Dual duplication of neurohypophysial hormones in an Australian marsupial: mesotocin, oxytocin, lysine vasopressin and arginine vasopressin in a single gland of the northern bandicoot (Isoodon macrourus)

Neurohypophysial hormones of an Australian marsupial, the Northern bandicoot (Isoodon macrourus), have been identified by their retention times in high-pressure reverse-phase liquid chromatography using two solvent systems and by their molar pressor or uterotonic activities. Two pressor peptides, arginine vasopressin and lysipressin, and two uterotonic peptides, mesotocin and oxytocin, have been characterized. Because mesotocin and arginine vasopressin have been identified in three other Australian marsupial families, it is assumed that a duplication of each ancestral gene occurred in Peramelidae and subsequent mutations in one copy led to the additional oxytocin and lysipressin. A similar dual duplication of neurohypophysial hormones has previously been discovered in the North-American opossum (Didelphis virginiana) so that the duplication propensity seems peculiar to marsupials in contrast to placental mammals.     

Structure, processing and evolution of the neurohypophysial hormone-neurophysin precursors

Neurohypophysial hormones and neurophysins are derived from common precursors processed during the axonal transport from the hypothalamus to the neurohypophysis. Two neurohormones, an oxytocin-like and a vasopressin-like, on one hand, two neurophysins, termed VLDV-and MSEL-neurophysins according to residues in positions 2, 3, 6 and 7, on the other, are usually found in vertebrate species. In contrast to placental mammals that have oxytocin and arginine vasopressin, marsupials have undergone a peculiar evolution. Two pressor peptides, lysipressin and vasopressin for American species, lysipressin and phenylpressin for Australian macropods, have been identified in individual glands and it is assumed that the primordial vasopressin gene has been duplicated in these lineages. On the other hand, the reptilian mesotocin is still present in Australian species instead of the mammalian oxytocin, while the North American opossum has both hormones and South American opossums have only oxytocin. The neurophysin domain of each precursor is encoded by 3 exons and different evolutionary rates have been found for the 3 corresponding parts of the protein. The central parts, encoded by the central exons, are evolutionarily very stable and nearly identical in the 2 neurophysins of a given species. Recurrent gene conversions have apparently linked the evolutions of the 2 precursor lineages. In mammals, the 3-domain precursor of vasopressin is processed in 2 stages: a first cleavage splitting off vasopressin and a second cleavage separating MSEL-neurophysin from copeptin. Two distinct enzymatic systems seem to be involved in these cleavages. Processing is usually complete at the level of the neurohypophysis, but an intermediate precursor encompassing MSEL -neurophysin and copeptin linked by an arginine residue has been characterized in guinea pig. In vitro processing of this intermediate through trypsin--Sepharose reveals cleavages only in the interdomain region. In non-mammalian tetrapods, such as birds and amphibians, mesotocin and vasotocin are associated with neurophysins in precursors similar to those found in mammals. However, processing of the vasotocin precursor seems to be different from the processing of the vasopressin precursor, with a single cleavage leading to the hormone release.     

Two multigene families for marsupial neurohypophysial hormones? Identification of oxytocin,mesotocin, lysipressin and arginine vasopressin in the North American opossum (Didelphis virginiana)

Oxytocin, mesotocin ([Ile⁸]-oxytocin), lysipressin ([Lys⁸]-vasopressin) and arginine vasopressin have been identified in the North American opossum ($\text{Didelphis virginiana}$) by amino acid composition and high pressure liquid chromatography. The same peptides with the exception of mesotocin have previously been found in two South American opossums ($\text{Didelphis marsupialis}$ and $\text{Philander opossum}$). Although a dual heterozygocity could also explain the simultaneous presence of oxytocin/mesotocin on one hand, lysipressin/arginine vasopressin on the other, it is assumed, from the results obtained with individual glands of Australian and South American marsupials, that distinct genes encode for the four peptides.     

Comparative Physiology of the Vasomotor Effects of Neurohypophysial Peptides in the Vertebrates

The neurohypophysial peptides are vasopressor or depressor inaction depending on the species. Isotocin, mesotocin and oxytocinconstrict the branchial vessels in fish and induce a reflexvasodilation in the systemic vasculature. The vasodilation haspersisted in some higher vertebrates and is particularly prominentin the snakes and birds where vasotocin and arginine vasopressinalso are vasodepressor but are much less potent than mesotocinand oxytocin. In other vertebrates including fish, vasotocinand vasopressin are pressor and exert their effects mainly onthe peripheral resistance. The newt, toads and soft-shell turtlegave pressor responses to all neurohypophysial peptides, withvasotocin showing the highest potency. The frogs, big-headedturtle and lizards were intermediate with vasotocin being pressor,mesotocin being pressor and oxytocin exhibiting a dual effect.     

Unique evolution of neurohypophysial hormones in cartilaginous fishes: possible implications for urea-based osmoregulation.

Most bony vertebrate species display a great evolutionary stability of their two neurohypophysial hormones, so that two molecular lineages, isotocin-mesotocin-oxytocin and vasotocin-vasopressin, have been traced from bony fishes to mammals. Chondrichthyes, in contrast, show a striking diversity of their oxytocin-like hormones, yet show a substantial decrease in vasotocin stored in neurohypophysis when compared to nonmammalian bony vertebrates. In the rays, glumitocin ([Ser(4),Gln(8)]-oxytocin) has been identified. In the spiny dogfish, aspargtocin ([Asn4]-oxytocin) and valitocin ([Val(8)]-oxytocin) have been characterized whereas in the spotted dogfish, asvatocin ([Asn(4),Val(8)]-oxytocin) and phasvatocin ([Phe(3),Asn(4),Val(8)]-oxytocin) have been found. Finally, in the holocephalian Pacific ratfish, oxytocin, the typical peptide of placental mammals, has been discovered. The duplication of the oxytocin-like hormone gene found in dogfishes has been observed only in some Australian and American marsupials. Cartilaginous fishes have developed an original urea-based osmoregulation involving a glutamine-dependent urea synthesis and blood urea retention through renal urea transporters. Furthermore, marine species use a rectal salt gland for sodium chloride excretion. Although vasopressin, in mammals, and vasotocin, in nonmammalian tetrapods, are clearly implied in water and salt homeostasis, the hormones involved in the blood osmotic pressure regulation of elasmobranchs are still largely unknown. It is suggested that the great diversity of oxytocin-like hormones in elasmobranchs expresses a release from an evolutionary receptor-binding constraint, so that amino-acid substitutions reflect neutral evolution. In contrast, the preservation of vasotocin suggests a selective pressure, which may be related to the regulation of renal urea transporter-recruitment mechanisms, as it has been shown for vasopressin in mammals. J. Exp. Zool. 284:475-484, 1999.     

Comparative Physiology of Neurohypophysial Hormone Action on the Vertebrate Oviduct-Uterus

The neurohypophysial hormone, arginine vasotocin, is depletedfrom the hypothalamus, and rises in concentration in the bloodduring oviposition in hens. The contractile responses of isolatedoviducts from birds, reptiles and amphibians are more sensitiveto arginine vasotocin than to oxytocin or mesotocin. This evidenceclearly indicates that arginine vasotocin is involved in parturitionor oviposition in nonmammalian tetrapods. Evidence for a physiologicalrole for specific neurohypophysial hormones in the regulationof oviduct—or in some cases ovarian — contractilityin fishes is unclear and occasionally contradictory. However,it appears unlikely that arginine vasotocin is involved in thefish species that have been investigated. It is evident that,much like the neurohypophysial hormones, the neurohypophysialhormone receptors of the vertebrate myometrium have undergoneevolutionary change.     

Sequence and organization of coelacanth neurohypophysial hormone genes: Evolutionary history of the vertebrate neurohypophysial hormone gene locus

Background  

The mammalian neurohypophysial hormones, vasopressin and oxytocin are involved in osmoregulation and uterine smooth muscle contraction respectively. All jawed vertebrates contain at least one homolog each of vasopressin and oxytocin whereas jawless vertebrates contain a single neurohypophysial hormone called vasotocin. The vasopressin homolog in non-mammalian vertebrates is vasotocin; and the oxytocin homolog is mesotocin in non-eutherian tetrapods, mesotocin and [Phe²]mesotocin in lungfishes, and isotocin in ray-finned fishes. The genes encoding vasopressin and oxytocin genes are closely linked in the human and rodent genomes in a tail-to-tail orientation. In contrast, their pufferfish homologs (vasotocin and isotocin) are located on the same strand of DNA with isotocin gene located upstream of vasotocin gene separated by five genes, suggesting that this locus has experienced rearrangements in either mammalian or ray-finned fish lineage, or in both lineages. The coelacanths occupy a unique phylogenetic position close to the divergence of the mammalian and ray-finned fish lineages.     

Precursors of mesotocin and vasotocin in birds: Identification of VLDV- and MSEL-neurophysins in chicken,goose, and ostrich

Precursors of neurohypophysial hormones are small proteins processed into nonapeptide hormones and neurophysins during axonal transport to the neurohypophysis. In mammals, oxytocin is associated with VLDV-neurophysin and vasopressin with MSEL-neurophysin. In birds, mesotocin and vasotocin are found instead of mammalian oxytocin and vasopressin. From goose, chicken and ostrich posterior pituitary glands, two types of neurophysins related to mammalian VLDV-and MSEL-neurophysins, respectively, have been identified by their N-terminal sequences. It is assumed that, as in mammals, hormonal peptide and the first 9 residues of the corresponding neurophysin are encoded by a common exon and that mesotocin and vasotocin, evolutionary predecessors of oxytocin and vasopressin, are associated in the precursors with VLDV-neurophysin and MSEL-neurophysin, respectively.     

Central cardiovascular effects of mammalian neurohypophyseal peptides in conscious rats

To confirm and extend the results of previous studies which demonstrated central cardiovascular effects of vasopressin in anesthetized rats, we determined blood pressure and heart rate changes for 30 minutes after intracerebroventricular injections of arginine vasopressin, arginine vasotocin and oxytocin in conscious rats. As compared to sham injections, significantly greater increases in either systolic or diastolic blood pressure were noted over the 30 minutes which followed the injection of 0.15, 1.0 or 10.0 nM of either vasopressin or vasotocin. In animals given vasopressin, plasma levels of the peptide were determined. There was a substantial increase in plasma vasopressin only after the highest dose. Overall blood pressure responses to doses of oxytocin as high as 100 nM were not significantly different than sham injections. Heart rate following both vasopressin and vasotocin was increased at 0.15 nM, was initially decreased then increased at 1.0 nM and was substantially decreased after the 10.0 nM dose. There was a significant increase in heart rate at the 10.0 nM and 100 nM doses of oxytocin. Dose response curves for systolic blood pressure and heart rate 20 minutes after injection were similar for vasopressin and vasotocin. We conclude that arginine vasopressin has significant central pressor and tachycardic effects in conscious rats, and it is related, at least in part, to the tail structure of the peptide, which is shared with arginine vasotocin.     

In vivo apparent peptide-receptor dissociation rate constants for arginine vasopressin analogs estimated from inhibition of rat pressor responses

Apparent pressor receptor dissociation rate constants for arginine vasopressin, arginine vasotocin, oxytocin, oxypressin, and [1-deamino, 9-D-alanineamide]arginine vasopressin were estimated by the following method. Two minutes after injection of a moderate dose of agonist into urethane-anesthetized rats prepared for recording mean blood pressure, a large dose of inhibitor was injected. Under these conditions, in the first few moments after inhibitor injection, there should be no rebinding of the agonist after it dissociates, because vacant receptors should be immediately occupied by inhibitor. The rate of the blood pressure drop at the initiation of inhibition was calculated and used as an estimate of the dissociation rate of the agonist. Apparent dissociation rate constants thus estimated were 1.1, 1.1, 6.9, 5.8, and 13.9 min-1 for arginine vasopressin, arginine vasotocin, oxytocin, oxypressin, and [1-deamino, 9-D-alanineamide]arginine vasopressin, respectively. These rate constants were inversely related to the pressor potencies (435, 250, 5, 3, and 0.7 U/mg, respectively) of these five compounds. Such a relationship is to be expected if decreased potency is in part due to a faster "off" rate from pressor receptors.     

Synthesis of new vasotocin analogues: effects on renal water and ion excretion in rats

Vasopressin and nonmammalian hormone vasotocin are known to increase the water permeability of mammalian collecting ducts, frog skin and the urinary bladder. Neurohypophysial nonapeptides have also been shown to interfere with the regulation of renal ion transport. The subject of this study was a search for vasopressin and vasotocin analogues with selective effects on renal water, sodium and potassium excretion. During this study, we synthesised the following peptides: 13 vasotocin analogues modified at positions 4 (Thr or Arg), 7 (Gly or Leu) and 8 (d ‐Arg, Lys or Glu); 4 vasopressin analogues modified at positions 4 and 8; and 9 peptides shortened or extended at the C‐terminal or with substitutions for Gly‐NH₂. Most of these peptides had mercaptopropionic acid (Mpa) instead of Cys in position 1. The effects of these nonapeptides on renal water, sodium and potassium transport were evaluated in in vivo experiments using Wistar rats. Some nonapeptides possessed antidiuretic, natriuretic and kaliuretic activities ([Mpa¹]‐arginine vasotocin, [Mpa¹, homoArg⁸]‐vasotocin, [Mpa¹, Thr⁴]‐arginine vasotocin and [Mpa¹, Arg⁴]‐arginine vasopressin). Substitutions at positions 4 and 8 increased the selectivity of peptide actions. The antidiuretic [d ‐Arg⁸]‐vasotocin analogues had no effects on sodium excretion. [Mpa¹, Arg⁴]‐arginine vasotocin was antidiuretic and kaliuretic but not natriuretic. [Mpa¹, Glu⁸]‐oxytocin had weak natriuretic activity without any effects on water and potassium transport. In accordance with the data obtained, synthesised vasotocin analogues could be good candidates for pharmaceuticals selectively regulating renal sodium and potassium transport, which is of clinical importance. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

The neurohypophysial hormones of the egg-laying mammals: identification of arginine vasopressin in the platypus (Ornithorhynchus anatinus)

Two neurohypophysial peptides have been purified from acetone desiccated posterior pituitary glands of the platypus (Ornithorhynchus anatinus) by molecular sieving and high-pressure liquid chromatography. A single pressor peptide, having an amino acid composition and a chromatographic retention time identical to those of arginine vasopressin, has been identified. A single oxytocic peptide has been isolated that ressembles oxytocin by its chromatographic retention time, but lack of material has prevented to obtain a correct amino acid composition. The pressor peptide is roughly four times more abundant than the oxytocic peptide. Neurohypophysial hormones of platypus seem similar to those of echidna, the other living prototherian, and to those of most placental mammals.     

Vasopressin receptor subtypes in dorsal hindbrain and renal medulla

We have investigated the ability of a series of synthetic vasopressin analogues and related peptides to compete with (3H)-arginine8 vasopressin for binding sites in rat renal medulla and dorsal hindbrain. In renal medulla, arginine8 vasopressin and deamino arginine8 vasopressin, a selective antidiuretic, were equipotent while two antagonists of the pressor action of arginine vasopressin were less potent. In the dorsal hindbrain, arginine8 vasopressin and the pressor antagonists were more potent than the synthetic antidiuretic. Potency profiles of these and other analogues suggest that the renal medulla and dorsal hindbrain vasopressin receptors represent different subtypes.     

Direct regulation of rat testicular steroidogenesis by neurohypophysial hormones. Divergent effects on androgen and progestin biosynthesis

The direct regulation of testis androgen and progestin biosynthesis by neurohypophysial hormones was investigated in a primary culture of rat testis cells. Treatment with arginine vasotocin (AVT; 10(-6) M) over a 10-day period inhibited the human chorionic gonadotropin (hCG)-stimulated testosterone accumulation while enhancing hCG-stimulated progesterone accumulation. Furthermore, treatment with increasing doses (10(-11) - 10(-6) M) of AVT by itself led to dose-dependent increases in the accumulation of pregnenolone (ED50: 8.0 +/- 0.2 X 10(-9) M) and progesterone (ED50: 1.6 +/- 0.3 X 10(-8) M) but not testosterone. Under blockade of pregnenolone metabolism using cyanoketone and spironolactone, AVT, like hCG, stimulated pregnenolone accumulation with an ED50 dose of 5.8 +/- 0.3 X 10(-9) M. Similar effects were observed with several related neurohypophysial hormones, but not with nine unrelated peptides. AVT, arginine vasopressin, and lysine vasopressin were about 100-fold more potent than mesotocin, valitocin, and oxytocin. Pressor (but not antidiuretic or oxytocic)-selective agonists of the neurohypophysial hormones also exerted dose-dependent stimulation of pregnenolone accumulation. Potent pressor (but not oxytocic)-selective antagonistic analogs of the neurohypophysial hormones prevented the AVT-stimulated accumulation of pregnenolone. Thus, the neurohypophysial hormones may exert a direct stimulatory effect on testis pregnenolone and progesterone biosynthesis via putative, pressor-selective recognition sites, and this progestin-stimulatory activity may be partly due to stimulation of steroidogenic steps preceding pregnenolone formation. Since the effective doses of neurohypophysial hormones in vitro are higher than the serum hormone levels, the present results suggest an intratesticular paracrine role for these peptides.     

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.     

Ontogeny of the bovine neurohypophysial hormone precursors. III. Identification of neurohormones, neurophysins and copeptin in the early bovine fetus

Neurohypophysial hormone precursors are small proteins processed into several fragments during axonal transport from hypothalamus to neurohypophysis. From 3-month-old fetal bovine pituitaries the three fragments of vasopressin precursor, arginine vasopressin, MSEL-neurophysin and copeptin, and the two fragments of oxytocin precursor, oxytocin and VLDV-neurophysin, have been isolated and characterized. These polypeptides are identical to those previously identified in the late fetus (7-9 months old) and in the adult. It is concluded that the same genes are expressed during fetal and adult lives, the vasopressin gene appearing roughly four times more active than the oxytocin gene in the early fetus. Vasotocin, mesotocin and additional neurophysin have not been detected in the early fetus.     

Antipyretic effect of arginine vasotocin in toads

Arginine vasotocin (AVT) is a nonmammalian analog of the mammalian hormone arginine vasopressin (AVP). These peptides are known for their antidiuretic and pressor effects. More recently, AVP has been recognized as an important antipyretic molecule in mammals. However, no information exists about the role of AVT in febrile ectotherms. We tested the hypothesis that AVT is an antipyretic molecule in the toad Bufo paracnemis. Toads equipped with a temperature probe were placed in a thermal gradient, and preferred body temperature was recorded continuously. A behavioral fever was observed after lipopolysaccharide (LPS) was injected systemically (200 microg/kg). Systemically injected AVT (300 pmol/kg) alone caused no significant change in body temperature, but abolished LPS-induced fever. Moreover, a smaller dose of AVT (10 pmol/kg), which did not affect LPS-induced fever when injected peripherally, abolished fever when injected intracerebroventricularly. We therefore conclude that AVT plays an antipyretic role in the central nervous system, by means of behavior, in an ectotherm, a fact consistent with the notion that AVT/AVP elicits antipyresis by reducing the thermoregulatory set point.     

Neurally Active Stress Peptide Inhibits Territorial Defense in Wild Birds

It is unclear whether the behavioral effects of peptides in laboratory studies always reflect natural conditions. Here we test whether we can detect measurable behavioral changes after rapidly injecting peptides into the brains of wild birds. We used a modified stereotaxic-like technique to inject corticotrophin-releasing factor (CRF) and arginine vasotocin (AVT, the nonmammalian form of arginine vasopressin), two hormones important in the stress response, into the brains of wild, freely behaving, male white-crowned sparrows (Zonotrichia leucophrys). We then monitored subsequent territorial behavior to determine whether CRF or AVT altered this behavior. Surprisingly, the potent stressors of capture and surgery did not eliminate territorial behavior, with many birds resuming territorial defense within 60–90 min after surgery. Centrally acting CRF, however, significantly reduced territorial defense whereas centrally acting AVT had no effect. These results indicate that the behavioral affects of peptides can be studied under natural conditions.