Susumu Hyodo

Vacuolar-type proton pump in the basolateral plasma membrane energizes ion uptake in branchial mitochondria-rich cells of killifish, Fundulus heteroclitus, adapted to a low ion environment

SUMMARY We examined the involvement of mitochondria-rich (MR) cells in ion uptake through gill epithelia in freshwater-adapted killifish Fundulus heteroclitus, by morphological observation of MR cells and molecular identification of the vacuolar-type proton pump (V-ATPase). MR cell morphology was compared in fish acclimated to defined freshwaters with different NaCl concentrations: low (0.1 mmol l-1)-, mid (1 mmol l-1)- and high (10 mmol l-1)-NaCl environments. MR cells, mostly located on the afferent-vascular side of the gill filaments, were larger in low- and mid-NaCl environments than in the high-NaCl environment. Electron-microscopic observation revealed that the apical membrane of well-developed MR cells in low- and mid-NaCl environments was flat or slightly projecting, and equipped with microvilli to expand the surface area exposed to these environments. On the other hand, in the high-NaCl environment, the apical membrane was invaginated to form a pit, and MR cells often formed multicellular complexes with accessory cells, although the NaCl concentration was much lower than that in plasma. We cloned and sequenced a cDNA encoding the A-subunit of killifish V-ATPase. The deduced amino acid sequence showed high identity with V-ATPase A-subunits from other vertebrate species. Light-microscopic immunocytochemistry, using a homologous antibody, revealed V-ATPase-immunoreactivity in Na+/K+-ATPase-immunoreactive MR cells in low-NaCl freshwater, whereas the immunoreactivity was much weaker in higher NaCl environments. Furthermore, immuno-electron microscopy revealed V-ATPase to be located in the basolateral membrane of MR cells. These findings indicate that MR cells are the site responsible for active ion uptake in freshwater-adapted killifish, and that basolaterally located V-ATPase is involved in the Na+ and/or Cl- absorbing mechanism of MR cells.

Differential production and regulation of gonadotropins (GTH I and GTH II) in the pituitary gland of rainbow trout, Oncorhynchus mykiss, during ovarian development

SummaryBiosynthesis of salmon gonadotropins, GTH I and GTH II, during ovarian development, were examined by means of in situ hybridization histochemistry and indirect immunocytochemistry. In rainbow trout pituitary glands, expression of GTH Iβ- and IIβ-subunit genes appeared separately in distinct cells (GTH I- and GTH II-cells), whereas the GTH α-subunit gene was expressed in both cell-types. In the GTH I-cells, coordinated increases in GTh, α and Iβ messenger ribonucleic acids (mRNAs) occurred coincident with the onset of vitellogenesis, indicating active synthesis of GTH I during vitellogenesis. In contrast, in the GTH II-cells, both GTH α-and IIβ-mRNA signals markedly increased from a later stage of vitellogenesis and persisted throughout oocyte maturation and ovulation, supporting the idea that GTH II is actively synthesized as a maturational GTH. GTH α-mRNA levels in the GTH I-cells selectively decreased prior to final oocyte maturation, although Iβ-mRNA levels remained elevated, thus suggesting a decline of biosynthesis of GTH I after vitellogenesis. These findings clarify how the synthesis of GTH I and GTH II are coordinated in the piscine pituitary, and indicate that the expression of GTH subunit genes during gametogenesis is regulated differentially in a cell-specific manner, both temporally and spatially.

Novel fish-derived adrenomedullin in mammals: structure and possible function.

Adrenomedullin (AM) has been recognized as a member of the calcitonin (CT)/CT gene-related peptide (CGRP) family. However, an independent AM family consisting of five paralogous peptides exists in teleost fish. Among them, the peptide named AM1 is an ortholog of mammalian AM as determined by the linkage analysis of orthologous genes and the presence of proAM N-terminal 20 peptide (PAMP)-like sequence in the prosegment. Since the peptides named AM2 and 3 are distinct from other members with respect to the precursor sequence, tissue distribution of the transcripts, and exon-intron organization, we searched for their mammalian orthologs from genome databases, which resulted in an identification of AM2 in human, rat, and mouse. AM2 was expressed abundantly in the submaxillary gland, kidney, and some vascular and digestive tissues of mice. AM2 injected in vivo induced potent cardiovascular and renal effects in mice. In the heart and kidney of mice, AM2 was localized in endothelial cells of the coronary vessels and in glomeruli and vasa recta, respectively. AM2 increased cAMP accumulation in cells expressing human CT receptor-like receptor (CRLR) and one of receptor activity-modifying proteins (RAMPs), but it was no more potent than CGRP and AM. AM2 was also less potent than CT in cells expressing CT receptor and RAMP. There remains a possibility that a new AM2-specific receptor or an additional RAMP that enables CRLR to be an AM2-specific receptor, exists in mammals.

Changes in expression of provasotocin and proisotocin genes during adaptation to hyper- and hypo-osmotic environments in rainbow trout

SummaryThe physiological roles of neurohypophysial hormones, vasotocin (VT) and isotocin (IT), are not yet clear in teleosts. Since information on responsiveness of hypothalamic neurosecretory neurons to environmental stimuli may contribute to an understanding of their physiological roles, effects of environmental hyper- and hypo-osmotic stimuli on expression of VT and IT precursor (proVT and proIT) genes in rainbow trout were investigated, using an in situ hybridization technique in which 46 mer synthetic oligonucleotides were used as hybridization probes. The probes corresponded to the mRNA loci encoding chum salmon proVT (-5 to 11) and proIT (-5 to 11), and were labeled at the 3′-end with 35S. Autoradiographic silver grains which represent the hybridization signals of proVT and proIT mRNAs were localized in both magnocellular and parvocellular neurons in the nucleus preopticus magnocellularis (NPOmg). Localizations of proVT and proIT hybridization signals coincided with those of VT- and IT-immunoreactive neurons in adjacent sections, and showed that proVT and proIT genes are expressed in separate neurons. The intensity of proVT hybridization signals as determined by grain counting in magnocellular neurons in the NPOmg was conspicuously decreased after transfer from fresh water (FW) to 80% seawater (SW). The proVT mRNA levels in SW trout were consistently lower than those of FW trout for up to 2 weeks. After return from 80% SW to FW, the proVT mRNA level increased, attaining the initial FW level. The proIT mRNA levels in SW trout were not statistically different from those in FW trout, except for the 1st day after transfer to SW. These results suggest that synthesis of proVT was elevated by transfer from higher to lower salinity, and that VT may have a physiological role in salmonid osmoregulation, especially in adaptation to a hypo-osmotic environment.

A facilitative urea transporter is localized in the renal collecting tubule of the dogfish Triakis scyllia.

SUMMARY Reabsorption of filtered urea by the kidney tubule is essential for retaining high levels of urea in body fluids of marine elasmobranchs. To elucidate the mechanisms of urea reabsorption, we examined the distribution of a facilitative urea transporter (UT) in the kidney of the dogfish Triakis scyllia. We isolated a cDNA encoding a UT that is homologous to the facilitative UT cloned from another dogfish species, Squalus acanthias. The Triakis UT mRNA is abundantly expressed in the kidney, while low levels of expression were detected in the brain and liver. In the dogfish kidney, each nephron makes four turns and traverses repeatedly between bundle zone and sinus zone. In the bundle zone, the resulting five tubular segments are arranged in a countercurrent loop fashion. Immunohistochemistry using specific antibodies raised against the cloned UT revealed that, among the nephron segments, the UT is expressed exclusively in the final segment of the bundle zone, i.e. in the collecting tubule of the Triakis kidney. In contrast to the limited localization of UT, the transport enzyme Na+/K+-ATPase is distributed in the basolateral membrane of numerous tubular segments both in the sinus zone and the bundle zone. However, in the collecting tubule, Na+/K+-ATPase immunoreactivity was not detected. The present study suggests that the collecting tubule is responsible for the reabsorption of urea in the marine elasmobranch kidney. Other countercurrent segments may contribute to production of a driving force for facilitative diffusion of urea through the UT.

Androgenic regulation of expression of β-tubulin messenger ribonucleic acid in motoneurons of the spinal nucleus of the bulbocavernosus

Expression of β‐tubulin mRNA was examined in androgen‐sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB) in adult male rats by in situ hybridization histochemistry using cDNA encoding mouse β‐tubulin. Hybridizable β‐tubulin mRNA was localized in the somata and proximal dendrites of SNB motoneurons. Removal of androgen by castration significantly reduced the expression level of β‐tubulin mRNA in the SNB motoneurons, whereas the change was prevented by testosterone treatment. On the contrary, castration or testosterone treatment did not induce any changes in the expression level of β‐tubulin mRNA in the androgen‐insensitive motoneurons of the retrodorsolateral nucleus. These results suggest that androgen regulates the expression of β‐tubulin gene in the SNB motoneurons and may provide evidence for the molecular mechanisms of hormonally‐induced neuronal plasticity in the SNB motoneurons.

Effect of androgen on the expression of gap junction and β-actin mRNAs in adult rat motoneurons

Expression of gap junction and beta-actin mRNAs was examined in androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB) in adult male rats by in situ hybridization histochemistry using complementary DNAs encoding rat liver gap junction protein (connexin 32) and chick beta-actin. Hybridizable gap junction and beta-actin mRNAs were localised on the somata and proximal dendrites of SNB motoneurons. Removal of androgen by castration significantly reduced the expression levels of both gap junction and beta-actin mRNAs in the SNB motoneurons, whereas these changes were prevented by testosterone treatment. On the contrary, castration or testosterone treatment did not induce any changes in the expression levels of gap junction and beta-actin mRNAs in the motoneurons of the retrodorsolateral nucleus (RDLN), which accumulate androgen less frequently and sparsely than those in the SNB. These results suggest that androgen regulates the expression of both gap junction and beta-actin genes in the SNB motoneurons and may provide evidence for the molecular mechanisms of hormonally induced neuronal plasticity in the SNB motoneurons.

Cytophysiology of gonadotropin-releasing-hormone neurons in chum salmon (Oncorhynchus keta) forebrain before and after upstream migration

Abstract.Cytophysiology of gonadotropin-releasing-hormone neurons in chum salmon (Oncorhynchus keta) was examined before and after upstream migration by an immunocytochemical technique with a specific antiserum to salmon gonadotropin-releasing hormone and an in situ hybridization technique with an oligonucleotide encoding salmon gonadotropin-releasing-hormone precursor (pro-salmon gonadotropin-releasing hormone). In the forebrain (olfactory nerve, olfactory bulb, telencephalon, and preoptic area), salmon gonadotropin-releasing hormone-immunoreactive neurons and neurons showing signals for pro-salmon gonadotropin-releasing-hormone mRNA were compared between fish from the coastal sea and those from the spawning ground. Neurons in the dorsal region of the olfactory nerve and in the ventral region of the transitional area between olfactory nerve and olfactory bulb showed strong salmon gonadotropin-releasing-hormone immunoreactivity and strong hybridization signals in fish from the coastal sea, but these activities and signals were not observed or were decreased in number in fish from the spawning ground. The neurons in the olfactory bulb, telencephalon, and preoptic area consistently revealed salmon gonadotropin-releasing-hormone immunoreactivity and hybridization signals, and the hybridization signals of salmon gonadotropin-releasing hormone in the telencephalon and the preoptic area were stronger in fish from the spawning ground than in those from the coastal sea. These findings suggest that salmon gonadotropin-releasing-hormone neurons in the olfactory nerve and the transitional area between olfactory nerve and olfactory bulb have different patterns of hormone production than those in the telencephalon and the preoptic area.

Vasotocin/V2-Type Receptor/Aquaporin Axis Exists in African Lungfish Kidney but Is Functional Only in Terrestrial Condition

The vasopressin/vasotocin (VT)-V2-type receptor (V2R)-aquaporin (AQP)-2 axis plays a pivotal role in renal water reabsorption in tetrapods. It is widely thought that this axis evolved with the emergence of the tetrapods, reflecting a requirement of water retention in terrestrial environment. Here we report that lungfish, the closest living relatives of tetrapods, already possess a system similar to the VT-V2R-AQP2 axis in the kidney, but the system is functional only in the terrestrial estivating condition. We cloned a novel AQP paralogous to AQP0. The water permeability of Xenopus oocytes was increased by injection with the AQP cRNA and was further facilitated by preincubation with cAMP. In the kidney of estivating lungfish, the AQP protein was localized on the apical plasma membrane of the late distal tubule and was colocalized with basolateral V2R. By contrast, we found only little expression of the AQP mRNA and protein in the kidney of lungfish in aquatic condition. The expression levels of mRNA and protein were dramatically increased during estivation and decreased again by reacclimation of estivating lungfish to water. The AQP mRNA levels positively correlated with the VT mRNA levels in the hypothalamus, suggesting that the AQP exerts tubular antidiuretic action under control of VT. Because the tetrapod AQP2/AQP5 lineage is considered to be evolved from duplication of an AQP0 gene, the paralogous AQP0 in the lungfish probably represents ancestral molecule for tetrapod AQP2.

Androgen Regulates Gene Expression of Cytoskeletal Proteins in Adult Rat Motoneurons

Expression of beta-actin and beta-tubulin mRNA was examined in androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB) in adult male rats by in situ hybridization histochemistry using complementary DNAs encoding chick beta-actin and mouse beta-tubulin, respectively. Both hybridizable beta-actin and beta-tubulin mRNAs were localized in the somata and proximal dendrites of SNB motoneurons. Removal of androgen by castration significantly reduced the expression levels of both beta-actin and beta-tubulin mRNAs in the SNB motoneurons, whereas the changes were prevented by testosterone treatment. In contrast, castration or testosterone treatment induced little or no change in the expression levels of these mRNAs in the much less androgen-sensitive motoneurons of the retrodorsolateral nucleus (RDLN). These results suggest that androgen regulates the expression of beta-actin and beta-tubulin genes in the SNB motoneurons and may provide evidence for the molecular mechanisms of hormonally induced neuronal plasticity in the SNB motoneurons.