urotensin-i and sauvagine

CRF system is comprised of 4 homologous lineages, 2 main receptors (CRF-R1 and CRF-R2), and a binding protein CRF-BP. The homologous lineages are corticotropin-releasing factor (CRF), urotensin I (UI)/sauvagine (SVG)/urocortin 1 (UCN1), urocortin 2 (UCN2), and urocortin 3 (UCN3), and UI, SVG, UCN1 are orthologous genes. CRF system genes are widely distributed in the brain and gastrointestinal tract, which may relate to feeding regulation. According the research progress about CRF system on mammals and non-mammals, this paper summarized the discovery, structure, tissue distribution, appetite regulation and mechanism of CRF system in animals, which can provide the reference for further research and production of feeding regulation and growth in mammals and fish species.

Topics: Amphibian Proteins; Animals; Appetite; Brain; Carrier Proteins; Corticotropin-Releasing Hormone; Feeding Behavior; Peptide Hormones; Urocortins; Urotensins

Peptides of the corticotropin-releasing factor (CRF) family are expressed throughout the central nervous system (CNS) and in peripheral tissues where they play diverse roles in physiology, behavior, and development. Current data supports the existence of four paralogous genes in vertebrates that encode CRF, urocortin/urotensin 1, urocortin 2 or urocortin 3. Corticotropin-releasing factor is the major hypophysiotropin for adrenocorticotropin, and also functions as a thyrotropin-releasing factor in non-mammalian species. In the CNS, CRF peptides function as neurotransmitters/neuromodulators. Recent work shows that CRF peptides are also expressed at diverse sites outside of the CNS in mammals, and we found widespread expression of CRF and urocortins, CRF receptors and CRF binding protein (CRF-BP) genes in the frog Xenopus laevis. The functions of CRF peptides expressed in the periphery in non-mammalian species are largely unexplored. We recently found that CRF acts as a cytoprotective agent in the X. laevis tadpole tail, and that the CRF-BP can block CRF action and hasten tail muscle cell death. The expression of the CRF-BP is strongly upregulated in the tadpole tail at metamorphic climax where it may neutralize CRF bioactivity, thus promoting tail resorption. Corticotropin-releasing factor and urocortins are also known to be cytoprotective in mammalian cells. Thus, CRF peptides may play diverse roles in physiology and development, and these functions likely arose early in vertebrate evolution.

Topics: Amphibian Proteins; Animals; Brain Chemistry; Carrier Proteins; Corticotropin-Releasing Hormone; Female; Humans; Male; Mammals; Peptide Hormones; Peptides; Pituitary Gland; Receptors, Corticotropin-Releasing Hormone; Tissue Distribution; Urocortins; Urotensins; Xenopus laevis

Topics: Adrenocorticotropic Hormone; Amphibian Proteins; Animals; Brain Chemistry; Catecholamines; Chemical Phenomena; Chemistry; Corticotropin-Releasing Hormone; Dogs; Drug Interactions; Haplorhini; Humans; Hydroxycorticosteroids; Hypothalamus; Immune Sera; Immunization, Passive; Nerve Tissue Proteins; Peptide Hormones; Peptides; Rabbits; Radioimmunoassay; Rats; Sheep; Urotensins

Corticotropin-releasing factor-binding protein (CRF-BP) is considered a key determinant for CRF receptor (CRF-R) activation by CRF and several related peptides. Earlier studies have shown that the CRF system is highly conserved in gene structures throughout evolution, yet little is known about the evolutionary conservation of its biological functions. Therefore, we address the functional properties of CRF-BP and CRF-Rs in a teleost fish (common carp; Cyprinus carpio L.). We report the finding of two similar, yet distinct, genes for both CRF-R1 and CRF-R2 in this species. The four receptors are differentially responsive to CRF, urotensin-I (UI), sauvagine, and urocortin-2 (Ucn-2) and -3 (Ucn-3) as shown by luciferase assays. In vitro, carp CRF-BP inhibits CRF- and UI-mediated activation of the newfound CRF-Rs, but its potency to do so varies between receptor and peptide ligand. This is the first paper to establish the functionality and physiological interplay between CRF-BP, CRF-Rs and CRF-family peptides in a teleostean species.

Topics: Amphibian Proteins; Animals; Carps; Carrier Proteins; Corticotropin-Releasing Hormone; Cyclic AMP; HEK293 Cells; Humans; Luciferases; Peptide Hormones; Protein Isoforms; Receptors, Corticotropin-Releasing Hormone; Recombinant Proteins; Urocortins; Urotensins

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

Corticotropin-releasing factor (CRF) receptors are members of the superfamily of G-protein coupled receptors that utilise adenylate cyclase and subsequent production of cAMP for signal transduction in many tissues. Activation of cAMP-dependent pathways, through elevation of intracellular cAMP levels is known to promote survival of a large variety of central and peripheral neuronal populations. Utilising cultured primary rat central nervous system neurons, we show that stimulation of endogenous cAMP signalling pathways by forskolin confers neuroprotection, whilst inhibition of this pathway triggers neuronal death. CRF and the related CRF family peptides urotensin I, urocortin, and sauvagine, which also induced cAMP production, prevented the apoptotic death of cerebellar granule neurons triggered by inhibition of phosphatidylinositol kinase-3 pathway activity with LY294002. These effects were negated by the highly selective CRF-R1 antagonist CP154,526. CRF even conferred neuroprotection when its application was delayed by up to 8 h following LY294002 addition. The CRF peptides also protected cortical and hippocampal neurons against death induced by beta-amyloid peptide (1-42), in a CRF-R1 dependent manner. In separate experiments, LY294002 reduced neuronal protein kinase B activity while increasing glycogen synthase kinase-3, whilst CRF (and related peptides) promoted phosphorylation of glycogen synthase kinase-3 without protein kinase B activation. Taken together, these results suggest that the neuroprotective activity of CRF may involve cAMP-dependent phosphorylation of glycogen synthase kinase-3.

Topics: 1-Methyl-3-isobutylxanthine; Amphibian Proteins; Amyloid beta-Peptides; Animals; Animals, Newborn; Apoptosis; Blotting, Western; Cell Survival; Cells, Cultured; Cerebellum; Cerebral Cortex; Chromatin; Chromones; Colforsin; Corticotropin-Releasing Hormone; Cyclic AMP; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Glycogen Synthase Kinase 3; Hippocampus; In Situ Nick-End Labeling; Inhibitory Concentration 50; L-Lactate Dehydrogenase; Mitogen-Activated Protein Kinase Kinases; Morpholines; Neurons; Neuroprotective Agents; Peptide Fragments; Peptide Hormones; Peptides; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Pyrimidines; Pyrroles; Rats; Rats, Sprague-Dawley; Receptors, Corticotropin-Releasing Hormone; Thionucleotides; Time Factors; Urocortins; Urotensins; Vasodilator Agents

Corticotropin-releasing hormone (CRH) plays a key role in the regulation of responses to stress. The presence of a high affinity binding protein for CRH (CRH-BP) has been reported in mammals. We have characterized the biochemical properties and expression of CRH-BP in the South African clawed frog, Xenopus laevis. Apparent inhibition constants (K(i[app])) for different ligands were determined by competitive binding assay. Xenopus CRH-BP (xCRH-BP) exhibited a high affinity for xCRH (K(i[app])=1.08 nM) and sauvagine (1.36 nM). Similar to rodent and human CRH-BPs, the frog protein binds urotensin I and urocortin with high affinity, and ovine CRH with low affinity. RT-PCR analysis showed that xCRH-BP is expressed in brain, pituitary, liver, tail, and intestine. Brain xCRH-BP mRNA is expressed at a relatively constant level throughout metamorphosis and increases slightly in the metamorphic frog. By contrast, the gene is strongly upregulated in the tail at metamorphic climax. Thus, regulation of xCRH-BP gene expression is tissue specific. Because xCRH-BP binds CRH-like peptides with high affinity the protein may regulated, the bioavailability of CRH in amphibia as it does in mammals.

Topics: Amino Acid Sequence; Amphibian Proteins; Animals; Base Sequence; Binding, Competitive; Blotting, Western; Brain; Carrier Proteins; Corticotropin-Releasing Hormone; Gene Expression Regulation, Developmental; Humans; Hydrogen-Ion Concentration; Larva; Mice; Molecular Sequence Data; Organ Specificity; Peptide Hormones; Peptides; RNA, Messenger; Tail; Urocortins; Urotensins; Xenopus laevis

The methionine residues in Tyr-corticotropin-releasing factor (CRF) and Tyr-sauvagine radioligands are subject to oxidation, which renders them biologically inactive. Therefore [Tyr(0,) Gln(1,) Leu(17)]sauvagine (YQLS), in which the methionine was replaced with leucine was synthesized and labeled with (125)Iodine using chloramine-T. Mass spectroscopy revealed that chloramine-T-treatment did not oxidize YQLS. (125)I-YQLS bound with high affinity to cells expressing the murine CRF receptor 1 (CRFR1), CRF receptor 2 (CRFR2), and the mouse brain regions known to express both CRF receptors. (125)I-YQLS chemically cross-linked to CRFR1. In conclusion, (125)I-YQLS is oxidation-resistant, high affinity radioligand that can be chemically cross-linked to the CRF receptors.

Topics: Amino Acid Sequence; Amphibian Proteins; Animals; Brain; Cells, Cultured; Chloramines; COS Cells; Cross-Linking Reagents; Cyclic AMP; DNA, Complementary; Dose-Response Relationship, Drug; Iodine; Leucine; Ligands; Mass Spectrometry; Methionine; Mice; Molecular Sequence Data; Oxygen; Peptide Hormones; Peptides; Protein Binding; Receptors, Corticotropin-Releasing Hormone; Sequence Homology, Amino Acid; Succinimides; Tyrosine; Urotensins; Vasodilator Agents

The corticotropin-releasing factor (CRF) receptor type 1 (CRFR1) contains five potential N-glycosylation sites: N38, N45, N78, N90, and N98. Cells expressing CRFR1 were treated with tunicamycin to block receptor glycosylation. The nonglycosylated receptor did not bind the radioligand and had a decreased cAMP stimulation potency in response to CRF. To determine which of the polysaccharide chain(s) is/are involved in ligand interaction, the polysaccharide chains were deleted using site-directed mutagenesis of the glycosylation consensus, N-X-S/T. Two sets of mutations were performed for each glycosylation site: N to Q and S/T to A, respectively. The single mutants Q38, Q45, Q78, Q90, Q98, A40, A47, A80, A92, and A100 and the double mutants A40/A47 and A80/A100 were well expressed, bound CRF, sauvagine (SVG), and urotensin-I (UTS-I) with a normal affinity, and increased cAMP accumulation with a high efficiency. In contrast, the combined mutations A80/A92/A100, A40/A80/A92/A100, and A40/A47/A80/A92/A100 had low levels of expression, did not bind the radioligand, and had a decreased cAMP stimulation. These data indicate the requirement for three or more polysaccharide chains for normal CRFR1 function.

Topics: 1-Methyl-3-isobutylxanthine; Amphibian Proteins; Animals; Binding Sites; Binding, Competitive; Cell Line; Consensus Sequence; Corticotropin-Releasing Hormone; Cyclic AMP; Electrophoresis, Polyacrylamide Gel; Glycosylation; Ligands; Mice; Molecular Weight; Mutagenesis, Site-Directed; Peptide Hormones; Peptides; Radioligand Assay; Receptors, Corticotropin-Releasing Hormone; Structure-Activity Relationship; Tunicamycin; Urotensins

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

We have assessed the utility of the Cytosensor microphysiometer for studying the pharmacology of recombinant CRF receptors. Chinese hamster ovary cells stably expressing the human CRF1 or CRF2 receptor were perfused in the Cytosensor with bicarbonate-free Hams F12 (pH 7.4) containing 0.2% bovine serum albumin. The rank order of potencies of agonist peptides were CRF = sauvagine = urocortin = urotensin at CRF1 (pEC50 values 11.16 +/- 0.17, 11.37 +/- 0.14, 11.43 +/- 0.09 and 11.46 +/- 0.13; n = 4), and urocortin = sauvagine > urotensin > CRF at CRF2 (pEC50 values 10.88 +/- 0.12, 10.44 +/- 0.05, 9.36 +/- 0.12 and 8.53 +/- 0.07; n = 7-9). alpha-Helical CRF (9-41) was a competitive antagonist at the CRF2 receptor (pK(B) = 6.99 +/- 0.08, n = 4), but was a partial agonist at the CRF1 receptor (pEC50 = 6.85 +/- 0.08, Emax = 33%, n = 3). CP 154,526 was a competitive antagonist at the CRF1 receptor (pK(B) = 8.17 +/- 0.05, n = 6), but was inactive at the CRF2 receptor. These data are consistent with established CRF receptor pharmacology and show that the Cytosensor is a viable method for assessing the functional activity of CRF-receptor agonists and antagonists.

Topics: Acid-Base Equilibrium; Amphibian Proteins; Animals; Cattle; CHO Cells; Corticotropin-Releasing Hormone; Cricetinae; Humans; Membrane Potentials; Peptide Fragments; Peptide Hormones; Peptides; Rats; Receptors, Corticotropin-Releasing Hormone; Urocortins; Urotensins; Vasodilator Agents

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

This study investigated whether sauvagine and urotensin I change [Ca2+]i in human epidermoid A-431 cells and whether these changes are correlated with their anti-edema properties in vivo. A-431 cells were used because they possess the corticotropin-releasing factor (CRF) receptor 2. Treatment with either sauvagine or urotensin I led to an immediate increase in [Ca2+]i, the magnitude of which depended on the concentration of the drug. Sauvagine was more effective than urotensin I, with a median effective concentration (EC50) of 1.4 +/- 0.2 fM, compared to an EC50 of 66 +/- 7 fM for urotensin I. Both were more effective at stimulating increases in [Ca2+]i than CRF (EC50 of 6.8 +/- 0.1 pM). There was a correlation between the EC50 for increasing [Ca2+]i and the median effective dose (ED50) for inhibiting edema induced by heating rat paw (r = 0.99). Removal of extracellular Ca2+ or incubation with La3+ eliminated the increase in [Ca2+]i stimulated by either peptide. Pretreatment with a CRF receptor antagonist reduced the increase in [Ca2+]i by these peptides. This occurred in an antagonist concentration-dependent manner, with median inhibitory concentrations (IC50) of 1.99 +/- 0.04 nM and 0.85 +/- 0.04 nM, respectively. Both pertussis toxin (an inhibitor of G proteins) and U-73122 (an inhibitor for inositol trisphosphate (InsP3) production) partially inhibited the increases. InsP3 was measured to determine whether these peptides mobilized Ca2+ from an InsP3-sensitive store. Both sauvagine and urotensin I increased InsP3. The InsP3 increases were inhibited by U-73 122 and CRF antagonist, but not by removal of external Ca2+. Both peptides elevated protein tyrosine phosphorylation. In summary, these peptides increase [Ca2+]i as a result of Ca2+ influx via CRF receptor-operated Ca2+ channels coupled to pertussis toxin-sensitive G proteins and a Ca2+ mobilization from InsP3-sensitive Ca2+ pools. Their in vivo effectiveness at inhibiting edema is related to their respective capacities to stimulate elevations of [Ca2+]i, supporting a role for intracellular Ca2+ in this process.

Topics: Amphibian Proteins; Calcium; Calcium Channel Blockers; Corticotropin-Releasing Hormone; Humans; Inosine Triphosphate; Peptide Fragments; Peptide Hormones; Peptides; Phosphorylation; Receptors, Corticotropin-Releasing Hormone; Tumor Cells, Cultured; Tyrosine; Urotensins

Immunocytochemistry was used to investigate the presence of corticotropin-releasing factor-like peptides in the interrenal (adrenal) glands of the bullfrog Rana catesbeiana by using specific antisera raised against synthetic nonconjugated rat/human corticotropin-releasing factor, urotensin I, and sauvagine. From these three antisera, covering a broad range of corticotropin-releasing factor-like immunoreactivities, only the sauvagine antiserum gave positive immunoreactivity. Sauvagine immunoreactivity was found in cortical cells grouped into cords in the renal zone of the interrenal gland. The central and subcapsular cords were less stained. Tyrosine hydroxylase-positive chromaffin cells were not sauvagine-immunoreactive. The immunoreactivity was abolished, in all cases, by previous immunoabsorption of the sauvagine antiserum with synthetic sauvagine (0.1 microM), but it was not eliminated by sucker (Catostomus commersoni) urotensin I, sole (Hippoglossoides elassodon) urotensin I, sucker corticotropin-releasing factor, rat/human corticotropin-releasing factor, or ovine corticotropin-releasing factor (0.1-10 microM). In a sauvagine radioimmunoassay, interrenal extracts displaced 125I-sauvagine from antiserum only partially, and not in parallel with the sauvagine standard curve. The results suggest that the sauvagine immunoreactivity in the R. catesbeiana interrenal gland may represent a novel sauvagine-like peptide.

Topics: Amphibian Proteins; Animals; Antibody Specificity; Corticotropin-Releasing Hormone; Epitopes; Female; Immunoenzyme Techniques; Interrenal Gland; Iodine Radioisotopes; Male; Peptide Hormones; Peptides; Radioimmunoassay; Rana catesbeiana; Urotensins; Vasodilator Agents

CRH is the principal mediator of the stress response in mammals. In addition to pituitary and central nervous system effects, peripheral effects of CRH have been observed involving the immune and cardiovascular systems. Two CRH receptor subtypes, CRH-R1 and CRH-R2, have been cloned and show significant amino acid homology (69%), but differ in their tissue distribution. CRH-R1 is expressed predominantly in the brain and pituitary, whereas the CRH-R2 subtype is highly expressed in heart and skeletal muscle. To investigate the role of CRH in cardiac signaling, we analyzed the effect of CRH on freshly isolated neonatal rat cardiomyocytes and murine atrial cardiomyocyte tumor cells, AT-1, which express CRH-R2 messenger RNA. We show that stimulation of these cells with CRH and the CRH-related peptides, sauvagine from frog and urotensin I from fish, elicits large increases in the intracellular level of cAMP. This stimulation is transient, reaching a maximum in 5-15 min in neonatal cardiomyocytes and in 2-4 min in AT-1 cells, followed by a rapid decline. We show that stimulation of AT-1 cells by these peptides is specific for CRH receptors, as the CRH antagonist, alpha-helical CRH-(9-41) inhibits cAMP increases. Furthermore, we show that CRH, sauvagine, and urotensin I stimulations are dose dependent in both neonatal cardiomyocytes and AT-1 cells. Sauvagine and urotensin I are more potent than CRH at stimulating an increase in intracellular cAMP in neonatal cardiomyocytes (EC50 = 1.74, 2.61, 6.42 nM, respectively) and AT-1 cells (EC50 = 16.2, 15.8, and 149 nM, respectively). This rank order is consistent with that previously demonstrated in CRH-R2-transfected HEK293 cells and parallels the in vivo vasodilatory activity of these peptides. In summary, this is the first evidence that CRH, sauvagine, and urotensin I act directly on cardiac myocytes to stimulate increases in intracellular cAMP, presumably through CRH-R2. In addition, these results indicate that cardiac myocytes may be an informative in vitro model to investigate the effects of CRH and its role in the cardiovascular response to stress.

Topics: Amphibian Proteins; Animals; Animals, Newborn; Corticotropin-Releasing Hormone; Cyclic AMP; Dose-Response Relationship, Drug; Kinetics; Mice; Mice, Transgenic; Myocardium; Peptide Fragments; Peptide Hormones; Peptides; Rats; Rats, Sprague-Dawley; Receptors, Corticotropin-Releasing Hormone; Tumor Cells, Cultured; Urotensins

The presence of corticotropin-releasing hormone (CRH) receptors in rat retinal membranes was investigated by using [125I-Tyro]-ovine CRH ([125I]oCRH) as radioligand. The receptor binding was rapid, reversible, saturable and specific. The [125I]oCRH binding was completely displaced by different CRH-related peptides with a rank order of potency similar to that displayed in stimulating rat retinal adenylyl cyclase activity. Two populations of binding sites were detected: one with high affinity (Kd = 1.7 nM) and the other with low-affinity (Kd = 130 nM). The GTP analogue guanosine 5'-O-(3'-thiotriphosphate) reduced the high-affinity binding and increased the relative proportion of sites with low-affinity. Incubation of rat retinal membranes with the RM/1 antibody, which recognizes the carboxyl-terminus of the alpha subunit of the G protein Gs, prevented the CRH stimulation of adenylyl cyclase. In immunoblots, the RM/1 antibody recognized an immunoreactive protein band of 45 kDa and a protein with a similar electrophoretic mobility was ADP-ribosylated by cholera toxin. These data provide evidence for the presence of specific CRH receptors in rat retina and contribute to define the CRH signalling system in this tissue.

Topics: Adenosine Diphosphate Ribose; Adenylyl Cyclases; Amphibian Proteins; Animals; Binding Sites; Binding, Competitive; Cholera Toxin; Corticotropin-Releasing Hormone; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Kinetics; Male; Peptide Hormones; Peptides; Rats; Rats, Sprague-Dawley; Receptors, Corticotropin-Releasing Hormone; Retina; Urotensins

In this study we have identified specific binding sites for corticotropin-releasing hormone (CRH) in human Y-79 retinoblastoma cell membranes by using 125I-Tyr-ovine CRH (125I-oCRH) as radioligand. Binding at 19 degrees C was rapid with steady state being reached within 20 min, reversible and linear with membrane protein concentration. The 125I-oCRH binding was enhanced by Mg2+ and inhibited by the GTP analogue guanosine 5'-O-(3'-thiotriphosphate). Y-79 cell membranes exhibited two populations of binding sites, a high-affinity site with an apparent dissociation constant (KD) of 1 nM and a low-affinity site with an apparent KD of 500 nM. 125I-oCRH binding was completely antagonized by human/rat CRH, [Met(O)21]oCRH, alpha-helical CRH9-41, urotensin I, and sauvagine with a rank order of potency similar to that displayed by CRH receptors of other tissues. These data describe for the first time the presence of specific CRH-binding sites in retinal cells. The Y-79 cell line may therefore constitute a valuable model in which to study CRH action on retinal cells.

Topics: Amphibian Proteins; Binding Sites; Corticotropin-Releasing Hormone; Eye Neoplasms; Humans; Peptide Hormones; Peptides; Retinoblastoma; Tumor Cells, Cultured; Urotensins; Vasodilator Agents

In the present study we have investigated whether sauvagine (SVG) and urotensin I (UT), two peptides displaying sequence homology with corticotropin-releasing factor (CRF), could affect synaptosomal tyrosine hydroxylase (TH) activity of mouse striatum in a manner similar to CRF. The enzyme activity was assayed in supernatants obtained following sonication and centrifugation of homogenates preincubated with the peptides. SVG and UT produced a concentration-dependent increase of TH activity with a half-maximal effect obtained at 5 and 10 nM, respectively. SVG and UT were as effective as CRF with maximal stimulations corresponding to 52-58% increase of basal enzyme activity, whereas the rank order of potency was SVG greater than UT = CRF. Kinetic analysis of TH activity versus low concentrations of the pterin co-factor (0.05-0.4 mM) indicated that the stimulations elicited by CRF, SVG and UT were associated with an increase in the Vmax of the enzyme form with high affinity for the co-factor. The CRF receptor antagonist alpha-helical CRF9-41 inhibited the effects of all 3 peptides. Moreover, the combined addition of CRF with either SVG or UT did not produce additive effects on TH activity. The stimulatory effects of CRF, SVG and UT were dependent on the concentration of extracellular free Ca2+, being minimal in a Ca2(+)-free medium and maximal at about 0.5 mM extracellular free Ca2+. These results indicate that SVG and UT can mimic the effect of CRF on synaptosomal TH by acting on a common receptor site associated with a Ca2(+)-dependent mechanism.

Topics: Adenylyl Cyclases; Amphibian Proteins; Animals; Corpus Striatum; Corticotropin-Releasing Hormone; In Vitro Techniques; Kinetics; Male; Mice; Peptide Hormones; Peptides; Sequence Homology, Nucleic Acid; Synaptosomes; Tyrosine 3-Monooxygenase; Urotensins

Corticotropin-releasing factor (CRF) stimulates rat retinal adenylate cyclase activity in a concentration-dependent manner. The half-maximal effect is obtained at 50 nM CRF and the maximal stimulation corresponds to approximately 90% increase of basal enzyme activity. The CRF effect is counteracted by the CRF antagonist alpha-helical CRF 9-41 with a Ki value of 40 nM. Other CRF-like peptides such as sauvagine and urotensin I are as effective as CRF with a rank order of potency of urotensin I greater than or equal to sauvagine greater than CRF. The sauvagine and urotensin I effects are not additive with that elicited by CRF. Moreover, the CRF stimulation is not additive with the increase of enzyme activity produced by vasoactive intestinal peptide or dopamine. The CRF effect is independent of the concentration of free Ca2+, is optimal at 5-10 mM MgCl2, and requires GTP. The results indicate that rat retinal adenylate cyclase is modulated by CRF via a receptor-mediated mechanism.

Topics: Adenylyl Cyclases; Amphibian Proteins; Animals; Corticotropin-Releasing Hormone; Cyclic AMP; Dopamine; Dose-Response Relationship, Drug; Magnesium; Male; Osmolar Concentration; Peptide Hormones; Peptides; Rats; Rats, Inbred Strains; Retina; Urotensins; Vasoactive Intestinal Peptide

The paws of pentobarbital-anesthetized rats were immersed in 48 or 58 degree C water for 5 min and the thermal inflammatory response to heat was measured by Evans blue dye leakage into the pawskin. Sauvagine, sucker fish urotensin I and human/rat corticotropin-releasing factor (CRF), homologous peptides belonging to the corticoliberin superfamily, injected i.v. 10 min before heat exposure, inhibited dye leakage with ED50 (nmol/kg) of 0.44, 1.5 and 5.9, respectively. The same rank order of potency was observed when the peptides were tested against the protein extravasation evoked by antidromic stimulation of the rat saphenous nerve. The alpha-helical CRF-(-9-41) antagonist, administered alone at 24 nmol/kg i.v., did not affect the heat-induced dye leakage into skin; but it attenuated the effects of h/rCRF, urotensin I and sauvagine. h/r[Tyr0]CRF, ovine[Nle21,Tyr32]CRF and h/r[Met(O)21,38]CRF, administered 24 nmol/kg i.v., did not affect the dye leakage response to heat. Peptides of the corticoliberin superfamily may have the unusual property of making small blood vessels less permeable when responding to injurious stimuli.

Topics: Amphibian Proteins; Animals; Anti-Inflammatory Agents; Capillary Permeability; Corticotropin-Releasing Hormone; Foot; Hot Temperature; Inflammation; Injections, Subcutaneous; Male; Peptide Hormones; Peptides; Rats; Rats, Inbred Strains; Skin; Steroids; Urotensins

Topics: Adipose Tissue; Amphibian Proteins; Animals; Body Temperature Regulation; Corticotropin-Releasing Hormone; Male; Peptide Hormones; Peptides; Rats; Rats, Inbred Strains; Urotensins; Vasodilator Agents

The localization of a sauvagine (SV)/urotensin I (UI)-like material in the brain of the dogfish, Scyliorhinus canicula, was studied by immunohistochemical techniques, employing an antiserum raised in rabbit against synthetic SV which widely cross-reacts with UI. Positive cell bodies and nerve fibers were identified in the dorsocaudal hypothalamic region of the tuberculum posterius, in the nucleus sacci vasculosi and nucleus tuberculi posterioris. A dense network of immunoreactive axons was shown in the whole tuberculum posterius. These findings support the view that SV/UI-like peptides may be involved in neuromodulatory functions throughout the brain of cartilaginous fish.

Topics: Amphibian Proteins; Animals; Brain; Dogfish; Female; Immunohistochemistry; Male; Peptide Hormones; Peptides; Sharks; Urotensins

The ability of corticoliberin (CRF), urotensin I, sauvagine, arginine-vasopressin (AVP), and mesotocin to stimulate ACTH release by frog anterior pituitary cells and alpha-melanotropin (MSH) by frog neurointermediate lobe was studied in vitro using a perifusion technique. CRF and AVP were found to be potent stimulators of ACTH secretion, whereas urotensin I and sauvagine were totally inactive. In opposition to recent findings in the rat. CRF did not modify alpha-MSH secretion by the frog neurointermediate lobe. Mesotocin, which is present in the parenchymal cells of the frog pars intermedia, had no effect on alpha-MSH release in vitro. No potentiation of CRF-induced ACTH release was observed when anterior pituitary cells were incubated with a combination of AVP and CRF. Together with the recent elucidation of a CRF-like molecule in the frog diencephalon, these results suggest that, in Amphibia, CRF and AVP exert their stimulatory action specifically on distal lobe corticotrophs.

Topics: Adrenocorticotropic Hormone; Amphibian Proteins; Animals; Arginine Vasopressin; Corticotropin-Releasing Hormone; In Vitro Techniques; Male; Melanocyte-Stimulating Hormones; Oxytocin; Peptide Hormones; Peptides; Pituitary Gland; Rana ridibunda; Ranidae; Urotensins

Three structurally related peptides, ovine corticotropin-releasing factor, sauvagine, and urotensin I are selective mesenteric vasodilators in dogs. To assess the possible benefit of these peptides in nonocclusive mesenteric ischemia, they were compared with a nonselective vasodilator, sodium nitroprusside, in the anesthetized dog. Mesenteric blood flow was reduced by approximately 30%, without lowering of systemic arterial pressure, by either digoxin or pericardial tamponade. In the digoxin model, i.v. infusions of corticotropin-releasing factor, sauvagine, and urotensin I restored intestinal vascular resistance and mesenteric blood flow to control values, without causing a fall in systemic arterial blood pressure. In the tamponade model, only urotensin I was assessed, and it produced the same restoration of hemodynamic variables. On the other hand, in both models, i.v. infusions of nitroprusside, which were effective in correcting intestinal vascular resistance, produced a fall in arterial blood pressure (presumably because of systemic dilatation), which prevented restoration of mesenteric blood flow. Intestinal oxygen uptake was not altered by tamponade, but was reduced by 23% in the digoxin model, where it was restored to control values by both the peptides and nitroprusside. The increased oxygen extraction seen in both models was corrected by the peptides but not by nitroprusside, suggesting that nitroprusside may have a direct and offsetting metabolic effect on the gut.

Topics: Amphibian Proteins; Animals; Blood Pressure; Cardiac Tamponade; Corticotropin-Releasing Hormone; Digoxin; Diuresis; Dogs; Female; Ischemia; Male; Mesenteric Arteries; Mesenteric Veins; Nitroprusside; Peptide Hormones; Peptides; Splanchnic Circulation; Urotensins; Vascular Resistance; Vasodilator Agents

Significant structural and biological homologies between urotensin I (UI), ovine hypothalamic corticotropin releasing factor (oCRF) and the frog skin peptide sauvagine (SVG) have been investigated and compared in fishes and mammals. In mammals, urotensin and the related peptides exert uniquely selective mesenteric vasodilatation, oCRF having approximately equal to 4% the activity of the other two. All three peptides are equipotent in stimulation of ACTH secretion in the rat in vivo and in vitro. UI is significantly more potent than the other two related peptides in stimulation of ACTH secretion in the goldfish pituitary. Immunocytochemical demonstration of UI not only in the caudal spinal cord but also in the brain, mainly in the lateral tuberal region and of an oCRF-like substance in the preoptic nucleus and pituitary, suggests that ACTH secretion in fishes may be controlled by two similar but distinct UI- or oCRF-like peptides.

Topics: Adrenocorticotropic Hormone; Amino Acid Sequence; Amphibian Proteins; Animals; Brain; Corticotropin-Releasing Hormone; Fishes; Hemodynamics; Hydrocortisone; Melanocyte-Stimulating Hormones; Peptide Hormones; Peptides; Urotensins; Water-Electrolyte Balance

Corticotropin-releasing factor (CRF), sauvagine (SVG), and urotensin I (UI) were tested for their effects on superior mesenteric blood flow in conscious dogs. Intravenous (iv) administration of CRF, SVG, and UI induced an immediate rise of mesenteric blood flow that was associated with a decrease in mean arterial pressure and an increase in heart rate. Intracerebroventricular (ICV) injection of SVG and UI, but not CRF, rapidly (within 5 min after injection) elicited a long (90 min) elevation of mesenteric blood flow. Central administration of these peptides induced a delayed rise in heart rate and slightly elevated mean arterial pressure. The finding that CRF given ICV did not increase mesenteric blood flow could not be explained by the release of vasoactive agents such as vasopressin, epinephrine, or norepinephrine. After injection of CRF, SVG, and UI, plasma concentrations of CRF-, SVG-, and UI-like immunoreactivity did not increase as determined by radioimmunoassay. These results indicate that SVG and UI, but not CRF, administered ICV produce a long increase of mesenteric blood flow in conscious dogs. Because iv SVG and UI decrease mean arterial pressure and ICV SVG and UI increase mean arterial pressure and do not cause an increase in SVG- and UI-like immunoreactivity in the peripheral circulation, it is proposed that SVG and UI injected into the third cerebral ventricle act within the central nervous system to increase superior mesenteric blood flow in the dog.

Topics: Amphibian Proteins; Animals; Blood Pressure; Brain; Corticotropin-Releasing Hormone; Diuresis; Dogs; Epinephrine; Heart Rate; Injections, Intraventricular; Male; Norepinephrine; Peptide Hormones; Peptides; Radioimmunoassay; Splanchnic Circulation; Time Factors; Urotensins; Vasopressins

Sauvagine (SV) is a forty amino acid peptide, isolated from the skin of the South American frog Phyllomedusa sauvagei and structurally related to fish Urotensin I (U1) and to mammalian corticotropin releasing factor (CRF). Intracerebroventricular (ICV) injections of SV (0.3-2.0 micrograms/rat), CRF (1.0-15.0 micrograms/rat) and U1 (0.5-2.0 micrograms/rat) inhibited feeding in 18 hr food deprived rats. By subcutaneous (SC) route, only SV (3.0-10.0 micrograms/kg) and U1 (10.0-20.0 micrograms/kg) exhibited anorexogenic effects, CRF being completely inactive up to a dose of 200 micrograms/kg. Vagotomy did not prevent the feeding inhibition by SC SV. In ICV injected rats, CRF increased grooming in comparison with both food deprived and satiated controls, while SV and U1 increased grooming only in comparison with fasted controls. Compared to satiated animals, food deprived rats when injected ICV with anorexogenic doses of the peptides showed decreased resting and increased moving. Rats given SC injections of SV and U1 significantly decreased grooming in comparison with both food deprived and satiated controls, while increased resting only in comparison with fasted controls. CRF by the SC route did not affect the behaviour of the rat.

Topics: Amphibian Proteins; Animals; Appetite; Corticotropin-Releasing Hormone; Feeding Behavior; Food Deprivation; Male; Peptide Hormones; Peptides; Rats; Satiation; Urotensins

Three synthetic peptides, ovine corticotropin-releasing factor, sauvagine, and urotensin I, contain homologous amino acid residues. In the anesthetized dog, all three produce marked and selective dilatation of the mesenteric circulation; none of the peptides influenced blood flow in the renal, femoral, carotid, or even celiac arteries. The mesenteric vasodilatation appeared to be unrelated to the common ability of these peptides to release corticotropin and beta-endorphin, and cannot be abolished or attenuated by conventional blocking agents or inhibitors. The unique action of these peptides suggests that there may be a related peptide in the intestine that acts to regulate intestinal blood flow. The peptides may also prove useful therapeutically in nonocclusive ischemia, if this unusual action is also present in humans.

Topics: Adrenocorticotropic Hormone; Amphibian Proteins; Animals; beta-Endorphin; Corticotropin-Releasing Hormone; Dogs; Endorphins; Hemodynamics; Hydrocortisone; Injections, Intravenous; Oxygen Consumption; Peptide Hormones; Peptides; Splanchnic Circulation; Urotensins; Vasodilation; Vasodilator Agents

The 41 amino acid peptide, corticotropin-releasing factor (CRF), isolated from ovine hypothalami has been shown to have sequence homologies with two other peptides, sauvagine (SA) and urotensin I (UT). All 3 of these peptides share some common biological properties in their ability to stimulate the release of ACTH from pituitary cells and cause vasodilation in defined vascular beds. CRF administered into the cerebral ventricles (i.c.v.) has previously been shown to have the additional property of being able to elicit behavior in the rat which has elements resembling the behavioral response to the stress of environmental novelty. The structurally related peptides SA and UT are shown here to have behavioral effects similar to those of CRF. When administered in doses of 0.5-2.0 micrograms i.c.v. 60 min prior to testing in a novel open-field environment, all 3 peptides cause a decrease in the amount of food consumed in the open field and a decrease in the mean amount eaten per approach to the food pedestal. They also decrease rearing and increase grooming. In addition, the 3 peptides caused the occurrence of a 'ballistic tail' phenomenon and a high frequency tremor in the forelimbs of several animals. The order of potency of the peptides in producing these effects was SA greater than or equal to CRF greater than UT. This order of potency differs from that previously reported for effects on vasodilation or on release of pituitary ACTH. The free acid C-terminus deaminated form of CRF (CRF-OH) failed to produce the profile of effects seen with the other peptides.

Topics: Amphibian Proteins; Animals; Behavior, Animal; Corticotropin-Releasing Hormone; Feeding Behavior; Grooming; Male; Motor Activity; Movement; Peptide Hormones; Peptides; Rats; Rats, Inbred Strains; Structure-Activity Relationship; Urotensins

Intraperitoneal injections of urotensin I, a CRF-like neuropeptide isolated from the caudal neurosecretory system of the teleost Catostomus commersoni, ovine CRF and sauvagine all produced significant increases in circulating levels of plasma cortisol in goldfish in which endogenous ACTH secretion was suppressed with betamethasone. In vitro, urotensin I was 2-3 times more potent than CRF or sauvagine in stimulating ACTH release from a superfused goldfish anterior pituitary cell column. These results demonstrate that urotensin I stimulates ACTH release in the goldfish, which suggests that urotensin I or a urotensin I-like peptide may serve as a CRF in teleost fishes.

Topics: Adrenocorticotropic Hormone; Amphibian Proteins; Animals; Corticotropin-Releasing Hormone; Cyprinidae; Dose-Response Relationship, Drug; Female; Goldfish; Hydrocortisone; In Vitro Techniques; Male; Peptide Hormones; Peptides; Pituitary Gland; Urotensins

Three recently isolated peptides, whose sequences have been determined--the corticotropin (adrenocorticotropic hormone)-releasing factor of ovine origin, sauvagine, from the skin of the frog Phyllomedusa sauvagei, and urotensin I from the teleost fish, Catostomus commersoni--show high (greater than 50%) sequence homology. CD spectra of the three peptides in trifluoroethanol indicate predominantly helical character for these peptides. Analysis of the secondary structures by the Chou-Fasman method predicts that the overall structural organization of the peptides is the same. All three possess a long internal helix, spanning about 25 residues, connected by a turn region to a COOH-terminal structural element that is an alpha-helix in corticotropin-releasing factor and urotensin I and a beta-sheet in sauvagine. The values for helical content estimated from the prediction method agree reasonably well with those computed from the CD spectra. This agreement as well as the CD spectra of corticotropin-releasing factor fragment 5-33 support the specific assignments of helical regions derived from the Chou-Fasman analysis. The three peptides exhibit significantly less helical structure in water than in trifluoroethanol as indicated by CD spectra. Hydrophilicity profiles provided comparison of the three peptides in terms of their overall hydrophilicity and the location of the regions of maximal hydrophilicity. A unique distribution of hydrophilic and hydrophobic residues within the internal helices is revealed by helical wheel analysis. Patches of both types of residues are formed following a heptad (four/three) rule. Since the two patches are shifted by one residue relative to one another, together they occupy only one face of the helical surface, a feature distinct from other amphiphilic structures.

Topics: Amphibian Proteins; Animals; Anura; Circular Dichroism; Corticotropin-Releasing Hormone; Fishes; Models, Molecular; Peptide Fragments; Peptide Hormones; Peptides; Protein Conformation; Sheep; Skin; Structure-Activity Relationship; Urotensins; Vasodilator Agents

Three synthetic peptides, urotensin I, sauvagine and ovine CRF, have less than 50% sequence homology. In the dog, all three produce selective mesenteric dilation, which appears to be solely responsible for a decrease in peripheral resistance and systemic arterial blood pressure. Urotensin I and sauvagine were found to be 5-10 times more potent than CRF.

Topics: Amphibian Proteins; Animals; Blood Pressure; Corticotropin-Releasing Hormone; Dogs; Hemodynamics; Peptide Hormones; Peptides; Sheep; Splanchnic Circulation; Urotensins; Vascular Resistance; Vasodilator Agents