Norifumi Konno

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.

The fifth neurohypophysial hormone receptor is structurally related to the V2-type receptor but functionally similar to V1-type receptors

The neurohypophysial peptides of the vasopressin (VP) and oxytocin (OT) families regulate salt and water homeostasis and reproduction through distinct G protein-coupled receptors. The current thinking is that there are four neurohypophysial hormone receptors (V1aR, V1bR, V2R, and OTR) in vertebrates, and their evolutionary history is still debated. We report the identification of a fifth neurohypophysial hormone receptor (V2bR) from the holocephalan elephant fish. This receptor is similar to conventional V2R (V2aR) in sequence, but induced Ca(2+) signaling in response to vasotocin (VT), the non-mammalian VP ortholog; such signaling is typical of V1-type receptors. In addition, V1aR, V1bR and OTR were also isolated from the elephant fish. Further screening revealed that orthologous V2bRs are widely distributed throughout the jawed vertebrates, and that the V2bR family is subdivided into two subfamilies: the fish specific type-1, and a type-2 that is characteristically found in tetrapods. Analysis suggested that the mammalian V2bR may have lost its function. Based on molecular phylogenetic, synteny and functional analyses, we propose a new evolutionary history for the neurohypophysial hormone receptors in vertebrates as follows: the first duplication generated V1aR/V1bR/OTR and V2aR/V2bR lineages; after divergence from the V2bR lineage, the V2aRs evolved to use cAMP as a second messenger, while the V2bRs retained the original Ca(2+) signaling system. Future studies on the role of V2bR in the brain, heart, kidney and reproductive organs, in which it is highly expressed, will open a new research field in VP/VT physiology and evolution.

African lungfish, Protopterus annectens, possess an arginine vasotocin receptor homologous to the tetrapod V2-type receptor.

SUMMARY In tetrapods, arginine vasopressin and its counterpart, arginine vasotocin (AVT), are involved in renal water conservation through vascular V1a-type and tubular V2-type receptors, and only the former has thus far been cloned in fish. We successfully cloned the V1a-type and V2-type AVT receptor from the kidney of the African lungfish, Protopterus annectens, and the deduced amino acid sequences exhibited high homology with amphibian V1a- and V2-type receptors, respectively. Functional analysis showed that AVT addition to CHO cells transfected with lungfish V1a-type receptor increased [Ca2+]i in a concentration-dependent manner, whereas CHO cells transfected with lungfish V2-type receptor responded with cAMP accumulation after AVT stimulation. Lungfish V2-type receptor mRNA was strongly expressed in the heart and kidney, while V1a-type receptor mRNA was ubiquitously expressed in all the tissues examined. In the kidney, immunohistochemistry using a specific antibody to lungfish V2-type receptor showed localization in the basolateral area of the cells in the late part of the distal tubules. Artificial estivation (EST) for 90 days significantly increased plasma osmolality and sodium and urea concentrations. There was no significant difference in the V2-type receptor mRNA and protein expression levels in the kidney between the freshwater and EST lungfish, while the AVT precursor mRNA level in the hypothalamus was remarkably higher in the EST lungfish. Our results indicate that African lungfish possess a functional V2-type receptor similar to that in tetrapods, suggesting that elevated plasma AVT during estivation exerts a renal tubular antidiuretic effect through the V2-type receptor expressed in the distal segments of lungfish kidney.

Effect of osmotic stress on expression of a putative facilitative urea transporter in the kidney and urinary bladder of the marine toad, Bufo marinus

SUMMARY Anuran amphibians accumulate a large amount of urea in their extracellular fluids to avoid a severe dehydration under dry and hyper-saline environments. To clarify the mechanisms of urea retention, we examined structure and distribution of the urea transporter (UT) in the kidney of the marine toad (Bufo marinus), and its expression in the kidney and urinary bladder following exposure to dry and hyper-saline conditions by means of cDNA cloning, semi-quantitative RT-PCR, immunoblot analysis and immunohistochemistry. The Bufo UT cDNA cloned from the kidney encodes a 390-amino-acid residue protein, which is 80% identical to Rana esculenta UT with the functional characteristics of a urea transporter. The Bufo UT mRNA was abundantly expressed in the kidney and urinary bladder, but not in the skin. In immunoblot analysis using a specific antibody raised against the Bufo UT, a 52 kDa protein similar to the glycosylated forms of mammalian UT-A2 (∼55 kDa) was detected in extracts from plasma membrane fractions of the kidney and urinary bladder. When toads were acclimated to dry and hyper-saline environments for 7 days, UT mRNA expression was upregulated in the kidney and urinary bladder and there was an elevated plasma urea concentration and osmolality. Immunohistochemistry showed that the UT was specifically localized on the apical membrane of the early distal tubule, known to be the diluting segment, in the kidney and the epithelial cells of urinary bladder. Immunoreactive cells were not detected along the late distal tubule, the connecting tubule or the collecting duct in the kidney. The present findings suggest that the Bufo UT probably contributes to urea transport in the kidney and urinary bladder in response to hyperosmotic stresses such as body fluid hypertonicity and dehydration.

Molecular cloning and characterization of V2-type receptor in two ray-finned fish, gray bichir, Polypterus senegalus and medaka, Oryzias latipes.

In tetrapods, vasopressin (VP) and vasotocin (VT) are involved in various aspects of physiology and behavior including osmoregulation, cardiovascular function, reproduction, stress response and social behavior. Pharmacological and molecular studies have identified three types of VP/VT receptors, V1a-type (V1aR), V1b-type (V1bR) and V2-type (V2R). On the other hand, only V1aR has so far been identified in teleosts. In the present study, we successfully cloned V2Rs from two ray-finned fish, gray bichir and medaka. Phylogenetic analysis showed that the cloned receptors belong to the V2R group of lobe-finned fish and tetrapods. The amino acid sequences of bichir V2R and medaka V2R were high identity (60-65.5% and 53.2-80.9%, respectively) with other known V2R members, respectively. Reverse transcriptase PCR revealed that ray-finned fish V2R transcripts have been detected in various tissues including brain, gill, heart, liver, kidney and reproductive organs, suggesting that ray-finned fish V2R might mediate multiple functions of VT. In functional analysis, the cells transfected with the cloned receptors responded with the accumulation of intracellular cAMP in a concentration-dependent manner following VT stimulation, but not respond with [Ca(2+)]i. Furthermore, pretreatment with mammalian V2R antagonist (OPC-31260) to the cells transfected with medaka V2R significantly inhibited an increase of the VT-induced intracellular cAMP. These results suggest that ray-finned fish possess a functional V2R linked to adenylate cyclase and the cAMP signaling pathway as well as V2Rs of lobe-finned fish and tetrapods. Thus, the present study suggests that functional V2R evolved prior to the divergence of the ray- and lobe-finned fish lineages.

Neuropeptide Y in tiger puffer (Takifugu rubripes): distribution, cloning, characterization, and mRNA expression responses to prandial condition.

Neuropeptide tyrosine (NPY) is a potent orexigenic neuropeptide implicated in feeding regulation in rodents. However, the involvement of NPY in feeding behavior has not well been studied in fish. Therefore, we investigated the role of NPY in food intake using a tiger puffer (Takifugu rubripes) model. We observed the distribution of NPY-like immunoreactivity in the brain. Neuronal cell bodies containing NPY were located in the telencephalon, hypothalamus, mesencephalon, and medulla oblongata, and their nerve fibers were also found throughout the brain. We cloned two cDNAs, encoding NPYa and NPYb orthologs, respectively, from the brain, and also confirmed two genes encoding these NPYs in the Takifugu genome database. We examined the distribution of these transcripts in the brain using real-time PCR. Levels of NPYa mRNA in the telencephalon, mesencephalon and hypothalamus were much higher than in the medulla oblongata and cerebellum, whereas levels of NPYb mRNA in the medulla oblongata were higher than in other regions. We also examined prandial effects on the expression level of these transcripts in the telencephalon and hypothalamus. NPYa mRNA levels in the hypothalamus, but not in the telencephalon, obtained from fish fasted for one week were higher than those in fish that had been fed normally. The level was decreased at 2 h after feeding. Levels of NPYb mRNA were not affected by prandial conditions. These results suggest that NPY is present throughout the brain, and that NPYa, but not NPYb, in the hypothalamus is involved in the feeding regulation in the tiger puffer.

Immunolocalization and mRNA expression of the epithelial Na+ channel α-subunit in the kidney and urinary bladder of the marine toad, Bufo marinus, under hyperosmotic conditions

The amiloride-sensitive epithelial sodium channel (ENaC) has previously been shown to be involved in the maintenance of body fluid volume and in Na+ absorption across the skin and urinary bladder in amphibians. However, the function and distribution of ENaC have not been clearly described in amphibian kidney. We therefore cloned the ENaC α-subunit cDNA from kidney of the marine toad, Bufo marinus. The ENaC mRNA and protein were abundantly expressed in the kidney and in the urinary bladder and ventral pelvic skin. In an immunohistochemical study, the ENaC α-subunit protein was specifically localized to the apical membrane of the principal cells but not the intercalated cells from the late distal tubule to the collecting duct in the kidney or in the apical area of cells of urinary bladder epithelia. When toads were acclimated to dry and hyper-saline environments, the levels of ENaC mRNA expression in the kidney and urinary bladder decreased under hyper-saline acclimation, but not under dry conditions. Immunohistochemical observations indicated that the levels of ENaC protein expression were much lower in the apical area of renal distal tubules and urinary bladder epithelia of hyper-saline acclimated toad compared with controls. The present study suggests that Bufo ENaC is significantly expressed and functions during Na+ reabsorption in the apical membrane domain in the distal nephron of normal and desiccated toads. Natriuresis may be caused by decreases in ENaC expression and its trafficking to the cell surface in the distal nephron, a response to prevent excessive Na+ reabsorption in hyper-saline-acclimated toads.

Ghrelin Receptor in Two Species of Anuran Amphibian, Bullfrog (Rana catesbeiana), and Japanese Tree Frog (Hyla japonica)

We have identified cDNA encoding a functional growth hormone secretagogue-receptor 1a (GHS-R1a, ghrelin receptor) in two species of anuran amphibian, the bullfrog (Rana catesbeiana), and the Japanese tree frog (Hyla japonica). Deduced receptor protein for bullfrog and Japanese tree frog (tree frog) was comprised of 374- and 371-amino acids, respectively. The two receptors shared 86% identity, and are grouped to the clade of the tetrapod homologs by phylogenetic analysis. In functional analyses, ghrelin and GHS-R1a agonists increased intracellular Ca2+ concentration in GHS-R1a-transfected-HEK293 cell, but ligand selectivity of ghrelin with Ser3 and Thr3 was not observed between the two receptors. Bullfrog GHS-R1a mRNA was mainly expressed in the brain, stomach, and testis. In the brain, the gene expression was detected in the diencephalon and mesencephalon, but not in the pituitary. Tree frog GHS-R1a mRNA was predominantly expressed in the gastrointestinal tract and ovary, but not detected in the pituitary. In bullfrog stomach but not the brain, GHS-R1a mRNA expression increased after 10 days of fasting. For tree frog, GHS-R1a mRNA expression was increased in the brain, stomach and ventral skin by 10 days of fasting, and in the stomach and ventral skin by a dehydration treatment. Intracerebroventricular injection of ghrelin in dehydrated tree frog did not affect water absorption from the ventral skin. These results suggest that ghrelin is involved in energy homeostasis and possibly in osmoregulation in frogs.

Orexin A enhances locomotor activity and induces anxiogenic-like action in the goldfish, Carassius auratus

Orexin acts as an orexigenic factor for the regulation of appetite and rhythmicity in rodents. In goldfish, intracerebroventricular (ICV) administration of orexin A has been shown to affect not only food intake, but also locomotor activity. However, as there is still no information regarding the effect of orexin A on emotional behavior in goldfish, we investigated the effect of orexin A on psychomotor activity in this species. Intracerebroventricular administration of synthetic orexin A at 2 and 4pmol/g body weight (BW) enhanced locomotor activity, and this enhancement by orexin A at 4pmol/g BW was attenuated by treatment with the orexin receptor 1 antagonist, SB334867, at 10pmol/g BW. Since intact goldfish prefer a black to a white background area, or the lower to the upper area of a tank, we used two types of preference tests (black/white and upper/lower tests) for measuring anxiety-like behavior in goldfish. Intracerebroventricular administration of orexin A at 4pmol/g BW shortened the time spent in the white background area, and increased the time taken to move from the lower to the upper area. This action of orexin A mimicked that of the central-type benzodiazepine receptor inverse agonist, FG-7142 (an anxiogenic agent), at 4pmol/g BW. The anxiogenic-like effect of orexin A was abolished by treatment with SB334867 at 10pmol/g BW. These results indicate that orexin A potently affects psychomotor activity in goldfish.

The epithelial sodium channel in the Australian lungfish, Neoceratodus forsteri (Osteichthyes: Dipnoi)

Epithelial sodium channel (ENaC) is a Na+-selective, aldosterone-stimulated ion channel involved in sodium transport homeostasis. ENaC is rate-limiting for Na+ absorption in the epithelia of osmoregulatory organs of tetrapods. Although the ENaC/degenerin gene family is proposed to be present in metazoans, no orthologues or paralogues for ENaC have been found in the genome databases of teleosts. We studied full-length cDNA cloning and tissue distributions of ENaCα, β and γ subunits in the Australian lungfish, Neoceratodus forsteri, which is the closest living relative of tetrapods. Neoceratodus ENaC (nENaC) comprised three subunits: nENaCα, β and γ proteins. The nENaCα, β and γ subunits are closely related to amphibian ENaCα, β and γ subunits, respectively. Three ENaC subunit mRNAs were highly expressed in the gills, kidney and rectum. Amiloride-sensitive sodium current was recorded from Xenopus oocytes injected with the nENaCαβγ subunit complementary RNAs under a two-electrode voltage clamp. nENaCα immunoreactivity was observed in the apical cell membrane of the gills, kidney and rectum. Thus, nENaC may play a role in regulating sodium transport of the lungfish, which has a renin–angiotensin–aldosterone system. This is interesting because there may have been an ENaC sodium absorption system controlled by aldosterone before the conquest of land by vertebrates.