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1.
J Neuroendocrinol ; 20(3): 279-89, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18194430

ABSTRACT

The hyperactivity of the brain renin-angiotensin system (RAS) has been implicated in the development and maintenance of hypertension in several types of experimental and genetic hypertension animal models. Among the main bioactive peptides of the brain RAS, angiotensin (Ang) II and Ang III display the same affinity for type 1 and type 2 Ang II receptors. Both peptides, injected intracerebroventricularly, similarly increase arginine vasopressin (AVP) release and blood pressure (BP); however, because Ang II is converted in vivo to Ang III, the identity of the true effector is unknown. We review new insights into the predominant role of brain Ang III in the control of BP, underlining the fact that brain aminopeptidase A (APA), the enzyme generating brain Ang III, may therefore be an interesting candidate target for the treatment of hypertension. This justifies the development of potent systemically active APA inhibitors, such as RB150, as prototypes of a new class of antihypertensive agents for the treatment of certain forms of hypertension. We also searched for a putative angiotensin receptor subtype specific for Ang III and isolated a seven transmembrane-domain G protein-coupled receptor corresponding to the receptor for apelin, a newly-discovered peptide isolated from bovine stomach. Apelin and its receptor are expressed in magnocellular vasopressinergic neurones in the hypothalamus. The central injection of apelin in lactating rats decreases the phasic electrical activity of vasopressinergic neurones and the systemic secretion of AVP, inducing water diuresis. Apelin is therefore a natural inhibitor of the antidiuretic effect of AVP. In addition, systemic administration of apelin decreases BP, improves cardiac contractility and reduces cardiac loading. The development of nonpeptide agonists of the apelin receptor may provide new therapeutic tools for treating water retention, hyponatraemia and cardiovascular diseases. Angiotensins and apelin thus exert opposing but complementary effects, and are thereby determinant for the maintenance of body fluid homeostasis and cardiovascular functions.


Subject(s)
Angiotensins/physiology , Intercellular Signaling Peptides and Proteins/physiology , Neurosecretory Systems/physiology , Animals , Apelin , Apelin Receptors , Blood Pressure/drug effects , Brain/metabolism , Brain/physiology , Cardiovascular Physiological Phenomena , Enzyme Inhibitors/pharmacology , Glutamyl Aminopeptidase/antagonists & inhibitors , Humans , Models, Biological , Rats , Receptors, G-Protein-Coupled/metabolism , Renin-Angiotensin System/physiology , Signal Transduction/physiology , Tissue Distribution , Water-Electrolyte Balance/physiology
2.
J Endocrinol Invest ; 28(5 Suppl): 2-9, 2005.
Article in English | MEDLINE | ID: mdl-16114267

ABSTRACT

The history of GH started with the pioneer clinical and anatomical observations of Pierre Marie, who described the symptoms of acromegaly in 1886. Progressively, histochemical and histophysiological methods made it possible to characterize most cell types responsible for normal or pathological pituitary hormone secretion. Although the methods applied were indirect, and hormonal function assigned to each cell type could only be inferred from correlations, the quality of the corresponding studies was such that most of their results proved correct. In the second half of the XXth century, biochemical methods and bioassays led, between 1943 and 1956, to the production from pituitary extracts of highly purified fractions containing somatotropin activity. The subsequent demonstration that hypothalamo-hypophyseal interactions are of a neurohumoral nature permitted isolation of neuropeptides, a new class of neurotransmitters, many of which turned into major therapeutic agents. Subsequent purification of hundreds of neuropeptides, many with hypophysiotropic activity, and mapping of neurons producing them permitted to shift from relatively simple theories, postulating that stimulatory and inhibitory peptides are sufficient to account for the physiological control of pituitary secretion to more complex models. These permitted to understand how complex neuronal networks can produce a fine tuning of multiple combinations of neuropeptides and neurotransmitters, which interact with each other to adapt hormonal secretion to discrete physiological and pathological conditions.


Subject(s)
Growth Hormone/history , Neurochemistry/history , Neurosciences/history , Animals , History, 19th Century , History, 20th Century , History, 21st Century , History, Ancient , Humans , Hypothalamo-Hypophyseal System , Linear Models , Nerve Net
3.
Endocrine ; 14(1): 1-8, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11322489

ABSTRACT

Growth hormone secretagogues (GHSs) act at distinct levels to control growth hormone (GH) secretion. At the pituitary level they reinforce or extend a tonic GH-releasing-hormone (GHRH)-induced activated state by mobilizing intracellular Ca2+ store. At the hypothalamic level GHS actions are more complex than originally anticipated. Chronic treatments with GHS result in loss of responsiveness to the secretagogues, an effect probably accounted for by indirect negative feedback of GHS stimulated plasma GH levels over GHRH release. Moreover, intracerebroventricular treatments with GHS can have paradoxical, inhibitory effects on GH secretion. Several mechanisms can account for such dual effects. GHS receptors were found to extend far beyond the arcuate nucleus and are mainly coexpressed by GHRH, somatostatin, and neuropeptide Y (NPY) neurons. Activation of GHRH neurons by GHS can be direct or indirect. Indeed using antisense strategy we found that sstl are physiological activators of arcuate GHRH neurons and we propose that activation of SRIH arcuate interneurons by GHS can increase GHRH neuron activity. Moreover, GHS can stimulate distinct populations of NPY neurons having opposite effects on GH secretion: arcuate NPY interneurons, act as indirect facilitators of GHRH release, whereas, on the contrary, a different subset of NPY neurons projecting to the periventricular hypothalamus (those also involved in mediating leptin effects on GH) seems able to activate SRIH release.


Subject(s)
Growth Hormone-Releasing Hormone , Growth Hormone/physiology , Hormones , Hypothalamus/physiology , Receptors, Cell Surface/agonists , Receptors, G-Protein-Coupled , Animals , Arcuate Nucleus of Hypothalamus/anatomy & histology , Arcuate Nucleus of Hypothalamus/metabolism , Calcium/metabolism , Growth Hormone/blood , Growth Hormone/metabolism , Growth Hormone-Releasing Hormone/physiology , Humans , Leptin/metabolism , Models, Animal , Nerve Net/metabolism , Nerve Net/physiology , Receptors, Cell Surface/physiology , Receptors, Ghrelin , Somatostatin/metabolism
4.
J Comp Neurol ; 429(3): 469-89, 2001 Jan 15.
Article in English | MEDLINE | ID: mdl-11116232

ABSTRACT

The forebrain and pituitary sites of synthesis of growth hormone secretagogue-receptor mRNA were identified in four adult lemurs (Microcebus murinus) by in situ hybridisation performed with a radiolabeled cRNA probe transcribed from human Growth Hormone Secretagogue-Receptor cDNA. The cRNA sense and antisense probes were hybridised to cryostat sections containing structures extending from the rostral hypothalamus to its caudal limit as defined by the mammillary bodies. The pituitary gland and areas adjacent to the hypothalamus were also analyzed. For comparative purposes, sections from five adult rats containing these structures were hybridised with the same probes. The results point to a widespread distribution of Growth Hormone Secretagogue-Receptor mRNA in the hypothalamus, hippocampal formation, and cerebellar cortex of both lemurs and rats. As in the rat, specific hybridisation was particularly dense in the arcuate nucleus. Significant species differences were observed in the periventricular nucleus, the ventromedial nucleus, the lateral hypothalamic area, and the pituitary gland. In contrast to the rat, the lemur exhibited marked labelling in the infundibular nucleus, the periventricular nucleus and the pars tuberalis of the pituitary gland, whereas no labeling was detectable in the ventromedial nucleus and the lateral hypothalamic area. These results are discussed in terms of difference between the control of growth hormone secretion, feeding behaviour and seasonal rhythmicity among murine species and primates.


Subject(s)
Hypothalamus/metabolism , Pituitary Gland/metabolism , RNA, Messenger/metabolism , Receptors, Cell Surface/metabolism , Receptors, G-Protein-Coupled , Animals , In Situ Hybridization , Lemur , Male , Rats , Rats, Sprague-Dawley , Receptors, Ghrelin
5.
Neuroendocrinology ; 72(3): 144-53, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11025408

ABSTRACT

The present work investigated whether neurotrophins could differentially affect in vitro growth and maturation of two related subsets of hypothalamic neurons, hypophysiotropic somatostatin (SRIH) neurons projecting from the periventricular area and arcuate SRIH interneurons. For this purpose, the hypothalamus of 17-day-old rat fetuses was sampled and separated into a ventral and a dorsal fragment containing respectively periventricular and arcuate regions. Each fragment was dissociated and seeded separately in defined medium. Brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3), two important members of the neurotrophin family involved in neuronal differentiation and plasticity, were added to the cultures at seeding time. After 6 or 11 days in vitro, neurons were labeled with an anti-SRIH antiserum and submitted to morphometric analysis. In parallel, SRIH mRNA was estimated by semiquantitative reverse-transcriptase-polymerase chain reaction, and neuronal SRIH content, basal and depolarisation-stimulated releases measured by radioimmunoassay. The response of control, non-labeled neurons was estimated by neuronal counts and by assaying glutamic acid decarboxylase, a marker of a large majority of hypothalamic neurons. BDNF markedly increased the size and the branching number of SRIH periventricular cell bodies. Expression of SRIH mRNA, as well as SRIH content and release into the culture medium, were also stimulated by the neurotrophin. Non-SRIH neurons were not affected by the treatment. Under the same conditions, arcuate neurons exhibited a weak, mostly transient response to BDNF. NT-3 was ineffective on either neuronal subset. Immunoneutralization of Trk receptors provided further evidence for BDNF effect specificity. The results indicate that BDNF is a selective activator of the differentiation of hypophysiotropic SRIH neurons in the periventricular area of the hypothalamus.


Subject(s)
Brain-Derived Neurotrophic Factor/metabolism , Cell Differentiation/physiology , Hypothalamus/drug effects , Hypothalamus/metabolism , Neurons/drug effects , Neurotrophin 3/metabolism , Somatostatin/metabolism , Animals , Arcuate Nucleus of Hypothalamus/cytology , Arcuate Nucleus of Hypothalamus/drug effects , Arcuate Nucleus of Hypothalamus/embryology , Arcuate Nucleus of Hypothalamus/metabolism , Brain-Derived Neurotrophic Factor/pharmacology , Cell Differentiation/drug effects , Cells, Cultured/cytology , Cells, Cultured/drug effects , Cells, Cultured/metabolism , Dose-Response Relationship, Drug , Fetus , Hypothalamus/cytology , Hypothalamus/embryology , Neurons/cytology , Neurons/metabolism , Neurotrophin 3/pharmacology , Rats , Rats, Sprague-Dawley , Receptor, trkB/immunology , Receptor, trkB/metabolism , Receptor, trkC/immunology , Receptor, trkC/metabolism
6.
Sci Eng Ethics ; 6(1): 41-8, 2000 Jan.
Article in English | MEDLINE | ID: mdl-11273435

ABSTRACT

Institutions in France are not yet well prepared to respond to allegations of scientific misconduct. Following a serious allegation in late 1997, INSERM, the primary organization for medical and health-related research in France, began to reflect on this subject, aided by scientists and jurists. The conclusions have resulted in establishing a procedure to be followed in cases of alleged misconduct, and also in reinforcing the application of good laboratory practices within each laboratory. Guidelines for authorship practices and scientific assessment must also be considered. Even though each institution must remain responsible for responding to allegations of scientific misconduct within its doors, INSERM would like to see national, European, and international co-ordination about the methods of such response.


Subject(s)
Biomedical Research , Government Regulation , Scientific Misconduct , Academies and Institutes , Advisory Committees , Ethics , Ethics, Research , Europe , France , Guidelines as Topic , International Cooperation
7.
Brain Res Dev Brain Res ; 118(1-2): 39-49, 1999 Dec 10.
Article in English | MEDLINE | ID: mdl-10611502

ABSTRACT

Coculture of adult pituitary intermediate lobe (IL) cells, a target for hypothalamic dopaminergic neurons, with fetal rat hypothalamic cells accelerate differentiation of dopaminergic neurons. This involves long range diffusible as well as additional factors which may be membrane-bound. To determine whether IL membrane-bound factors contribute to the differentiating effect of IL cells, IL membranes were added to dispersed fetal hypothalamic neurons. This stimulated the outgrowth of dopaminergic neurites and elevated TH levels. Limited trypsin proteolysis of IL cell surface abolished the effect on TH levels. Addition of adenohypophyseal membranes was ineffective. Joint treatment with IL membranes, and medium conditioned (CM) over IL cells, produced the same effect on TH levels as did coculture with the same number of IL cells. The results demonstrate that IL cells express on their surface a membrane-bound factor promoting differentiation of fetal dopaminergic neurons in vitro; this factor acts in addition to diffusible activities.


Subject(s)
Dopamine/metabolism , Hypothalamus/embryology , Neurons/cytology , Neurons/metabolism , Pituitary Gland/physiology , Animals , Cell Differentiation/physiology , Coculture Techniques , Culture Media, Conditioned , Extracellular Matrix/physiology , Fetus/cytology , Hypothalamus/cytology , Pituitary Gland/cytology , Rats , Rats, Wistar
8.
Neuroendocrinology ; 70(5): 343-52, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10567860

ABSTRACT

Somatotropes and GC cells, a GH-producing cell line, exhibit [Ca(2+)](i) oscillations that result from rhythmic Ca(2+) action potentials. Determination of this operating mode required simultaneous recording of both parameters by fura-2 imaging and patch-clamp techniques. In order to test whether patch recording induces artificial alteration of the [Ca(2+)](i) oscillatory pattern, we recorded separately or simultaneously [Ca(2+)](i) and membrane potential. In the absence of any other stimulation, seal formation in patch-clamp recording evoked by itself a 2.5- to 4-fold persistent increase in basal [Ca(2+)](i), speeded up their frequency (from 0.03-0.17 to 0.4 Hz) and changed their pattern to a tonic mode. Patch-induced [Ca(2+)](i) increase was reproduced by mechanical contact between the pipette and the membrane. It was reduced by nifedipine, a blocker of L-type Ca(2+) channels, as well as by removal of external Na(+). It was fully blocked by external Ca(2+) removal or gadolinium. All patch-clamp-induced perturbations were reversed by membrane hyperpolarization. We propose that patch-clamp recording evokes Ca(2+) entry through L-type Ca(2+) channels either directly, or indirectly via membrane depolarization. This shows that patch recordings in endocrine cells showing mechanosensitivity have to be interpreted with caution, and explains why long-lasting patch recordings are so difficult to obtain.


Subject(s)
Calcium Channels, L-Type/physiology , Calcium/metabolism , Pituitary Gland/cytology , Pituitary Gland/physiology , Animals , Biological Clocks , Calcium Channel Blockers/pharmacology , Cells, Cultured , Fluorescent Dyes , Fura-2/analogs & derivatives , Gadolinium/pharmacology , Gonadotropin-Releasing Hormone/pharmacology , Male , Nifedipine/pharmacology , Patch-Clamp Techniques , Pituitary Gland/drug effects , Rats , Rats, Wistar , Stress, Mechanical
9.
Eur J Neurosci ; 11(2): 617-24, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10051762

ABSTRACT

We have previously demonstrated that differentiation of hypothalamic dopaminergic (DA) neurons can be induced in culture by their pituitary intermediate lobe target cells, through both membrane and diffusible factors. We also showed that subpopulations of DA neurons from the arcuate nucleus only, not the periventricular area, can respond to the target. Here we investigated the possibility that both neuronal subsets could also respond differentially to brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT3). Addition of NT3, but not BDNF, enhanced growth and branching of neurites, tyrosine hydroxylase (TH) as well as increasing levels of cultured arcuate DA neurons. Conversely, BDNF, but not NT3, affected the same parameters in cultured periventricular DA neurons. The neurotrophins thus affect DA neurons in a structure and neuronal type-selective manner, since general neuronal markers were not affected by either neurotrophin. Neurotrophin effects were reversed by addition of specific antibodies directed against them or their respective receptors, TrkB or TrkC. By themselves, the antibodies inhibited development of DA neurons below that of control cultures, suggesting involvement of endogenous neurotrophins. BDNF and NT3 were indeed found in both arcuate and periventricular neurons and in the intermediate lobe. BDNF was always present as the mature peptide. The mature form of NT3 was only detected in the periventricular area; a precursor-like heavier form was present in all tissues studied. The present data suggest that NT3, but not BDNF, could participate in the differentiating action of intermediate lobe cells on arcuate DA neurons.


Subject(s)
Arcuate Nucleus of Hypothalamus/cytology , Brain-Derived Neurotrophic Factor/pharmacology , Dopamine/physiology , Nerve Growth Factors/pharmacology , Neurons/cytology , Animals , Arcuate Nucleus of Hypothalamus/chemistry , Arcuate Nucleus of Hypothalamus/embryology , Blotting, Western , Brain-Derived Neurotrophic Factor/analysis , Brain-Derived Neurotrophic Factor/genetics , Cell Differentiation/drug effects , Cell Survival/drug effects , Cell Survival/physiology , Dendrites/drug effects , Dendrites/physiology , Fetus/cytology , Gene Expression/physiology , Nerve Growth Factors/analysis , Nerve Growth Factors/genetics , Neurons/chemistry , Neurons/enzymology , Neurotrophin 3 , Organ Culture Techniques , Paraventricular Hypothalamic Nucleus/chemistry , Paraventricular Hypothalamic Nucleus/cytology , Paraventricular Hypothalamic Nucleus/embryology , Phosphopyruvate Hydratase/analysis , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Tyrosine 3-Monooxygenase/analysis
10.
Interv Neuroradiol ; 5(4): 285-8, 1999 Dec 20.
Article in English | MEDLINE | ID: mdl-20670525
11.
Mol Cell Endocrinol ; 142(1-2): 99-117, 1998 Jul 25.
Article in English | MEDLINE | ID: mdl-9783907

ABSTRACT

Exposure of the gonadotrope cells to gonadotropin-releasing hormone (GnRH) reduces their responsiveness to a new GnRH stimulation (homologous desensitization). The time frame as well as the mechanisms underlying this phenomenon are yet unclear. We studied in a gonadotrope cell line (alphaT3-1) the effects of short as well as long term GnRH pretreatments on the GnRH-induced phospholipases-C (PLC), -A2 (PLA2) and -D (PLD) activities, by measuring the production of IP3, total inositol phosphates (IPs), arachidonic acid (AA) and phosphatidylethanol (PEt) respectively. We demonstrated that although rapid desensitization of GnRH-induced IP3 formation did not occur in these cells, persistent stimulation of cells with GnRH or its analogue resulted in a time-dependent attenuation of GnRH-elicited IPs formation. GnRH-induced IPs desensitization was potentiated after direct activation of PKC by the phorbol ester TPA, suggesting the involvement of distinct mechanisms in the uncoupling exerted by either GnRH or TPA on GnRH-stimulated PI hydrolysis. The levels of individual phosphoinositides remained unchanged under any desensitization condition applied. Interestingly, while the GnRH-induced PLA2 activity was rapidly desensitized (2.5 min) after GnRH pretreatments, the neuropeptide-evoked PLD activation was affected at later times, indicating an important time-dependent contribution of these enzymatic activities in the sequential events underlying the GnRH-induced homologous desensitization processes in the gonadotropes. Under GnRH desensitization conditions, TPA was still able to induce PLD activation and to further potentiate the GnRH-evoked PLD activity. AlphaT3-1 cells possess several PKC isoforms which, except PKCzeta, were differentially down-regulated by TPA (PKCalpha, betaII, delta, epsilon, eta) or GnRH (PKCbetaII, delta, epsilon, eta). In spite of the presence of PKC inhibitors or down-regulation of PKC isoforms by TPA, the desensitizing effect of the neuropeptide on GnRH-induced IPs, AA and PEt formation remained unchanged. In conclusion, in alphaT3-1 cells the GnRH-induced homologous desensitization affects the GnRH coupling with PLC, PLA2 and PLD by mechanism(s) which do not implicate TPA-sensitive PKC isoforms, but likely reflect time-dependent modification(s) on the activation processes of the enzymes.


Subject(s)
Gonadotropin-Releasing Hormone/pharmacology , Gonadotropins, Pituitary/metabolism , Pituitary Gland/drug effects , Signal Transduction , Arachidonic Acid/biosynthesis , Cell Line , Drug Tolerance , Enzyme Activation/drug effects , Glycerophospholipids/biosynthesis , Inositol 1,4,5-Trisphosphate/biosynthesis , Inositol Phosphates/biosynthesis , Isoenzymes/metabolism , Kinetics , Phosphatidylinositols/metabolism , Phospholipase D/metabolism , Phospholipases A/metabolism , Phospholipases A2 , Pituitary Gland/metabolism , Sodium Fluoride/pharmacology , Tetradecanoylphorbol Acetate/pharmacology , Type C Phospholipases/metabolism
12.
Eur J Neurosci ; 10(1): 204-12, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9753128

ABSTRACT

We have previously shown that somatostatin can either enhance or decrease AMPA/kainate receptor-mediated responses to glutamate in mouse-dissociated hypothalamic neurones grown in vitro. To investigate whether this effect is due to differential activation of somatostatin (SRIF) receptor subtypes, we compared modulation of the response to glutamate by SRIF with that induced by CH-275 and octreotide, two selective agonists of sst1 and sst2/sst5 receptors, respectively. Somatostatin either significantly decreased (49%) or increased (30%) peak currents induced by glutamate, and was ineffective in the remaining cells. Only the decreased response was obtained with octreotide, whereas only increased responses were elicited by CH-275 (47 and 35% of the tested cells, respectively). Mean amplitude variations under somatostatin or octreotide on the one hand, and under somatostatin or CH-275 on the other hand, were equivalent. Pertussis toxin pretreatment significantly decreased the number of cells inhibited by somatostatin or octreotide, but had no effect on the frequency of neurones showing increased sensitivity to glutamate during somatostatin or CH-275 application. About half of the neurones tested by single cell reverse transcriptase polymerase chain reaction (RT-PCR) expressed only one sst receptor (sst1 in 26% and sst2 in 22% of studied cells). Out of the remaining neurones, 34% displayed neither sst1 nor sst2 mRNAs, whereas 18% showed a simultaneous expression of both mRNA subtypes. Expression of sst1 or sst2 mRNA subtypes matched totally with the effects of somatostatin on sensitivity to glutamate in 79% of the neurones processed for PCR after recordings. These data show that pertussis toxin-insensitive activation of the sst1 receptor subtype mediates somatostatin-induced increase in sensitivity to glutamate, whereas decrease in the response to glutamate is linked to pertussis toxin-sensitive activation of the sst2 receptor subtype.


Subject(s)
Glutamic Acid/pharmacology , Hypothalamus/chemistry , Receptors, Somatostatin/metabolism , 6-Cyano-7-nitroquinoxaline-2,3-dione/pharmacology , Animals , Excitatory Amino Acid Antagonists/pharmacology , Hormones/pharmacology , Hypothalamus/drug effects , Hypothalamus/physiology , Membrane Potentials/drug effects , Mice , Octreotide/pharmacology , Patch-Clamp Techniques , Pertussis Toxin , Polymerase Chain Reaction , RNA, Messenger/analysis , Receptors, N-Methyl-D-Aspartate/analysis , Receptors, N-Methyl-D-Aspartate/physiology , Receptors, Somatostatin/chemistry , Receptors, Somatostatin/genetics , Somatostatin/analogs & derivatives , Somatostatin/pharmacology , Virulence Factors, Bordetella/pharmacology
13.
J Physiol ; 508 ( Pt 3): 883-905, 1998 May 01.
Article in English | MEDLINE | ID: mdl-9518740

ABSTRACT

1. Cells derived from a rat pituitary tumour (GC cell line) that continuously release growth hormone behave as endogenous pacemakers. In simultaneous patch clamp recordings and cytosolic Ca2+ concentration ([Ca2+]i) imaging, they displayed rhythmic action potentials (44.7 +/- 2.7 mV, 178 +/- 40 ms, 0.30 +/- 0.04 Hz) and concomitant [Ca2+]i transients (374 +/- 57 nM, 1.0 +/- 0.2 s, 0.27 +/- 0.03 Hz). 2. Action potentials and [Ca2+]i transients were reversibly blocked by removal of external Ca2+, addition of nifedipine (1 microM) or Ni2+ (40 microM), but were insensitive to TTX (1 microM). An L-type Ca2+ current activated at -33.6 +/- 0.4 mV (holding potential (Vh), -40 mV), peaked at -1.8 +/- 1.3 mV, was reduced by nifedipine and enhanced by S-(+)-SDZ 202 791. A T/R-type Ca2+ current activated at -41.7 +/- 2.7 mV (Vh, -80 or -60 mV), peaked at -9.2 +/- 3.0 mV, was reduced by low concentrations of Ni2+ (40 microM) or Cd2+ (10 microM) and was toxin resistant. Parallel experiments revealed the expression of the class E calcium channel alpha1-subunit mRNA. 3. The K+ channel blockers TEA (25 mM) and charybdotoxin (10-100 nM) enhanced spike amplitude and/or duration. Apamin (100 nM) also strongly reduced the after-spike hyperpolarization. The outward K+ tail current evoked by a depolarizing step that mimicked an action potential reversed at -69. 8 +/- 0.3 mV, presented two components, lasted 2-3 s and was totally blocked by Cd2+ (400 microM). 4. The slow pacemaker depolarization (3.5 +/- 0.4 s) that separated consecutive spikes corresponded to a 2- to 3-fold increase in membrane resistance, was strongly Na+ sensitive but TTX insensitive. 5. Computer simulations showed that pacemaker activity can be reproduced by a minimum of six currents: an L-type Ca2+ current underlies the rising phase of action potentials that are repolarized by a delayed rectifier and Ca2+-activated K+ currents. In between spikes, the decay of Ca2+-activated K+ currents and a persistent inward cationic current depolarize the membrane, activate the T/R-type Ca2+ current and initiate a new cycle.


Subject(s)
Biological Clocks/physiology , Growth Hormone/metabolism , Pituitary Neoplasms , Action Potentials/drug effects , Action Potentials/physiology , Animals , Antisense Elements (Genetics) , Apamin/pharmacology , Barium/pharmacokinetics , Cadmium/pharmacology , Calcium/metabolism , Calcium/pharmacokinetics , Calcium Channel Blockers/pharmacology , Calcium Channels/physiology , Charybdotoxin/pharmacology , Computer Simulation , Cytosol/metabolism , Dihydropyridines/pharmacology , Growth Hormone-Releasing Hormone/metabolism , Nickel/pharmacology , Nifedipine/pharmacology , Patch-Clamp Techniques , Peptides/pharmacology , Potassium/metabolism , Rats , Ryanodine Receptor Calcium Release Channel/physiology , Sodium/pharmacology , Spider Venoms/pharmacology , Tetraethylammonium/pharmacology , Tetrodotoxin/pharmacology , Tumor Cells, Cultured/chemistry , Tumor Cells, Cultured/physiology , omega-Agatoxin IVA , omega-Conotoxin GVIA
14.
Endocrinology ; 139(5): 2272-7, 1998 May.
Article in English | MEDLINE | ID: mdl-9564833

ABSTRACT

In the present study, we tested whether 17beta-estradiol (E2)-induced PRL sensitivity to somatostatin-14 (SRIF) involves selective up-regulation of discrete somatostatin receptor subtypes (ssts) in primary cultures of female rat pituitary cells. The efficacy of the endogenous peptide SRIF to inhibit GH and PRL secretion and cAMP accumulation was compared with those of octreotide (OCT), BIM-23052, BIM-23056, and BIM-23268, which have been reported to be relatively selective for rat sst2, sst3, and sst5. Experiments were performed in steroid-depleted media supplemented or not with 1 nM E2 for 96 h. SRIF, OCT, and BIM-23052 inhibited cAMP accumulation and GH release independently of E2. In contrast, all three agonists affected PRL release in E2-treated cultures only. Inhibition of cAMP accumulation by SRIF, OCT, and BIM-23052 was enhanced by exposure of cells to E2. The rank of potency of the agonists, OCT = SRIF > BIM-23052, was similar for GH and PRL inhibition. BIM-23268 was a weak agonist on GH, but not on PRL, secretion. BIM-23056 had no effect on the release of either hormone, but slightly inhibited cAMP formation in E2-treated cells. To verify whether SRIF receptor gene expression correlated with these observations, messenger RNA (mRNA) transcripts corresponding to the five ssts were measured by quantitative RT-PCR in the presence or absence of E2. Control cells expressed predominantly sst2 and sst3 transcripts; sst1 mRNA was present in moderate amounts, whereas sst4 and sst5 were only weakly expressed. E2 had a differential effect on distinct ssts; it increased mRNA concentrations corresponding to sst2 and sst3, and decreased that of sst1. These results indicate that sst2 and sst3 receptors are the major somatostatin receptors expressed in the female rat pituitary, and that both of them are positively regulated by estradiol. However, the capacity of lactotropes to respond to SRIF after exposure to E2 seems to depend more upon E2-induced up-regulation of the sst2 than of the sst3 receptor subtype.


Subject(s)
Estradiol/pharmacology , Gene Expression/drug effects , Growth Hormone/metabolism , Pituitary Gland, Anterior/drug effects , Prolactin/metabolism , Receptors, Somatostatin/genetics , Animals , Cells, Cultured , Colforsin/pharmacology , Cyclic AMP/metabolism , Female , Octreotide/pharmacology , Pituitary Gland, Anterior/metabolism , Polymerase Chain Reaction , RNA, Messenger/metabolism , Rats , Rats, Wistar , Somatostatin/agonists , Somatostatin/pharmacology
15.
Cell Mol Neurobiol ; 18(1): 101-23, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9524732

ABSTRACT

1. Regulation of pulsatile secretion of growth hormone (GH) relies on hypothalamic neuronal loops, major transmitters involved in their operation are growth hormone releasing hormone (GHRH) synthetized mostly in arcuate nucleus (ARC) neurons, and somatostatin (SRIH), synthetized both in hypothalamus periventricular (PVe) and ARC neurons. 2. Neurons synthetizing both peptides can inhibit each other in a reciprocal manner. Other neuropeptides synthetized in ARC neurons, such as galanin, or in ARC interneurons, such as neuropeptide Y (NPY), are able to modulate synthesis and release of GHRH and SRIH into the hypothalamohypophyseal portal system. 3. In addition, the hitherto uncharacterized endogenous ligand of the recently cloned growth hormone releasing peptide receptor, expressed mostly in the ARC, triggers GH release, presumably by actions on ARC interneurons. 4. Thyroid, gonadal, and adrenal steroid hormones also affect the GHRH-SRIH balance; a differential distribution of sex steroid receptors in the ARC and the PVe is likely to account for the different pattern of GH secretion in male and female animals. 5. Growth hormone itself is able to inhibit the amplitude of GH secretory episodes and to increase their frequency, by entering the brain (presumably by receptor-mediated internalization at the level of the choroid plexus) and acting subsequently on ARC neurons. 6. At the pituitary level, major neurotransmitters regulating GH cells act on receptors of the VIP/PACAP/GHRH family and of the somatostatin family, in particular, sst2 and sst3. Those are coupled to accumulation of cAMP as a second messenger. 7. In addition, patch-clamp experiments and measurement of intracellular Ca2+ indicate that GH cells present characteristic, GHRH-dependent, but self-maintained Ca2+ spikes and [Ca2+]i transients, which reflect adaptive mechanisms to constraints of episodic release. 8. Recent data on transcription factors affecting GH gene expression and somatotrope differentiation are also summarized. 9. Regulation and differentiation of somatotropes also depend upon paracrine processes within the pituitary itself and involve growth factors and several neuropeptides, for instance, vasoactive intestinal peptide, angiotensin 2, endothelin, and activin. 10. Finally, characteristic changes occur in the GH secretory pattern under discrete, pathological conditions, such as abnormal growth and dwarfism, diabetes, and acromegaly, as well as during inflammatory processes.


Subject(s)
Growth Hormone/metabolism , Hypothalamo-Hypophyseal System/physiology , Animals , Humans
16.
Neuroendocrinology ; 68(6): 386-94, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9873202

ABSTRACT

Under control incubation conditions, gonadotropin-releasing hormone (GnRH) binds only a fraction of its receptors in rat-cultivated pituitary cells. Unmasking of the remaining receptors, which have been termed 'cryptic', requires drug- or peptide-induced protein kinase activation. Spontaneous masking however is not observed on pituitary cells sampled from castrated male rats, suggesting the presence of an intrinsic unmasking factor. Many endogenous factors could theoretically account for the effect. Here we attempted to identify the factor involved by taking advantage of their differential dependency upon second messengers and transduction cascades. Spontaneous unmasking of GnRH binding was found reversed by pertussis toxin (PTX), an inhibitor of alphai and alphao subunits of heterotrimeric G proteins, and by U73122, a phospholipase C (PLC) inhibitor. In contrast, desensitization of protein kinase C (PKC) or inhibition of tyrosine kinase by herbimycin were ineffective. Among endogenous pituitary factors able to unmask GnRH receptors in pituitary cells from normal male rats, as EGF, NPY or opiate peptides, only the latter were found to correspond to this transduction profile. In an attempt to characterize the pharmacology of opiate effects, naloxone (10 microM), a poorly selective opiate antagonist, restored masking of GnRH binding in cells from castrates. Only the delta antagonist naltrindole (1 microM) was able to mimick the action of naloxone. Conversely, when tested on cells from intact animals, morphine (10 microM), as well as dslet (1 microM) and met-ENK (10 nM), preferential delta agonists, but not dago and beta-endorphin or U50488 H and dynorphin, respectively micro and kappa agonists, were able to suppress masking. Among opioid peptides endogenous to the pituitary, only met-ENK was able to unmask cryptic receptors, an effect antagonized by naltrindole. We conclude that an opiate delta receptor subtype is endogenously activated in the pituitary of castrated male rats to prevent masking of GnRH binding.


Subject(s)
Orchiectomy , Pituitary Gland/metabolism , Receptors, LHRH/metabolism , Receptors, Opioid, delta/metabolism , Animals , Antimetabolites/pharmacology , Cells, Cultured , Culture Media, Conditioned , Ligands , Male , Neuropeptide Y/pharmacology , Opioid Peptides/pharmacology , Pituitary Gland/physiology , Rats , Signal Transduction/physiology
17.
J Immunoassay ; 18(4): 335-56, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9358340

ABSTRACT

A competitive enzyme immunoassay for rat growth hormone (rGH) has been developed using polyclonal anti-rGH antibodies and an acetylcholinesterase (EC 3.1.1.7.) enzymatic tracer coupled covalently with rGH. The assay was performed in 96-well microtiter plates coated with rabbit polyclonal anti-goat immunoglobulin antibodies. Molecular sieve filtration and Western blot analysis revealed a single immunoreactive peak for rat plasma or pituitary extracts. Cross-reactivity with other rat pituitary hormones or human GH was less than 1%. Assay of samples in a concentration range of 0.7 to 69 ng/ml by enzyme immunoassay and radioimmunoassay were well correlated (r = 0.87 and 0.85 respectively for plasma and culture medium samples). Intra- and inter-assay variations in plasma were 4 (n = 24) and 14% (n = 9) respectively. Minimal detectable amounts of rGH were 0.6 ng/ml. A two-site immunometric assay also developed with the same antibodies allowed a detection threshold of 0.25 ng/ml.


Subject(s)
Growth Hormone/analysis , Immunoenzyme Techniques , Acetylcholinesterase/metabolism , Animals , Binding, Competitive , Cross Reactions , Culture Media/chemistry , Growth Hormone/blood , Growth Hormone/immunology , Human Growth Hormone/analysis , Human Growth Hormone/immunology , Humans , Molecular Weight , Pituitary Gland, Anterior/chemistry , Rabbits , Rats , Recombinant Proteins/analysis , Sensitivity and Specificity
18.
J Neurochem ; 68(6): 2273-80, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9166719

ABSTRACT

Somatostatin (SRIF) receptor subtypes (sst) were characterized in hypothalamic neurons and astrocytes by quantitative reverse transcription-polymerase chain reaction and radioreceptor assays using [125I-Tyr0,D-Trp8]SRIF-14 as a ligand in ionic conditions discriminating between SRIF-1 (sst2, -3, and -5 receptors) and SRIF-2 (sst1 and -4 receptors) binding sites. In neurons, sstl mRNA levels were twofold higher than those of sst2, and sst3-5 expression was only minor. Astrocytes expressed 10-fold less sst mRNAs than neurons, which corresponded mostly (80%) to sst2. SRIF-1 binding site radioautography indicated that 10% of hypothalamic neurons were labelled on both cell bodies and neuritic processes, as were 35% of astrocytes. On neuronal and glial membranes, SRIF-14 and octreotide, an sst2/sst3/sst5-selective analogue, completely displaced SRIF-1 binding, whereas des-AA(1,2,5)[D-Trp8,IAmp9]SRIF (CH-275), an sst1-selective analogue, was ineffective. Using SRIF-2 conditions, only SRIF-14 and CH-275 displaced the binding on neurons. No SRIF-2 binding was observed on glia. SRIF-14 and octreotide inhibited forskolin-stimulated adenylyl cyclase activity in neurons and glia, whereas CH-275 was effective in neurons only. In patch-clamp experiments, SRIF-14 modulated the glutamate sensitivity of hypothalamic neurons with either synergistic or antagonistic effects; CH-275 was only stimulatory and octreotide inhibitory. It is concluded that hypothalamic neurons express primarily sst1 and sst2, sst2 predominates in astrocytes, and both receptors induce distinct biological effects.


Subject(s)
Astrocytes/chemistry , Hypothalamus/cytology , Neurons/chemistry , Receptors, Somatostatin/genetics , Adenylyl Cyclase Inhibitors , Adenylyl Cyclases/metabolism , Animals , Astrocytes/cytology , Astrocytes/enzymology , Autoradiography , Binding Sites/physiology , Cells, Cultured , Colforsin/pharmacology , Fetus/cytology , Gene Expression Regulation, Developmental/physiology , Glutamic Acid/pharmacology , Hormones/metabolism , Hormones/pharmacology , Iodine Radioisotopes , Mice , Neurons/cytology , Neurons/enzymology , Octreotide/metabolism , Octreotide/pharmacology , Patch-Clamp Techniques , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Polymerase Chain Reaction , RNA, Messenger/metabolism , Receptors, Somatostatin/metabolism , Sensitivity and Specificity , Somatostatin/analogs & derivatives , Somatostatin/metabolism , Somatostatin/pharmacology
19.
J Physiol ; 499 ( Pt 3): 613-23, 1997 Mar 15.
Article in English | MEDLINE | ID: mdl-9130158

ABSTRACT

1. The effects of brief applications of growth hormone-releasing hormone (GHRH) to male rat somatotrophs in culture were analysed with the perforated patch clamp technique to record changes in potential or with fura-2 imaging techniques to measure variations of cytosolic Ca2+ concentration ([Ca2+]i). 2. Silent somatotrophs (n = 61) had a mean resting potential of -37 +/- 1 mV and a mean basal [Ca2+]i of 30 +/- 4 nM. Brief GHRH applications (30 nM, 40 s) triggered rhythmic action potentials (23.6 +/- 0.9 mV, 613 +/- 82 ms, 0.21 +/- 0.02 Hz) and [Ca2+]i increase (to 352 +/- 30 nM) followed by rhythmic [Ca2+]i transients (to 138 +/- 6 nM) that persisted up to 90 min after the last GHRH application. Both action potentials and [Ca2+]i transients were totally and reversibly blocked by removing external Ca2+ or Na+ or by adding inorganic Ca2+ channel blockers or nifedipine (3 microM). 3. Somatostatin (1-300 nM), carbamylcholine (0.1-1 microM) and muscarine (0.1-1 microM) each had a dose-dependent inhibitory effect, from a decrease of Ca2+ spike duration and frequency to a complete block of the GHRH-evoked action potentials. 4. The present results show that somatotrophs in culture have intrinsic membrane properties that allow them to sustain a pacemaker activity and subsequent long-lasting sequences of [Ca2+]i oscillations triggered by short pulses of GHRH and inhibited by somatostatin and muscarinic agonists.


Subject(s)
Biological Clocks/physiology , Calcium/metabolism , Gonadotropin-Releasing Hormone/physiology , Growth Hormone/metabolism , Pituitary Gland, Anterior/metabolism , Action Potentials , Animals , Carbachol/administration & dosage , Carbachol/pharmacology , Dose-Response Relationship, Drug , Fluorescent Dyes/metabolism , Fura-2/metabolism , Male , Muscarinic Agonists/administration & dosage , Muscarinic Agonists/pharmacology , Osmolar Concentration , Potassium/pharmacology , Pulsatile Flow , Rats , Rats, Wistar , Somatostatin/administration & dosage , Somatostatin/pharmacology , Tetrodotoxin/administration & dosage , Tetrodotoxin/pharmacology
20.
J Endocrinol ; 152(3): 431-6, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9071964

ABSTRACT

Involvement of endogenous corticotropin releasing hormone (CRH) in the regulation of spontaneous growth hormone (GH) secretion was investigated. A CRH antagonist, alpha helical CRH 9-41, was intracerebroventricularly infused for 36 h at a rate of 1 microgram/0.5 microliter/h to freely moving, cannulated adult male rats. Serial blood samples were drawn every 20 min for the last 8 hours of alpha helical CRH 9-41 infusion. The treatment induced a marked increase in GH peak amplitude without affecting either trough levels or numbers of peaks. In parallel, levels of growth hormone releasing hormone (GHRH) mRNA in the arcuate nucleus, but not of somatotropin release inhibiting hormone (SRIH) mRNA in the periventricular and arcuate nuclei, were increased. These data suggest that, in addition to its action in the stress-induced inhibition of GH secretion through regulation of periventricular SRIH neurons, CRH can also act as a modulator of endogenous GH secretion through regulation of arcuate GHRH neurons. Whether the modulatory effects of CRH on GHRH neurons are direct or indirect remains to be established.


Subject(s)
Arcuate Nucleus of Hypothalamus/metabolism , Corticotropin-Releasing Hormone/administration & dosage , Growth Hormone-Releasing Hormone/genetics , Growth Hormone/metabolism , Hormone Antagonists/administration & dosage , Peptide Fragments/administration & dosage , RNA, Messenger/metabolism , Adrenocorticotropic Hormone/metabolism , Animals , Cerebral Ventricles , Corticotropin-Releasing Hormone/pharmacology , Hormone Antagonists/pharmacology , In Situ Hybridization , Male , Peptide Fragments/pharmacology , RNA, Messenger/analysis , Radioimmunoassay , Rats , Rats, Wistar , Somatostatin/genetics
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