bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of Cholecystokinin in Food Intake: III. Oestradiol Potentiates the Inhibitory Effect of Cholecystokinin Octapeptide on Food Intake in Ovariectomized Rats
Authors: Lindén A. , Uvnäs‐Moberg K. , Forsberg G. , Bednar I. , Södersten P.
Published: September 2006

The role of Cholecystokinin in a model of hypophagia, oestradiol‐treated Ovariectomized rats, was investigated. Implantation of oestradiol‐filled constant‐release implants in rats made obese by ovariectomy potentiated the inhibitory effect of intraperitoneal injection of Cholecystokinin octapeptide on food intake after 24 h of food deprivation. The alterations in the concentration of Cholecystokinin in pjasma and of cholecystokinin‐like immunoreactivity in cerebrospinal fluid produced by deprivation of food for 24 h and subsequent food intake for 1 h were unaffected by the oestradiol treatment as was the amount of food consumed during 1 h. Oestradiol‐treated rats deprived of food for 6 h, however, consumed less food during a 15‐min test than controls. Treatment with oestradiol blunted the decrease in the concentration of cholecystokinin‐like immunoreactivity in the cerebrospinal fluid in response to 6 h of food deprivation. No alterations in the concentration of Cholecystokinin in plasma occurred after this period of food deprivation and subsequent feeding during 15 min in either oestradiol‐treated or control rats. Thus, treatment with oestradiol enhances responsivity to exogenous Cholecystokinin octapeptide and changes the response of endogenous levels of cholecystokinin‐like immunoreactivity in the cerebrospinal fluid to a short period of food deprivation. It is suggested that these effects are caused by an action of oestradiol on Cholecystokinin pathways in the brain. The results support the suggestion that hunger in the rat is inversely related to the decrease in the concentration of cholecystokinin‐like immunoreactivity in the cerebrospinal fluid.
Interaction of Opiate Peptides with Dopamine Effects on Prolactin Secretion and Membrane Electrical Properties in Anterior Pituitary Cells from Lactating Rats
Authors: Enjalbert A. , Israel J. M. , Zhang J. , Kordon C. , Vincent J. D.
Published: September 2006

Met‐enkephalin and β‐endorphin induced a partial reversion of the dopamine inhibition of prolactin release from pituitary cells of lactating rats in primary culture. This effect of opiate peptides was dose‐dependent with an EC50 of 40 ± 8 nM and 45 ± 7 nM and maximal blockade of dopamine inhibition of 60% and 68% for Met‐enkephalin and β‐endorphin, respectively. Naloxone antagonized the effect of Met‐enkephalin with an EC50 of 22 ± 12 nM. Furthermore, this Met‐enkephalin effect on dopamine inhibition of prolactin secretion appeared non‐competitive since it reduced maximal inhibition without affecting the apparent affinity of dopamine. Finally, it should be noted that the two opiate peptides had no effect on spontaneous prolactin release.

In electrophysiological experiments, local ejection of dopamine on tested cells induced an hyperpolarization concomitant with an increase of the membrane conductance. Ejection of Met‐enkephalin or β‐endorphin alone did not modify the electrical properties of the cells (resting potential, membrane conductance and excitability). In contrast, both peptides blocked in a reversible manner the dopamine‐induced electrical responses. These effects were antagonized by naloxone. However, this interaction of opiatepeptides with dopamine electrical response was not observed on all cells tested. We conclude that the blocking effect of opiates on dopamine‐induced hyperpolarization may account, at least in part, for the ability of these peptides to interact with dopamine inhibition of prolactin release.
Estrogens Modulate the Responsiveness of in vivo Recorded Striatal Neurons to lontophoretic Application of Dopamine in Rats: Role of D1 and D22 Receptor Activation
Authors: Demotes‐Mainard J. , Arnauld E. , Vincent J. D.
Published: September 2006

Estrogens are known to affect the functioning of the extrapyramidal motor system. Their actions concern both pre‐ and postsynaptic components of the dopaminergic nigrostriatal neurotransmission. Postsynaptically, estradiol alters the electrophysiological responses of striatal neurons to dopamine application. The aim of this study was to determine the respective roles of D1 and D2 receptor subtypes in this modification of dopamine action. Eighty‐three spontaneously firing caudate neurons of ovariectomized female rats were extracellularly recorded during iontophoretic ejection of dopamine or dopaminergic agonists or antagonists. Between 5 and 10 h after a single 17β‐estradiol injection (60 μg/kg), the predominant effect of dopamine was an activation of firing (63%), and the distribution of responses significantly differed from controls and from neurons recorded less than 5 h after estradiol injection (P<0.001) on which dopamine mainly elicited inhibitory effects (71% and 63%, respectively). The excitatory effects of dopamine on this subset of spontaneously firing striatal neurons were blocked by the D1 antagonist SCH 23390 (56%), whereas the inhibitory actions were antagonized by sulpiride (62%), whose isolated application often had an excitatory effect (54%).

The distributions of the responses to specific D1 or D2 agonists were not altered by estrogen treatment: the predominant effect of the D2 agonist RU 24213 was in all groups a reduction of firing rate (54%), and the D1 agonist SKF 38393 mainly induced either a decrease in mean firing rate (21%) or a biphasic effect consisting on an excitation‐inhibition sequence (53%).

These results suggest that estradiol does not qualitatively alter the coupling of D1 or D2 receptors to their electrophysiological effectors, but rather quantitatively changes the ratio between the D1 and D2 receptor‐mediated components of dopamine actions, reinforcing the D1 and/or attenuating the D2 receptor‐mediated component.
Specific Physiological Regulation of Luteinizing Hormone Secretory Events Throughout the Human Menstrual Cycle: New Insights into the Pulsatile Mode of Gonadotropin Release
Authors: Sollenberger Michael J. , Carlsen Elisabeth C. , Johnson Michael L. , Veldhuis Johannes D. , Evans William S.
Published: September 2006

To investigate the physiological regulation of luteinizing hormone (LH) secretory events and the endogenous clearance of this hormone, we applied multiple‐parameter deconvolution analysis to serum LH concentration‐time series obtained from normal women during three phases of the menstrual cycle. The number of significant LH secretory bursts (/24 h) was maximal in the late follicular (LF) phase (27 ± 1.6; mean ± SEM), minimal in the mid‐luteal (ML) phase (10 ±1.0) and intermediate in the early follicular (EF) phase (18 ± 1.4). Similarly, the half‐duration of the secretory impulse (min) was different at each phase of the cycle with values of 6.5±1.0, 3.5±0.9 and 11 ± 1.1 during the EF, LF and ML phases, respectively. In contrast, there were no cycle‐dependent differences in the LH half‐life or in the total daily secretion of LH.

When maximal secretory impulse amplitudes were examined, a putative bimodal distribution was found in the ML but not the EF or LF phases. The amplitudes for the large ML impulses, the LF and EF impulses and the small ML impulses were 0.95 ± 0.05, 070 ± 0.03, 0.43 ± 0.02 and 0.26 ± 0.02, respectively. The mass (mlU/ml) of hormone secreted within bursts was minimal in the LF phase (2.1 ± 0.1), maximal in the large ML impulses (10.2 ± 0.5) and intermediate in the EF (2.8 ± 0.1) and small ML (3.1 ±0.3) secretory impulses.

There was no evidence of tonic (i.e. inter‐secretory burst) LH secretion during any phase of the menstrual cycle. The early morning hours of the EF phase were characterized by fewer secretory bursts of greater amplitude. During the ML phase, autocorrelation analysis of inter‐secretory burst intervals revealed a negative association indicating that high frequency events both precede and follow secretory pauses. In addition, secretory burst amplitude and both the preceding and following inter‐secretory burst interval was correlated to secretory burst amplitude.

These new data on the nature of regulated LH secretion indicate that specific facets of spontaneous LH secretory events are controlled throughout the menstrual cycle. Such observations offer a basis for defining altered secretory dynamics in a variety of pathophysiologic situations.
The Effects of Rat Corticotrophin‐Releasing Factor‐41 Peptide Fragments on Bioassay and Immunoassay Determination of Corticotrophin‐Releasing Factor‐41 Levels
Authors: Milton N. G. N. , Nicholson S. A. , Self C. H. , Hillhouse E. W.
Published: September 2006

Peptide fragments of rat corticotrophin‐releasing factor‐41 (CRF‐41) containing amino‐acid residues 21–33 antagonized the 5 nmol/l CRF‐41‐stimulated adrenocorticotrophin secretion from the adult rat pituitary gland in vitro. The CRF 6‐33 sequence had antagonistic effects at equimolar (5 nmol/l) concentrations which were not observed at high (50 nmol/l) concentrations whilst the CRF 21–41 sequence had effects only at high (50 nmol/l) concentrations. Similar effects were observed with CRF 6‐33 on basal release of adrenocorticotrophin. Peptide fragments elevated radioimmunoassay measurement of CRF‐41 whilst inhibiting measurement of CRF‐41 in a two‐site enzyme amplified immunometric assay. The inhibitory effects of peptide fragments in the enzyme amplified immunometric assay could be removed by dilution to within the lower end of the standard curve or by increasing the concentration of antibody bound to the solid phase. These inhibitory effects mimic those of peptide fragments on basal adrenocorticotrophin release seen in a rat pituitary gland in vitro bioassay indicating that such two‐site immunoassay determinations bear closer relation to bioactivity than those obtained using radioimmunoassay.
Differential Mechanisms of Dopamine and Somatostatin Inhibition of Prolactin Secretion from Anterior Pituitary Cells
Authors: Musset F. , Bertrand P. , Priam M. , Kordon C. , Enjalbert A.
Published: September 2006

The relative potencies of dopamine and somatostatin to inhibit prolactin secretion by pituitary cells in primary culture were compared. Hormone secretion was evaluated under basal conditions as well as after challenging it with thyrotropin‐releasing hormone, vasoactive intestinal peptide or with drugs affecting either the activity of adenylate cyclase (forskolin), and protein kinase C (phorbol 12 myristate 13 acetate), or eliciting Ca2+ fluxes in the cell by various ways (A23187, a Ca2+ ionophore, the dihydropyridine agonist BAY‐K‐8644, or K+ depolarization which activates voltage‐sensitive Ca2+ channels). In order to test whether all effects of dopamine and somatostatin were mediated by inhibitory guanosine triphosphate binding proteins, the experiments were systematically carried out in the presence or absence of pertussis toxin, an agent which selectively uncouples given subsets of G proteins from corresponding receptors.

Dopamine markedly inhibited basal as well as thyrotropin‐releasing hormone‐, vasoactive intestinal peptide‐, forskolin‐ and BAY‐K‐8644‐stimulated release of prolactin. In contrast, dopamine was only able to induce partial inhibition of hormone release when secretion was triggered by tumour‐promoting activator, A23187 or K+ depolarization. Under all conditions tested, inhibition by somatostatin was significant, but of limited amplitude.

Pertussis toxin completely reversed the effects of somatostatin. In contrast, complete reversal of dopamine effects by pertussis toxin was only achieved after hormone stimulation by tumour‐promoting activator, alone or with A23187. Under all other conditions a residual dopamine inhibition was maintained in the presence of the toxin. The amplitude of this residual toxin resistant inhibition was comparable in all other cases to that observed for unstimulated (basal) prolactin release.

It is concluded that: 1) As expected, dopamine is a potent inhibitor of secretory processes involving cyclic AMP accumulation or voltage‐sensitive Ca2+ channel activation. In contrast, the amine is only a partial inhibitor of exocytosis resulting from non‐voltage‐sensitive Ca2+ channel‐gated increase in Ca2+ or direct activation of protein kinase C. 2) Somatostatin is a partial inhibitor of prolactin under all conditions tested. Dopamine and all somatostatin effects are mediated by pertussis toxin‐sensitive G proteins. However, a small, but significant, proportion of dopamine inhibition is resistant to pertussis toxin and can thus be assumed to involve a distinct mode of action. This alternate mechanism of dopamine inhibition operates under all conditions except after treatment with tumour‐promoting activator, suggesting that it can be inactivated by protein kinase C stimulation.
Effect of Exogenous Arginine Vasopressin on Adrenocorticotropin and Cortisol Release in Myotonie Dystrophy Patients: Delayed Responses of Normal Magnitude
Authors: Grice Jeffrey E. , Jackson Jane , Hewett Margot , Penfold Paula J. , Jackson Richard V.
Published: September 2006

We administered intramuscular arginine vasopressin (AVP) to ten normal controls and eight myotonic dystrophy patients. By measuring plasma AVP levels in five of the myotonics and all the normals, we showed that absorption and distribution of AVP was not delayed or significantly reduced in myotonics. The magnitude of the mean plasma adrenocorticotropin (ACTH) response to AVP in the myotonics was not different from that of normals, although it was significantly delayed (mean peak time, 37.5±4.9 versus 17.0 ± 3.2 min). We propose that this delay was caused by a significantly reduced ACTH secretion rate in myotonics, because the maximum rate of detection of ACTH in plasma is reduced in myotonics (0.6 ± 0.2 versus 1.7 ± 0.5 pmol/L/min), whose corticotropes, while having the same capacity to respond to the AVP stimulus, are slower to attain that capacity. The mean integrated cortisol response (AUC) was significantly smaller for myotonics (8072 ± 2017 versus 13049±1630 nmol.min/L). This may be due to the slower rate of ACTH delivery to the adrenal in myotonics. The timing of the adrenal response does not appear to be impaired in myotonic dystrophy, with the cortisol peak following the ACTH peak by approximately 15 min in both groups. The normal magnitude ACTH response to AVP in myotonics is in contrast to that seen to ACTH secretagogues acting via corticotropin‐ releasing hormone‐initiated pathways, where a rapid hypersecretion is seen. We propose a mechanism of defective calcium transport to account for these observations.
The Effect of Gonadal Steroids on Vasoactive Intestinal Peptide Concentration and Release from Mediobasal Hypothalamus and the Anterior Pituitary Gland
Authors: Lasaga Mercedes , Duvilanski Beatriz Haydee , Seilicovich Adriana , Afione Sandra , Diaz Maria del Carmen , Pisera Daniel , Traktenberg Ruth , Debeljuk Luciano
Published: September 2006

The effects of chronic administration of sex steroids on the content of vasoactive intestinal peptide (VIP) in the mediobasal hypothalamus and anterior pituitary were studied in adult rats.

Gonadectomy had no effect on VIP concentration in the mediobasal hypothalamus or anterior pituitary gland. Estradiol benzoate (1 μg/100 g body wt/day) administered for 10 days decreased mediobasal hypothalamus VIP concentration of ovariectomized rats whereas it produced no change in mediobasal hypothalamus VIP content of orchidectomized rats. Testosterone propionate (100 μg/100 g body wt/day) administration decreased mediobasal hypothalamus VIP content in both sexes. Estradiol administration caused an increase whereas testosterone treatment resulted in a decrease in anterior pituitary VIP levels in both sexes. The effect of chronic administration of the sex steroids on VIP release from the mediobasal hypothalamus and anterior pituitary was also investigated. Estradiol increased evoked VIP release from the mediobasal hypothalamus and decreased mediobasal hypothalamus VIP content whereas testosterone decreased both mediobasal hypothalamus release and content. Chronic treatment with estradiol enhanced anterior pituitary VIP release and content while testosterone decreased both parameters studied. The data indicate that anterior pituitary VIP content is under the control of gonadal hormones and that the increased anterior pituitary VIP found after estradiol administration may be due to an augmented release from the mediobasal hypothalamus and probably an increase in anterior pituitary VIP synthesis.
Neonatal Exposure to Gonadal Hormones Affects the Development of Monoamine Systems in Rat Cortex
Authors: Stewart Jane , Kühnemann Sylvia , Rajabi Heshmat
Published: September 2006

The effects of neonatal gonadectomy of male, and testosterone propionate treatment of female rat pups on levels of monoamines and metabolites in the cerebral cortex were assessed using high‐performance liquid chromatography with electrochemical detection. In Experiment 1, pups were killed at 0, 4, 10 and 21 days of age and the anterior and posterior portions of cortex in each hemisphere were removed. At 21 days of age the levels of dopamine in anterior cortex were higher in males and testosterone propionate‐treated females than in females and gonadectomized males. However, dopaminergic activity developed earlier in females than in males and the gonadal hormone manipulations shifted the pattern of development to that of the other sex. In Experiment 2, the effects of these same gonadal hormone manipulations on the uptake, metabolism and storage capacity of catecholamine neurons in the cingulate, agranular insular, parietal and occipital cortex were estimated at 4 and 10 days of age by considering the difference between measured catecholamines in animals pretreated with vehicle or 2.5 mg/kg reserpine and then given 100 mg/kg L‐DOPA. Again, the data indicated earlier development of catecholamine neurons in females, especially in the agranular insular cortex. Dopamine was found to account for group differences; for when dopamine levels alone were considered it was found that, at 4 days of age, females had the highest levels in every area with the exception of the occipital cortex where gonadectomized males had equally high levels. These data suggest a mechanism that might account for sex differences in the development of specific cortical regions.
An Enkephalinergic Mechanism for the Hypothalamic Effect of Atrial Natriuretic Peptide to Inhibit Luteinizing Hormone Secretion
Authors: Huang Fong‐Lee S. , Samson Willis K.
Published: September 2006

Previous studies have demonstrated a hypothalamic site of action of atrial natriuretic peptide (ANP) to inhibit luteinizing hormone and an opioid mechanism has been suggested. We have identified the paraventricular nucleus as at least one locus of action of ANP since site‐specific injection of 0.1 nmol, bilaterally into this nucleus, but not the medial preoptic nuclei or arcuate nucleus, resulted in a significant inhibition of plasma levels in conscious, ovariectomized rats 90 and 120 min later. This paraventricular site of action suggested an involvement of endogenous enkephalins. The stable enkephalin analog, [D‐Pen2.5]enkephalin, when injected into the third ventricle in a dose of 1.0 nmol, produced a significant and transient inhibition of luteinizing hormone secretion similar to that seen following injection of 2.0 nmol ANP. Pretreatment of the rats 15 min before peptide injection with the selective 5 opioid antagonist naltrindole (50 μg/2 μl saline) completely prevented the luteinizing hormone‐inhibiting effects of both ANP and the enkephalin analog, δ opioid blockade failed to prevent the prolactin‐inhibiting effect of ANP but did reverse the prolactin‐stimulating effect of [D‐Pen2.5]enkephalin. Our results suggest a paraventricular nucleus site of action of ANP in addition to probable effects in the median eminence and indicate the possible enkephalinergic mediation of the peptide's ability to inhibit luteinizing hormone secretion.
In vivo Infusion of Adrenaline Stimulates Corticotropin‐Releasing Hormone‐Producing Neurons when given Centrally but not Distally
Authors: Barbanel Gérard , Ixart Guy , Assenmacher Ivan
Published: September 2006

There is evidence that adrenaline stimulates the release of corticotropin‐releasing hormone (CRH‐41) from hypothalamic neurons. This study was carried out to ascertain the effects of adrenaline on the perikarya and/or distal terminals of these cells. All experiments were performed on unrestrained rats having chronic intracerebroventricular cannulas in the lateral ventricle or intracerebral cannulas in the paraventricular nucleus and, additionally, a push‐pull median eminence cannula. Adrenaline (1.4 μg adrenaline bitartrate in 5 μI vehicle for intracerebroventricular infusion, or 0.7 μg in 0.25 μI for intracerebral infusion) was infused over 2 min, and 15‐min median eminence perfusate samples were collected over 2 to 3 h. In another experiment the median eminence was directly perfused for 30 min with a total of 1.2 μg (n = 4) or 12 μg (n = 3) adrenaline per rat. Intracerebroventricular or intracerebral adrenaline induced a swift, short‐lived (15 min) CRH‐41 surge which reached 7 to 10 times the basal level. Direct perfusion of the median eminence with adrenaline (around the nerve endings of CRH‐41‐producing neurons) did not stimulate CRH‐41 release and higher amine concentrations even tended to depress the neuropeptide release. The stimulatory effect of adrenaline on the corticotropic axis appears, therefore, to be restricted to the central dendro‐perikaryal region of CRH‐41‐producing neurons.
Intrahypothalamic Mu‐, not Delta‐ or Kappa‐Opioid Receptor Activation Causes Growth Hormone Secretion
Authors: Willoughby John O. , Kapoor Ranjna , Mackenzie Lorraine
Published: September 2006

The possible effects of opioid receptor agonists on growth hormone (GH)‐releasing factor or somatostatin neurons were examined by measuring the effects of localized intracerebral injections of mu‐, delta‐ and kappa‐selective agonists on GH secretion. Serial GH concentrations were measured in plasma in unanaesthetized male rats chronically prepared with venous and intracerebral cannulae, before and after treatment with bilateral intracerebral injections of opioid agonists in the preoptic anterior hypothalamic area and medial basal hypothalamus.

In the medial basal hypothalamus, injections of the mu‐agonist DAGO (Tyr‐D‐Ala‐Gly‐(Me)Phe‐Gly‐ol) caused dose‐responsive increases in GH, the maximally effective dose being 0.001 nmoles. Injection of 10,000‐fold higher doses of the delta‐agonist DPDPE ([D‐Pen, D‐Pen]enkephalin) and the kappa‐agonist U50,488H were also effective in stimulating GH secretion. In the preoptic anterior hypothalamic area, DAGO caused dose‐responsive increases in GH, the maximally effective dose being 0.01 nmoles. U50.488H was ineffective at 1,000‐fold higher doses while DPDPE was effective at 100‐ to 1,000‐fold higher doses.

We conclude that hypothalamic mu‐opioid receptor activation on or near somatostatin or GH‐releasing factor neurons causes GH secretion. Opioids capable of acting on other opioid receptors may also stimulate GH secretion, though only at doses that seem likely to affect mu‐receptors.
Oxytocin in the Bed Nucleus of the Stria Terminalis and Lateral Septum Facilitates Bursting of Hypothalamic Oxytocin Neurons in Suckled Rats
Authors: Moos F. , Ingram C. D. , Wakerley J. B. , Guerné Y. , Freund‐Mercier M. J. , Richard Ph.
Published: September 2006

Several regions of the forebrain possess high densities of oxytocin (OT)‐binding sites including the bed nucleus of the stria terminalis (BST) and lateral septum (LS). In order to examine whether these regions participate in the central facilitation of the milk ejection reflex by OT, microinjections of OT (1 ng in 100 nl containing Janus Green dye) were made into the BST (13 tests) or LS (9 tests) of anaesthetized, suckled rats, while recording the electrical activity of OT neurons in the contralateral supraoptic nucleus. Histological localization of injection sites using Janus Green demonstrated that all BST injections were close to the anterior commissure, and LS injections were all located in the ventral division of the LS. Film autoradiographic visualization of OT‐binding sites (in 7 tests using [125I]OT antagonist) confirmed that the BST and LS injections were located within regions of high OT binding. Injections into both regions facilitated the milk ejection reflex by increasing either the frequency and/or amplitude of OT neuron bursts, or by triggering bursts in animals that previously had shown no milk ejection responses; the mean number of milk ejections in the 30 min before and after injection increasing from 1.6·0.5 to 3.6·0.5 for BST and from 1.5·0.6 to 3.9·0.4 for LS. The OT microinjections had a more variable effect on background activity of OT neurons, increasing firing in some cases and not in others. This facilitatory effect was similar to that induced by microinjections into the lateral ventricle, but was smaller and delayed compared to that induced by injection into the third ventricle (9 tests), possibly due to unilateral activation of target sites. The facilitatory effect was unlikely to have been due to diffusion of OT into the ventricle, since injections into control sites (striatum and thalamus) at similar distances from the ventricle (9 tests) had no facilitatory effect (number of bursts during 30 min before and after injection; 2.2·0.5 and 1.8·0.5, respectively). These data suggest that limbic structures (BST and LS) participate in the action of central OT on the pattern of milk ejections in the suckled rat.
Hypothalamic Gonadotropin‐Releasing Hormone Secretion: Methodological Aspects of in vitro Systems
Authors: Wibullaksanakul Sunee , Handelsman David J.
Published: September 2006

The 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

Using 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

In 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

Lactating 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

A 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

Photoperiodic 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

In 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

This 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

Parturition 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

Inhibin, 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

Previous 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.