Gilles Michel

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 (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 (Didelphis marsupialis and 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.

One-step processing of the amphibian vasotocin precursor: structure of a frog (Rana esculenta) big neurophysin

Vasotocin-associated neurophysin (MSEL-neurophysin) from the frog Rana esculenta has been isolated and sequenced through tryptic and staphylococcal proteinase peptides and cyanogen bromide fragments. This protein appears homologous to the mammalian vasopressin-associated neurophysin with a C-terminal glycopeptide extension homologous to the mammalian copeptin. In contrast to the two-step processing of mammalian vasopressin/MSEL-neurophysin/copeptin precursor, a single cleavage is therefore involved in the processing of the amphibian vasotocin/neurophysin precursor. It appears that the physiological release of the vasopressin-like hormone from the N-terminal end of the protein precursor is not dependent upon a previous trimming of the C-terminal copeptin-like moiety.

Neurohypophyseal hormones as evolutionary tracers: Identification of oxytocin, lysine vasopressin, and arginine vasopressin in two South American opossums (Didelphis marsupialis and Philander opossum)

The neurohypophyseal hormones of two South American opossums (Didelphis marsupialis and Philander opossum) were isolated by molecular sieving and preparative high-pressure liquid chromatography (HPLC). One oxytocin-like and two vasopressin-like peptides were found in each species. These peptides have been identified by their amino acid composition and by their retention time in HPLC. Oxytocin, lysine vasopressin, and arginine vasopressin have been characterized in both species. Lysine vasopressin is roughly as abundant as arginine vasopressin. Comparison is made with Australian marsupials Macropodidae and Phalangeridae, and possible evolutionary mechanisms are discussed.

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.

Evolutionary specificity of hydrins, new hydroosmotic neuropeptides: occurrence of hydrin 2 (vasotocinyl-Gly) in the toad Bufo marinus but not in the viper Vipera aspis

Hydrin 2 (vasotocinyl‐Gly), a hydroosmotic peptide resulting from differential processing of provasotocin and recently identified in frog neurohypophysis, has been looked for in the pituitary gland of an exotic toad (Bufo marinus) and of a reptile (Vipera aspis). Hydrin 2 has been found in the amphibian but not in the reptile. This result confirms the evolutionary specificity of hydrin 2 that has been identified only in frogs and toads but not in birds and reptiles. Occurrence of hydrin 2 is explained by its regulatory function on the water permeability of the skin of anurans.

Molecular evolution of neurohypophysial hormones in relation to osmoregulation: the two fish options

In vertebrates, water and osmolyte homeostasis is controlled by at least three hormonal systems: the hypothalamo-neurohypophysial system, the renin-angiotensin-aldosterone system and the atrial natriuretic factor system. The first system implies neuroendocrine reflexes involving an afferent neural limb from osmo- and baroreceptors to hypothalamus and an efferent endocrine limb from secretory neurons to target cell receptors and transduction to intracellular effectors, namely water and sodium channels. Evolution can affect any level of this molecular cascade. Whereas virtually all vertebrate species have two neurohypophysial hormones, an oxytocin-like and a vasopressin-like peptides, the most primitive vertebrates, Cyclostomata (lampreys and hagfishes) possess a single peptide, vasotocin, so that an early gene duplication occurred before the emergence of fishes, about 400 million years ago. The remarkable evolutionary stability of neurohypophysial hormones in bony vertebrates allows us to trace two main lineages: isotocin-mesotocin-oxytocin and vasotocin-vasopressin. In contrast, in cartilaginous fishes, the oxytocin-like peptides display a great evolutionary diversity: we have identified glumitocin in rays, aspargtocin and valitocin in the spiny dogfish, asvatocin and phasvatocin in the spotted dogfish, and oxytocin in the chimaera. Whereas bony vertebrates regulate their blood osmotic pressure (about 250–450 mOsm kg-1 H2O) essentially through salts, cartilaginous fishes use urea as the main osmolyte for adjusting their osmotic pressure above the external medium (about 1050 mOsm kg-1 H2O for marine fishes). The hypothesis is made that the stability of neurohypophysial hormones in bony vertebrates is due to their implication in ion-based osmoregulation (selective evolution), whereas in Chondrichthyes the occurrence of urea-based osmoregulation has relieved the hormones from this function and therefore made them free to vary (neutral evolution).

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.

Differential processing of provasotocin: Relative increase of hydrin 2 (vasotocinyl-Gly) in amphibians able to adapt to an arid environment

Hydrin 2 (vasotocinyl-Gly) is an intermediate in pro-vasotocin processing found, along with vasotocin, only in the neurohypophysis of anuran amphibians. It increases cutaneous water permeability in the frog and is likely involved in neuroendocrine control of osmoregulation. The relative amounts of vasotocin and hydrin 2 stored in neurohypophysis have been measured on the one hand in amphibian species known not to adapt in dry areas, on the other hand in two species, Bufo regularis (Africa) and Bufo viridis (Near-East) able to survive in an arid environment. In the first group, the proportions of the two peptides are approximately equal whereas in the two toads the molar ratio hydrin 2 to vasotocin reaches 2. The ratio does not appear to vary significantly when these toads are either submitted to dehydration or placed in saline solutions. Predominance of hydrin 2 suggests an adaptive decrease of the activity of the alpha-amidating enzymatic system involved in the conversion of vasotocinyl-Gly into mature amidated vasotocin.

An amphibian two-domain ‘big’ neurophysin: conformational homology with the mammalian MSEL-neurophysin/copeptin intermediate precursor shown by trypsin-Sepharose proteolysis

A ‘big’ frog (Rana esculenta) neurophysin, encompassing sequences homologous to mammalian MSEL‐neurophysin and copeptin, has been passed through a trypsin‐Sepharose column in order to compare its conformation with that of the two‐domain intermediate precursor isolated from guinea pig. Whereas the polypeptide possesses 8 arginine residues, only two cleavages were observed located in a putative inter‐domain sequence (at Arg‐94 and Arg‐114). Because free vasotocin has been isolated from the frog, it is assumed that pro‐vasotocin has a three‐domain conformation similar to that of pro‐vasopressin but processing in amphibians involves only one step rather than two steps as in mammals.

Particular processing of pro-opiomelanocortin in Xenopus laevis intermediate pituitary Sequencing of α- and β-melanocyte-stimulating hormones

α‐ and β‐melanocyte‐stimulating hormones (α‐MSH and β‐MSH) have been isolated from Xenopus laevis neurointermediate pituitary and microsequenced. Intracellular α‐MSH is not N‐acetylated after proteolytic processing of pro‐opiomelanocortin in contrast to mammalian α‐MSHs. There is a high preservation of the melanotropic amino acid sequence common to all MSHs although in Xenopus β‐MSH a histidine residue replaces the glutamic acid residue found in position 8 of mammalian β‐MSHs.