Identification and expression of two oxytocin/vasopressin-related peptides in the cuttlefish Sepia officinalis

Two novel members of the oxytocin/vasopressin superfamily have been identified in the cephalopod Sepia officinalis. Oxytocin/vasopressin gene sequences were cloned by Race PCR. The two precursors we identified exhibit the classical organization of OT/VP superfamily precursors: a signal peptide followed by a nonapeptide and a neurophysin domain. The neurophysin domain is entirely conserved for the cuttlefish precursors, but the nonapeptides and the signal peptides differ. The first nonapeptide, called sepiatocin, is highly homologous to Octopus vulgaris octopressin. The second nonapeptide, called pro-sepiatocin, shows sequence homologies with a Crustacean oxytocin/vasopressin-like peptide identified in Daphnia culex and with a novel form of oxytocin described in New World monkeys. The expression of pro-sepiatocin is restricted to the supraesophageal and subesophageal masses of the brain whereas sepiatocin is expressed in the entire central nervous system. Sepiatocin, as described for octopressin, modulates the contractile activity of several muscles such as penis, oviduct and vena cava muscles; this suggests its involvement in reproduction and blood circulation. Pro-sepiatocin is released in the hemolymph; it is a neurohormone able to target numerous peripheral organs.     

EGFP-Tagged Vasopressin Precursor Protein Sorting Into Large Dense Core Vesicles and Secretion From PC12 Cells

1. Hypothalamic magnocellular neurons synthesize, store, and secrete large quantities of the neuropeptides, vasopressin (VP) and oxytocin (OT), which are synthesized as protein precursors also containing proteins called neurophysins. These protein precursors are sorted through the regulated secretory pathway (RSP), packaged into large dense core vesicles LDCVs, and their peptide products are secreted from nerve terminals in the posterior pituitary.2. It has been hypothesized that this efficient packaging is dependent on the interaction of the peptide with neurophysin in a complex that forms the granule core. To test this, PC12 cells were transfected with vasopressin precursor DNA constructs that either contained or deleted the neurophysin moiety and tagged with enhanced green fluorescent protein (EGFP) as reporters. The intracellular routing and secretion of the EGFP-tagged VP precursor proteins were studied by in differentiated PC12 cells by fluorescence microscopy, electron microscopic immunocytochemistry, and fluorescent imaging techniques.3. The data showed that only when the neurophysin was present in the VP precursor construct did the fluorescent fusion protein become routed to the RSP and get efficiently packaged into LDCVs and secreted. These data are consistent with the view that routing of the precursor to LDCVs requires the amino acids that encode the intravesicular chaperone, neurophysin.     

Comparative aspects of invertebrate neuropeptides

1. We searched for bioactive peptides, most of which were considered to be neuropeptides, in various animals of several phyla. These peptides were compared with each other and with peptides identified by many other investigators. Consequently, we found that structures of neuropeptides are generally conserved in each phylum. 2. We also found some exceptional interesting aspects. First, there are a number of peptide groups whose members are distributed among several phyla. Second, there are many structural similarities between molluscan and annelidan peptides as if molluscs and annelids were the animals in a phylum. Third, certain toxic peptides of invertebrates are closely related to vertebrate neuropeptides. 3. In addition to the above phylogenetic aspects, we found some other interesting aspects. A wide structural variety of members of a peptide group is generally found in invertebrate species. Invertebrate muscles seem to be generally regulated not only by some or several classical non-peptidic neuromediators but also by various peptidic neuromediators. Peptides containing a D-amino acid residue are not rare.     

Identification and localization of material with gastrin-like immunoreactivity in the neural ganglion of a protochordate, Ciona intestinalis

Little is known of the identity of gastrin/cholecystokinin (CCK)-like peptides in protochordates. These animals are at a level of organization corresponding to that from which the vertebrate line arose; in order to shed light on the origins of gastrin/CCK-like peptides, we have studied by immunochemical methods these peptides in a protochordate, Ciona intestinalis. In radioimmunoassay, boiling water extracts of the neural ganglion reacted with C-terminal specific gastrin/CCK antibodies, but not N-terminal or intact G17 specific antibodies. Of particular importance was the fact that a gastrin antibody which reacts weakly with CCK8 showed full activity with the Ciona material, suggesting that it resembles the C-terminus of gastrin. A single major peak was found by gel filtration and HPLC. In immunohistochemistry, nerve cell bodies were found in the cortical regions of the ganglion, and abundant fibres ramified in the central neuropile. We conclude that peptides of the gastrin/CCK series occur in nervous tissue in protochordates, and that while they are distinguishable from known forms of both gastrin and CCK, they resemble C-terminal fragments of the mammalian gastrins.     

Immunohistochemical evidence for the presence, localization and partial coexistence of insulin, insulin-like growth factor I and relaxin in the protochordate Ciona intestinalis

The occurrence and coexistence of peptides of the insulin-like growth factor (IGF)/insulin superfamily were investigated in the ovary and gastro-intestinal tract of the protochordate Ciona intestinalis. Antisera specific for mammalian IGF-I, insulin and relaxin were used in a double-immunofluorescence method on paraffin sections and with an immunogold technique on consecutive semi-thin sections. IGF-I and relaxin immunoreactions but no insulin immunoreactions occurred in the ovary and were confined to medium-sized and mature follicle cells. Two subpopulations of reacting follicular cells were present: those containing only IGF-I immunoreactivity (5%) and those containing IGF-I and relaxin immunoreactivities (95%). In the gastro-intestinal tract, IGF-I and insulin immunoreactions coexisted, whereas no relaxin immunoreactions were obtained. Gel chromatography and radioimmunoassay in Ciona ovary revealed IGF-I immunoreactivity in two peaks with apparent molecular masses of approximately 16 kDa and 3 kDa. The present results indicate that (1) the same IGF-I-related peptide probably occurs in gastro-intestinal tract and ovary, (2) three different members of the insulin/IGF family of peptides are probably present in protochordates, (3) different types of coexistence of these peptides seem to exist in protochordates, i.e. an IGF-I-related peptide and an insulin-related peptide in the digestive tract and, as shown previously, in central nervous system, and the IGF-I-related peptide and relaxin in the ovary, (4) an IGF-I-related peptide and relaxin may be involved in oocyte maturation in the protochordate ovary.     

Absence of negative feedback by oxytocin on release from magnocellular neurones in conscious rats.

Peripheral administration of vasopressin (VP) was previously shown to exert a negative feedback influence on its own release and on the release of oxytocin (OT). In this study we examined the possible influence that OT has on the function of hypothalamic magnocellular neurones. Oxytocin was administered intraperitoneally and its effects on release from VP neurones and from OT neurones were determined as indexed by plasma concentrations of vasopressin-associated neurophysin ([VP-RNP]) and oxytocin-associated neurophysin ([OT-RNP]) under basal conditions and conditions of high plasma osmolality (Posm) induced by acute salt loading. Studies were performed on conscious, chronically instrumented Long-Evans rats. Oxytocin (1 nmol or 10 nmol) dissolved in 1 mL of 0.9% saline was administered intraperitoneally to animals 1 h before they received an intravenous infusion of hypertonic saline over 60 min at a rate designed to raise Posm by approximately 0.75 mosmol.min-1. Intraperitoneal injection of vehicle or 1 nmol of OT did not significantly alter [VP-RNP], [OT-RNP], or basal Posm. Administration of 10 nmol OT also had no effect on [VP-RNP] or [OT-RNP], but this dose of peptide significantly lowered basal Posm (299 +/- 2 to 290 +/- 2 mosmol/kg H2O, p less than 0.001). Both doses of OT did not significantly alter the responsiveness of VP neurones to hyperosmotic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recognition properties of antisense peptides to Arg8-vasopressin/bovine neurophysin II biosynthetic precursor sequences

We studied the interaction properties of synthetic antisense (AS) peptides encoded in the antisense strand of DNA corresponding to the N-terminal 20-residue sequence of the biosynthetic precursor of Arg8-vasopressin (AVP) and its binding protein bovine neurophysin II (BNPII). Binding affinities of sense polypeptides AVP and BNPII with AS peptides were measured by analytical affinity chromatography, in each case by the extent of chromatographic retardation of a soluble polypeptide interactor on an affinity matrix containing the other interactor as the immobilized species. Chromatographically calculated dissociation constants ranged from 10(-3) to 10(-6) M. Experiments were carried out to define the selectivity and underlying forces involved in the AS peptide interactions. For AS peptide elutions on sense peptide affinity supports, reduced binding affinity with increasing 1-propanol concentration and ionic strength suggested the presence of both ionic and hydrophobic contributions to AS peptide/immobilized sense peptide recognition. This same conclusion was reached with the antisense peptides as the immobilized species and measurement of elution of sequence-simplified, truncated, and charge-depleted forms of sense peptides. Immobilized AS 20-mer affinity matrix differentially retarded AVP versus oxytocin (OT) and BNPII versus BNPI (the neurophysin related biosynthetically to OT) and was used to separate these polypeptides from acid extracts of bovine posterior pituitaries. In addition, immobilized AS 12-mer corresponding to AVP-Gly-Lys-Arg could be used to separate AVP from OT. The results confirm that antisense peptides recognize sense peptides with significant selectivity in the AVP/BNPII precursor case.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tachykinin and tachykinin receptor of an ascidian, Ciona intestinalis: evolutionary origin of the vertebrate tachykinin family

Tachykinins (TKs) are the most prevalent vertebrate brain/gut peptides. In this study, we originally identified authentic TKs and their receptor from a protochordate, Ciona intestinalis. The Ciona TK (Ci-TK) precursor, like mammalian gamma-preprotachykinin A (gamma-PPTA), encodes two TKs, Ci-TK-I and -II, including the -FXGLM-NH(2) vertebrate TK consensus. Mass spectrometry of the neural extract revealed the production of both Ci-TKs. Ci-TK-I contains several Substance P (SP)-typical amino acids, whereas a Thr is exceptionally located at position 4 from the C terminus of Ci-TK-II. The Ci-TK gene encodes both Ci-TKs in the same exon, indicating no alternative generation of Ci-TKs, unlike the PPTA gene. These results suggested that the alternative splicing of the PPTA gene was established during evolution of vertebrates. The only Ci-TK receptor, Ci-TK-R, was equivalently activated by Ci-TK-I, SP, and neurokinin A at physiological concentrations, whereas Ci-TK-II showed 100-fold less potent activity, indicating that the ligand selectivity of Ci-TK-R is distinct from those of vertebrate TK receptors. Ci-TK-I, like SP, also elicited the typical contraction on the guinea pig ileum. The Ci-TK gene was expressed in neurons of the brain ganglion, small cells in the intestine, and the zone 7 in the endostyle, which corresponds to the vertebrate thyroid gland. Furthermore, the Ci-TK-R mRNA was distributed in these three tissues plus the gonad. These results showed that Ci-TKs play major roles in sexual behavior and feeding in protochordates as brain/gut peptides and endocrine/paracrine molecules. Taken together, our data revealed the biochemical and structural origins of vertebrate TKs and their receptors.     

Complement in urochordates: cloning and characterization of two C3-like genes in the ascidian Ciona intestinalis

The recent identification of complement components in deuterostome invertebrates has indicated the presence of a complement system operating via an alternative pathway in echinoderms and tunicates and via a MBL-mediated pathway thus far identified only in tunicates. Here, we report the isolation of two C3-like genes, CiC3-1 and CiC3-2, from blood cell total RNA of the ascidian Ciona intestinalis. The deduced amino acid sequences of both Ciona C3-like proteins exhibit a canonical processing site for alpha and beta chains, a thioester site with an associated catalytic histidine and a convertase cleavage site, thus showing an overall similarity to the other C3 molecules already characterized. Southern blotting analysis indicated that each gene is present as a single copy per haploid genome. In situ hybridization experiments showed that both CiC3-1 and CiC3-2 are expressed in one type of blood cell, the compartment cells. Two polyclonal antibodies, raised against two deduced peptide sequences in the alpha chain of CiC3-1 and CiC3-2, allowed the identification by Western blot of a single band in the blood serum, of about M(r)150,000. A phylogenetic tree, based on the alignment of CiC3-1 and CiC3-2 with molecules of the alpha(2)-macroglobulin superfamily, indicated that the Ciona C3s form a cluster with Halocynthia roretzi C3. The phylogenetic analysis also suggested that the duplication event from which the CiC3-1 and CiC3-2 genes originated occurred in the urochordate lineage after the separation of the Halocynthia and Ciona ancestor.     

Conopressin-T from Conus tulipa reveals an antagonist switch in vasopressin-like peptides

We report the discovery of conopressin-T, a novel bioactive peptide isolated from Conus tulipa venom. Conopressin-T belongs to the vasopressin-like peptide family and displays high sequence homology to the mammalian hormone oxytocin (OT) and to vasotocin, the endogenous vasopressin analogue found in teleost fish, the cone snail's prey. Conopressin-T was found to act as a selective antagonist at the human V 1a receptor. All peptides in this family contain two conserved amino acids within the exocyclic tripeptide (Pro7 and Gly9), which are replaced with Leu7 and Val9 in conopressin-T. Whereas conopressin-T binds only to OT and V 1a receptors, an L7P analogue had increased affinity for the V 1a receptor and weak V2 receptor binding. Surprisingly, replacing Gly9 with Val9 in OT and vasopressin revealed that this position can function as an agonist/antagonist switch at the V 1a receptor. NMR structures of both conopressin-T and L7P analogue revealed a marked difference in the orientation of the exocyclic tripeptide that may serve as templates for the design of novel ligands with enhanced affinity for the V 1a receptor.     

Precursors in the biosynthesis of vasopressin and oxytocin in the rat. Characteristics of all the components in high-performance liquid chromatography.

A reverse-phase high performance liquid-chromatography (h.p.l.c.) protocol has been developed, whereby all the major known posterior-pituitary components that are derived from the processing of pro-oxytocin and pro-vasopressin can be separated one from another. Thus, in a single chromatographic step, it has been possible to separate vasopressin (VP), oxytocin (OT), oxytocin-neurophysin (rOT-Np), vasopressin-neurophysin (rVP-Np) and vasopressin-glycopeptide (rVP-GP) from acid extracts of the neurointermediate lobes of rat pituitary glands. All these peptides except rVP-GP were labelled in the neural lobe by 24h after a hypothalamic injection of [35S]cysteine, whereas all except VP were labelled by 24h after a similar injection of [3H]leucine. Three major labelled proteins were isolated from 20 min [35S]cysteine-injected rats when extracts of the supraoptic nucleus were subjected to Sephadex G-75 chromatography, h.p.l.c. and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Immunoprecipitation with antisera raised against rat neurophysins, VP and OT revealed 21000- and 19000-mol.wt. common precursors to VP and rVP-Np and a 15000-mol.wt. common precursor to OT and rOT-Np. Some immunoreactive rVP-Np could occasionally be detected in the Vo of Sephadex G-75 chromatograms of Wistar rat supraoptic-nucleus extracts, but no evidence of [35S]neurophysin in this fraction was obtained from h.p.l.c. fingerprinting of its S-carboxymethylated tryptic digests. Radioimmunoassay for rVP-Np and rOT-Np revealed that about 70-80% of the total recovered immunoreactive neurophysin (IR-Np) in the supraoptic nucleus eluted from Sephadex G-75 and h.p.l.c. in the positions of rVP-Np and rOT-Np. Evidence is presented for an approx. 20000-mol.wt. rOT-Np in both Wistar and Brattleboro rats and for an approx. 20000-mol.wt. component in the Brattleboro rat that is recognized by vasopressin-neurophysin antisera.     

Evidence for conservation of the vasopressin/oxytocin superfamily in Annelida

Annetocin is a structurally and functionally oxytocin-related peptide isolated from the earthworm Eisenia foetida. We present the characterization of the annetocin cDNA. Sequence analyses of the deduced precursor polypeptide revealed that the annetocin precursor is composed of three segments: a signal peptide, an annetocin sequence flanked by a Gly C-terminal amidation signal and a Lys-Arg dibasic processing site, and a neurophysin domain, similar to other oxytocin family precursors. The proannetocin showed 37.4-45.8% amino acid homology to other prohormones. In the neurophysin domain, 14 cysteines and amino acid residues essential for association of a neurophysin with a vasopressin/oxytocin superfamily peptide were conserved, suggesting that the Eisenia neurophysin can bind to annetocin. Furthermore, in situ hybridization experiments demonstrated that the annetocin gene is expressed exclusively in neurons of the central nervous system predicted to be involved in regulation of reproductive behavior. These findings confirm that annetocin is a member of the vasopressin/oxytocin superfamily. This is the first identification of the cDNA encoding the precursor of an invertebrate oxytocin-related peptide and also the first report of the identification of an annelid vasopressin/oxytocin-related precursor.     

Neurohumoral effects of orphanin FQ/nociceptin: relevance to cardiovascular and renal function

Orphanin FQ/Nociceptin (OFQ/N) is a peptide whose structure resembles that of the endogenous opioid peptides (endorphins). OFQ/N and its receptor are distributed in neural tissue and brain regions involved in the regulation of pituitary hormone release. Functional studies have shown that this peptide evokes a unique pattern of cardiovascular and renal excretory responses. This review will focus on the neural and humoral effects of OFQ/N and how this peptide may participate in the regulation of cardiovascular and renal function.     

A genomewide analysis of genes for the heat shock protein 70 chaperone system in the ascidian Ciona intestinalis

Molecular chaperones play crucial roles in various aspects of the biogenesis and maintenance of proteins in the cell. The heat shock protein 70 (HSP70) chaperone system, in which HSP70 proteins act as chaperones, is one of the major molecular chaperone systems conserved among a variety of organisms. To shed light on the evolutionary history of the constituents of the chordate HSP70 chaperone system and to identify all of the components of the HSP70 chaperone system in ascidians, we carried out a comprehensive survey for HSP70s and their cochaperones in the genome of Ciona intestinalis. We characterized all members of the Ciona HSP70 superfamily, J-proteins, BAG family, and some other types of cochaperones. The Ciona genome contains 8 members of the HSP70 superfamily, all of which have human and protostome counterparts. Members of the STCH subfamily of the HSP70 family and members of the HSPA14 subfamily of the HSP110 family are conserved between humans and protostomes but were not found in Ciona. The Ciona genome encodes 36 J-proteins, 32 of which belong to groups conserved in humans and protostomes. Three proteins seem to be unique to Ciona. J-proteins of the RBJ group are conserved between humans and Ciona but were not found in protostomes, whereas J-proteins of the DNAJC14, ZCSL3, FLJ13236, and C21orf55 groups are conserved between humans and protostomes but were not found in Ciona. J-proteins of the sacsin group seem to be specific to vertebrates. There is also a J-like protein without a conserved HPD tripeptide motif in the Ciona genome. The Ciona genome encodes 3 types of BAG family proteins, all of which have human and protostome counterparts (BAG1, BAG3, and BAT3). BAG2 group is conserved between humans and protostomes but was not found in Ciona, and BAG4 and BAG5 groups seem to be specific to vertebrates. Members for SIL1, UBQLN, UBADC1, TIMM44, GRPEL, and Magmas groups, which are conserved between humans and protostomes, were also found in Ciona. No Ciona member was retrieved for HSPBP1 group, which is conserved between humans and protostomes. For several groups of the HSP70 superfamily, J-proteins, and other types of cochaperones, multiple members in humans are represented by a single counterpart in Ciona. These results show that genes of the HSP70 chaperone system can be distinguished into groups that are shared by vertebrates, Ciona, and protostomes, ones shared by vertebrates and protostomes, ones shared by vertebrates and Ciona, and ones specific to vertebrates, Ciona, or protostomes. These results also demonstrate that the components of the HSP70 chaperone system in Ciona are similar to but simpler than those in humans and suggest that changes of the genome in the lineage leading to humans after the separation from that leading to Ciona increased the number and diversity of members of the HSP70 chaperone system. Changes of the genome in the lineage leading to Ciona also seem to have made the HSP70 chaperone system in this species slightly simpler than that in the common ancestor of humans and Ciona.     

M-Ras evolved independently of R-Ras and its neural function is conserved between mammalian and ascidian, which lacks classical Ras

The Ras family small GTPases play a variety of essential roles in eukaryotes. Among them, classical Ras (H-Ras, K-Ras, and N-Ras) and its orthologues are conserved from yeast to human. In ascidians, which phylogenetically exist between invertebrates and vertebrates, the fibroblast growth factor (FGF)-Ras-MAP kinase signaling is required for the induction of neural system, notochord, and mesenchyme. Analyses of DNA databases revealed that no gene encoding classical Ras is present in the ascidians, Ciona intestinalis and Halocynthia roretzi, despite the presence of classical Ras-orthologous genes in nematode, fly, amphioxus, and fish. By contrast, both the ascidians contain single genes orthologous to Mras, Rras, Ral, Rap1, and Rap2. A single Mras orthologue exists from nematode to mammalian. Thus, Mras evolved in metazoans independently of other Ras family genes such as Rras. Whole-mount in situ hybridization showed that C. intestinalis Mras orthologue (Ci-Mras) was expressed in the neural complex of the ascidian juveniles after metamorphosis. Knockdown of Ci-Mras with morpholino antisense oligonucleotides in the embryos and larvae resulted in undeveloped tails and neuronal pigment cells, abrogation of the notochord marker brachyury expression, and perturbation of the neural marker Otx expression, as has been shown in the experiments of the FGF-Ras-MAP kinase signaling inhibition. Mammalian Ras and M-Ras mediate nerve growth factor-induced neuronal differentiation in rat PC12 cells by activating the ERK/MAP kinase pathway transiently and sustainedly, respectively. Activated Ci-M-Ras bound to target proteins of mammalian M-Ras and Ras. Exogenous expression of an activated Ci-M-Ras in PC12 cells caused ERK activation and induced neuritogenesis via the ERK pathway as do mammalian M-Ras and Ras. These results suggest that the ascidian M-Ras orthologue compensates for lacked classical Ras and plays essential roles in neurogenesis in the ascidian.     

Ascidians as a vertebrate-like model organism for physiological studies of Rho GTPase signaling

GTPases of the Rho family are evolutionarily conserved proteins that control cell shape dynamics during physiological processes as diverse as cell migration and polarity, axon outgrowth and guidance, apoptosis and phagocytosis. In mammals, 18 Rho proteins are distributed in 7 subfamilies. Rho, Rac and Cdc42 are the best-characterized ones, benefiting from the use of worm and drosophila, which only express these 3 subfamilies. An additional model would therefore help understand the physiological role of other mammalian subfamilies. We identified in genome databases the complete Rho family of two ascidians, Ciona intestinalis and Ciona savignyi, and showed that these families contain single ancestors of most mammalian Rho subfamilies. In Ciona intestinalis, all Rho genes are expressed and display specific developmental variations of mRNA expression during tadpole formation. Although C. intestinalis expresses five additional Rac compared to the closely related Ciona savignyi, only two appeared fully active in functional assays. Last, we identified in Ciona intestinalis database more than 50 Rho regulators (RhoGEFs and RhoGAPs) and 20 effector targets, whose analysis further supports the notion that Rho signaling components are of comparable complexity in mammals and ascidians. Since the tadpole of ascidians combines vertebrate-like developmental features with reduced cell number, particularly adapted to evolutionary and developmental biology studies, our data advocate this model for physiological studies of Rho signaling pathways.     

Discovery of sea urchin NGFFFamide receptor unites a bilaterian neuropeptide family

Neuropeptides are ancient regulators of physiology and behaviour, but reconstruction of neuropeptide evolution is often difficult owing to lack of sequence conservation. Here, we report that the receptor for the neuropeptide NGFFFamide in the sea urchin Strongylocentrotus purpuratus (phylum Echinodermata) is an orthologue of vertebrate neuropeptide-S (NPS) receptors and crustacean cardioactive peptide (CCAP) receptors. Importantly, this has facilitated reconstruction of the evolution of two bilaterian neuropeptide signalling systems. Genes encoding the precursor of a vasopressin/oxytocin-type neuropeptide and its receptor duplicated in a common ancestor of the Bilateria. One copy of the precursor retained ancestral features, as seen in highly conserved vasopressin/oxytocin–neurophysin-type precursors. The other copy diverged, but this took different courses in protostomes and deuterostomes. In protostomes, the occurrence of a disulfide bridge in neuropeptide product(s) of the precursor was retained, as in CCAP, but with loss of the neurophysin domain. In deuterostomes, we see the opposite scenario—the neuropeptides lost the disulfide bridge, and neurophysin was retained (as in the NGFFFamide precursor) but was subsequently lost in vertebrate NPS precursors. Thus, the sea urchin NGFFFamide precursor and receptor are ‘missing links’ in the evolutionary history of neuropeptides that control ecdysis in arthropods (CCAP) and regulate anxiety in humans (NPS).     

Distribution of oxytocin-like and vasopressin-like immunoreactivities within the central nervous system of the cuttlefish, <Emphasis Type="Italic">Sepia officinalis</Emphasis>

We have investigated the distribution of oxytocin/vasopressin (OT/VP) superfamily peptides in the central nervous system (CNS) of the cuttlefish, Sepia officinalis, by using antibodies raised against mammalian OT and VP. Several populations of OT-like and VP-like immunoreactive cell bodies and fibers were widely distributed in cerebral structures involved in learning processes (vertical lobe complex, optic lobes), behavioral communication (peduncle, lateral basal and chromatophore lobes), feeding behavior (inferior frontal, brachial and buccal lobes), sexual activity (dorsal basal, subpedunculate, olfactory lobes), and metabolism (visceral lobes). The two most remarkable findings of this study were the occurrence of OT-like immunoreactivity in many amacrine cells of the vertical lobe and the dense accumulation of VP-like immunoreactive cell bodies in the subpedunculate 1 lobe. No double-immunolabeled cell bodies or fibers were found in any lobes of the CNS, indicating, for the first time in a decapod cephalopod mollusc, the existence of distinct oxytocinergic-like and vasopressinergic-like systems. The widespread distribution of the immunoreactive neurons suggests that these OT-like and VP-like peptides act as neurotransmitters or neuromodulators. This research was supported by grants from the “Région Basse-Normandie” (FRANCE) and the LARC-Neurosciences network (FRANCE).     

Genomics reveal ancient forms of stanniocalcin in amphioxus and tunicate

Stanniocalcin (STC) is present throughout vertebrates, including humans, but a structure for STC has not been identified in animals that evolved before bony fish. The origin of this pleiotropic hormone known to regulate calcium is not clear. In the present study, we have cloned three stanniocalcins from two invertebrates, the tunicate Ciona intestinalis and the amphioxus Branchiostoma floridae. Both species are protochordates with the tunicates as the closest living relatives to vertebrates. Amphioxus are basal to both tunicates and vertebrates. The genes and predicted proteins of tunicate and amphioxus share several key structural features found in all previously described homologs. Both the invertebrate and vertebrate genes have four conserved exons. The predicted length of the single pro-STC in Ciona is 237 amino acids and the two pro-hormones in amphioxus are 207 and 210 residues, which is shorter than human pro-STCs at 247 and 302 residues due to expansion of the C-terminal region in vertebrate forms. The conserved pattern of 10 cysteines in all chordate STCs is crucial for identification as amphioxus and tunicate amino acids are only 14-23% identical with human STC1 and STC2. The 11th cysteine, which is the cysteine shown to form a homodimer in vertebrates, is present only in amphioxus STCa, but not in amphioxus STCb or tunicate STC, suggesting the latter two are monomers. The expression of stanniocalcin in Ciona is widespread as shown by RT-PCR and by quantitative PCR. The latter method shows that the highest amount of STC mRNA is in the heart with lower amounts in the neural complex, branchial basket, and endostyle. A widespread distribution is present also in mammals and fish for both STC1 and STC2. Stanniocalcin is a presumptive regulator of calcium in both Ciona and amphioxus, although the structure of a STC receptor remains to be identified in any organism. Our data suggest that amphioxus STCa is most similar to the common ancestor of vertebrate STCs because it has an 11th cysteine necessary for dimerization, an N-glycosylation motif, although not the canonical one in vertebrate STCs, and similar gene organization. Tunicate and amphioxus STCs are more similar in structure to vertebrate STC1 than to vertebrate STC2. The unique features of STC2, including 14 instead of 11 cysteines and a cluster of histidines in the C-terminal region, appear to be found exclusively in vertebrates.     

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.