sauvagine and astressin

Corticotropin-Releasing Factor Receptors (CRFRs) are class B1 G-protein-coupled receptors, which bind peptides of the corticotropin releasing factor family and are key mediators in the stress response. In order to dissect the receptors' binding specificity and enable structural studies, full-length human CRFR1α and mouse CRFR2β as well as fragments lacking the N-terminal extracellular domain, were overproduced in E. coli. The characteristics of different CRFR2β-PhoA gene fusion products expressed in bacteria were found to be in agreement with the predicted ones in the hepta-helical membrane topology model. Recombinant histidine-tagged CRFR1α and CRFR2β expression levels and bacterial subcellular localization were evaluated by cell fractionation and Western blot analysis. Protein expression parameters were assessed, including the influence of E. coli bacterial hosts, culture media and the impact of either PelB or DsbA signal peptide. In general, the large majority of receptor proteins became inserted in the bacterial membrane. Across all experimental conditions significantly more CRFR2β product was obtained in comparison to CRFR1α. Following a detergent screen analysis, bacterial membranes containing CRFR1α and CRFR2β were best solubilized with the zwitterionic detergent FC-14. Binding of different peptide ligands to CRFR1α and CRFR2β membrane fractions were similar, in part, to the complex pharmacology observed in eukaryotic cells. We suggest that our E. coli expression system producing functional CRFRs will be useful for large-scale expression of these receptors for structural studies.

Topics: Amphibian Proteins; Animals; Blotting, Western; Cell Membrane; Corticotropin-Releasing Hormone; Culture Media; Detergents; Escherichia coli; Genetic Vectors; Humans; Kinetics; Ligands; Mammals; Mice; Peptide Fragments; Peptide Hormones; Protein Binding; Protein Sorting Signals; Protein Structure, Tertiary; Receptors, Corticotropin-Releasing Hormone; Recombinant Fusion Proteins; Solubility

Urocortin 3 (Ucn3) is a new member of the corticotropin-releasing factor (CRF) peptide family and is considered to be a specific and endogenous ligand for CRF type 2 receptors (CRF2Rs). The presence of CRF(2)Rs has been reported in the nucleus tractus solitarius (NTS) of the rat. It was hypothesized that the activation of CRF2Rs in the medial NTS (mNTS) may play a role in cardiovascular regulation. This hypothesis was tested in urethane-anesthetized, artificially ventilated, adult male Wistar rats. Microinjections (100 nl) of Ucn3 (0.03, 0.06, 0.12, and 0.25 mM) into the mNTS of anesthetized rats elicited decreases in mean arterial pressure (MAP: 5.0 +/- 1.0, 21.6 +/- 2.6, 20.0 +/- 2.8, and 12.7 +/- 3.4 mmHg, respectively) and heart rate (HR: 7.8 +/- 2.6, 46.2 +/- 9.3, 34.5 +/- 8.4, and 16.6 +/- 4.9 beats/min, respectively). Microinjections of artificial cerebrospinal fluid (100 nl) into the mNTS did not elicit cardiovascular responses. Maximum decreases in MAP and HR were elicited by 0.06 mM concentration of Ucn3. Cardiovascular responses to Ucn3 were similar in unanesthetized midcollicular decerebrate rats. A bilateral vagotomy completely abolished Ucn3-induced bradycardia. The decreases in MAP and HR elicited by Ucn3 (0.06 mM) were completely blocked by astressin (1 mM; nonselective CRFR antagonist) and K41498 (5 mM; selective CRF2R antagonist). Microinjections of Ucn3 (0.06 mM) into the mNTS decreased the efferent greater splanchnic nerve activity. After the blockade of CRF2Rs in the mNTS, a Ucn3-induced decrease in the efferent sympathetic nerve discharge was abolished. These results indicate that Ucn3 microinjections into the mNTS exerted excitatory effects on the mNTS neurons via CRF2Rs, leading to depressor and bradycardic responses.

Topics: Amphibian Proteins; Anesthesia, General; Animals; Blood Pressure; Bradycardia; Corticotropin-Releasing Hormone; Decerebrate State; Dose-Response Relationship, Drug; Heart; Heart Rate; Hypotension; Male; Microinjections; Peptide Fragments; Peptide Hormones; Rats; Rats, Wistar; Receptors, Corticotropin-Releasing Hormone; Respiration, Artificial; Solitary Nucleus; Splanchnic Nerves; Urocortins; Vagotomy

Previously, we showed that corticotropin-releasing factor (CRF) injected i.p. mimicked epithelial responses to stress, both stimulating ion secretion and enhancing permeability in the rat colon, and mast cells were involved. However, the ability of CRF-sensitive mucosal/submucosal loops to regulate intestinal barrier and the participation of resident mast cells are unclear.. We examined colonic epithelial responses to stress-like peptides in Wistar-Kyoto (WKY), and mast cell-deficient (Ws/Ws) and their +/+ littermate control rats in distal segments mounted in Ussing chambers. Short-circuit current (ion secretion), flux of horseradish peroxidase (macromolecular permeability), and the release of rat mast cell protease II were measured in response to CRF [10(-6) to 10(-8)M] or sauvagine [10(-8) to 10(-10)M] in tissues pretreated with astressin, doxantrazole, or vehicle.. Stress-like peptides (sauvagine > CRF) induced a dose-dependent increase in short-circuit current (maximal at 30 min), and significantly enhanced horseradish peroxidase flux and protease II release in WKY. Epithelial responses were inhibited by both astressin and doxantrazole, and significantly reduced in tissues from Ws/Ws rats.. The stress mediators CRF and sauvagine modulate barrier function in the rat colon acting on mucosal/submucosal CRF receptor-bearing cells, through mast cell-dependent pathways.

Topics: Amphibian Proteins; Animals; Colon; Corticotropin-Releasing Hormone; Dose-Response Relationship, Drug; Enteric Nervous System; Epithelium; Intestinal Mucosa; Ion Transport; Male; Mast Cells; Neuropeptides; Peptide Fragments; Peptide Hormones; Rats; Rats, Inbred WKY; Rats, Mutant Strains; Receptors, Corticotropin-Releasing Hormone; Signal Transduction; Statistics, Nonparametric; Stress, Physiological; Thioxanthenes; Xanthones

The objective of this study was to investigate the role of corticotropin-releasing factor receptors 1 (CRF(1)) and 2 (CRF(2)) in anxiety-like behavior and learning of C57BL/6J mice after exposure to a stressful stimulus. When C57BL/6J mice were exposed to immobilization (1 h) serving as stressful stimulus, context- and tone-dependent fear conditioning were impaired if the training followed immediately after immobilization. The stress-induced impairment of context-dependent fear conditioning was prevented by specific blockade of CRF(2) of the lateral septum (LS) with anti-sauvagine-30. Immobilization did not only affect conditioned fear, but also enhanced, through CRF(2) of the LS, anxiety-like behavior determined with the elevated plus maze. Recovery from stress-induced anxiety and impairment of context-dependent fear conditioning was observed after 1 h delay of training and required hippocampal CRF(1), as indicated by the finding that this recovery was prevented by blockade of intrahippocampal CRF(1). It was concluded that exposure to a stressor initially affected both anxiety-like behavior and contextual conditioned fear through septal CRF(2), while the later activation of hippocampal CRF(1) resulted in the return to baseline levels of both processes. Intraventricular injection of mouse urocortin 2, a CRF(2)-selective agonist, removed the stress-induced anxiety and learning impairment, but did not reduce the activation of the hypothalamic pituitary adrenal axis indicative of the hormonal stress response. We propose that the enhanced anxiety is the component of the stress response responsible for the memory deficit.

Topics: Adrenocorticotropic Hormone; Amphibian Proteins; Animals; Antibodies; Anxiety; Autoradiography; Behavior, Animal; Conditioning, Classical; Corticotropin-Releasing Hormone; Dose-Response Relationship, Drug; Fear; Immobilization; Male; Maze Learning; Memory Disorders; Mice; Mice, Inbred C57BL; Peptide Fragments; Peptide Hormones; Peptides; Receptors, Corticotropin-Releasing Hormone; Septal Nuclei; Stress, Physiological; Time Factors; Urocortins

The corticotropin-releasing factor (CRF) type 1 receptors (CRF(1)) from human (hCRF(1)) and Xenopus (xCRF(1)) differ from one another by their agonist- and antagonist-binding preference. While the agonist-binding site of the xCRF(1) receptor has been mapped, the amino acids that mediate binding of the potent peptide antagonist astressin are unknown. By constructing receptor chimeras followed by site-directed mutagenesis, the astressin-binding site of the xCRF(1) receptor was located between residues 76 and 83. This region partially overlaps with the agonist-selective domain of the xCRF(1) receptor (residues 76-89). Mutagenesis of the amphibian residues Gln(76), Gly(81) and Val(83) to the human sequence (Arg(76)Asn(81)Gly(83)) generated a receptor mutant that bound astressin with even higher affinity than the native hCRF(1) receptor. An amino acid doublet (Glu(70)Tyr(71)) that is conserved in the xCRF(1) and hCRF(2(a)) receptor after incorporation into the hCRF(1) receptor sequence was found to facilitate antagonist binding up to 15-fold higher. In agreement with the binding data, astressin was a more potent functional antagonist at receptors expressing the Glu(70)Tyr(71) motif. These data show that the agonist- and antagonist-binding sites of the hCRF(1) receptor partially overlap and that two amino acids within the N terminus of the hCRF(1) receptor negatively influence binding and functional antagonism of astressin.

Topics: Amino Acid Sequence; Amino Acid Substitution; Amphibian Proteins; Animals; Binding Sites; Binding, Competitive; Cell Line; Cell Membrane; Corticotropin-Releasing Hormone; Cyclic AMP; Epithelial Cells; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptide Fragments; Peptide Hormones; Peptides; Protein Binding; Radioligand Assay; Receptors, Corticotropin-Releasing Hormone; Recombinant Proteins; Sequence Homology, Amino Acid; Sheep; Transfection; Urocortins; Xenopus

Peptide ligands bind the CRF(1) receptor by a two-domain mechanism: the ligand's carboxyl-terminal portion binds the receptor's extracellular N-terminal domain (N-domain) and the ligand's amino-terminal portion binds the receptor's juxtamembrane domain (J-domain). Little quantitative information is available regarding this mechanism. Specifically, the microaffinity of the two interactions and their contribution to overall ligand affinity are largely undetermined. Here we measured ligand interaction with N- and J-domains expressed independently, the former (residues 1-118) fused to the activin IIB receptor's membrane-spanning alpha-helix (CRF(1)-N) and the latter comprising residues 110-415 (CRF(1)-J). We also investigated the effect of nonpeptide antagonist and G-protein on ligand affinity for N- and J-domains. Peptide agonist affinity for CRF(1)-N was only 1.1-3.5-fold lower than affinity for the whole receptor (CRF(1)-R), suggesting the N-domain predominantly contributes to peptide agonist affinity. Agonist interaction with CRF(1)-J (potency for stimulating cAMP accumulation) was 12000-1500000-fold weaker than with CRF(1)-R, indicating very weak direct agonist interaction with the J-domain. Nonpeptide antagonist affinity for CRF(1)-J and CRF(1)-R was indistinguishable, indicating the compounds bind predominantly the J-domain. Agonist activation of CRF(1)-J was fully blocked by nonpeptide antagonist, suggesting antagonism results from inhibition of agonist-J-domain interaction. G-protein coupling with CRF(1)-R (forming RG) increased peptide agonist affinity 92-1300-fold, likely resulting from enhanced agonist interaction with the J-domain rather than the N-domain. Nonpeptide antagonists, which bind the J-domain, blocked peptide agonist binding to RG, and binding of peptide antagonists, predominantly to the N-domain, was unaffected by R-G coupling. These findings extend the two-domain model quantitatively and are consistent with a simple equilibrium model of the two-domain mechanism: (1) The N-domain binds peptide agonist with moderate-to-high microaffinity, substantially increasing the local concentration of agonist and so allowing weak agonist-J-domain interaction. (2) Agonist-J-domain interaction is allosterically enhanced by receptor-G-protein interaction and inhibited by nonpeptide antagonist.

Topics: Amphibian Proteins; Aniline Compounds; Animals; Binding, Competitive; Cell Line; Corticotropin-Releasing Hormone; Extracellular Space; GTP-Binding Proteins; Humans; Ligands; Models, Chemical; Peptide Fragments; Peptide Hormones; Peptides; Protein Binding; Protein Structure, Tertiary; Pyrazoles; Pyrimidines; Pyrroles; Rats; Receptors, Corticotropin-Releasing Hormone; Triazines; Urocortins

The mouse corticotropin-releasing factor (CRF) type 2a receptor (CRF2(a)) splice variant was cloned by a PCR-based approach. The corresponding cDNA was found to encode a 411-amino acid polypeptide with highest sequence homology to the rat CRF2(a) receptor. By semiquantitative reverse transcriptase PCR (RT-PCR) analysis, the CRF2(b) mRNA was mainly found in the heart and skeletal muscle with only low level expression in the brain. In contrast, CRF2(a) mRNA was restricted to the brain with major expression sites in the cortex, hippocampus, hypothalamus and telencephalon. Binding and cyclic AMP stimulation studies showed a similar ligand selective profile for both mCRF2 receptor splice variants. A notable exception however, was urotensin I which displayed a approximately 3-fold higher affinity for the CRF2(a) receptor and also stimulated cyclic AMP production in mCRF2(a)-transfected cells with a approximately 3-fold higher potency than in mCRF2(b)-transfected cells. These data show that the mouse like other mammalian species expresses two ligand-selective CRF2 receptor splice variants and that the mCRF2(a) receptor is the predominant central CRF2 receptor in the mouse.

Topics: Alternative Splicing; Amino Acid Sequence; Amphibian Proteins; Animals; Brain; Cell Line; Cloning, Molecular; Corticotropin-Releasing Hormone; Cyclic AMP; Gene Expression Profiling; Humans; Mice; Molecular Sequence Data; Peptide Fragments; Peptide Hormones; Peptides; Protein Binding; Receptors, Corticotropin-Releasing Hormone; Sequence Alignment; Transfection; Urocortins; Urotensins

Mechanisms of nonpeptide ligand action at family B G protein-coupled receptors are largely unexplored. Here, we evaluated corticotropin-releasing factor 1 (CRF(1)) receptor regulation by nonpeptide antagonists. The antagonist mechanism was investigated at the G protein-coupled (RG) and uncoupled (R) states of the receptor in membranes from Ltk(-) cells expressing the cloned human CRF(1) receptor. R was detected with the antagonist (125)I-astressin with 30 microM guanosine 5'-O-(3-thiotriphosphate present, and RG detected using (125)I-sauvagine. At the R state, nonpeptide antagonists antalarmin, NBI 27914, NBI 35965, and DMP-696 only partially inhibited (125)I-astressin binding (22-32% maximal inhibition). NBI 35965 accelerated (125)I-astressin dissociation and only partially increased the IC(50) value of unlabeled sauvagine, CRF, and urocortin for displacing (125)I-astressin binding (by 4.0-7.1-fold). Reciprocal effects at the R state were demonstrated using [(3)H]NBI 35965: agonist peptides only partially inhibited binding (by 13-40%) and accelerated [(3)H]NBI 35965 dissociation. These data are quantitatively consistent with nonpeptide antagonist and peptide ligand binding spatially distinct sites, with mutual, weak negative cooperativity (allosteric inhibition) between their binding. At the RG state the compounds near fully inhibited (125)I-sauvagine binding at low radioligand concentrations (79-94 pM). NBI 35965 did not completely inhibit (125)I-sauvagine binding at high radioligand concentrations (82 +/- 1%, 1.3-2.1 nM) and slowed dissociation of (125)I-sauvagine and (125)I-CRF. The antagonist effect at RG is consistent with either strong allosteric inhibition or competitive inhibition at one of the peptide agonist binding sites. These findings demonstrate a novel effect of R-G interaction on the inhibitory activity of nonpeptide antagonists: Although the compounds are weak inhibitors of peptide binding to the R state, they strongly inhibit peptide agonist binding to RG. Strong inhibition at RG explains the antagonist properties of the compounds.

Topics: Amphibian Proteins; Animals; Binding Sites; Corticotropin-Releasing Hormone; Humans; Peptide Fragments; Peptide Hormones; Peptides; Pyrazoles; Rats; Receptors, Corticotropin-Releasing Hormone; Triazines; Tritium

The heptahelical receptors for corticotropin-releasing factor (CRF), CRFR1 and CRFR2, display different specificities for CRF family ligands: CRF and urocortin I bind to CRFR1 with high affinity, whereas urocortin II and III bind to this receptor with very low affinities. In contrast, all the urocortins bind with high affinities, and CRF binds with lower affinity to CRFR2. The first extracellular domain (ECD1) of CRFR1 is important for ligand recognition. Here, we characterize a bacterially expressed soluble protein, ECD1-CRFR2beta, corresponding to the ECD1 of mouse CRFR2beta. The K(i) values for binding to ECD1-CRFR2beta are: astressin = 10.7 (5.4-21.1) nm, urocortin I = 6.4 (4.7-8.7) nm, urocortin II = 6.9 (5.8-8.3) nm, CRF = 97 (22-430) nm, urocortin III = sauvagine >200 nm. These affinities are similar to those for binding to a chimeric receptor in which the ECD1 of CRFR2beta replaces the ECD of the type 1B activin receptor (ALK4). The ECD1-CRFR2beta possesses a disulfide arrangement identical to that of the ECD1 of CRFR1, namely Cys(45)-Cys(70), Cys(60)-Cys(103), and Cys(84)-Cys(118). As determined by circular dichroism, ECD1-CRFR2beta undergoes conformational changes upon binding astressin. These data reinforce the importance of the ECD1 of CRF receptors for ligand recognition and raise the interesting possibility that different ligands having similar affinity for the full-length receptor may, nevertheless, have different affinities for microdomains of the receptor.

Topics: Amino Acid Sequence; Amphibian Proteins; Animals; Circular Dichroism; Corticotropin-Releasing Hormone; Ligands; Mice; Molecular Sequence Data; Peptide Fragments; Peptide Hormones; Peptides; Protein Conformation; Rats; Receptors, Corticotropin-Releasing Hormone; Recombinant Fusion Proteins; Urocortins

Previous corticotropin releasing factor 1 (CRF1) receptor characterization has been performed using radiolabeled agonists, which bind predominantly the receptor-G-protein complex. The pharmacological profile of other receptor states, and their abundance, remain poorly characterized. Here we investigated the affinity states of the CRF1 receptor heterologously expressed in Ltk- cells and endogenously expressed in rat cerebellum. In L-CRF1 cell membranes, three agonist affinity states were detected: a very-high affinity receptor-G-protein complex state (eliminated by GTPgammaS) bound by [125I]sauvagine (43 pM, RG); a high affinity state insensitive to GTPgammaS bound by [125I]sauvagine (1.4 nM, termed RO); and a low affinity G-protein-uncoupled state detected by sauvagine displacement of [125I]astressin, a labeled antagonist (120 nM, R). The relative abundance of RG:RO:R was 18%:16%:66%. All three states were demonstrated in rat cerebellum with similar relative abundance (15%:16%:69%). The R state bound CRF with low affinity (270-330 nM), displayed a novel rank order of ligand affinity, and represented the majority of the receptor population in both receptor preparations. This study provides a framework to identify CRF1 receptor conformational states in various receptor preparations.

Topics: Amino Acid Sequence; Amphibian Proteins; Animals; Binding, Competitive; Cell Line; Cell Membrane; Cerebellum; Corticotropin-Releasing Hormone; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Iodine Radioisotopes; Ligands; Molecular Sequence Data; Peptide Fragments; Peptide Hormones; Peptides; Protein Conformation; Rats; Receptors, Corticotropin-Releasing Hormone; Recombinant Proteins; Sequence Homology, Amino Acid

A novel photoactivatable analog of antisauvagine-30 (aSvg-30), a specific antagonist for corticotropin-releasing factor (CRF) receptor, type 2 (CRF2), has been synthesized and characterized. The N-terminal amino-acid d-Phe in aSvg-30 [d-Phe11,His12]Svg(11-40) was replaced by a phenyldiazirine, the 4-(1-azi-2,2,2-trifluoroethyl)benzoyl (ATB) residue. The photoactivatable aSvg-30 analog ATB-[His12]Svg was tested for its ability to displace [125I-Tyr0]oCRF or [125I-Tyr0]Svg from membrane homogenates of human embryonic kidney (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 (rCRF1) or mouse CRF receptor, type 2beta (mCRF2beta). Furthermore, the ability of ATB-[His12]Svg(12-40) to inhibit oCRF- or Svg-stimulated cAMP production of transfected HEK 293 cells expressing either rCRF1 (HEK-rCRF1 cells) or mCRF2beta (HEK-mCRF2beta cells) was determined. Unlike astressin and photo astressin, ATB-[His12]Svg(12-40) showed high selective binding to mCRF2beta (Ki = 3.1 +/- 0.2 nm) but not the rCRF1 receptor (Ki = 142.5 +/- 22.3 nm) and decreased Svg-stimulated cAMP activity in mCRF2beta-expressing cells in a similar fashion as aSvg-30. A 66-kDa protein was identified by SDS/PAGE, when the radioactively iodinated analog of ATB-[His12]Svg(12-40) was covalently linked to mCRF2beta receptor. The specificity of the photoactivatable 125I-labeled CRF2beta antagonist was demonstrated with SDS/PAGE by the finding that this analog could be displaced from the receptor by antisauvagine-30, but not other unrelated peptides such as vasoactive intestinal peptide (VIP).

Topics: Amino Acid Sequence; Amphibian Proteins; Animals; Aziridines; Azirines; Benzoates; Binding, Competitive; Cell Line; Cell Membrane; Corticotropin-Releasing Hormone; Cyclic AMP; Humans; Iodine Radioisotopes; Kidney; Ligands; Mice; Molecular Sequence Data; Peptide Fragments; Peptide Hormones; Peptides; Photochemistry; Rats; Receptors, Corticotropin-Releasing Hormone; Substrate Specificity; Transfection

The abundance of a histidine residue at position 185 (His(185)) of the human corticotropin-releasing factor (CRF) type 2 alpha receptor (hCRF(2alpha)) was investigated. His(185) has only been reported in hCRF(2); CRF(2) proteins from other species and all CRF(1) receptors encode an arginine (Arg(185)) at the corresponding position. Cloning of partial and full-length hCRF(2) cDNAs from a variety of neuronal and peripheral tissues revealed the existence of receptor molecules encoding Arg(185) only. Sequence analysis of the hCRF(2) gene verified the existence of Arg(185) also on genomic level. Full-length cDNAs encoding either the His(185) (R2H(185)) or the Arg(185) (R2R(185)) variants of hCRF(2alpha) were stably expressed in HEK293 cells and tested for ligand binding properties. In displacement studies R2H(185) and R2R(185) displayed a similar substrate specificity, human and rat urocortin, and the peptide antagonists astressin and alpha-helical CRF((9-41)) were bound with high affinity whereas human and ovine CRF were low-affinity ligands. Significant differences were observed for sauvagine and urotensin I, which bound with 3-fold (sauvagine) and 9-fold (urotensin I) higher affinity to R2R(185). These data indicate that hCRF(2), like all vertebrate CRF(1) and CRF(2) proteins encodes an arginine residue at the junction between extracellular domain 2 and transmembrane domain 3 and that this amino acid plays a role for the discrimination of some CRF peptide ligands.

Topics: Amino Acid Sequence; Amphibian Proteins; Animals; Arginine; Base Sequence; Binding, Competitive; Brain; Cell Line; Corticotropin-Releasing Hormone; DNA; DNA, Complementary; Gene Expression Regulation; Histidine; Humans; Ligands; Membranes; Molecular Sequence Data; Myocardium; Peptide Fragments; Peptide Hormones; Peptides; Radioligand Assay; Rats; Receptors, Corticotropin-Releasing Hormone; Retina; RNA, Messenger; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Urotensins

CRF exerts a key neuroregulatory control on the function of the hypothalamic-pituitary-adrenal axis. These effects are thought to be mediated primarily through activation of Gs-coupled plasma membrane receptors. In the present study, we investigated the effects of activation of CRF receptors by sauvagine on signaling pathways that converge on phosphorylation of the transcription factor calcium/cAMP response element-binding protein (CREB). Studies were undertaken using CHO cell lines transfected with either rat CRF-1 or CRF-2alpha receptors. Signaling pathways were investigated using immunocytochemical, Western blot, and imaging techniques. Treatment with sauvagine increased phosphorylation of p42/p44, but not of p38 or stress-activated protein kinase (SAPK)/JUN N-terminal kinase (JNK) mitogen-activated protein (MAP) kinases correlating with increased p42/p44 MAP kinase activity. Mobilization of intracellular Ca2+ stores was observed in cells treated with high concentrations (100 nM, 1 microM) of sauvagine. A time- and dose-dependent increase in phosphorylation of the transcription factor CREB was observed in cultures treated with sauvagine. Phosphorylation of CREB occurred at lower concentrations of sauvagine than those required to mobilize intracellular calcium stores, and phosphorylation was not blocked by the mitogen-activated protein kinase kinase inhibitor PD98059 at a concentration (1 microM) that fully inhibited phosphorylation of MAP kinase. Cotreatment of cultures with the protein kinase A inhibitor H89 (10 microM) blocked fully the stimulatory actions of sauvagine (0.1 nM, 1 nM) on phosphorylation of CREB, but not those on phosphorylation of MAP kinase. Phosphorylation of MAP kinase was partially blocked by the phosphoinositide 3-kinase inhibitor LY294002 (5 microM) and by the phosphoinositide-phospholipase C inhibitor U73122 (10 microM). These data demonstrate that cAMP-, Ca2+-, and MAP kinase-dependent signaling pathways are activated by stimulation of CRF-1 and CRF-2alpha receptors. However, in these cells, only protein kinase A-dependent pathways contribute significantly to enhanced phosphorylation of CREB. These represent the first reported observations of CRF receptor-mediated phosphorylation of the transcription factor CREB and activation of MAP kinase signal transduction pathways.

Topics: Amphibian Proteins; Animals; Blotting, Western; Calcium; CHO Cells; Corticotropin-Releasing Hormone; Cricetinae; Cyclic AMP Response Element-Binding Protein; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Immunohistochemistry; Mitogen-Activated Protein Kinase 1; Peptide Fragments; Peptide Hormones; Peptides; Phosphorylation; Rats; Receptors, Corticotropin-Releasing Hormone; Signal Transduction

Corticotropin-releasing factor (CRF)-related peptides exhibit different affinity for the receptor subtypes 1 and 2 cloned in the rat brain. We investigated, in conscious rats, the effects of intracisternal (i.c.) injection of CRF (rat/human) on the 5-h rate of gastric emptying of a solid nutrient meal (Purina chow and water ad libitum for 3 h) and the CRF receptor subtype involved. CRF, urotensin I (suckerfish), and sauvagine (frog) injected i.c. inhibited gastric emptying in a dose-dependent manner, with ED50 values of 0.31, 0.13, and 0.08 microgram/rat, respectively. Rat CRF-(6-33) (0.1-10 micrograms i.c.) had no effect. The nonselective CRF1 and CRF2 receptor antagonist, astressin, injected i.c. completely blocked the inhibitory effect of i.c. CRF, urotensin I, and sauvagine with antagonist-to-agonist ratios of 3:1, 10:1, and 16:1, respectively. The CRF1-selective receptor antagonist NBI-27914 injected i.c. at a ratio of 170:1 had no effect. These data show that central CRF and CRF-related peptides are potent inhibitors of gastric emptying of a solid meal with a rank order of potency characteristic of the CRF2 receptor subtype affinity (sauvagine > urotensin I > CRF). In addition, the reversal by astressin but not by the CRF1-selective receptor antagonist further supports the view that the CRF2 receptor subtype is primarily involved in central CRF-induced delayed gastric emptying.

Topics: Amphibian Proteins; Animal Feed; Animals; Brain; Corticotropin-Releasing Hormone; Gastric Emptying; Injections, Intraventricular; Male; Peptide Fragments; Peptide Hormones; Peptides; Rats; Rats, Sprague-Dawley; Receptors, Corticotropin-Releasing Hormone; Urotensins