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1.
Neuroscience ; 162(1): 14-22, 2009 Aug 04.
Article in English | MEDLINE | ID: mdl-19362130

ABSTRACT

We have previously reported that repeated central administration of sub-anxiogenic doses of the corticotropin releasing factor 1 (CRF(1)) agonist Cortagine, termed "priming," elicits a phenotype of increased anxiety-like behaviors in the elevated plus maze (EPM) and open-field test, and enhanced retention of contextual conditioned fear in C57BL/6J mice. Observed behavioral changes were functionally coupled to CRF(1)-mediated elevated central cholecystokinin (CCK) tone in discrete brain regions. However, the changes in gene expression that mediated "priming"-induced behavioral and concurrent molecular changes in specific brain regions remained unknown. In the present study, a complementary DNA microarray analysis was used to investigate gene expression profiles in the hippocampus and prefrontal cortex (PFC) of C57BL/6J mice following the "priming" procedure. Here, we report that chronic stimulation of CRF(1), by i.c.v. administration of 10 ng Cortagine for five days, brought about alterations in the expression of a wide range of hippocampal (31 genes) and PFC (18 genes) genes, implicated in anxiety and aversive memory formation. These expression changes involved genes associated with signal transduction, neurotransmitter secretion, synaptic transmission, myelination, and others involved in the transport, biosynthesis, and binding of proteins. In particular, several genes of the protein kinase A (PKA) and protein kinase C (PKC) signaling cascades, known to be involved in synaptic plasticity, such as neurogranin, calmodulin 3, and the PKA regulatory subunit 1 b were found to be upregulated in the PFC and hippocampus of CRF(1) agonist "primed" mice. Moreover, we show pharmacologically that one of the newly implicated memory regulatory elements, diazepam-binding inhibitor (DBI) is functionally involved in hippocampus-dependent enhancement of contextual fear, a cardinal phenotypic feature of the "primed" mice. Finally, an interaction network mapping of the altered genes and their known interacting partners identified additional molecular candidates responsible for CRF(1)-mediated hypersensitive fear circuitry.


Subject(s)
Diazepam Binding Inhibitor/metabolism , Fear/physiology , Gene Expression Regulation/physiology , Hippocampus/metabolism , Prefrontal Cortex/metabolism , Receptors, Corticotropin-Releasing Hormone/metabolism , Animals , Anxiety/genetics , Anxiety/metabolism , Central Nervous System Agents/administration & dosage , Conditioning, Classical/drug effects , Conditioning, Classical/physiology , Corticotropin-Releasing Hormone/administration & dosage , Electroshock , Fear/drug effects , Gene Expression Regulation/drug effects , Hippocampus/drug effects , Male , Maze Learning/drug effects , Maze Learning/physiology , Mice , Mice, Inbred C57BL , Models, Genetic , Oligonucleotide Array Sequence Analysis , Prefrontal Cortex/drug effects , Recombinant Fusion Proteins/administration & dosage , Reverse Transcriptase Polymerase Chain Reaction
2.
Mol Psychiatry ; 14(3): 291-307, 2009 Mar.
Article in English | MEDLINE | ID: mdl-18195718

ABSTRACT

Corticotropin-releasing factor (CRF) and cholecystokinin (CCK), two highly colocalized neuropeptides, have been linked to the etiology of stress-related anxiety disorders. Recent evidence points to the possibility that some of the anxiogenic effects of the central CCK system take place through interplay with the CRF system. The aim of the present study was to examine the effects of chronic, mild activation of CRF receptor 1 (CRF(1)) on the central CCK system of the C57BL/6J mouse. As shown by in situ hybridization, real-Time PCR and immunohistochemistry, 5 days of intracerebroventricular (i.c.v.) injections of a subeffective dose (2.3 pmol) of cortagine, a CRF(1)-selective agonist, resulted in an increase in CCK mRNA levels and CCK(2) receptor immunoreactivity in several brain regions, such as amygdala and hippocampus, known to be involved in the regulation of anxiety. Mice with elevated endogenous central CCK tone exhibited significantly higher anxiety-like behaviors in the open-field task and elevated plus maze, and enhanced conditioned fear. These behavioral changes were reversed by i.c.v. administration of the CCK(2)-selective antagonist LY225910, after 5 days of priming with cortagine. Under the same conditions, the intraperitoneal administration of the CRF(1) antagonist antalarmin was ineffective. This result indicated that once the CCK system was sensitized by prior CRF(1) activation, it exhibited its anxiogenic effects, without influence by CRF(1), possibly because of its observed downregulation. In sum, our results provide a novel model for the interaction of the CRF and CCK systems contributing to the development of hypersensitive emotional circuitry.


Subject(s)
Anxiety/metabolism , Cholecystokinin/metabolism , Conditioning, Classical/physiology , Exploratory Behavior/physiology , Receptors, Corticotropin-Releasing Hormone/physiology , Amygdala/drug effects , Amygdala/metabolism , Animals , Brain/drug effects , Brain/metabolism , Cholecystokinin/genetics , Conditioning, Classical/drug effects , Corticotropin-Releasing Hormone/administration & dosage , Dose-Response Relationship, Drug , Exploratory Behavior/drug effects , Fear , Hippocampus/drug effects , Hippocampus/metabolism , Hormone Antagonists/pharmacology , Injections, Intraventricular , Mice , Mice, Inbred C57BL , Quinazolinones/pharmacology , RNA, Messenger/analysis , Receptor, Cholecystokinin B/antagonists & inhibitors , Receptor, Cholecystokinin B/genetics , Receptor, Cholecystokinin B/metabolism , Receptors, Corticotropin-Releasing Hormone/agonists , Recombinant Fusion Proteins/administration & dosage , Stress, Physiological/physiology
3.
Mini Rev Med Chem ; 7(1): 55-64, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17266638

ABSTRACT

One of the most remarkable features of the mammalian central nervous system is its ability to store large amounts of information for periods approaching a lifetime. However, during the aging process cognitive domains, such as long-term (declarative) memory and working memory decline in some, but by far not all individuals. It is essential to understand the physiological changes that cause memory decline and also to elucidate why preserved memory abilities vary so greatly across individuals and memory tasks. A generally accepted hypothesis has been that long-lasting activity-dependent changes in the efficacy of synaptic transmission in the mammalian brain are considered to be of fundamental importance for the storage of information. There is now a more detailed understanding of the changes in neuronal plasticity during aging at the molecular and systems levels. This review discusses recent findings on age-related changes in neuronal plasticity, which have opened up novel sites of action for therapeutic intervention.


Subject(s)
Aging/physiology , Memory Disorders/physiopathology , Neuronal Plasticity , Acetylcholine/metabolism , Adenosine/metabolism , Aging/metabolism , Animals , Calcium Channels, L-Type/metabolism , Humans , Memory Disorders/drug therapy , Memory Disorders/metabolism , Nerve Growth Factors/metabolism , Potassium Channels/metabolism , Reactive Oxygen Species/metabolism
4.
Endocrinology ; 148(4): 1524-38, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17194738

ABSTRACT

Corticotropin-releasing factor (CRF) affects catecholamine production both centrally and peripherally. The aim of the present work was to examine the presence of CRF, its related peptides, and their receptors in the medulla of human and rat adrenals and their direct effect on catecholamine synthesis and secretion. CRF, urocortin I (UCN1), urocortin II (UCN2), and CRF receptor type 1 (CRF1) and 2 (CRF2) were present in human and rat adrenal medulla as well as the PC12 pheochromocytoma cells by immunocytochemistry, immunofluorescence, and RT-PCR. Exposure of dispersed human and rat adrenal chromaffin cells to CRF1 receptor agonists induced catecholamine secretion in a dose-dependent manner, an effect peaking at 30 min, whereas CRF2 receptor agonists suppressed catecholamine secretion. The respective effects were blocked by CRF1 and CRF2 antagonists. CRF peptides affected catecholamine secretion via changes of subplasmaliminal actin filament polymerization. CRF peptides also affected catecholamine synthesis. In rat chromaffin and PC12 cells, CRF1 and CRF2 agonists induced catecholamine synthesis via tyrosine hydroxylase. However, in human chromaffin cells, activation of CRF1 receptors induced tyrosine hydroxylase, whereas activation of CRF2 suppressed it. In conclusion, it appears that a complex intraadrenal CRF-UCN/CRF-receptor system exists in both human and rat adrenals controlling catecholamine secretion and synthesis.


Subject(s)
Adrenal Glands/drug effects , Catecholamines/metabolism , Corticotropin-Releasing Hormone/pharmacology , Receptors, Corticotropin-Releasing Hormone/physiology , Adrenal Glands/metabolism , Animals , Catecholamines/biosynthesis , Cells, Cultured , Chromaffin Cells/metabolism , Female , Humans , PC12 Cells , Rats , Rats, Sprague-Dawley , Receptors, Corticotropin-Releasing Hormone/agonists , Receptors, Corticotropin-Releasing Hormone/antagonists & inhibitors , Receptors, Corticotropin-Releasing Hormone/metabolism , Tissue Distribution , Tyrosine 3-Monooxygenase/genetics , Tyrosine 3-Monooxygenase/metabolism , Urocortins
5.
Nervenarzt ; 77(12): 1492-4, 2006 Dec.
Article in German | MEDLINE | ID: mdl-17102989

ABSTRACT

We report a previously healthy 47-year-old woman suffering acute embolic stroke in the left middle cerebral artery distribution. A filiform hypermobile mass along the line of the aortic valve representing a Lambl's excrescence was first identified by transesophageal echocardiography. Lambl's excrescences are structures attached to the edges of cardiac valves that have been associated with stroke, although their exact pathogenetic mechanisms have not yet been established. Further, treatment after cryptogenic stroke associated with presence of a valvular strand is based on platelet inhibition. Following currently available evidence, surgery cannot be generally recommended for this condition.


Subject(s)
Aortic Valve , Echocardiography, Transesophageal , Heart Valve Diseases/complications , Infarction, Middle Cerebral Artery/diagnosis , Magnetic Resonance Angiography , Diagnosis, Differential , Female , Heart Valve Diseases/diagnosis , Humans , Middle Aged
6.
Neuroscience ; 134(4): 1113-22, 2005.
Article in English | MEDLINE | ID: mdl-16039799

ABSTRACT

The role of corticotropin-releasing factor in autonomic regulation of heart rate, heart rate variability and behavior responses was investigated in two genetic mouse models: corticotropin-releasing factor receptor 1-deficient mice, and corticotropin-releasing factor-transgenic mice overexpressing corticotropin-releasing factor. Heart rate was recorded by radio-telemetry during novelty exposure and auditory fear conditioning. Locomotor activity and freezing served as behavioral indices. Locomotor activity and heart rate were invariably increased in response to novelty exposure in both corticotropin-releasing factor receptor 1-deficient mice and littermate wild-type controls. The heart rate responses during retention of conditioned auditory fear and the exponential relationship between heart rate and heart rate variability were unaffected by genotype. Moreover, conditioned fear responses inferred from multiple behavioral measures including freezing did not differ between corticotropin-releasing factor receptor 1-deficient and corticotropin-releasing factor receptor 1 wild-type control mice. Corticotropin-releasing factor-transgenic mice exhibited markedly reduced locomotor activity during novelty exposure when compared with littermate wild-type controls. Baseline and novelty-driven heart rate was slightly elevated in corticotropin-releasing factor-transgenic mice, whereas the novelty-induced increase of heart rate was not different between genotypes. In contrast, corticotropin-releasing factor-transgenic mice did not display a heart rate response indicative of conditioned auditory fear. It is concluded that corticotropin-releasing factor receptor 1-deficiency does not affect heart rate adjustment and behavioral responses to acute fearful stimuli. The resiliency of behavioral and cardiovascular patterns elevation argues against the involvement of corticotropin-releasing factor receptor 1 in acute emotional regulation on these two functional levels despite an absent corticosterone elevation in corticotropin-releasing factor receptor 1-deficient mice. It is hypothesized that the lack of a conditioned heart rate response in corticotropin-releasing factor-transgenic mice is attributable to an impairment of cognitive function. The results are compared with those of corticotropin-releasing factor receptor 2-deficient mice, and the role of the corticotropin-releasing factor system in cardiovascular regulation is discussed.


Subject(s)
Behavior, Animal/physiology , Heart Rate/physiology , Receptors, Corticotropin-Releasing Hormone/metabolism , Stress, Psychological/physiopathology , Animals , Conditioning, Classical/physiology , Electrocardiography , Fear/physiology , Mice , Mice, Transgenic , Motor Activity/physiology
7.
Z Kardiol ; 93(12): 944-53, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15599569

ABSTRACT

UNLABELLED: The purpose of this study was to evaluate whether the underlying mechanism of mitral regurgitation influences the reliability of the proximal flow con- vergence method to assess the regurgitant volume. Furthermore, the mode of imaging the flow convergence region and different correction algorithms for calculation of the regurgitant volume were compared. METHODS: Regurgitant volume was assessed in 45 patients (age 61+/-13 years) with organic (n=19) and functional (n=26) mitral regurgitation by the proximal flow convergence method for aliasing velocities between 14 and 64 cm/s using two-dimensional color Doppler imaging. Different correction and calculation algorithms were compared. In addition, regurgitant volume was determined using color Doppler M-mode for an aliasing velocity of 28 cm/s. The quantitative Doppler method was used as reference. RESULTS: In organic mitral regurgitation correlation coefficients (mean differences) between the proximal flow convergence method and the reference method were 0.25-0.43/ 0.58-0.67 (46-111 ml/15-17 ml) before/after geometric correction of the regurgitant volume for the aliasing velocities investigated. The correlation coefficient (mean difference) using color Doppler M-mode imaging was 0.68 (85 ml). The corresponding values in functional mitral regurgitation were 0.74-0.88/0.74-0.88 (-5-8 ml/-7-5 ml) for two-dimensional color Doppler and 0.88 (-1 ml) for M-mode imaging. CONCLUSIONS: The regurgitant volume was overestimated by the proximal flow convergence method in organic mitral regurgitation irrespective of the application of different correction algorithms or the use of color Doppler M-mode. A sufficiently reliable determination of the regurgitant volume by the proximal flow convergence method was possible in functional mitral regurgitation. In that case a simplified calculation of the regurgitant volume based on the proximal flow convergence method was feasible.


Subject(s)
Blood Flow Velocity/physiology , Blood Volume/physiology , Echocardiography, Doppler, Color/statistics & numerical data , Mitral Valve Insufficiency/diagnostic imaging , Adult , Aged , Aged, 80 and over , Algorithms , Female , Humans , Linear Models , Male , Middle Aged , Mitral Valve/diagnostic imaging , Mitral Valve/physiopathology , Mitral Valve Insufficiency/physiopathology , Reproducibility of Results , Statistics as Topic
8.
Mol Psychiatry ; 9(2): 174-83, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14581933

ABSTRACT

Stress insults intensify fear memory; however, the mechanism(s) facilitating this physiological response is still unclear. Here, we report the molecular, neurophysiological and behavioral findings attributing much of this effect to alternative splicing of the acetylcholinesterase (AChE) gene in hippocampal neurons. As a case study, we explored immobilization-stressed mice with intensified fear memory and enhanced long-term potentiation (LTP), in which alternative splicing was found to induce overproduction of neuronal 'readthrough' AChE-R (AChE-R). Selective downregulation of AChE-R mRNA and protein by antisense oligonucleotides abolished the stress-associated increase in AChE-R, the elevation of contextual fear and LTP in the hippocampal CA1 region. Reciprocally, we intrahippocampally injected a synthetic peptide representing the C-terminal sequence unique to AChE-R. The injected peptide, which has been earlier found to exhibit no enzymatic activity, was incorporated into cortical, hippocampal and basal nuclei neurons by endocytosis and retrograde transport and enhanced contextual fear. Compatible with this hypothesis, inherited AChE-R overexpression in transgenic mice resulted in perikaryal clusters enriched with PKCbetaII, accompanied by PKC-augmented LTP enhancement. Our findings demonstrate a primary role for stress-induced alternative splicing of the AChE gene to elevated contextual fear and synaptic plasticity, and attribute to the AChE-R splice variant a major role in this process.


Subject(s)
Acetylcholinesterase/genetics , Alternative Splicing/physiology , Fear/physiology , Long-Term Potentiation/genetics , Memory/physiology , Stress, Physiological/genetics , Acetylcholinesterase/metabolism , Animals , Gene Expression Regulation, Enzymologic/physiology , Hippocampus/physiology , Mice , Neuronal Plasticity/genetics , Protein Kinase C/metabolism , Protein Kinase C beta , RNA, Messenger/genetics , Stress, Physiological/physiopathology
9.
Recept Channels ; 8(3-4): 163-77, 2002.
Article in English | MEDLINE | ID: mdl-12529934

ABSTRACT

The biology of corticotropin-releasing factor (CRF) finds increasing interest in the scientific community because of the neuromodulatory actions of CRF on brain functions such as learning, anxiety, feeding, and locomotion. Additional actions on immunumodulation and apoptosis have recently been discovered. All actions of CRF are mediated by G protein-coupled receptors, which trigger different, sometimes opposite actions in different regions of the central nervous system. The CRF system exhibits considerable plasticity by the involvement of numerous different ligands, splice variants, and transductional couplings. The generation of multiple splice variants is facilitated by the intron exon structure of the CRF receptor genes.


Subject(s)
Receptors, Corticotropin-Releasing Hormone/metabolism , Amino Acid Sequence , Animals , Apoptosis/physiology , Behavior/physiology , Binding Sites , Carrier Proteins/metabolism , Corticotropin-Releasing Hormone/antagonists & inhibitors , GTP-Binding Proteins/metabolism , Humans , Ligands , Molecular Sequence Data , Neurons/metabolism , Receptors, Corticotropin-Releasing Hormone/genetics , Sequence Alignment , Signal Transduction
10.
Brain Res Mol Brain Res ; 94(1-2): 15-24, 2001 Oct 19.
Article in English | MEDLINE | ID: mdl-11597761

ABSTRACT

Phosphorylation of the cAMP response element binding protein (pCREB) triggered by associative learning was monitored immunohistochemically in different areas of the mouse brain during a 6-h interval, starting immediately after training. One trial context-dependent fear conditioning was employed as a learning paradigm. Training consisted of contextual exposure followed by shock. Control groups consisted of naïve mice, mice exposed to the context alone and mice exposed to an immediate shock in the context. For all trained mice, the time course of CREB phosphorylation in hippocampus, parietal cortex and amygdaloid nuclei exhibited a biphasic pattern. The early phase was between 0 and 30 min, and the late phase was between 3 and 6 h after training. The animals exposed to context followed by an electric shock, as well as those exposed to an immediate electric shock, exhibited significantly higher pCREB levels than the mice subjected to context alone. During the late phase, the pCREB levels were highest in the mice exposed to the context followed by shock. It was observed that CREB phosphorylation and Fos production followed different regional and stimulus-dependent patterns. It is suggested that the early phase of pCREB increase may be related to stress-related behaviors, whereas the late phase may rather relate to memory consolidation.


Subject(s)
Brain Chemistry/physiology , Cyclic AMP Response Element-Binding Protein/metabolism , Fear/physiology , Proto-Oncogene Proteins c-fos/biosynthesis , Amygdala/physiology , Animals , Association Learning/physiology , Conditioning, Psychological/physiology , Dentate Gyrus/physiology , Electroconvulsive Therapy , Male , Memory/physiology , Mice , Mice, Inbred C57BL , Parietal Lobe/physiology , Phosphorylation , Proto-Oncogene Proteins c-fos/analysis
11.
Neuropharmacology ; 41(4): 507-16, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11543771

ABSTRACT

Corticotropin releasing factor (CRF) represents an early chemical signal in the stress response and modulates various brain functions through G protein-coupled receptors. Two CRF receptor subtypes, CRF(1) and CRF(2), have been identified. Since the physicochemical properties of CRF receptor antagonists might influence their biological potency, the peptidic antagonists astressin, alpha-helical CRF(9-41) (alpha-helCRF) and antisauvagine-30 (aSvg-30) have been analyzed. The rank order of solubility of these compounds in artificial cerebrospinal fluid (aCSF, pH 7.4) was aSvg-30>alpha-helCRF>>astressin, whereas the rank order of relative lipophilicity as determined with RP-HPLC was alpha-helCRF>astressin>aSvg-30. The calculated isoelectric points were 4.1 (alpha-helCRF), 7.4 (astressin) and 10.0 (aSvg-30). According to Schild analysis of the CRF receptor-dependent cAMP production of transfected HEK cells, aSvg-30 exhibited a competitive antagonism and displayed a 340 fold selectivity for mCRF(2 beta) receptor. For astressin, however, the pharmacodynamic profile could not be explained by a simple competitive mechanism as indicated by Schild slopes >1 for rCRF(1) or mCRF(2 beta) receptor. Behavioral experiments demonstrated that after i.c.v. injection, alpha-helCRF reduced oCRF-induced anxiety-like behavior in the elevated plus-maze, whereas astressin, despite its higher in vitro potency, did not. These findings could be explained by different physicochemical properties of the antagonists employed.


Subject(s)
Behavior, Animal/drug effects , Corticotropin-Releasing Hormone/pharmacology , Neuroprotective Agents/pharmacology , Peptide Fragments/pharmacology , Animals , Anxiety/psychology , Cells, Cultured , Chromatography, High Pressure Liquid , Corticotropin-Releasing Hormone/antagonists & inhibitors , Corticotropin-Releasing Hormone/chemistry , Cyclic AMP/metabolism , Male , Membranes/drug effects , Membranes/metabolism , Mice , Mice, Inbred BALB C , Neuropeptides/pharmacology , Neuroprotective Agents/chemistry , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/chemistry , Receptors, Corticotropin-Releasing Hormone/antagonists & inhibitors , Solubility
12.
Protein Sci ; 10(10): 2050-62, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11567096

ABSTRACT

Rat corticotropin-releasing factor receptor 1 (rCRFR1) was produced either in transfected HEK 293 cells as a complex glycosylated protein or in the presence of the mannosidase I inhibitor kifunensine as a high mannose glycosylated protein. The altered glycosylation did not influence the biological function of rCRFR1 as demonstrated by competitive binding of rat urocortin (rUcn) or human/rat corticotropin-releasing factor (h/rCRF) and agonist-induced cAMP accumulation. The low production rate of the N-terminal domain of rCRFR1 (rCRFR1-NT) by transfected HEK 293 cells, was increased by a factor of 100 in the presence of kifunensine. The product, rCRFR1-NT-Kif, bound rUcn specifically (K(D) = 27 nM) and astressin (K(I) = 60 nM). This affinity was 10-fold lower than the affinity of full length rCRFR1. However, it was sufficiently high for rCRFR1-NT-Kif to serve as a model for the N-terminal domain of rCRFR1. With protein fragmentation, Edman degradation, and mass spectrometric analysis, evidence was found for the signal peptide cleavage site C-terminally to Thr(23) and three disulfide bridges between precursor residues 30 and 54, 44 and 87, and 68 and 102. Of all putative N-glycosylation sites in positions 32, 38, 45, 78, 90, and 98, all Asn residues except for Asn(32) were glycosylated to a significant extent. No O-glycosylation was observed.


Subject(s)
Disulfides/chemistry , Receptors, Corticotropin-Releasing Hormone/physiology , Amino Acid Sequence , Animals , Cells, Cultured , Gene Deletion , Glycosylation , Humans , Ligands , Mass Spectrometry , Molecular Sequence Data , Protein Sorting Signals/physiology , Protein Structure, Secondary , Protein Structure, Tertiary/physiology , Rats , Receptors, Corticotropin-Releasing Hormone/chemistry , Receptors, Corticotropin-Releasing Hormone/genetics , Receptors, Corticotropin-Releasing Hormone/metabolism
13.
Proc Natl Acad Sci U S A ; 98(20): 11142-7, 2001 Sep 25.
Article in English | MEDLINE | ID: mdl-11572971

ABSTRACT

In view of the observation that corticotropin-releasing factor (CRF) affects several brain functions through at least two subtypes of G protein-dependent receptors and a binding protein (CRFBP), we have developed synthetic strategies to provide enhanced binding specificity. Human/rat CRF (h/rCRF) and the CRF-like peptide sauvagine (Svg), differing in their affinities to CRFBP by two orders of magnitude, were used to identify the residues determining binding to CRFBP. By amino acid exchanges, it was found that Ala(22) of h/rCRF was responsible for this peptide's high affinity to CRFBP, whereas Glu(21) located in the equivalent position of Svg prevented high affinity binding to CRFBP. Accordingly, [Glu(22)]h/rCRF was not bound with high affinity to CRFBP in contrast to [Ala(21)]Svg, which exhibited such high affinity. Furthermore, the affinity of both peptides to either CRF receptor (CRFR) subtype was not reduced by these replacements, and their subtype preference was not changed. Thus, exchange of Ala and Glu and vice versa in positions 22 and 21 of h/rCRF and Svg, respectively, serves as a switch discriminating between CRFBP and CRFR. On the basis of this switch function, development of new specific CRF agonists and antagonists is expected to be facilitated. One application was the modification of the CRF antagonist astressin (Ast), whose employment in animal experiments is limited by its low solubility in cerebrospinal fluid. Introduction of Glu residues into Ast generated with [Glu(11,16)]Ast an acidic astressin, which efficiently antagonized in vivo the CRFR1-dependent reduction of locomotion induced by ovine CRF without detectable binding to CRFBP.


Subject(s)
Carrier Proteins/metabolism , Corticotropin-Releasing Hormone/chemistry , Corticotropin-Releasing Hormone/metabolism , Peptides/pharmacology , Receptors, Corticotropin-Releasing Hormone/chemistry , Receptors, Corticotropin-Releasing Hormone/metabolism , Amino Acid Sequence , Amphibian Proteins , Animals , Binding, Competitive , Carrier Proteins/chemistry , Cell Line , Cyclic AMP/metabolism , Drug Design , Glutamic Acid , Humans , Models, Molecular , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Hormones , Peptides/chemistry , Protein Structure, Secondary , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/drug effects , Recombinant Proteins/metabolism , Structure-Activity Relationship , Transfection
14.
Neurosci Lett ; 303(2): 123-6, 2001 May 04.
Article in English | MEDLINE | ID: mdl-11311508

ABSTRACT

Studies employing classical fear conditioning (FC) and inhibitory avoidance (IA), two procedurally different aversive tasks, provide different insight into the neuronal mechanism(s) underlying fear learning. We examined whether immediate post-training injections of catecholaminergic drugs modulate memory consolidation in one-trial FC, as has been demonstrated in one-trial IA. Neither epinephrine (0.1, 0.3, 1.0 mg/kg intraperitoneally) nor amphetamine (1.0, 2.0 mg/kg) modulates FC to tone or context, as indicated by freezing in rats. Similarly, epinephrine (0.1, 1.0 mg/kg) and beta-adrenergic antagonists (sotalol and propranolol; 2 mg/kg) also failed to modulate FC in mice. These results indicate that FC is not susceptible to memory modulation by catecholaminergic drugs in the manner described in IA tasks.


Subject(s)
Adrenergic Agonists/pharmacology , Avoidance Learning/physiology , Catecholamines/agonists , Catecholamines/metabolism , Conditioning, Psychological/physiology , Fear/physiology , Acoustic Stimulation/adverse effects , Adrenergic beta-Antagonists/pharmacology , Amphetamine/pharmacology , Amygdala/drug effects , Amygdala/metabolism , Animals , Avoidance Learning/drug effects , Behavior, Animal/drug effects , Behavior, Animal/physiology , Conditioning, Psychological/drug effects , Drug Administration Schedule , Electric Stimulation/adverse effects , Epinephrine/pharmacology , Fear/drug effects , Male , Memory/drug effects , Memory/physiology , Rats , Rats, Long-Evans
15.
Arch Immunol Ther Exp (Warsz) ; 49(1): 33-8, 2001.
Article in English | MEDLINE | ID: mdl-11266088

ABSTRACT

Corticotropin-releasing factor (CRF) was originally identified as a hypothalamic peptide which stimulates secretion of the hypophyseal adrenocorticotropin hormone. CRF exhibits its actions through G protein-dependent seven membrane domain receptors. Two subtypes of CRF receptors (CRF-R1 and CRF-R2) have been characterized thus far. CRF and its receptors were found in a number of brain regions, where they function by neuromodulation and also in several peripheral organs. Besides CRF, another naturally occurring CRF-like peptide, urocortin, has been characterized. In the immune system, CRF and CRF-R1 were so far detected at both mRNA and protein levels in several lymphoid organs and at sites of inflammation. Locally injected CRF was shown to modulate the severity of inflammation. This effect was not only a result of hemodynamic changes known to be induced by CRF or by activation of the hypothalamo-pituitary-adrenal axis, as CRF-binding sites were also found on immune cells. CRF was shown to directly modulate secretion of cytokines and neuropeptides, proliferation, chemotaxis and degranulation of purified macrophage and lymphocyte populations in vitro. Functional CRF-R was more recently demonstrated also on polymorphonuclear cells and significant amounts of CRF were shown to be produced in lymphoid organs, or delivered to lymphoid organs by peripheral nerves. Taken together, the experimental results obtained so far strongly point to the importance of CRF as a signaling molecule in lymphoid tissues and at the sites of inflammation.


Subject(s)
Adjuvants, Immunologic/physiology , Corticotropin-Releasing Hormone/immunology , Animals , Humans , Hypothalamo-Hypophyseal System/immunology , Ligands , Pituitary-Adrenal System/immunology , Receptors, Corticotropin-Releasing Hormone/immunology
16.
Mol Pharmacol ; 59(3): 485-92, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11179443

ABSTRACT

The influence of an upstream open reading frame (ORF) in the 5'-untranslated region (UTR) of the mRNA on corticotropin-releasing hormone receptor type 1 (CRHR1) translation was studied in constructs containing the 5'-UTR of CRHR1, with or without an ATG-to-ATA mutation in the upstream ORF, and the main ORF of luciferase or CRHR1. Upstream mutation in luciferase constructs increased luciferase activity when transfected into COS-7 or AtT20 cells compared with the native 5'-UTR. Transfection of CRHR1 constructs containing the upstream mutation into AtT20 or LVIP2.0zc reporter cells, resulted in higher (125)I-Tyr-oCRH binding and corticotropin-releasing hormone-stimulated cAMP production, without changes in CRHR1 mRNA levels (measured by RNase protection assay). In vitro translation of luciferase or CRHR constructs with or without mutation of the upstream ATG, and Western blot analysis with anti-luciferase and anti-CRHR1 antibodies confirmed that mutation of the upstream ATG increases translation of the main ORF. The mechanism by which the upstream ORF inhibits translation may involve translation of the upstream peptide, because in vitro translation, or transfection into LVIP2.0zc cells of a fusion construct of the upstream ORF and green fluorescent protein (GFP) yielded a band consistent with the molecular size of GFP protein. The study shows that the upstream AUG in 5'-UTR of CRHR1 mRNA inhibits receptor expression by inhibiting mRNA translation and suggests the short open reading frame in the 5'-UTR plays a role in regulating translation of the CRH receptor.


Subject(s)
5' Untranslated Regions/genetics , Codon, Initiator/genetics , Receptors, Corticotropin-Releasing Hormone/genetics , Animals , Base Sequence , COS Cells , DNA, Complementary/analysis , Luciferases/metabolism , Molecular Sequence Data , Mutation , Nucleic Acid Conformation , Protein Biosynthesis , RNA, Messenger/chemistry , Receptors, Corticotropin-Releasing Hormone/metabolism
17.
Peptides ; 22(1): 47-56, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11179597

ABSTRACT

Little is known on the structural ligand requirements for corticotropin-releasing factor binding protein (CRFBP) of the rat used as an important experimental animal. To obtain such information recombinant rat CRFBP was produced in stably transfected HEK 293 cells. The primary structure and posttranslational processing of purified rat CRFBP was established by peptide mapping using HPLC combined with mass spectrometric analysis. Rat CRFBP was pharmacologically characterized employing a competition binding assay with tritium-labeled rat urocortin. The rank order of declining affinity of various CRF analogs was urotensin-I, human/rat CRF (h/rCRF), rat urocortin, sauvagine (Svg), and ovine CRF in agreement with the rank order found for human CRFBP. In contrast to astressin, the CRF receptor 2-selective antagonist anti-sauvagine-30 did not show any detectable specific binding to rat CRFBP. The significance of residues 10 to 12 and 21 to 24 of Svg for its low affinity binding was established by changing these residues of Svg to those of h/rCRF. The corresponding residues 22 to 25 of h/rCRF represented the ARAE motif determined to be crucial for binding in agreement with reported data on human CRFBP. Residues 11 to 13 of CRF introduced into Svg also enhanced the affinity to rat CRFBP.


Subject(s)
Peptides/pharmacology , Receptors, Corticotropin-Releasing Hormone/agonists , Amino Acid Sequence , Amphibian Proteins , Animals , Humans , Ligands , Molecular Sequence Data , Peptide Hormones , Peptides/chemistry , Rats , Receptors, Corticotropin-Releasing Hormone/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology
18.
J Neurosci ; 20(22): 8515-27, 2000 Nov 15.
Article in English | MEDLINE | ID: mdl-11069959

ABSTRACT

The present study examined the involvement of the 5-HT(1A) receptors in classical fear conditioning using the 5-HT(1A) agonist 8-hydroxy-2-(di-n-propyloamino)tetralin hydrobromide (8-OH-DPAT) and the selective "silent" 5-HT(1A) receptor antagonist (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclo- hexane carboxamide trihydrochloride (WAY 100635). The drugs were administered both subcutaneously and bilaterally into the dorsal hippocampus of male C57BL/6J mice. The training was performed in a single trial in which a tone was followed by a footshock. The retention of context- and tone-dependent fear was examined in separate tests conducted either 1 or 24 hr after training. Subcutaneous 8-OH-DPAT (0.1-1.0 mg/kg), when injected before but not after training, caused a dose-dependent impairment of contextual fear in both 1 and 24 hr tests, whereas tone-dependent fear was less affected. Pretraining intrahippocampal injections of 5.0 microg but not 1.0 microg 8-OH-DPAT caused a severe deficit in contextual fear when tested 24 hr after training. When injected both subcutaneously and intrahippocampally, 8-OH-DPAT induced the 5-HT syndrome, indicative of postsynaptic 5-HT(1A) receptor activation at the dose ranges that impaired fear conditioning. However, the behavioral changes induced by 8-OH-DPAT at the time of training could not account for inhibitory effects of 8-OH-DPAT on fear conditioning. Neither subcutaneous (0.03 mg/kg) nor intrahippocampal (0.5 microg per mouse) WAY 100635 altered context- or tone-dependent fear. However, subcutaneous WAY 100635 blocked both the 5-HT syndrome and the impairment of fear conditioning induced by subcutaneous or intrahippocampal 8-OH-DPAT. In contrast, intrahippocampal WAY 100635 blocked the impairment caused by intrahippocampal but not subcutaneous 8-OH-DPAT, indicating the involvement of extrahippocampal 5-HT(1A) receptors in fear conditioning. It is concluded that the deficits in fear conditioning induced by 8-OH-DPAT are a result of postsynaptic 5-HT(1A) receptor activation that interferes with learning processes operating at acquisition but not consolidation. Furthermore, the dorsohippocampal 5-HT(1A) receptors play an important but not exclusive role in the limbic circuitry subserving contextual fear conditioning.


Subject(s)
Conditioning, Classical/physiology , Fear/physiology , Receptors, Serotonin/metabolism , 8-Hydroxy-2-(di-n-propylamino)tetralin/administration & dosage , Acoustic Stimulation , Analysis of Variance , Animals , Behavior, Animal/drug effects , Conditioning, Classical/drug effects , Dose-Response Relationship, Drug , Drug Administration Routes , Electroshock , Hippocampus/drug effects , Hippocampus/physiology , Injections, Subcutaneous , Male , Memory/drug effects , Mice , Mice, Inbred C57BL , Microinjections , Piperazines/administration & dosage , Pyridines/administration & dosage , Receptors, Serotonin/drug effects , Receptors, Serotonin, 5-HT1 , Retention, Psychology/drug effects , Retention, Psychology/physiology , Serotonin Antagonists/administration & dosage , Serotonin Receptor Agonists/administration & dosage
19.
Behav Brain Res ; 116(2): 157-68, 2000 Dec 05.
Article in English | MEDLINE | ID: mdl-11080547

ABSTRACT

The effects of acute injections of the NMDA receptor antagonist APV on one-trial fear conditioning of C57BL/6J mice were investigated in a time, dose (0.4-3.2 microg) and region-specific manner. Conditioned fear was determined by the assessment of freezing and the computer-controlled measurement of inactivity. Additionally, conditioned heart rate responses were evaluated in the tone-dependent memory test performed in the home cage with the help of implanted ECG transmitters. Injections of APV into the dorsal hippocampus (i.h.) 15 min before training, impaired dose-dependent contextual fear conditioning without discrimination between contextual foreground (unsignaled shock) and background (signaled shock) conditioning as tested 24 h after training. Short-term memory analyzed 1 h after training was also impaired. The observation that a smaller dose of APV was required for intracerebroventricular than for i.h. injection to achieve a similar memory impairment, pointed to the involvement of extrahippocampal NMDA receptors. APV treatment did not affect conditioned tone-dependent fear as indicated by unchanged freezing and heart rate responses of the freely moving mice. The results indicated the contribution of hippocampal and extrahippocampal NMDA receptors to multisensory contextual information processing during acquisition.


Subject(s)
Association Learning/drug effects , Conditioning, Classical/drug effects , Fear/drug effects , Hippocampus/drug effects , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Valine/analogs & derivatives , Animals , Dose-Response Relationship, Drug , Injections, Intraventricular , Male , Memory, Short-Term/drug effects , Mice , Mice, Inbred C57BL , Valine/pharmacology
20.
J Neurochem ; 75(2): 749-54, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10899951

ABSTRACT

In the present study we investigated the modulation of hypothalamic NMDA receptor-mediated currents by cyclic AMP-dependent protein kinase (PKA) using the two-electrode voltage-clamp technique in Xenopus oocytes injected with rat hypothalamic mRNA. Application of forskolin, which activates PKA by means of cyclic AMP stimulation, caused a transient increase of NMDA-induced currents, whereas the inactive forskolin analogue 1,9-dideoxyforskolin had no effect. Incubation of oocytes with a membrane-permeable analogue of cyclic AMP, 8-bromoadenosine 3',5' -cyclic monophosphate, potentiated NMDA responses even more prominently than with forskolin. NMDA-induced currents recorded from Xenopus oocytes injected with cRNA encoding the NMDA receptor subunits NR1, NR2A, and/or NR2B, mainly found in rat hypothalamus, were not affected by PKA activation but were increased by protein kinase C (PKC) stimulation. It is interesting that inhibition of endogenous protein phosphatase 1 and/or 2A by calyculin A resulted in a similar enhancement of hypothalamic NMDA-induced currents. Preinjection of oocytes with calyculin A impeded the PKA- but not the PKC-mediated potentiation of hypothalamic NMDA-induced currents. We propose the involvement of an additional third messenger in the PKA effect, which acts most likely via the inhibition of tonically active protein phosphatase 1 and/or 2A.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Hypothalamus/metabolism , N-Methylaspartate/pharmacology , Phosphoprotein Phosphatases/metabolism , Receptors, N-Methyl-D-Aspartate/physiology , Sulfonamides , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Animals , Colforsin/pharmacology , Enzyme Inhibitors/pharmacology , Female , Isoquinolines/pharmacology , Kinetics , Marine Toxins , Oocytes/drug effects , Oocytes/physiology , Oxazoles/pharmacology , Protein Kinase C/metabolism , Protein Phosphatase 1 , RNA, Messenger/genetics , Rats , Rats, Wistar , Recombinant Proteins/metabolism , Xenopus laevis
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