hydrin-2 and hydrin-1

In a previous study, we showed that corticotropin-releasing factor (CRF) is the major thyroid-stimulating hormone (TSH)-releasing factor in the bullfrog (Rana catesbeiana) hypothalamus. Our findings prompted us to ascertain whether CRF or arginine vasotocin (AVT), a known adrenocorticotropic hormone (ACTH) secretagogue in several vertebrates, is the main stimulator of the release of ACTH from the bullfrog pituitary. Both the frog CRF and AVT stimulated the release of immunoassayable ACTH from dispersed anterior pituitary cells in vitro in a concentration-dependent manner. AVT, however, exhibited far more potent ACTH-releasing activity than CRF. Although CRF by itself weakly stimulated ACTH release, it acted synergistically with AVT to enhance the release of ACTH markedly. Mesotocin and AVT-related peptides such as hydrin 1 and hydrin 2 showed relatively weak ACTH-releasing activity. Subsequently, cDNAs encoding the bullfrog AVT V1a-type and V1b-type receptors were molecularly cloned. Reverse transcriptase-PCR using specific primers revealed that the anterior lobe of the pituitary predominantly expressed AVT V1b-type receptor mRNA but scarcely expressed AVT V1a-type receptor mRNA. Abundant signals for V1b-type receptor mRNA in the corticotropes were also detected by in situ hybridization. The results obtained by the experiments with the bullfrog pituitary indicate that AVT acts as the main ACTH-releasing factor through the AVT V1b-type receptor and that CRF acts synergistically with AVT to enhance the release of ACTH.

Topics: Adrenocorticotropic Hormone; Amino Acid Sequence; Animals; Base Sequence; Corticotropin-Releasing Hormone; Fluorescent Antibody Technique; Pituitary Gland, Anterior; Rana catesbeiana; Receptors, Vasopressin; RNA, Messenger; Time Factors; Vasotocin

The ventral pelvic skin of the tree frog Hyla japonica expresses two kinds of arginine vasotocin (AVT)-stimulated aquaporins (AQP-h2 and AQP-h3), which affect the capacity of the frog's skin to absorb water. As such, it can be used as a model system for analyzing the molecular mechanisms of water permeability. We investigated AQP dynamics and water permeability in the pelvic skin of H. japonica following challenge with AVT, hydrins (intermediate peptides of pro-AVT) and beta-adrenergic effectors. In the in vivo experiment, both AQP-h2 and AQP-h3 proteins were translocated to the apical plasma membrane in the principal cells of the first-reacting cell (FRC) layer in the pelvic skin following challenge with AVT, hydrin 1 and hydrin 2, thereby increasing the water permeability of the pelvic skin. The beta-adrenergic receptor agonist isoproterenol (IP) and its anatagonist propranolol (PP) in combination with AVT or hydrins were used as challenge in the in vitro experiment. IP increased water permeability whereas PP inhibited it, and both events were well correlated with the translocation of the AQPs to the apical membrane. In the PP+AVT-treated skins, labels for AQP-h2 and AQP-h3 were differentially visible among the principal cells; the apical plasma membrane of some cells was labeled while others were not, indicating that the response of PP or AVT is different from cell to cell. These results provide morphological evidence that the principal cells of the FRC layers may have two kinds of receptors: a V2 receptor and beta-adrenergic receptor.

Topics: Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Animals; Anura; Aquaporins; Isoproterenol; Male; Permeability; Propranolol; Skin; Time Factors; Vasotocin; Water

Regions of specialization for water absorption across the skin of Bufonid and Ranid anurans were identified by immunohistochemistry and Western blot analysis, using antibodies raised against arginine vasotocin (AVT)-stimulated aquaporins (AQPs) that are specific to absorbing regions of Hyla japonica. In Bufo marinus, labeling for Hyla urinary bladder-type AQP (AQP-h2), which is also localized in the urinary bladder, occurred in the ventral surface of the hindlimb, pelvic, and pectoral regions. AQP-h2 was not detected in any skin regions of Rana catesbeiana, Rana japonica, or Rana nigromaculata. Hyla ventral skin-type AQP (AQP-h3), which is found in the ventral skin but not the bladder of H. japonica, was localized in the hindlimb, pelvic, and pectoral skins of Bufo marinus, in addition to AQP-h2. AQP-h3 was also localized in ventral skin of the hindlimb of all three Rana species and also in the pelvic region of R. catesbiana. Messenger RNA for AQP-x3, a homolog of AQP-h3, could be identified by RT-PCR from the hindlimb, pectoral, and pelvic regions of the ventral skin of Xenopus laevis, although AVT had no effect on water permeability. In contrast, 10(-8) M AVT-stimulated water permeability and translocation of AQP-h2 and AQP-h3 into the apical membrane of epithelial cells in regions of the skin of species where they had been localized by immunohistochemistry and Western blot analysis. Finally, water permeability of the hindlimb skin of B. marinus and all the Rana species was stimulated by hydrins 1 and 2 to a similar level as seen for AVT. The present data demonstrate species differences in the occurrence, distribution, and regulation of AQPs in regions of skin specialized for rapid water absorption that can be associated with habitat and also phylogeny.

Topics: Animals; Anura; Aquaporin 2; Aquaporin 3; Blotting, Western; Bufonidae; Female; Hindlimb; Immunohistochemistry; Male; Pelvis; Permeability; Protein Transport; Ranidae; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin; Skin Absorption; Species Specificity; Vasotocin; Water; Xenopus laevis

The biological properties of vasotocin, hydrin 1 (vasotocinyl-Gly-Lys-Arg) and hydrin 2 (vasotocinyl-Gly), in particular the hydro-osmotic activities on the frog skin, the frog urinary bladder and the frog kidney, have been compared. Hydrins are as active or more active than vasotocin on the first two organs but they are virtually devoid of antidiuretic activity in the rat and the frog, in contrast to vasotocin. It appears that where the oxytocin ring (residues 1-6), present in the three peptides, is necessary for the action on the three organs, the C-terminal amidated group of vasotocin is necessary for the renal receptor but not for the skin and bladder receptors. It is known that amphibians have two types of vasotocin receptors, V1 and V2, homologous to the vascular/hepatic V1 and the renal V2 vasopressin receptors of mammals, respectively. We suggest that adaptation has led to specialization of (at least) two subtypes of hydro-osmotic V2 receptors, the renal subtype on which vasotocin is mainly active for the reabsorption of tubular water, and the skin/bladder subtype on which hydrin 2 is specifically involved in ensuring the rehydration of the animal. Cooperative evolution might have created in anuran Amphibia, on the one hand, two hydro-osmotic peptides, vasotocin and hydrin 2, derived from a single precursor through differential processing; and on the other hand, two corresponding receptors in kidney and skin for internal and external water recovery.

Topics: Amino Acid Sequence; Animals; Kidney; Molecular Sequence Data; Osmosis; Rana esculenta; Rats; Receptors, Pituitary Hormone; Skin; Structure-Activity Relationship; Urinary Bladder; Vasotocin; Water

The neurohypophysis of Xenopus and that of Ranidae and Bufonidae contain hydrin 1 (vasotocinyl-Gly-Lys-Arg) and hydrin 2 (vasotocinyl-Gly), respectively. In order to test the aldosterone-releasing activity of arginine vasotocin (AVT) and hydrin 1, purification of these peptides from an acid-extract of the neurointermediate lobe of Xenopus laevis was performed using an ODS-silica cartridge and reverse-phase and ion-exchange HPLC columns. As a result, an additional AVT-related peptide was newly found. Amino-acid analysis revealed that this peptide is vasotocinyl Gly-Lys (AVT-GK). The aldosterone-releasing activity of AVT-GK was equivalent to that of hydrin 1 (AVT-GKR) and lower than that of AVT. Like AVT and AVT-GKR, AVT-GK were effective in stimulating water flux from the isolated urinary bladder of the toad. Since AVT-GK is regarded as an intermediate between hydrin 1 and hydrin 2 in terms of its C-terminal form, it was designated hydrin 1'.

Topics: Aldosterone; Amino Acid Sequence; Amino Acids; Animals; Bufonidae; Molecular Sequence Data; Pituitary Gland, Posterior; Ranidae; Urinary Bladder; Vasotocin; Xenopus

Cardiovascular and renal actions of hydrins and arginine vasotocin (AVT) in frogs (Rana tigrina and Rana catesbeiana) were examined in view of their relationships as precursor/prohormone-hormones in the anuran amphibian. In vitro hydrins and AVT respectively vasorelaxed and vasoconstricted KCl-preconstricted femoral artery in R. tigrina. AVT also produced dose-dependent contractions on untreated preparations; hydrins had no effect on these preparations. Both the hydrins and AVT were positively inotropic and chronotropic, with AVT being more potent. In vivo hydrins and AVT were anti-diuretic except at high doses, when AVT became diuretic. The hydrins and AVT were vasodepressor and pressor, respectively, in R. catesbeiana. These data suggest that the addition of one to three amino acids onto the AVT molecule reverses the contractile response in the vascular smooth muscle. With this dissociation of the vascular effect from that of the renal, the hydrins might play a physiological role in water balance of frogs.

Topics: Animals; Blood Pressure; Cardiovascular Physiological Phenomena; Cardiovascular System; Diuresis; Female; Femoral Artery; Heart Rate; Kidney; Male; Myocardial Contraction; Potassium Chloride; Rana catesbeiana; Ranidae; Vasoconstriction; Vasodilation; Vasotocin

From neurointermediate pituitary glands of Xenopus laevis and Rana esculenta, previously unreported peptides termed hydrins, active on water permeability of frog urinary bladder and frog skin (Brunn or "water-balance" effect), have been isolated and sequenced. These peptides seem to be derived from the pro-vasotocin-neurophysin precursor. Hydrin 1, found in Xenopus, has been identified as vasotocin C-terminally extended with the Gly-Lys-Arg sequence; hydrin 2, found in Rana, has been identified as vasotocin C-terminally extended with glycine. Hydrin 2 has been detected in several Ranidae (R. esculenta, Rana temporaria, Rana pipiens) and Bufonidae (Bufo bufo, Bufo ictericus) and appears to have a large distribution in terrestrial or semiaquatic anurans. Hydrins, in contrast to vasotocin, are not active on rat uterus or rat blood pressure. They are absent from other vasotocin-bearers such as birds and could be involved specifically in water-electrolyte regulation of amphibians.

Topics: Acclimatization; Amino Acid Sequence; Animals; Molecular Sequence Data; Oxytocin; Pituitary Gland, Posterior; Protein Processing, Post-Translational; Rana esculenta; Species Specificity; Vasotocin; Water-Electrolyte Balance; Xenopus laevis