Isolation and amino acid sequence of urotensin II from the sturgeon Acipenser ruthenus.

Urotensin II (UII) peptides have previously been isolated from the urophysis (the neurohemal organ of the caudal neurosecretory system) of several teleost fish, and the UII amino acid sequences have been determined. Chondrostean fish, such as the Acipenseridae (sturgeon), though without a distinct urophysis, also have a caudal neurosecretory system, which has been indicated by bioassay and immunological evidence to contain UII-like peptides. In the present studies, we investigated by UII radioimmunoassay the UII-like peptides in the spinal cord of three Acipenser species, and isolated and sequenced UII from one of them. As expected, much more UII immunoreactivity (UII-IR) was found in caudal than in anterior spinal cord extracts. In addition, caudal extracts from A. ruthenus were found to contain much more UII-IR (whether determined on a UII-IR/weight or UII-IR/fish basis) than those from the larger A. stellatus and A. guldenstadti. UII was therefore isolated from A. ruthenus and its amino acid sequence was shown to be H-Gly-Ser-Thr-Ser-Glu-Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH. This sequence is identical at positions 6-11 (the disulfide ring) with the known teleost UII peptides, and has acidic and hydrophobic amino acids at positions 5 and 12, respectively, as do the teleost UII peptides. Overall sequence identity with the various forms of teleost UII was 58-83%.     

Occurrence of two distinct urotensin II-related peptides in zebrafish provides new insight into the evolutionary history of the urotensin II gene family

The urotensin II (UII) family is currently known to consist of two paralogous peptides, namely UII and UII-related peptide (URP). In contrast to UII, which has been identified in all vertebrate classes so far, URP has only been characterized in tetrapods. We report here the occurrence of two distinct URP genes in teleosts, which we have named URP1 and URP2. Synteny analysis revealed that teleost URP1 and URP2 genes and tetrapod URP genes represent three distinct paralog genes that, together with the UII gene, probably arose from the two rounds of tetraploidization, which took place early in vertebrate evolution. The absence of URP in fish indicates that the corresponding gene has been lost in the teleost lineage, whereas it is likely that both the URP1 and URP2 genes have been lost in the tetrapod lineage. Quantitative RT-PCR analysis revealed that the URP2 gene is mainly expressed in the spinal cord and the brain in adult zebrafish. In situ hybridization experiments showed that in zebrafish embryos, URP2 mRNA-containing cells are located in the floor plate of the neural tube. In adult, URP2-expressing cells occur in close contact with the ventral side of the ependymal canal along the whole spinal cord, whereas in the brain, they are located below the fourth ventricle. These URP-expressing cells may correspond to cerebrospinal fluid-contacting neurons. In conclusion, our study reveals the occurrence of four distinct UII paralogous systems in vertebrates that may exert distinct functions, both in tetrapods and teleosts.     

Purification and characterization of urotensin II and parvalbumin from an elasmobranch fish, Scyliorhinus canicula (common dogfish).

The caudal portion of the spinal cord of elasmobranch fish incorporates a diffuse neuroendocrine system. Using an antiserum raised against urotensin II from a teleost fish (goby) to facilitate purification, a peptide structurally related to urotensin II was isolated in pure form from an extract of neuroendocrine tissue from the spinal cord of the European common dogfish, Scyliorhinus canicula. The primary structure of the peptide was established as: Asn-Asn-Phe-Ser-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val. The amino acid sequence was confirmed by chemical synthesis. A comparison of this sequence with those of the known teleost urotensin II peptides shows that the cyclic region of the molecule has been fully conserved between species and suggests that the presence of an acidic residue at position 5 and a hydrophobic residue at position 12 are important features for the biological activity of the peptide. The dogfish spinal cord extract also contained a high concentration of the calcium-binding protein, parvalbumin and the amino acid sequence at its NH2 terminus [residues (1-50)] was determined.     

Characterization of the true ortholog of the urotensin II-related peptide (URP) gene in teleosts

It has been recently established that the urotensin II (UII) family consists of four distinct paralogs in bony vertebrates, namely UII, and the three UII-related peptides (URPs) called URP, URP1 and URP2. These four peptides are encoded by genes which arose from the two rounds of tetraploidization (2R) which took place early during vertebrate evolution. Up to now, three of them, UII, URP1 and URP2, have been identified in teleosts, while only two, UII and URP, have been reported in tetrapods. The fact that fish URP has not been found in previous studies led to the suggestion that the corresponding gene had been lost in the teleost lineage. In the present study, we show that this view is not correct. A search of the most recent release of the Ensembl genome database led us to identify a novel UII/URP-like gene in teleosts. Using synteny analysis, we demonstrate that this gene corresponds to the true ortholog of the tetrapod URP gene. Molecular cloning of the corresponding cDNA in medaka revealed that URP gene encodes a putative peptide, with the primary structure GEPCFWKYCV. In stickleback, tilapia and takifugu, URP exhibited the same sequence while, in tetraodon, it differed by only one amino acid substitution Gly ? Ser. In zebrafish, URP appeared totally divergent at its N-terminus with the structure DDTCFWKYCV. In conclusion, the occurrence of a true URP in teleosts shows that the quartet of UII-related genes which arose from 2R has been integrally preserved in this lineage.     

Cloning and sequence analysis of cDNA encoding urotensin I precursor.

The primary structure of the precursor of urotensin I, a neuropeptide hormone from the caudal neurosecretory system of the carp Cyprinus carpio, has been determined by analyzing the nucleotide sequence of cloned DNA complementary to the mRNA encoding it. The precursor consists of 145 amino acid residues and the carboxyl terminus represents the 41-amino acid sequence of urotensin I, preceded by Lys-Arg and followed by Gly-Lys. Sequence homology as well as similar organization of the precursors of urotensin I and mammalian corticotropin-releasing factors suggest that they are evolutionarily related. RNA transfer blot analysis indicates that mRNA encoding the precursor of urotensin I is present only in the spinal cord and not in the brain, intestine, liver, or kidney of the carp.     

Cloning of cDNAs encoding amphibian bombesin: evidence for the relationship between bombesin and gastrin-releasing peptide.

Bombesin is a tetradecapeptide originally isolated from frog skin; its mammalian homologue is the 27-amino acid peptide gastrin-releasing peptide (GRP). cDNAs encoding GRP have been cloned from diverse species, but little is yet known about the amphibian bombesin precursor. Mass spectrometry of HPLC-separated skin exudate from Bombina orientalis was performed to demonstrate the existence of authentic bombesin in the skin of this frog. A cDNA library was prepared from the skin of B. orientalis and mixed oligonucleotide probes were used to isolate cDNAs encoding amphibian bombesin. Sequence analysis revealed that bombesin is encoded in a 119-amino acid prohormone. The carboxyl terminus of bombesin is flanked by two basic amino acids; the amino terminus is not flanked by basic amino acids but is flanked by a chymotryptic-like cleavage site. Northern blot analysis demonstrated similarly sized bombesin mRNAs in frog skin, brain, and stomach. Polymerase chain reaction was used to show that the skin and gut bombesin mRNAs encoded the identical prohormones. Prohormone processing, however, differed between skin and gut. Chromatography showed the presence of only authentic bombesin in skin whereas gut extracts contained two peaks of bombesin immunoreactivity, one consistent in size with bombesin and one closer in size to mammalian GRP. Thus the same bombesin prohormone is processed solely to bombesin in skin but is processed to a peptide similar in size to bombesin and to a peptide similar in size to mammalian GRP in stomach.     

A homologous radioimmunoassay for the measurement of urotensin II in the euryhaline flounder, Platichthys flesus

A sensitive and specific homologous radioimmunoassay (RIA) has been developed to measure tissue and circulating levels of the fish caudal neurosecretory system neuropeptide, urotensin II (UII), in the euryhaline flounder Platichthys flesus. A polyclonal antiserum was raised against flounder UII in rabbit; UII-125I was produced by the iodogen method and purified by HPLC. Antiserum specificity to flounder UII was demonstrated through lack of cross-reactivity with several small peptides and parallelism with standard curves for serial dilutions of UII in plasma and urophysial extracts. Biological activity of the peptide measured by UII RIA was confirmed by bioassay. Plasma intra- and interassay coefficients of variation were 9 and 18% (n = 5 and n = 3), respectively, nonspecific binding constituted 4.6% (+/-1.42%, n = 8) of total counts, and the limit of RIA detectability was estimated as 1.5 x 10(-16) M UII/assay tube. Plasma samples were subject to a reversed-phase liquid chromatography purification protocol which had an extraction efficiency of 63% (+/-10%, n = 6) and showed consistent recovery of UII over a range of plasma volumes and peptide concentrations. Plasma UII concentrations in seawater (SW)-adapted flounder (3.80 +/- 0.77 x 10(-11) M, n = 7) were significantly higher than those in freshwater (FW)-adapted fish (1.10 +/- 0.15 x 10(-11) M, n = 7). This variation coincided with differences in plasma osmolality and Na+ levels. No differences were found, however, between urophysial UII concentrations in SW-adapted (3.71 +/- 1.78 x 10(-10) M UII/gland, n = 7) and FW-adapted (2.53 +/- 1.33 x 10(-10) M UII/gland, n = 7) flounder.     

Elevated expression of urotensin II and its receptor in diabetic cardiomyopathy

OBJECTIVE: Urotensin II (UII) is a somatostatin-like peptide recently identified to have several cardiovascular effects, including potent vasoactive, cardiac inotropic and chronotropic properties. Our aim was to determine the degree of expression of UII and UII receptor (UT) in the myocardium of rats with streptozotocin (STZ)-induced diabetes. METHODS: Real-time polymerase chain reaction, Western blot, and immunohistochemistry were used to determine the degree of expression and location of UII and UT in the myocardium of STZ-induced diabetic rats. RESULTS: UII and UT expression were significantly enhanced in the myocardium of rats with diabetes compared with healthy controls on both messenger RNA and protein levels. Both UII and UT protein expression were mainly concentrated in the cardiomyocytes, endothelial cells, cardiac fibroblasts, and smooth muscle cells of diabetic cardiomyopathy (DCM). CONCLUSIONS: Our results suggest a possible role for the UII/UT system in the pathophysiology of DCM.     

Increased urotensin I and II immunoreactivity in the urophysis of Gillichthys mirabilis transferred to low salinity water

The function of the fish caudal neurosecretory system is uncertain, but a role in osmoregulation has been suggested by many investigators. Our objective was to determine if acclimation to water of different salinity has an effect on immunoreactive patterns and staining intensities of the two caudal neuropeptides, urotensins I (UI) and II (UII), in Gillichthys mirabilis. Five fish, originally maintained in seawater, were transferred to deionized fresh water (FW), and five were transferred to new seawater (SW). After 24 hr spinal cords were removed and fixed, FW and SW spinal cords were paired in blocks to receive identical treatment, and cryostat sections were double immunostained for both peptides using a double sequential immunofluorescence procedure. FW spinal cord exhibited increased staining intensities for both UI and UII in their urophyses (the neurohemal organ) compared to the SW spinal cords. The magnitude of intensity difference appeared greater for UI than for UII. In addition, the FW urophyses had more loci displaying intense, perivascular UI immunoreactivity than the SW urophyses. Thus, it appears that environmental salinity has an effect on the urophysial content of UI and UII in this euryhaline fish. The increased immunoreactivity in FW fish could reflect increased synthesis and storage, decreased release of the stored peptides, or decreased peptide degradation.     

Comparative genomics provides evidence for close evolutionary relationships between the urotensin II and somatostatin gene families

Although urotensin II (UII) and somatostatin 1 (SS1) exhibit some structural similarities, their precursors do not show any appreciable sequence identity and, thus, it is widely accepted that the UII and SS1 genes do not derive from a common ancestral gene. The recent characterization of novel isoforms of these two peptides, namely urotensin II-related peptide (URP) and somatostatin 2 (SS2)/cortistatin (CST), provides new opportunity to revisit the phylogenetic relationships of UII and SS1 using a comparative genomics approach. In the present study, by radiation hybrid mapping and in silico sequence analysis, we have determined the chromosomal localization of the genes encoding UII- and somatostatin-related peptides in several vertebrate species, including human, chicken, and zebrafish. In most of the species investigated, the UII and URP genes are closely linked to the SS2/CST and SS1 genes, respectively. We also found that the UII-SS2/CST locus and the URP/SS1 locus are paralogous. Taken together, these data indicate that the UII and URP genes, on the one hand, and the SS1 and SS2/CST genes, on the other hand, arose through a segmental duplication of two ancestral genes that were already physically linked to each other. Our results also suggest that these two genes arose themselves through a tandem duplication of a single ancestral gene. It thus appears that the genes encoding UII- and somatostatin-related peptides belong to the same superfamily.     

The rat melanin-concentrating hormone messenger ribonucleic acid encodes multiple putative neuropeptides coexpressed in the dorsolateral hypothalamus

The melanin-concentrating hormone (MCH) is a cyclic neuropeptide, first isolated from salmon pituitary glands, which regulates melanin dispersion in the skin and perhaps the activity of the pituitary-adrenal axis in teleost fish. We have recently purified and characterized rat MCH (rMCH) and report here the cloning and sequencing of specific MCH cDNA isolated from a rat hypothalamic library. The sequence of rMCH found by DNA sequencing confirms the sequence deduced from the purified peptide. rMCH is located at the C-terminus of a protein precursor of 165 amino acid residues. Comparison of the amino acid sequence of prepro-MCH and that of the Aplysia peptide-A prohormone suggests that these proteins as well as other precursors may be evolutionarily related. Besides rMCH, two putative neuropeptides, termed NGE and NEI, might be generated from the same precursor. The rMCH precursor shared sequence identities with human GH-releasing factor and mammalian CRF in the regions encoding NGE and NEI. By immunohistochemical studies we have established that the amidated C-terminus of NEI is recognized by some alpha MSH and rat CRF antisera and that the C-terminal portion of NGE is responsible for the cross-reactivity revealed with one hGRF-(1-37) antiserum. Our results explain the staining of a discrete population of dorso-lateral hypothalamic neurons by heretofore seemingly unrelated antisera and provide evidence for the production of multiple novel neuropeptides from a common precursor.     

Molecular characterization and expression of urotensin II and its receptor in the flounder (Platichthys flesus): a hormone system supporting body fluid homeostasis in euryhaline fish

Urotensin II (UII) is a potent vasoconstrictor in mammals, but the source of circulating UII remains unclear. Investigations of the caudal neurosecretory system (CNSS), considered the major source of UII in fish, alongside target tissue expression of UII receptor (UT), can provide valuable insights into this highly conserved regulatory system. We report UII gene characterization, expression of the first fish UT, and responses to salinity challenge in flounder. The 12-aa UII peptide shares 73% sequence identity with pig and human UII. Flounder UT receptor shares 56.7% identity with rat. Although the CNSS is the major site of UII expression, RT-PCR revealed expression of UII and UT in all tissues tested. Around 30-40% of large CNSS Dahlgren cells expressed UII, alone or in combination with urotensin I and/or corticotrophin releasing hormone. Immunolocalization of UT in osmoregulatory tissues (gill, kidney) was associated with vascular elements. There were no consistent differences in CNSS UII expression or plasma UII between seawater (SW)- and freshwater (FW)-adapted fish, although gill and kidney UT expression was lower in FW animals. After acute transfer from SW to FW, plasma UII and kidney and gill UT expression were reduced, whereas UT expression in kidney was increased after reverse transfer. UII appears to be more important to combat dehydration and salt-loading in SW than the hemodilution faced in FW. Potentially, altered target tissue sensitivity through changes in UT expression, is an important physiological controlling mechanism, not only relevant for migratory fish but also likely conserved in mammals.     

Active chloride transport by the skin of a marine teleost is stimulated by urotensin I and inhibited by urotensin II

The caudal neurosecretory peptide, urotensin I (UI), stimulated in vitro short-circuit current (I_sc) across skin from the marine teleost Gillichthys mirabilis. UI reversed the previous inhibition of I_sc by both epinephrine and urotensin II (UII), and stimulated untreated skins that had low I_sc. UII and epinephrine reduced I_sc and net flux of chloride by decreasing chloride efflux; a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IMX), stimulated I_sc and chloride net flux by increasing both unidirectional fluxes. The results suggest that UI can antagonize the effects of UII and epinephrine on active chloride transport by chloride-secreting cells of marine teleosts.     

cDNA cloning of the beta subunit of teleost thyrotropin.

cDNA clones encoding the beta subunit of thyrotropin (thyroid-stimulating hormone; TSH) were isolated from a cDNA library made from the pituitaries of immature rainbow trout and sequenced. The precursor of rainbow trout TSH beta consists of 147 aa, which can be cleaved into a signal peptide (20 aa) and a mature protein (127 aa) containing one potential N-glycosylation site and 12 cysteine residues. The protein showed highest homology with human TSH beta (51%) and lesser homology with human follitropin (42%), human lutropin (32%), and salmon gonadotropin (31-33%) beta subunits. The identification of TSH in addition to two gonadotropins (gonadotropins I and II) in the teleost fish suggests that the divergence of three kinds of glycoprotein hormones from an ancestral molecule took place earlier than the time of divergence of teleosts from the main line of evolution leading to tetrapods. Northern blot analysis showed that the expression of the rainbow trout TSH beta gene is specific to the pituitary gland and is significantly higher in immature fish than in mature fish, suggesting that TSH plays some role in the biological processes of immature fish.     

Identification of a cDNA encoding a novel amphibian growth hormone-releasing peptide and localization of its transcript

Recently, we identified in the bullfrog brain a novel neuropeptide with a C-terminal Leu-Pro-Leu-Arg-Phe-NH(2) sequence. This amphibian neuropeptide was shown to stimulate growth hormone (GH) release in vitro and in vivo and so was designated frog GH-releasing peptide (fGRP). In this study, we cloned a cDNA encoding fGRP from the bullfrog brain by a combination of 3' and 5' rapid amplification of cDNA ends (RACE). The deduced fGRP precursor consisted of 221 amino acid residues, encoding one fGRP and three putative fGRP-related peptides that included Leu-Pro-Xaa-Arg-Phe-NH(2) (Xaa=Leu or Gln) at their C-termini. All these peptide sequences were flanked by a glycine C-terminal amidation signal and a single basic amino acid on each end as an endoproteolytic site. Northern blot analysis detected a single band of approximately 1.0 kb, indicating that no alternatively spliced forms were present. Such an apparent migration was in agreement with the estimated length of the cDNA, 902 bp. In situ hybridization further revealed the cellular localization of fGRP mRNA in the suprachiasmatic nucleus in the hypothalamus. In addition to fGRP, its related peptides may be hypothalamic factors involved in pituitary hormone secretion.     

Identification of 26RFa, a hypothalamic neuropeptide of the RFamide peptide family with orexigenic activity

A neuropeptide was isolated from a frog brain extract by HPLC purification and characterized by mass spectrometry. This 26-aa neuropeptide, which belongs to the RFamide peptide family, was designated 26RFa, and its primary structure was established as VGTALGSLAEELNGYNRKKGGFSFRF-NH2. Research in databases revealed the presence of sequences homologous to frog 26RFa in the human genome and in rat ESTs. On the basis of this sequence information, the cDNAs encoding the human and rat 26RFa precursors were cloned. The two preproteins show a similar organization, with the 26RFa sequence located in the C-terminal region of the precursor. Human preprotein (prepro)-26RFa encodes an additional putative RFamide peptide that is not found in the rat precursor. The primary structures of human, rat, and frog 26RFa exhibit approximately 80% identity, and the C-terminal octapeptide has been fully conserved from amphibians to mammals. In situ hybridization histochemistry revealed that, in the rat brain, the 26RFa gene is exclusively expressed in the ventromedial hypothalamic nucleus and in the lateral hypothalamic area. 26RFa induced a dose-dependent stimulation in cAMP production by rat pituitary cells in vitro and markedly increased food intake in mice. The conservation of the primary structure of 26RFa during vertebrate evolution, the discrete localization of the mRNA encoding its precursor in hypothalamic nuclei involved in the control of feeding behavior, and the observation that 26RFa possesses orexigenic properties indicate that this neuropeptide may play important biological functions.     

Human renal carcinoma expresses two messages encoding a parathyroid hormone-like peptide: evidence for the alternative splicing of a single-copy gene.

A peptide secreted by tumors associated with the clinical syndrome of humoral hypercalcemia of malignancy was recently purified from human renal carcinoma cell line 786-0. The N-terminal amino acid sequence of this peptide has considerable similarity with those of parathyroid hormone (PTH) and of peptides isolated from human breast and lung carcinoma (cell line BEN). In this study we obtained the nucleotide sequence of a 1595-base cDNA complementary to mRNA encoding the PTH-like peptide produced by 786-0 cells. The cDNA contains an open reading frame encoding a leader sequence of 36 amino acids and a 139-residue peptide, in which 8 of the first 13 residues are identical to the N terminus of PTH. Through the first 828 bases the sequence of this cDNA is identical with one recently isolated from a BEN cell cDNA library; however, beginning with base 829 the sequences diverge, shortening the open reading frame by 2 amino acids. Differential RNA blot analysis revealed that 786-0 cells express two major PTH-like peptide mRNAs with different 3' untranslated sequences, one of which hybridizes with the presently described sequence and the other one with that reported for the BEN cell PTH-like peptide cDNA. Primer-extension analysis of 786-0 poly(A)+ RNA together with Southern blot analysis of human DNA confirmed the presence of a single-copy gene coding for multiple mRNAs through alternate splicing. In addition, the 3' untranslated sequence of the cDNA described here has significant similarity to the c-myc protooncogene.     

Auxin-binding protein located in the endoplasmic reticulum of maize shoots: molecular cloning and complete primary structure.

We previously purified an auxin-binding protein (ABP) from the microsomal fraction of maize shoots (Zea mays L. cv. Golden Cross Bantam). In the present study cDNA clones derived from mRNAs encoding the ABP were isolated and sequenced. The nucleotide sequence of the 822-base-pair cDNA includes a 603-base-pair open reading frame. RNA blot hybridization analysis indicated a single mRNA species of approximately 1.0 kilobase. The predicted precursor of ABP is composed of 201 amino acid residues and has a molecular weight of 21,976. The NH2-terminal sequence of 38 residues is hydrophobic and may be a signal peptide for translocation of the ABP across the membrane of the endoplasmic reticulum. The mature ABP, composed of 163 residues with a molecular weight of 18,352, contains a potential N-glycosylation site (Asn-Thr-Thr), and the COOH-terminal tetrapeptide (Lys-Asp-Glu-Leu) may be a signal for retention of the ABP in the lumen of the endoplasmic reticulum.     

New insight into the molecular evolution of the somatostatin family

The present review describes the molecular evolution of the somatostatin family and its relationships with that of the urotensin II family. Most of the somatostatin sequences collected from different vertebrate species can be grouped as the products of at least four loci. The somatostatin 1 (SS1) gene is present in all vertebrate classes from agnathans to mammals. The SS1 gene has given rise to the somatostatin 2 (SS2) gene by a segment/chromosome duplication that is probably the result of a tetraploidization event according to the 2R hypothesis. The somatostatin-related peptide cortistatin, first identified in rodents and human, is the counterpart of SS2 in placental mammals. In fish, the existence of two additional somatostatin genes has been reported. The first gene, which encodes a peptide usually named somatostatin II (SSII), exists in almost all teleost species investigated so far and is thought to have arisen through local duplication of the SS1 gene. The second gene, which has been characterized in only a few teleost species, encodes a peptide also named SSII that exhibits a totally atypical structure. The origin of this gene is currently unknown. Nevertheless, because the two latter genes are clearly paralogous genes, we propose to rename them SS3 and SS4, respectively, in order to clarify the current confusing nomenclature. The urotensin II family consists of two genes, namely the urotensin II (UII) gene and the UII-related peptide (URP) gene. Both UII and URP exhibit limited structural identity to somatostatin so that UII was originally described as a "somatostatin-like peptide". Recent comparative genomics studies have revealed that the SS1 and URP genes, on the one hand, and the SS2 and UII genes, on the other hand, are closely linked on the same chromosomes, thus confirming that the SS1/SS2 and the UII/URP genes belong to the same superfamily. According to these data, it appears that an ancestral somatostatin/urotensin II gene gave rise by local duplication to a somatostatin ancestor and a urotensin II ancestor, whereupon this pair was duplicated (presumably by a segment/chromosome duplication) to give rise to the SS1-UII pair and the SS2-URP pair.