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. These two peptides together with two related CRF-like peptides, namely, sauvagine and urocortin, were also tested in cAMP assays; for cells expressing the salmon CRF(1) receptor, EC(50) values for the stimulation of cAMP production were between 4.5+/-1.8 and 15.3+/-3.1 nM. For the salmon CRF(2) receptor, the corresponding values were: rat/human CRF, 9.4+/-0.4 nM; urotensin I, 21.2+/-2.1 nM; sauvagine, 0.7+/-0.1 nM; and urocortin, 2.2+/-0.7 nM. We have also functionally coupled the O. keta CRF(1) receptor, in Xenopus laevis oocytes, to the endogenous Ca(2+)-activated chloride conductance by co-expression with the G-protein alpha subunit, G(alpha16). The EC(50) value for channel activation by rat/human CRF (11.2+/-2.6 nM) agrees well with that obtained in cAMP assays (15.3+/-3.1 nM). We conclude that although sauvagine is 13- and 30-fold more potent than rat/human CRF and urotensin I, respectively, in activating the salmon CRF(2) receptor, neither receptor appears able to discriminate between the native ligands CRF and urotensin I.

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. It is postulated that ccTRH-R2 and rat TRH-R2 are members of the same TRH-R subfamily 2. Functional expression of ccTRH receptors in human embryonic kidney cells and in Xenopus laevis oocytes demonstrated that both ccTRH receptors were fully functional in both systems. Oocytes expressing either receptor responded to the application of TRH by an induction of membrane chloride currents, indicating that ccTRH-R of both subtypes are coupled to the inositol phosphate/calcium pathway. The analysis of genomic clones revealed, for the first time, both similarities and differences in the structure of TRH-R subtype genes. Both ccTRH-R genes contained an intron within the coding region at the beginning of transmembrane domain (TM) 6. The position of this intron is highly conserved, as it was found at an identical position in the human TRH-R1 gene. The ccTRH-R2 gene contained an additional intron at the end of TM 3 that was not found in any of the TRH-R1 genes identified so far. The analysis of the gene structure of ccTRH-R and the amino acid sequence comparisons of mammalian and teleost TRH-R of both subtypes suggest that TRH receptors have been highly conserved during the course of vertebrate evolution. A common ancestral TRH receptor gene that could be found much earlier in evolution, possibly in invertebrates, might be the origin of ccTRH-R genes.

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 = 0.63 +/- 0.15 nM) and for the nonselective antagonist naloxone (Kd = 3.1 +/- 1.3 nM). The receptor had negligible affinity for U50488 and [D-Pen2, D-Pen5]enkephalin (DPDPE), which are kappa- and delta-opioid receptor selective agonists, respectively. Stimulation of transfected cells with 1 microM DAMGO lowered forskolin-induced cAMP levels, an effect that could be reversed by naloxone. Experiments in Xenopus oocytes have demonstrated that the fish opioid receptor can, in an agonist-dependent fashion, activate a coexpressed mouse G-protein-gated inward-rectifying potassium channel (GIRK1). The identification of six distinct fish opioid receptor-like proteins suggests that additional mammalian opioid receptors remain to be identified at the molecular level. Furthermore, our data indicate that the mu-opioid receptor arose very early in evolution, perhaps before the appearance of vertebrates, and that the pharmacological and functional properties of this receptor have been conserved over a period of approximately 400 million years implying that it fulfills an important physiological role.

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. These results are complemented by a molecular model of the vasotocin receptor obtained by aligning its sequence with those of other G-protein coupled receptors as well as that of bacteriorhodopsin. The model indicates that amino acid residues of transmembrane regions II-VII that are located close to the extracellular surface also contribute to the binding of vasotocin.

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. Abundant SVG-ir varicose fibres were seen in the granulosa layer of the cerebellum, the nucleus isthmi, and the obex of the spinal cord. SVG-ir fibres were also seen by the alar plate of the rombencephalon. In the diencephalon, the antiserum stained parvocellular neurons of the preoptic nucleus (PON) which extended their dendrites into the cerebro-spinal fluid (CSF) of the third ventricle and projected their ependymofugal fibres to the zona externa (ZE) of the median eminence. Immunopositive fibres were also present in the medial forebrain bundle at the chiasmatic field, the posterior thalamus, the pretectal gray, and the ventrocaudal hypothalamus. In the telencephalon (forebrain), SVG-ir fibres were seen in the medial septum, the lateral septum, and the amygdala. The SVG immunoreactivity could not be detected after using the SVG antiserum previously immunoabsorbed with synthetic SVG (0.1 microM), but immunoblock of the antiserum with sucker (Catostomus commersoni) urotensin I (sUI), sole (Hippoglossoides elassodon) urotensin I, sucker CRF, rat/human CRF, or ovine CRF (0.1-10 microM) did not eliminate visualization of the immunoreactivity. In radioimmunoassay, the SVG antiserum did not crossreact with sUI, or the SVG fragments SVG1-16, SVG16-27, and SVG26-34, but it recognized the C-terminal fragment SVG35-40. Crossreaction with mammalian ovine CRF and rat/human CRF was negligible. Both hypothalamic and mesencephalic extracts gave parallel displacement curves to SVG. The results suggest the presence in the bullfrog brain of a SVG-like neuropeptide, i.e., a peptide of the CRF family, that either is SVG or shares high homology with the C-terminus of that peptide. The function of this neuropeptide in amphibians is not known at this time, but based on its anatomical distribution to the ZE it could affect the release of adrenocorticotropin (ACTH) or other substances from the amphibian pars distalis. Involvement of the SVG-like peptide in behavioural (forebrain), visual (thalamus-tegmentum mesencephali-pretectal gray-optic tectum), motor coordination (cerebellum), and autonomic (spinal) functions, as well as an undefined interaction with the CSF in the bullfrog, seems likely.

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. 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.

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. Oocytes that express the cloned receptor respond to the application of isotocin by an induction of membrane chloride currents indicating that it is coupled to the inositol phosphate/calcium pathway. The isotocin receptor (ITR) can also be activated by vasotocin, mesotocin, oxytocin and Arg-vasopressin, although these have lower potencies than isotocin. ITR-encoding mRNA has been detected in brain, intestine, bladder, skeletal muscle, lateral line, gills and kidney indicating that this receptor may mediate a variety of physiological functions.

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. The pharmacological properties of the expressed AVT receptor show that it represents, or is closely related to, an ancestral nonapeptide receptor: oxytocin, aspargtocin, mesotocin, and vasopressin activated the receptor, but other members of the vasopressin/oxytocin family tested showed little or no potency; antagonists of the mammalian vasopressin V1 and oxytocin receptors blocked the AVT response. Comparison of AVT receptor sequences spanning transmembrane domains two to five, deduced by cloning cDNAs from the Pacific salmon Oncorhynchus kisutch, the cave-dwelling fish Astyanax fasciatus, and the anuran Xenopus laevis, with those of their mammalian counterparts emphasizes amino acid residues that are involved in hormone binding. The presence of a 5.0-kb transcript in various teleost tissues (pituitary, liver, gills, swim bladder, and lateral line) points to a physiological role for the fish AVT receptor in metabolic, osmoregulatory, and sensory processes.

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. In the hypothalamus, corticotropin-immunoreactive parvicellular perikarya were found in the infundibular nucleus and in dendritic projections to the infundibular recess. In addition, ependymofugal corticotropin-immunoreactive fibres were found to terminate in the ventral hypothalamus. Corticotropin releasing factor-immunoreactive neurons were found in the rostral portion of the ventral hypothalamus (tuberal nucleus), and in the vicinity of the rostral aspect of the lateral recess. These cells projected to the dorsal hypothalamus, the ventral hypothalamus, the median eminence, the anterior and posterior telencephalon, the tegmentum mesencephali, and the pars nervosa of the pituitary. An affinity-purified UI antiserum failed to stain the sturgeon hypothalamus. Corticotrophs in the rostral pars distalis of the pituitary were also corticotropin-immunoreactive. In the neurointermediate lobe, only about 50% of cells of the pars intermedia appeared to be corticotropin-positive, the rest appeared unstained. These results suggest that the presence of corticotropin-like and corticotropin releasing factor-like peptides in the brain is a relatively early event in vertebrate evolution, already occurring in Chondrostean/Actinopterigian fishes, as exemplified by A. ruthenus. The close spatial relationship between corticotropin releasing factor immunoreactivity and corticotropin immunoreactivity in the ventral hypothalamus of A. ruthenus supports a possible interaction between the two systems in that area of the sturgeon brain. The pars intermedia might be an important site for corticotropin synthesis, even though the possibility cannot be excluded that the antiserum was recognizing the proopiomelanocortin molecule. The occurrence of corticotropin releasing factor immunoreactivity in the region of median eminence/pars intermedia of the sturgeon suggests that the sturgeon corticotropin releasing factor might regulate the adenohypophyseal release of proopiomelanocortin products in the same manner as in other vertebrates. The presence of extrahypothalamic corticotropin releasing factor-immunoreactive projections suggests further neuromodulatory functions for this peptide in A. ruthenus.

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. Immunoreactive fibres were present in the neuropile of the abdominal, pedal, pleural and cerebral ganglia, but not in the buccal ganglion. The identification of these neurons provides a morphological localization for vasopressin-like substances detected previously in CNS extracts of Aplysia californica. In addition, the possibility of electrophysiological studies involving the immunoreactive neurons identified in the present paper will allow a more direct approach to study the physiological role of vasopressin-like peptides in Aplysia.

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%.

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. The hagfish element, called Tes1, is flanked by inverted terminal repeats representing an example of the existence of a typical inverted terminal-repeat transposon within vertebrates. The presence of Tc1-like elements in nematodes, Drosophila, and cyclostomes indicates that these genetic elements have a much broader phylogenetic distribution than hitherto expected.

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)

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. Further analysis of the major AVT-IR peak showed it to be indistinguishable, in three distinct solvent systems, from the sea snail venom peptide Lys-conopressin G, but to be different from the vertebrate peptides [Arg8]VP (AVP), [Lys8]VP (LVP), AVT, oxytocin (OT), mesotocin, isotocin, aspargtocin, glumitocin, and valitocin, from the sea snail venom peptide Arg-conopressin S, and from the peptides [Lys8]VT and [Gln8]OT. In addition, the carboxymethylated (CM) A. kurodai peptide had the same HPLC retention time as CM-Lys-conopressin G. The HPLC/RIA results suggest that (i) based on the properties of the solvent systems used, the A. kurodai peptide has two basic amino acids (like the conopressins but unlike the vertebrate peptides), and (ii) there is a high probability that the A. kurodai peptide is identical with Lys-conopressin G.

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). 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.

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. 2. In the absence of extracellular Ca2+, 10(-7) M phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, induced a small but sustained contraction of the rat thoracic aorta strips within 60 min. Preincubation of the strips with 10(-7) M PMA for 60 min in Ca(2+)-free buffer, did not affect the 10(-8) M ET-1-induced contraction, but decreased the 5 x 10(-8) M phorbol 12,13-dibutyrate (PDB)-, or the 10(-7) M PMA-induced contraction, and potentiated the contraction induced by 10(-8) M urotensin II. Preincubation with 10(-8) M ET-1 (which induced maximum contraction) for 25 min in Ca(2+)-free buffer did not change the subsequent contraction induced by PMA (10(-7) M) or urotensin II (10(-8) M) but gave a somewhat lower maximum tension than in non-treated strips. 3. Calyculin-A, a potent inhibitor of phosphatase, also induced a contraction of the Ca(2+)-depleted muscle strips in Ca(2+)-free buffer. Preincubation of the strips with ET-1 (10(-8) M) or PMA (10(-7) M) decreased the calyculin-A (3 x 10(-8) M)-induced contraction.4. These results suggest that ET-1 may induce phosphorylation of an unknown protein either without an increase in myoplasmic Ca2 + concentration or, alternatively, with mobilization of intracellular Ca2+ from noradrenaline- and caffeine-insensitive Ca2 + sources, through a mechanism different from that of phorbol ester.

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). In contrast, urotensin I (U I) encoding mRNA is equally present in both tissues. In urophysectomized fish, U I mRNA is elevated especially in LTN tissue, while CRF mRNA levels remain more or less constant in the PON and LTN regions.

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)

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). A hydropathy profile predicts a secondary structure of the Na+/K+ ATPase alpha-subunit with at least eight membrane-spanning domains. Northern and southern blot analyses suggest the existence of two distinct Na+/K+ ATPase alpha-subunit genes in the sucker genome.

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. The interaction of PHM with the vasoconstrictor amines, norepinephrine, histamine, and serotonin, was examined using isolated strips of the bovine middle cerebral artery. PHM shifted the concentration-response curves for vasocontraction by norepinephrine and histamine to the right but did not affect the vasocontraction induced by serotonin. These results suggest that PHM may have a role in the regulation of the cerebral circulation.