Blockade of endogenous opioid activity during the preparturient increase in pituitary‐adrenal activity in foetal sheep, results in a reduction in plasma concentrations of ACTH. These results therefore provide evidence for the tonic regulation of foetal pituitary‐adrenal activity by endogenous opioids during the final days prior to birth in the sheep.
Localization of Inhibin Alpha‐Subunit Immunoreactivity in the Rat Adrenal Cortex
Authors: Merchenthaler Istvân , Culler Michael D. , Negro‐Vilar Andres 
Published: September 2006

AbstractInhibin, a water soluble, non‐steroidal glycoprotein hormone that inhibits follicle‐stimulating hormone secretion, is produced by the gonads and several non‐gonadal tissues (placenta, pituitary and brain). This study describes the presence of inhibin α‐chain‐immunoreactive cells in the adrenal cortex of intact adult male rats and the effects of hypophysectomy and adrenocorticotropin treatment on the expression of inhibin in that gland. In intact rats, the majority of the immunoreactive cells were found scattered in the zona glomerulosa and reticularis. The zona fasciculata contained only a few cells immunopositive for the inhibin α‐chain. No immunoreactive cells were found in the medulla. Hypophysectomy resulted in a dramatic reduction in α‐subunit immunoreactivity with only scattered cells observed in the zona glomerulosa and reticularis (0 to 2 cells/section). No immunoreactive cells were found in the zona fasciculata or the medulla. Following supraphysiologic adrenocorticotropin administration to hypophysectomized animals, the number of inhibin α‐chain‐immunoreactive cells significantly increased in each zone of the adrenal cortex. The most prominent changes were seen in the zona reticularis. Interestingly, in the adrenocorticotropin‐treated animals scattered immunopositive cells were also present in the medulla. Our results suggest that inhibin‐related peptide(s) exist within the adrenal gland and that the expression of these peptides is regulated by adrenocorticotropin. Inhibin‐related peptide(s) may, therefore, play a paracrine and/or endocrine role in the adrenal function.
Long‐Term Inhibition of Stress‐Induced Adrenocorticotropin Release by Intracerebral Administration of a Monoclonal Antibody to Rat Corticotropin‐Releasing Factor Together with Ricin A Chain and Monensin
Authors: Menzaghi Frédérique , Burlet Arlette , Van Oers Joep W. A. M. , Tilders Fred J. H. , Nicolas Jean‐Pierre , Burlet Claude 
Published: September 2006

AbstractPrevious studies have shown that microinjection of cytotoxic lgG2a monoclonal antibody to rat/human corticotropin‐releasing factor (CRF‐MAb) into the hypothalamic paraventricular nucleus of Long Evans rats resulted in antibody uptake into specific neurons of the paraventricular nucleus. We tested the hypothesis that this neuronal uptake may allow non‐linked toxins to enter into specific neurons. The effects of central or peripheral administration of a mix containing CRF‐MAb and cellular toxins (toxin/ CRF‐MAb) on plasma adrenocorticotropin levels were determined before and after exposure to ether stress in freely moving rats. Peripheral injection (jugular vein) of the toxin/CRF‐MAb mix or injection into the supraoptic nuclei did not affect resting or stress‐induced adrenocorticotropin secretion. In contrast, bilateral injection of the same mix into the paraventricular nucleus or the lateral ventricle caused a consistent 70% reduction of the ether stress‐induced adrenocorticotropin release but had no significant effect on the resting adrenocorticotropin levels. The blocking effect of intracerebroventricular administration disappeared after 24 h whereas the blockade persisted for at least 15 days after local injection into the paraventricular nucleus. The injection into the paraventricular nucleus of CRF‐MAb without toxins restricted the inhibitory effects to 24 h. These data suggest that administration of a mix of toxins added to a specific lgG2a monoclonal antibody to CRF resulted in a long‐term interference with the control of adrenocorticotropin secretion. This new approach may be of use for elucidating the physiological roles of different central peptidergic systems.
Effects of N‐Methyl‐D,L‐Aspartate on Beta‐Endorphin and Prolactin Secretion in Rams Exposed to Long or Short Days
Authors: Lincoln G. A. , Wu F. C. W. 
Published: September 2006

AbstractIn a previous study we demonstrated that the injection of the excitatory amino‐acid N‐methyl‐D, L‐aspartate (NMDA) stimulates an acute increase in the peripheral blood concentrations of luteinizing hormone in Soay rams, and this response varies with the photoperiodically‐induced reproductive cycle. To extend these observations, we have now measured the changes in the blood concentrations of β‐endorphin and prolactin in the same animals to establish whether NMDA stimulates the secretion of other pituitary hormones. Groups of adult Soay rams were exposed to alternating 16‐weekly periods of long and short days to induce a long‐term cycle in the endogenous secretion of β‐endorphin (maximum under short days) and prolactin (maximum under long days). NMDA injected intravenously caused a dose‐dependent increase in the blood plasma concentrations of β‐endorphin (over the range 1 to 20 mg/kg NMDA). The initial increase in β‐endorphin occurred within 2 to 4 min with peak levels after 20 to 100 min. The magnitude of the β‐endorphin response was greatest following exposure to long days when the endogenous secretion of β‐endorphin was low, while the duration of the response was greatest following exposure to short days when the endogenous secretion of β‐endorphin was high. NMDA injected intravenously also caused a dose‐dependent increase in the blood plasma concentrations of prolactin but this only occurred following exposure to long days when the endogenous secretion of prolactin was high. At this time the initial increase in prolactin concentrations occurred within 2 to 4 min after the injection of NMDA as for β‐endorphin but the values continued to increase for 2 to 4 h. In a separate experiment, it was shown that pretreatment of the rams with dexamethasone (synthetic glucocorticoid, 133.4 μg/kg iv) blocked the β‐endorphin response to NMDA but had no effect on the prolactin response. This indicates that NMDA stimulates the secretion of β‐endorphin from the corticotrophs probably acting centrally to induce the release of corticotrophin‐releasing hormone and/or arginine vasopressin, while NMDA acts through separate mechanisms to affect the secretion of prolactin.The overall results show that NMDA can be used as a probe to investigate the neuroendocrine control of β‐endorphin and prolactin in addition to luteinizing hormone as described previously. The multiple responses are likely to represent the effects of NMDA acting on the hypothalamus to induce the acute release of peptides and other hormones into the pituitary portal blood system to affect the corticotrophs, lactotrophs and gonadotrophs. The variation in responsiveness to NMDA related to the photoperiodic cycle may reflect changes in the synthesis and storage of the hypothalamic hormones and the influence of other neural systems. Alternatively, the changes in the secretion of endogenous excitatory amino‐acids and NMDA receptors may constitute part of the neuroendocrine mechanism relaying the effects of photoperiod.