Cloning and functional pharmacology of two corticotropin-releasing factor receptors from a teleost fish.

Although it is well established that fish possess corticotropin-releasing factor (CRF) and a CRF-like peptide, urotensin I, comparatively little is known about the pharmacology of their cognate receptors. Here we report the isolation and functional expression of two complementary DNAs (cDNAs), from the chum salmon Oncorhynchus keta, which encode orthologues of the mammalian and amphibian CRF type 1 (CRF(1)) and type 2 (CRF(2)) receptors. Radioligand competition binding experiments have revealed that the salmon CRF(1) and CRF(2) receptors bind urotensin I with approximately 8-fold higher affinity than rat/human CRF.

Cloning of two thyrotropin-releasing hormone receptor subtypes from a lower vertebrate (Catostomus commersoni): functional expression, gene structure, and evolution.

A PCR approach was used to clone thyrotropin-releasing hormone receptors (TRH-R) from the brain and anterior pituitary of the teleost Catostomus commersoni (cc), the white sucker. Two distinct TRH-R, designated ccTRH-R1 and ccTRH-R2, were identified. ccTRH-R1 was similar to mammalian TRH-R of the subtype 1, whereas ccTRH-R2 exhibited the highest identity (61% at the amino acid level) with the recently discovered rat TRH-R2.

Opioid receptors from a lower vertebrate (Catostomus commersoni): sequence, pharmacology, coupling to a G-protein-gated inward-rectifying potassium channel (GIRK1), and evolution.

The molecular evolution of the opioid receptor family has been studied by isolating cDNAs that encode six distinct opioid receptor-like proteins from a lower vertebrate, the teleost fish Catostomus commersoni. One of these, which has been obtained in full-length form, encodes a 383-amino acid protein that exhibits greatest sequence similarity to mammalian mu-opioid receptors; the corresponding gene is expressed predominantly in brain and pituitary. Transfection of the teleost cDNA into HEK 293 cells resulted in the appearance of a receptor having high affinity for the mu-selective agonist [D-Ala2, MePhe4-Gly-ol5]enkephalin (DAMGO) (Kd = 0.

Mutational analysis and molecular modeling of the nonapeptide hormone binding domains of the [Arg8]vasotocin receptor.

To identify determinants that form nonapeptide hormone binding domains of the white sucker Catostomus commersoni [Arg8]vasotocin receptor, chimeric constructs encoding parts of the vasotocin receptor and parts of the isotocin receptor have been analyzed by [(3,5-3H)Tyr2, Arg8]vasotocin binding to membranes of human embryonic kidney cells previously transfected with the different cDNA constructs and by functional expression studies in Xenopus laevis oocytes injected with mutant cRNAs. The results indicate that the N terminus and a region spanning the second extracellular loop and its flanking transmembrane segments, which contains a number of amino acid residues that are conserved throughout the nonapeptide receptor family, contribute to the affinity of the receptor for its ligand. Nonapeptide selectivity, however, is mainly defined by transmembrane region VI and the third extracellular loop.

Hypothalamic and extrahypothalamic sauvagine-like immunoreactivity in the bullfrog (Rana catesbeiana) central nervous system.

In the present study, immunocytochemistry and radioimmunoassay were used to investigate the presence of sauvagine in both hypothalamic and extrahypothalamic areas of the central nervous system (CNS) of the bullfrog (Rana catesbeiana) using a specific antiserum raised against synthetic non-conjugated sauvagine (SVG), a frog (Phyllomedusa sauvagei) skin peptide of the corticotropin-releasing factor (CRF) family. Sauvagine-immunoreactive (SVG-ir) bipolar neurons were found in the nucleus of the fasciculus longitudinalis medialis located in the rostral mesencephalic tegmentum. In the tectal mesencephalon, beaded SVG-ir fibres were present in the optic tectum, and in the torus semicircularis.

Presence of sauvagine-like epitopes in the interrenal gland of the bullfrog Rana catesbeiana.

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.

Teleost isotocin receptor: structure, functional expression, mRNA distribution and phylogeny.

A cDNA encoding a receptor for the oxytocin-related peptide isotocin has been identified by screening a lambda gt11 library constructed from poly(A)+ RNA of the hypothalamic region of the teleost Catostomus commersoni. The probe used was obtained by PCR amplification of white sucker genomic DNA using degenerate primers based on conserved sequences in the mammalian receptor counterparts. The full-length cDNA specifies a polypeptide of 390 amino acid residues that displays the typical hydrophobicity profile of a seven transmembrane domain receptor and which exhibits greatest similarity to mammalian oxytocin receptors.

Structure, function, and phylogeny of [Arg8]vasotocin receptors from teleost fish and toad.

[Arg8]Vasotocin (AVT) is considered to be the most primitive known vertebrate neurohypophyseal peptide of the vasopressin/oxytocin hormone family and may thus be ancestral to all the other vertebrate peptide hormones. The molecular evolution of the corresponding receptor family has now been studied by cloning an AVT receptor, consisting of 435 amino acid residues, from the teleost fish, the white sucker Catostomus commersoni. Frog oocytes injected with the AVT receptor-encoding cRNA respond to the application of AVT, but not to its structural and functional counterpart isotocin, by an induction of membrane chloride currents indicating the coupling of the AVT receptor to the inositol phosphate/calcium pathway.

Comparative localization of corticotropin and corticotropin releasing factor-like peptides in the brain and hypophysis of a primitive vertebrate, the sturgeon Acipenser ruthenus L.

The sturgeon is a primitive actinopterigian fish that, unlike modern teleosts, possess a portal vascular system that connects a true median eminence with the anterior pituitary as in mammals. The occurrence and localization of corticotropin and corticotropin releasing factor-like immunoreactivies were examined in the brain of the sturgeon (Acipenser ruthenus L.) by immunocytochemistry with antisera raised against synthetic non-conjugated human corticotropin, and rat/human corticotropin releasing factor.

Localization of vasopressin-like immunoreactivity in the CNS of Aplysia californica.

Chromatographic and immunological evidence indicates that a vasopressin-like peptide might be present in the CNS of Aplysia californica, and that this peptide may be involved in modulating the behaviour of the gill. Immunocytochemical techniques using antisera raised against various vasopressin-like peptides were used to localize the sites containing these peptides in the CNS of Aplysia. Vasopressin-like immunoreactivity was found to be restricted to one single neuron in the abdominal ganglion and two small neurons located bilaterally in each pedal ganglion.

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.

Presence of a member of the Tc1-like transposon family from nematodes and Drosophila within the vasotocin gene of a primitive vertebrate, the Pacific hagfish Eptatretus stouti.

Molecular cloning of the vasotocin gene of a cyclostome, the Pacific hagfish Eptatretus stouti, reveals, in contrast to other known members of the vertebrate vasopressin/oxytocin hormone gene family, an unusual exon-intron organization. Although the location of three exons and two introns is conserved, an additional intron is present 5' of the coding region of the hagfish gene. The third intron, which is greater than 14 kilobase pairs in size, contains on the opposite DNA strand to that encoding vasotocin an open reading frame exhibiting striking similarity to the putative transposase of Tc1-like nonretroviral mobile genetic DNA elements, so far reported only from nematodes and Drosophila.

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.

A vasotocin-like peptide in Aplysia kurodai ganglia: HPLC and RIA evidence for its identity with Lys-conopressin G.

The presence of a vasopressin (VP)- or vasotocin (VT)-like peptide in the central nervous system of the gastropod mollusc Aplysia has been indicated previously. In the case of Aplysia californica, HPLC and RIA evidence suggested the peptide was VT-like but not identical with the nonmammalian vertebrate peptide [Arg8]VT (AVT). In the present study, anterior ganglia extracts from the related species Aplysia kurodai were analyzed by HPLC followed by RIA.

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

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).

Contraction of rat thoracic aorta strips by endothelin-1 in the absence of extracellular Ca2+.

1. Endothelin-1 (ET-1) caused a concentration-dependent contraction of helical strips from rat thoracic aorta in the absence of extracellular Ca2+. The Ca(2+)-depleted muscle strips, prepared by three repeated applications of 10(-2) M caffeine or 10(-6) M noradrenaline in Ca(2+)-free buffer, were contracted by 10(-8) M ET-1 in the same manner as non-treated strips.

Corticotropin-releasing factor (CRF) gene family in the brain of the teleost fish Catostomus commersoni (white sucker): molecular analysis predicts distinct precursors for two CRFs and one urotensin I peptide.

Molecular cloning experiments indicate the presence of two distinct CRF genes in the sucker genome encoding independent 162-amino-acid precursors, which both consist of a signal sequence, succeeded by a cryptic peptide and subsequently by the hormone moiety. The two 41-amino-acid CRF peptides differ by an Ala-->Val substitution at amino acid position 28. CRF transcripts are primarily found in the sucker pre-optic nucleus (PON), to a much lesser extent in the lateral tuberal nucleus (LTN).

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.

Sodium and potassium ATPase of the teleost fish Catostomus commersoni. Sequence, protein structure and evolutionary conservation of the alpha-subunit.

The alpha-subunit of a Na+/K+ ATPase has been cloned by analysing a lambda gt11 library constructed from polyA+ RNA from the hypothalamic region of the teleost fish Catostomus commersoni (white sucker). The cDNA clone consists of 3853 bp and predicts a protein of 1027 amino-acid residues. Alignment of the sucker sequence with protein sequences previously published for alpha-subunits from various species reveals a high degree of homology throughout the entire sequence containing five potential sites for N-glycosylation, a phosphorylation site and a site for binding fluorescein 5'-isothiocyanate (FITC).

Cerebral vascular effects of peptide histidine methionine in canine and bovine species.

Peptide histidine methionine (PHM) is a neuropeptide with structural homology to vasoactive intestinal peptide (VIP), itself a putative vasodilatory neurotransmitter. Intra-arterial administration of PHM caused a transient, dose-dependent increase in canine vertebral artery blood flow in vivo. PHM was less potent in this effect than VIP.

Contraction of porcine ocular ciliary muscle by epidermal growth factor-urogastrone.

Epidermal growth factor-urogastrone (EGF) caused a concentration-dependent contractile response in porcine ocular ciliary muscle preparations. The half-maximal contraction was observed at 23 ng/ml EGF (4 nM). The contractile action of EGF, which was not abolished by the removal of extracellular calcium, was not affected by atropine, tetrodotoxin, phentolamine and indomethacin.

Isolation, Amino-Acid Sequence, Synthesis and Biological Properties of Urotensin I from Hippoglossoides elassodon.

Abstract A 41-residue urotensin I neuropeptide (H-UI) was isolated from urophyses of the marine teleost Hippoglossoides elassodon (the flathead sole). The peptide was recognized by its partial cross-reactivity in a radioimmunoassay developed for Catostomus (sucker) Ul (S-UI), and was purified by reversed-phase high-performance liquid chromatography. The amino-acid sequence was shown to be H-Ser-Glu-Glu-Pro-Pro-Met-Ser-lle-Asp-Leu-Thr-Phe-His-Met-Leu-Arg-Asn-Met-lle-His-Arg-Ala-Lys-Met-Glu-Gly-Glu-Arg-Glu-Gln-Ala-Leu-lle-Asn-Arg-Asn-Leu-Leu-Asp-Glu-Val-NH(2).

Vasopressin and oxytocin in rat brain in response to prostaglandin fever.

Urethan-anesthetized rats were used to identify effective stimuli for the release of the peptides arginine vasopressin (AVP) and oxytocin into the ventral septal area (VSA) of the brain. Febrile responses to intracerebroventricular injection of prostaglandin E1 (PGE1) were observed in rats whose body temperatures were maintained at 35, 37, or 39 degrees C. Microinjection of the AVP antagonist d(CH2)5Tyr(Me)AVP into the VSA enhanced fever only when PGE1 administration was associated with a significant rise in body temperature.

Relaxation of isolated bovine coronary arteries by vasoactive intestinal peptide.

The relaxant action of vasoactive intestinal peptide (VIP) was investigated using helical strips of four major branches of bovine coronary arteries. The concentration of VIP causing 50 percent of maximal relaxation ranged from 23 to 90 nM. Preincubation of arterial strips with VIP shifted the concentration-response curves for contractions elicited by potassium chloride or prostaglandin F2 alpha to the right.

CRF, urotensin I, and sauvagine stimulate the release of POMC-derived peptides from goldfish neurointermediate lobe cells.

In teleost fishes, the melanotropes of the neurointermediate lobe of the pituitary gland release numerous peptides--adrenocorticotropin (ACTH), melanotropin (MSH), lipotropin (LPH), corticotropin-like intermediate lobe peptide (CLIP), and endorphin--which are derived from the precursor molecule proopiomelanocortin. Superfused, isolated, dispersed goldfish neurointermediate lobe cell columns were used to investigate the release of immunoreactive (ir) alpha-MSH and ir ACTH from goldfish melanotropes. Stimulation of neurointermediate lobe cell columns with pulses of the structurally homologous peptides, Catostomus urotensin I (UI), ovine corticotropin-releasing factor (oCRF), or sauvagine, produced a significant increase in the concomitant release of ir alpha-MSH and ir ACTH.