Identification of an urotensin I-like peptide in the pituitary of the lungfish Protopterus annectens: immunocytochemical localization and biochemical characterization

In the present study we have investigated the localization and biochemical characteristics of urotensin I (UI)-like and urotensin II (UII)-like immunoreactive peptides in the central nervous system (CNS) and pituitary of the lungfish, Protopterus annectens, by using antisera raised against UI from the white sucker Catostomus commersoni and against UII from the goby Gillichythys mirabilis. UI-like immunoreactive material was found within the melanotrope cells of the intermediate lobe of the pituitary. By contrast, no UI-immunoreactive structures were found in the brain. No UII-like peptides structurally similar to goby UII were found in the brain and pituitary of P. annectens. The UI-immunoreactive material localized in the pituitary was characterized by combining reversed-phase high-performance liquid chromatography (HPLC) analysis and radioimmunological detection. The UI-like immunoreactivity contained in a pituitary extract eluted as a single peak with a retention time intermediate between those of sucker UI and rat corticotropin-releasing factor (CRF). Control tests on adjacent sections of pituitary showed that the UI antiserum cross-reacted with the frog skin peptide sauvagine, but lungfish UI did not co-elute with synthetic sauvagine on HPLC. On the contrary, no cross-reaction was observed between the UI antiserum and CRF or alpha-melanocyte-stimulating hormone (alpha-MSH). The occurrence of an UI-like peptide in the intermediate lobe of the pituitary of P. annectens suggests that, in lungfish, this peptide may act as a classic pituitary hormone or may be involved in the control of melanotrope cell secretion.     

Urotensin I-like immunoreactivity in the central nervous system of Aplysia californica.

In the present study the occurrence and localization of urotensin I (UI, a corticotropin releasing factor-like peptide) in the CNS of Aplysia californica were investigated by immunocytochemistry and radioimmunoassay. The RIA cross-reactivity pattern indicated that the UI antiserum used recognized an epitope in the C-terminal region of the UI, but it did not cross-react with mammalian corticotropin-releasing factor (CRF) and partially recognized sauvagine (SVG, a frog CRF-like peptide). The use of CRF-specific and sauvagine-specific antisera failed to give positive immunostaining. The application of UI antiserum (which does not cross-react with CRF in RIA) gave a positive staining, which was blocked by synthetic sucker (Catostomus commersoni) UI, but not by rat/human CRF (10 microM). On the basis of immunostaining and RIA parallel to fish UI displacement curves of cerebral ganglia extracts, the unknown UI/CRF-like substance in the Aplysia ganglia is likely to have greater homology with sucker UI than with the known CRF peptides. Urotensin I-immunoreactive (UI-ir) neurons were seen mainly in the F neuron clusters, located in the midline and rostrodorsal portion of the cerebral ganglia. Few UI-ir neurons were also found in the C and D neuron clusters of the cerebral ganglia, as well as in the left pleural and abdominal ganglia. In addition, numerous fine and coarse, and beaded UI-ir fibers were found in the cerebral commissure. UI-ir fibers were also seen in the neuropile of the buccal, pedal and pleural ganglia, and abdominal ganglion. A cuff-like arrangement of UI-ir fibers was seen in the supralabial nerves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution and coexistence of urotensin I and urotensin II peptides in the cerebral ganglia of Aplysia californica.

Urotensin I (UI) and urotensin II (UII) were demonstrated in the cerebral ganglia of Aplysia californica by applying immunocytochemical and radioimmunoassay procedures. Sequential analysis of adjacent sections of the cerebral ganglia of Aplysia demonstrated that the UI-immunoreactive (UI-IR) neurons of the F cluster of the cerebral ganglia also contained UII immunoreactivity (UII-IR). Both UI-IR and UII-IR were also observed in a cuff-like arrangement of fibers surrounding the proximal portion of the supralabial nerve, as well as in a few fibers in the anterior tentacular nerves. The UI-IR perikarya of the cerebral ganglia appeared to project to the entire CNS of Aplysia, but the UII-IR fibers appeared only in the neuropile and commissure of the cerebral ganglia. The UI-IR staining was abolished by previous immunoabsorption of the UI antiserum with sucker (Catastomus commersoni) UI, but not with ovine corticotropin-releasing factor (CRF), rat/human CRF, or goby (Gillichthys mirabilis) UII. Immunostaining with UII antiserum was quenched by goby UII, but not by sucker UII-A, UII-B, UII-A(6-12), or carp (Cyprinus carpio) UII-alpha and UII-gamma. The UII staining was not abolished by UI or somatostatin. The F cluster was not stained when a somatostatin antiserum was applied. Radioimmunoassay of dilutions of cerebral ganglia extract, using UII antiserum, revealed a parallel displacement curve to synthetic goby UII.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro and in vivo ACTH-releasing activity of ovine CRF,sauvagine and urotensin I

The ability of three homologous peptides, ovine corticotropin-releasing factor (CRF), sauvagine and urotensin I, to release ACTH was examined in vitro and in vivo. All three peptides exhibit statistically equivalent potencies to stimulate ACTH secretion both by cultured pituitary cells and in pharmacology blocked rats. These results suggest that all three peptides evolved from a common mammalian precursor that possessed high hypophysiotropic potency.     

In situ hybridization of corticotropin-releasing factor-encoding messenger RNA in the hypothalamus of the white sucker,Catostomus commersoni

Summary In situ hybridization procedure with a ³²P-labelled synthetic oligonucleotide probe was used to detect corticotropin-releasing factor-encoding messenger RNA (CRF mRNA) in the hypothalamus of the white sucker, Catostomus commersoni. Adjacent sections were immunostained by a sucker CRF-specific antiserum. CRF mRNA-containing neurons were identified by autoradiography in the magnocellular and parvocellular subdivisions of the preoptic nucleus (PON). Many of these neurons were also immunostained by sucker antiserum, showing the same distribution patterns. These results confirm the presence of CRF mRNA and CRF peptide in the white sucker hypothalamus and support the view that the magnocellular and parvocellular neurons of the PON may be involved in the control of adrenocorticotropic hormone secretion from the pituitary in the white sucker.     

Distribution of corticotropin releasing factor-like immunoreactivity in the rat brain by immunohistochemistry and radioimmunoassay: comparison and characterization of ovine and rat/human CRF antisera

The distribution of corticotropin releasing factor (CRF)-like immunoreactivity in the rat brain has been demonstrated by immunohistochemistry and radioimmunoassay using 4 different antisera. Two antisera were directed against synthetic ovine CRF, two antisera were directed against synthetic rat/human CRF. Immunohistochemistry revealed that there are discrete regions where CRF immunoreactive cell bodies are seen with all 4 antisera (e.g., the paraventricular nucleus, the dorsolateral tegmental nucleus) whereas there are cells observed only with one rat CRF antiserum (e.g., in the cortex) or terminal fields observed only with ovine CRF antisera (e.g., the spinal trigeminal tract, the substantia gelatinosa, the spinal cord). Radioimmunoassay showed different cross reactivity of the antisera with synthetic ovine or rat/human CRF and sauvagine, however, there was no cross reactivity with a variety of other peptides. Tissue values of CRF obtained by RIA of micropunched brain nuclei with the 4 antisera were frequently dissimilar suggesting that different antisera recognize different substances. High performance liquid chromatography and radioimmunoassay of brain tissue samples, revealed that there is more than one form of CRF-like immunoreactivity present. There is indirect evidence that there exists at least one peptide in the rat brain, prominent in the medulla and the spinal cord, which cross reacts with antisera directed to ovine CRF only.     

Urocortins as cardiovascular peptides

Urocortins (Ucn) 1, 2 and 3, human homologues of fish urotensin I, form the corticotropin-releasing factor (CRF) family, together with CRF, urotensin I and sauvagine. Ucn 3 is a novel member of this family and is a specific ligand for CRF type 2 receptor. CRF type 2 receptor is thought to mediate the stress-coping responses, such as anxiolysis, anorexia, vasodilatation, a positive inotropic action on myocardium and dearousal. Endogenous ligands for the CRF type 2 receptor expressed in the cardiovascular tissues, such as the myocardium, have long been unknown. We have shown expression of Ucn 3 as well as Ucn 1 in the human heart. Ucn 3 is also expressed in the kidney, particularly distal tubules. Studies in various rat tissues showed that high concentrations of immunoreactive Ucn 3 were found in the pituitary gland, adrenal gland, gastrointestinal tract, ovary and spleen in addition to the brain, heart and kidney. These observations suggest that Ucn 3 is expressed in various tissues including heart and kidney, and may regulate the circulation in certain aspects of stress and diseases, such as inflammation. Ucn 1 and 3 appear to have important pathophysiological roles in some cardiovascular diseases.     

Immunocytochemical Demonstration of Serotonin and Neuropeptides in the Nervous System of Gyrodactylus salaris (Monogenea)

A whole mount immunocytochemical (ICC) method has been used for the investigation of immunoreactivity (IR) to the molluscan cardioactive peptide FMRF-amide, to 9 vertebrate neuropeptides—leu-enkephalin, growth hormone-releasing factor (GRF), urotensin I, urotensin II, bovine pancreatic peptide (BPP), β-endorphin, substance P, secretin and insulin—and to the bioamine 5-HT in the nervous system (NS) of the trematode Gyrodactylus salaris belonging to the taxon Monogenea. Positive results were obtained using antisera to FMRF-amide, leu-enkephalin, urotensin I, GRF and to 5-HT. The present results are the first documentation of the presence of neuroregulatory peptides and a bioamine in the nervous system (NS) of Monogenea. Differences in the distribution pattern of the IR to the different antisera indicate that different subsets of neurons are revealed. In addition, details of the basic neuroanatomical pattern in monogeneans are confirmed by the whole mount ICC method used in this study. Negative results were obtained with antisera to urotensin II, substance P, β-endorphin, secretin, insulin and bovine pancreatic polypeptide (BPP).     

Cortisol inhibits the ACTH-releasing activity of urotensin I, CRF and sauvagine observed with superfused goldfish pituitary cells

The structurally homologous peptides urotensin I, ovine CRF and sauvagine stimulate the release of immunoreactive ACTH from a superfused dispersed goldfish anterior pituitary cell column. The addition of cortisol to the superfusion buffer resulted, following a latent period, in a decrease in basal release of ACTH from the pituitary cell column and a diminution in the ACTH-releasing activities of urotensin I, CRF and sauvagine. The removal of cortisol from the superfusion buffer resulted in a slow recovery of basal ACTH release and a recovery of the ACTH-releasing activities of urotensin I, CRF and sauvagine. These results are supportive of the view that urotensin I, or a urotensin I-like peptide, serves as a physiological regulator of ACTH release in teleost fishes.     

Regulation of MSH release from the neurointermediate lobe of Xenopus laevis by CRF-like peptides

Immunocytochemical studies showed the presence of a fiber system containing a CRF-like peptide in the median eminence and in the neural lobe of the pituitary gland of Xenopus laevis. During in vitro superfusion of neurointermediate lobe tissue, CRF, sauvagine and urotensin I induced a rapid and dose-dependent stimulation of secretion of MSH and endorphin. Tissue of white-background adapted animals displayed a remarkably higher sensitivity to CRF and sauvagine than tissue from animals that were adapted to a black background. During superfusion of isolated melanotrope cells in suspension, it was shown that CRF and sauvagine exerted their effect directly on the melanotrope cell. We therefore conclude that there is morphological and biochemical evidence to consider a CRF-like peptide as a physiological MSH-releasing factor.     

Differential profile of CRF receptor distribution in the rat stomach and duodenum assessed by newly developed CRF receptor antibodies

Peripheral corticotropin-releasing factor (CRF) receptor ligands inhibit gastric acid secretion and emptying while stimulating gastric mucosal blood flow in rats. Endogenous CRF ligands are expressed in the upper gastrointestinal (GI) tissues pointing to local expression of CRF receptors. We mapped the distribution of CRF receptor type 1 (CRF1) and 2 (CRF2) in the rat upper GI. Polyclonal antisera directed against the C-terminus of the CRF receptor protein were generated in rabbits and characterized by western blotting and immunofluorescence using CRF1- and CRF2-transfected cell lines and in primary cultured neurons from rat brain cortex. A selective anti-CRF1 antiserum (4467a-CRF1) was identified and used in parallel with another antiserum recognizing both CRF1 and CRF2 (4392a-CRF1&2) to immunostain gastric tissue sections. Antiserum 4467a-CRF1 demonstrated specific immunostaining in a narrow zone in the upper oxyntic gland within the stomach corpus. Conversely, 4392a-CRF1&2 labeled cells throughout the oxyntic gland and submucosal blood vessels. Pre-absorption with the specific antigen peptide blocked immunostaining in all experiments. Doublestaining showed co-localization of 4392a-CRF1&2 but not 4467a-CRF1 immunoreactivity with H/K-ATPase and somatostatin immunostaining in parietal and endocrine cells of the oxyntic gland. No specific staining was observed in the antrum with either antisera, whereas only antiserum 4392a-CRF1&2 showed modest immunoreactivity in the duodenal mucosa. Finally, co-localization of CRF2 and urocortin immunoreactivity was found in the gastric glands. These results indicate that both CRF receptor subtypes are expressed in the rat upper GI tissues with a distinct pattern and regional differences suggesting differential function.     

Presence of Corticotropin-Releasing Factor-Stimulated Adenylate Cyclase Activity in Rat Retina

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.     

Isolation and amino acid sequence of urotensin I, a vasoactive and ACTH-releasing neuropeptide, from the carp (Cyprinus carpio) urophysis

Urotensin I (UI), a 41-residue mammalian hypotensive and fish or mammalian corticotropin-releasing peptide, isolated from 0.1 N HCI extracts of urophyses of the carp (Cyprinus carpio) was purified and the amino acid sequence was determined to be: H-Asn-Asp-Asp-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu- Arg-Asn-Met-Ile-Glu-Met-Ala-Arg-Asn-Glu-Asn-Gln-Arg-Glu-Gln-Ala-Gly-Leu-Asn-Arg-Lys-Tyr-Leu-Asp-Glu-Val-NH2. When the extraction procedure included heating at 100 degrees C for 15 min, UI was cleaved at a highly acid labile Asp-Pro bond to give the fully active UI (4-41). Urotensin I shows close structural and biological homology with the recently isolated ovine hypothalamic corticotropin-releasing factor (CRF) and the frog skin peptide sauvagine and thus may be considered an evolutionary prototype of unique mammalian-hypotensive and vertebrate corticotropin-releasing factors.     

Ion transport systems in the kidney and urinary bladder of two Antarctic teleosts, Chionodraco hamatus and Trematomus bernacchii

Na⁺-K⁺-Cl^- cotransport and Na⁺/K⁺ATPase were studied by immunohistochemistry in the kidney and urinary bladder of Trematomus bernacchii and Chionodraco hamatus. The activity was correlated to the density of mitochondria. The first segment of the renal proximal tubule was more active than the second one. In T. bernacchii and the temperate marine teleost Pagellus bogaraveo, the immunoreactivity for the antibody to cotransporters and to the !-subunit of the sodium pump was stronger than in the icefish. This difference indicates in the kidney of the icefish, a weaker secretory activity, a consequent lower osmolarity in the lumen and lower water loss, which correlates well with the need for a greater blood volume in the icefish. The epithelium of the urinary bladder in T. bernacchii, where intense immunostaining was observed, was composed of columnar cells. In C. hamatus the columnar cells, where the immunostaining was weaker, lined only a portion of the urinary bladder, the other region being composed of cuboidal cells.     

Immunoreactivity to the pancreatic polypeptide family in the nervous system of the adult human blood fluke,Schistosoma mansoni

Summary The presence and distribution of neuropeptides belonging to the pancreatic polypeptide family have been demonstrated by an indirect immunofluorescence technique in the nervous systems of adult male and female Schistosoma mansoni. Seven antisera of differing regional specificity to pancreatic polypeptide (PP), peptide YY (PYY) and neuropeptide Y (NPY) were employed on both whole-mount and cryostat-sectioned material. Positive immunoreactivity (IR) was obtained with all antisera except an N-terminally-directed antiserum to NPY. In the CNS, immunoreactivity was restricted to cell bodies and nerve fibres in the anterior ganglia, central commissure and dorsal and ventral nerve cords of both sexes, whereas, in the PNS, positive-IR was present in the plexuses innervating the subtegumental musculature and the oral and ventral suckers. Intense immunoreactivity was observed in a plexus of nerve fibres and cell bodies in the lining of the gynaecophoric canal and in fine nerve fibres innervating the dorsal tubercles of the male. In contrast, in the female, strong immunoreactivity was evident in nerve plexuses innervating the lining of the ovovitelline duct and in the wall of the ootype, but most notably in a cluster of cells in the region of Mehlis' gland. Results suggest that molecules with C-terminal homology to the PP-family are present in S. mansoni. These peptides would appear to be important regulatory molecules in the parasite's nervous system and may play a role in the control of egg production.     

Current Studies on Urotensins

Recent studies on urotensins I and II and experiments on urophysialfunction are described. Chemical and pharmacological studies showed that: (i) The isoelectricpoints of urotensins I and II are pI 5.5 ± 0.05 and 5.9± 0.05, respectively; (ii) two active peaks of urotensinI are obtained by gel chromatography of HCl-extracted urophyseson Bio-Gel P⁶, one corresponding to a substance of mol wt 4000,the other, mol wt 2000; (iii) the rat-hypotensive activity ofurotensin I affects peripheral blood vessels at an undeterminedreceptor site(s) and is not mediated by cholinergic, adrenergic,or histaminergic receptors or the corresponding transmittersubstances; and (iv) urotensins I and II increase blood pressureand urine flow in the American eel (Anguilla rostrata). Urophysial content of urotensin II in the white sucker (Catostomuscommersoni) was high during about 3 months preceding spawningand was considerably lower during the remainder of the year.Pituitary content of isotocin is significantly higher in malesuckers after spawning as compared with the content before spawning.The reverse was found on urophysial content of urotensin IIin the male fish.     

Isolation and characterization of CRF-related diuretic hormones from the whitelined sphinx moth Hyles lineata

We have isolated and characterized two diuretic hormones (DH), Hylli-DH41 and Hylli-DH30, from extracts of whole heads of the lepidopteran Hyles lineata. We monitored the isolation by measuring the ability of fractions to affect levels of cyclic AMP production by Malpighian tubules of Manduca sexta maintained in vitro. These DH are related to a family of vertebrate neuropeptides which includes sauvagine, corticotropin-releasing factor (CRF), and urotensin I. Both Hylli-DH41 (RMPSLSIDLPMSVLRQKLSLE KERKVQALRAAANRNFLNDI-NH2) and Hylli-DH30 (SFSVNPAVEILQHRYMEKVAQNNRNFLNRV-NH2) show extremely high similarity with two DH from the tobacco hornworm M. sexta. This is not surprising because both H. lineata and M. sexta are sphingid moths. The discovery of these DH provides a third example of two CRF-related DH occurring in one insect species.     

A comparative immunocytochemical study of the hyperglycaemic, moult-inhibiting and vitellogenesis-inhibiting neurohormone family in three species of decapod Crustacea

Eyestalks of the palinuran species Jasus lalandii and Panulirus homarus, and the brachyuran species Carcinus maenas, were examined with antisera raised against purified crustacean hyperglycaemic hormone (cHH) of the astacidean species Homarus americanus and Procambarus bouvieri, as well as the brachyuran species Cancer pagurus. Other antisera used in this investigation were raised against purified moult-inhibiting hormone (MIH) of C. pagurus and vitellogenesis-inhibiting hormone (VIH) of H. americanus. Positive immunoreactions to all the antisera were localised in perikarya of the X-organ and the axon terminals in the sinus gland of all the crustaceans investigated. These results illustrate the existence of an immunological similarity, detectable at the immunocytochemical level, between the cHH/MIH/VIH neurohormones of the Astacidae, Palinura and Brachyura infraorders. Furthermore, results from consecutive tissue sections indicate that cHH, MIH and VIH are co-localised in a subpopulation of X-organ neurons.     

Sauvagine-like and corticotropin-releasing factor-like immunoreactivity in the brain of the bullfrog (Rana catesbeiana)

Summary Immunocytochemical methods were used to investigate the occurrence and distribution of sauvagine, corticotropin-releasing factor-, or urotensin I-like immunoreactivities (SVG-ir, CRF-ir, UI-ir, respectively) in the bullfrog (Rana catesbeiana) brain, using specific antisera raised against non-conjugated SVG, ovine CRF, rat/human CRF, and UI. In the hypothalamus, SVG-ir was found in the magnocellular perikarya, in the dorsal and ventral regions of the preoptic nucleus, and in the hypothalamo-hypophyseal projections to the external zone as well as the internal zone of the median eminence, to pars nervosa, and in fibres running from the pars nervosa to the pars intermedia of the pituitary. In contrast, CRF-ir was found only in parvocellular perikarya, mainly localized in the rostro-ventral part of the preoptic nucleus, with fine processes protruding through the ependyma of the third ventricle, fibre projections terminating in the anterior preoptic area and in the neuropil of the periventricular gray, and a caudal projection to the external zone of the median eminence. No CRF-ir staining was seen in the pars nervosa and pars intermedia. The use of UI-specific antisera failed to give a positive response in the frog brain. It is concluded that, in the frog brain, two anatomically different CRF-like (or SVG-like) systems co-exist, comparable to the reported co-existence of UI-ir and CRF-ir neuronal systems in fish brain.