Prolactin osmoregulatory function in fishes and its projection on mammals

Prolactin evolution and key role in fish osmoregulation were reviewed. Comparison of fish and mammalian prolactin was made in respect of its structure, producing tissues, regulation of pituitary secretion. Peculiarities of prolactin receptor structure and prolactin-induced signal cascades, tissue distribution and regulation of prolactin receptor expression were compared in fishes and mammals. Data on mechanisms of prolactin action on ionoconservation in teleost fishes at the level of gills, kidney, intestine, and skin were presented. The facts of prolactin participation in the regulation of water and salt balance in mammals were observed. The existence of fundamentally similar mechanisms of osmoregulatory prolactin action in fishes and mammals was accumed and algorithm of their investigation was suggested.     

Biological properties of prolactin-releasing peptide analogs with a modified aromatic ring of a C-terminal phenylalanine amide

Prolactin-releasing peptide (PrRP)-induced secretion of prolactin is not currently considered a primary function of PrRP, but the development of late-onset obesity in both PrRP and PrRP receptor knock-out mice indicates the unique anorexigenic properties of PrRP. In our recent study, we showed comparable potencies of peptides PrRP31 and PrRP20 in binding, intracellular signaling and prolactin release in pituitary RC-4B/C cells, and anorexigenic effect after central administration in fasted mice. In the present study, eight analogs of PrRP20 with C-terminal Phe amide modified with a bulky side chain or a halogenated aromatic ring revealed high binding potency, activation of mitogen-activated protein kinase/extracellular-regulated kinase (MAPK/ERK1/2) and cAMP response element-binding protein (CREB) and prolactin release in RC-4B/C cells. In particular, [PheNO₂³¹]PrRP20, [1-Nal³¹]PrRP20, [2-Nal³¹]PrRP20 and [Tyr³¹]PrRP20 showed not only in vitro effects comparable or higher than those of PrRP20, but also a very significant and long-lasting anorexigenic effect after central administration in fasted mice. The design of potent and long-lasting PrRP analogs with selective anorexigenic properties promises to contribute to the study of food intake disorders.     

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.     

Evolutionary history of the calcitonin gene-related peptide family in vertebrates revealed by comparative genomic analyses

The calcitonin gene-related peptide (CGRP) family is composed of CGRP, amylin and adrenomedullin (AM) in mammals. In teleost fish, AM forms an independent subfamily of five members (AM1–5), which inspired us to trace the evolutionary history of the CGRP family throughout vertebrates by comparative genomic approach. Linkage mapping and synteny analyses of the CGRP family genes in medaka, Oryzias latipes, revealed that AM1/CGRP, AM2/amylin, and AM5 genes were located on respective proto-chromosomes before the divergence of teleost lineage. In teleost fish, additional whole genome duplication generated AM1/4, CGRP1/2, AM2/3, but one of the duplicated amylin and AM5 genes was silenced. In mammals, the amylin or AM2 gene was translocated to different chromosomes, while the CGRP gene was multiplied in tandem to generate CGRP-,β, and recently identified calcitonin receptor-stimulating peptide genes. Based on these data, we identified a novel AM5 gene in several mammalian species as we previously did for AM2.     

Characterization of prolactin-releasing peptide: binding, signaling and hormone secretion in rodent pituitary cell lines endogenously expressing its receptor

The recently discovered prolactin-releasing peptide (PrRP) binds to the PrRP receptor and is involved in endocrine regulation and energy metabolism. However, its main physiological role is currently unknown. Two biologically active isoforms of PrRP exist: the 31 (PrRP31) and the 20 (PrRP20) amino acid forms, which both contain a C-terminal Phe amide sequence. In the present study, the PrRP receptor was immunodetected in three rodent tumor pituitary cell lines: GH3, AtT20 and RC-4B/C cells. The saturation binding of radioiodinated PrRP31 to intact cells demonstrated a K_d in the 10⁻⁹ M range and a B_max in the range of tens of thousands binding sites per cell. For binding to RC-4B/C cells, both PrRP31 and PrRP20 competed with ¹²⁵I-PrRP31 with a similar K_i. The C-terminal analog PrRP13 showed lower binding potency compared to PrRP31 and PrRP20. All PrRP analogs increased the phosphorylation of MAPK/ERK1/2 (mitogen-activated phosphorylase/extracellular-regulated kinase) and CREB (cAMP response element-binding protein) in RC-4B/C cells. Additionally, prolactin release was induced by the PrRP analogs in a dose-dependent manner in RC-4B/C cells. Finally, food intake after intracerebroventricular administration of PrRP analogs in fasted mice was followed. Both PrRP31 and PrRP20 decreased food intake, but PrRP13 did not show significant effect. Studies on pituitary cell lines expressing the PrRP receptor are more physiologically relevant than those on cells transfected with the receptor. This cell type can be used as a model system for pharmacological studies searching for PrRP antagonists and stable effective PrRP agonists, as these drugs may have potential as anti-obesity agents.     

Tissue distribution, hormone regulation and evidence for a human homologue of the estrogen-inducible Xenopus laevis vitellogenin mRNA binding protein

17β-estradiol induces the synthesis of massive amounts of the hepatic mRNA encoding the Xenopus laevis egg yolk precursor protein, vitellogenin. Vitellogenin mRNA exhibits a half life of approx. 500 h when 17β-estradiol is present, and 16 h after removal of 17β-estradiol from the culture medium. We recently reported that Xenopus liver contains a protein, which is induced by 17β-estradiol and binds with a high degree of specificity to a binding site in a segment of the 3′-untranslated region (3′-UTR) of vitellogenin mRNA implicated in 17β-estradiol stabilization of vitellogenin mRNA. To determine if this mRNA binding protein was specific to this system, or if it was present elsewhere, and regulated by other steroids, we examined the tissue distribution and androgen regulation of this protein. Substantial amounts of the vitellogenin 3′-UTR binding protein were found in several Xenopus tissues including testis, ovary and muscle. In the absence of hormone treatment, lung and intestine contained minimal levels of the mRNA binding protein. Testosterone administration induced the vitellogenin 3′-UTR RNA binding protein in several tissues. Additionally, we found a homologous mRNA binding protein in MCF-7, human breast cancer cells. Although the MCF-7 cell protein was not induced by 17β-estradiol, the MCF-7 cell mRNA binding protein appears to be closely related to the Xenopus protein since: (i) the human and Xenopus proteins elicit gel shifted bands with the same electrophoretic mobility using the vitellogenin mRNA 3′-UTR binding site; (ii) The human and Xenopus proteins exhibit similar binding specificity for the vitellogenin 3′-UTR RNA binding site; and (iii) RNA from MCF-7 cells is at least as effective as RNA from control male Xenopus liver in blocking the binding of the Xenopus and human proteins to the vitellogenin mRNA 3′-UTR binding site. Its broad tissue distribution and regulation by both 17β-estradiol and testosterone suggests that this mRNA binding protein may play a significant role in steroid hormone regulation of mRNA metabolism in many vertebrate cells.     

Singular contributions of fish neuroendocrinology to mammalian regulatory peptide research

During the past 20 years, several bioactive peptides have been identified in teleost fishes that subsequently have been shown to play important regulatory roles in mammalian physiology. The urophysis, corpuscles of Stannius and Brockmann body are anatomical structures particular to fish that have no obvious counterpart in mammals. Extracts and/or cDNA libraries prepared from these tissues have been used to identify for the first time urotensin II (U-II), urotensin-I (U-I), stanniocalcin and glucagon-like peptide-1 (GLP-1). Although U-II and U-I were originally regarded as exclusively the products of the teleost urophysis, the peptides have a wide phylogenetic distribution across the vertebrate lineage, including mammals. U-II is localized to motor neurones in the human spinal cord and is a potent vasoconstrictor that may be implicated in the pathogenesis of heart failure. The human ortholog of urotensin-I is urocortin which is synthesized in selected regions of the brain and is the endogenous ligand for the CRF type 2 receptor. Urocortin is believed to important in mediating the effects of stress on appetite. Stanniocalcin is involved in maintaining calcium and phosphate homeostasis in teleost fish. An ortholog of stanniocalcin has a widespread distribution in mammalian tissues and is postulated to regulate renal phosphate excretion and to protect neurons against damage during cerebral ischemia. The biological actions and therapeutic potential of GLP-1 in humans are now fully appreciated but the peptide was first identified as a domain in a preproglucagon cDNA prepared from anglerfish Brockmann bodies. In contrast to mammalian preproglucagons, GLP-1 is present in anglerfish preproglucagon as the bioactive, truncated sequence [corresponding to human GLP-1(7-37)] rather than the inactive, N-terminally extended form [corresponding to GLP-1(1-37)]. Failure to appreciate the significance of this fact retarded progress in the field for several years.     

Phosphoinositide 3-kinase is involved in Xenopus and Labrus melanophore aggregation

Melanophores are pigmented cells capable of quick colour changes through coordinated transport of their intracellular pigment granules. We demonstrate the involvement of phosphoinositide 3-kinase (PI3-K) in Xenopus and Labrus aggregation by the use of the PI3-K inhibitor, LY-294002. In Xenopus, wortmannin-insensitive PI3-K was found to be essential for the aggregation, mitogen-activated protein kinase (MAPK) activation and tyrosine phosphorylation of a 280-kDa protein, and for the maintenance of low cyclic adenosine 3′:5′-monophosphate (cAMP) during the aggregated state. Pre-aggregated cells disperse completely to LY-294002 at 50–100 μM, involving a transient elevation in cAMP due to adenylate cyclase (AC) stimulation or to inhibition of cyclic nucleotide phosphodiesterase (PDE). The inactive analogue LY-303511 did not induce dispersion at the same concentrations. PDE4 and/or PDE2 was found to be involved in melanosome aggregation. The similar kinetics of LY-294002 and various PDE inhibitors indicates that the elevation of cAMP might be due to inhibition of PDE. In Labrus melanophores, LY-294002 had a less dramatic effect, probably due to less dependence on PDE in regulation of cAMP levels. In Xenopus aggregation, we suggest that melatonin stimulation of the Mel1c receptor via G_βγ activates PI3-K that, directly or indirectly via MAPK, activates PDE.     

Ancient vertebrate conserved noncoding elements have been evolving rapidly in teleost fishes

Vertebrate genomes contain thousands of conserved noncoding elements (CNEs) that often function as tissue-specific enhancers. In this study, we have identified CNEs in human, dog, chicken, Xenopus, and four teleost fishes (zebrafish, stickleback, medaka, and fugu) using elephant shark, a cartilaginous vertebrate, as the base genome and investigated the evolution of these ancient vertebrate CNEs (aCNEs) in bony vertebrate lineages. Our analysis shows that aCNEs have been evolving at different rates in different bony vertebrate lineages. Although 78-83% of CNEs have diverged beyond recognition ("lost") in different teleost fishes, only 24% and 40% have been lost in the chicken and mammalian lineages, respectively. Relative rate tests of substitution rates in CNEs revealed that the teleost fish CNEs have been evolving at a significantly higher rate than those in other bony vertebrates. In the ray-finned fish lineage, 68% of aCNEs were lost before the divergence of the four teleosts. This implicates the "fish-specific" whole-genome duplication in the accelerated evolution and the loss of a large number of both copies of duplicated CNEs in teleost fishes. The aCNEs are rich in tissue-specific enhancers and thus many of them are likely to be evolutionarily constrained cis-regulatory elements. The rapid evolution of aCNEs might have affected the expression patterns driven by them. Transgenic zebrafish assay of some human CNE enhancers that have been lost in teleosts has indicated instances of conservation or changes in trans-acting factors between mammals and fishes.     

Molecular cloning and expression study of Xenopus latent TGF-beta binding protein-1 (LTBP-1)

Latent transforming growth factor β binding protein-1 (LTBP-1) is important in regulating the localization and activation of transforming growth factor β. In this paper is reported the isolation of the full-length Xenopus LTBP-1 cDNA from screening a neurula embryo cDNA library. Sequence analysis of XLTBP-1 cDNA revealed an open reading frame of 4518 bp encoding a 1398 amino acid protein with a molecular mass of 154.1 kDa and an isoelectric point of 4.65. The Xenopus XLTBP-1 shares 61 and 65% amino acid identity with the mouse and human LTBP-1, respectively. It contains 17 epidermal growth factor-like motifs and four eight-cysteine repeats (8-Cys). RNase protection assay revealed that XLTBP-1 is a maternal and zygotic gene, while whole-mount in situ hybridization analysis performed on embryos at different stages showed that during early Xenopus development, XLTBP-1 mRNA is expressed in the Spemann organizer, prechordal and chordal mesoderm, and later on in the organizer derived tissues. These findings suggest an important role for XLTBP-1 in embryo axis formation.     

Prolactin-releasing peptide is essential to maintain the prolactin level and osmotic balance in freshwater teleost fish

We administered prolactin-releasing peptide (PrRP) or anti-PrRP antiserum to goldfish in fresh water and analyzed their effects on prolactin and osmoregulatory mechanisms. The pituitary mRNA level of prolactin increased by PrRP but decreased by anti-PrRP. The rate of water inflow in the gills decreased by PrRP and increased by anti-PrRP, showing that PrRP restricts branchial water permeability, as also restricted by prolactin. PrRP also expanded the mucous cell layers on the scales, which may restrict efficiently water inflow by the mucous system. Eventually, the plasma osmotic pressure decreased by anti-PrRP. We conclude that PrRP is essential to maintain prolactin levels and osmotic balance in fresh water.     

Regioselective metabolism of phenanthrene in Atlantic cod (Gadus morhua): studies on the effects of monooxygenase inducers and role of cytochromes P-450

The polycyclic aromatic hydrocarbon phenanthrene was converted mainly (>90%) to the 1,2-dihydrodiol when metabolized in vivo by the marine teleost cod. This is also found in other bony fishes, but contrary to what is known from cartilaginous fish, crustaceans and mammals, where the K-region 9,10-dihydrodiol is the main metabolite. When liver microsomal preparations from differently pretreated cod were incubated with phenanthrene in vitro, the metabolic profile was dramatically different from the in vivo pattern, as shown by gas chromatography—mass spectrometry. The microsomes from untreated, phenanthrene, phenobarbital and pregnenolone-16-carbonitrile-treated cod converted phenanthrene mainly, but to a varying extent, to the 9,10-dihydrodiol. Treatment with β-naphthoflavone (BNF), however, resulted in a large increase in the oxidation at the 1,2-position, along with a four- to seven-fold increase in specific activity. The major cytochrome P-450 isozyme purified from BNF-treated cod liver (P-450c) showed highest activity with phenanthrene (a turnover of 0.18 nmol/min per nmol P-450), but with about equal selectivity for the 1,2- and 9,10-region of the substrate in a reconstituted system with phospholipid and NADPH-cytochrome P-450 reductase. The low regioselectivity was also observed as a lack of regioselective inhibition of microsomal phenanthrene metabolism with antiserum to cod P-450c. Two of the minor isozymes, cod cytochromes P-450b and d, showed a similar turnover to P-450c, but with a stronger selectivity for the 1,2-position (55–60%). The results indicate that other control systems, in addition to the content of individual P-450-forms in the regulatory systems, in addition to the content of individual P-450-forms in the endoplasmic reticulum, are involved in the in vivo transformation of phenanthrene by cod to the 1,2-dihydrodiol metabolite.     

Correlations between coding and contiguous non-coding sequences in isochore families from vertebrate genomes

Many years ago compositional correlations were found to hold between coding and contiguous non-coding sequences. These correlations were essentially studied in whole genomes of mammals, which are characterized by strong compositional heterogeneities. Here we investigated whether these correlations also hold within the much more homogeneous isochore families. This point was checked not only in the case of mammals, but also in that of phylogenetically distant vertebrates, which are characterized by very different compositional patterns. Indeed, these are remarkably different in cold- and warm-blooded vertebrates. Fish genomes, for instance, are much more homogeneous than those of mammals and birds. The compositional correlations between coding sequences and the corresponding introns, or their 5′ and 3′ flanking regions, were studied in the isochore families of the fully sequenced genomes from four fishes (Brachydanio rerio, Oryzias latipes, Gasterosteus aculeatus and Tetraodon nigroviridis), human and chicken.