Metabolic Water As a Route for Water Acquisition in Vertebrates Inhabiting Dehydrating Environments.

Vertebrates have expanded their habitats during evolution, which accompanies diversified routes for water acquisition. Water is acquired by oral intake and subsequent absorption by the intestine in terrestrial and marine animals which are subjected to constant dehydration, whereas most water is gained osmotically across body surfaces in freshwater animals. In addition, a significant amount of water, called metabolic water, is produced within the body by the oxidation of hydrogen in organic substrates. The importance of metabolic water production as a strategy for water acquisition has been well documented in desert animals, but its role has attracted little attention in marine animals which also live in a dehydrating environment. In this article, the author has attempted to reevaluate the role of metabolic water production in body fluid regulation in animals inhabiting desiccating environments. Because of the exceptional ability of their kidney, marine mammals are thought to typically gain water by drinking environmental seawater and excreting excess NaCl in the urine. On the other hand, it is established that marine teleosts drink seawater to enable intestinal water and ion absorption, and the excess NaCl is excreted by branchial ionocytes. In addition to the oral route, we suggest through experiments using eels that water production by lipid metabolism is an additional route for water acquisition when they encounter seawater. It seems that metabolic water production contributes to counteract dehydration before mechanisms for water regulation are reversed from excretion in freshwater to acquisition in seawater.

Gene duplication of C-type natriuretic peptide-4 (CNP4) in teleost lineage elicits subfunctionalization of ancestral CNP.

The diversified natriuretic peptide (NP) family, consisting of four CNPs (CNP1-4), ANP, BNP, and VNP, has been identified in the eel. Here, we successfully cloned additional cnp genes from the brain of eel (a basal teleost) and zebrafish (a later branching teleost). The genes were identified as paralogues of cnp4 generated by the third round of whole genome duplication (3R) in the teleost lineage, thereby being named eel cnp4b and zebrafish cnp4-like, respectively. To examine the histological patterns of their expressions, we employed a newly developed in situ hybridization (ISH) chain reaction using short hairpin DNAs, in addition to conventional ISH. Eel cnp4b was expressed in the medulla oblongata, while mRNAs of eel cnp4a (former cnp4) were localized in the preoptic area. In the zebrafish brain, cnp4-like mRNA was undetectable, while the known cnp4 was expressed in both the preoptic area and medulla oblongata. Together with the different mRNA distribution of cnp4a and cnp4b in eel peripheral tissues determined by RT-PCR and ISH, it is suggested that subfunctionalization by duplicated cnp4s in ancestral teleosts has been retained only in basal teleosts. Intriguingly, cnp4b-expressing neurons in the glossopharyngeal-vagal motor complex of the medulla oblongata were co-localized with choline acetyltransferase, suggesting an involvement of Cnp4b in swallowing and respiration functions that are modulated by the vagus. Since teleost Cnp4 is an ortholog of mammalian CNP, the identified localization of teleost Cnp4 will contribute to future studies aimed at deciphering the physiological functions of CNP.

Circulating isotocin, not angiotensin II, is the major dipsogenic hormone in eels.

Angiotensin II (AngII) is generally known as the most important dipsogenic hormone throughout vertebrates, while two other neurohypophysial hormones, vasopressin and oxytocin, are not dipsogenic in mammals. In this study, we found that systemic isotocin, but not vasotocin, is the potent dipsogenic hormone in eels. When injected intra-arterially into conscious eels, isotocin, vasotocin and AngII equally increased ventral aortic pressure dose dependently at 0.03-1.0 nmol kg-1, but only isotocin induced copious drinking. The dipsogenic effect was dose dependent and occurred significantly at as low as 0.1 nmol kg-1. By contrast, a sustained inhibition of drinking occurred after AngII injection, probably due to baroreflexogenic inhibition. No such inhibition was observed after isotocin injection despite similar concurrent hypertension. The baroreceptor may exist distal to the gill circulation because the vasopressor effect occurred at both ventral and dorsal aorta after AngII but only at ventral aorta after isotocin. By contrast, intra-cerebroventricular (i.c.v.) injection of isotocin had no effect on drinking or blood pressure, but AngII increased drinking and aortic pressure dose dependently at 0.03-0.3 nmol per eel. Lesioning of the area postrema (AP), a sensory circumventricular organ, abolished drinking induced by peripheral isotocin, but not i.c.v. AngII. Collectively, isotocin seems to be a major circulating hormone that induces swallowing through its action on the AP, while AngII may be an intrinsic brain peptide that induces drinking through its action on a different circumventricular site, possibly a recently identified blood-brain barrier-deficient structure in the antero-ventral third ventricle of eels, as shown in birds and mammals.

Evolution of the membrane/particulate guanylyl cyclase: From physicochemical sensors to hormone receptors.

Guanylyl cyclase (GC) is an enzyme that produces 3',5'-cyclic guanosine monophosphate (cGMP), one of the two canonical cyclic nucleotides used as a second messenger for intracellular signal transduction. The GCs are classified into two groups, particulate/membrane GCs (pGC) and soluble/cytosolic GCs (sGC). In relation to the endocrine system, pGCs include hormone receptors for natriuretic peptides (GC-A and GC-B) and guanylin peptides (GC-C), while sGC is a receptor for nitric oxide and carbon monoxide. Comparing the functions of pGCs in eukaryotes, it is apparent that pGCs perceive various environmental factors such as light, temperature, and various external chemical signals in addition to endocrine hormones, and transmit the information into the cell using the intracellular signaling cascade initiated by cGMP, e.g., cGMP-dependent protein kinases, cGMP-sensitive cyclic nucleotide-gated ion channels and cGMP-regulated phosphodiesterases. Among vertebrate pGCs, GC-E and GC-F are localized on retinal epithelia and are involved in modifying signal transduction from the photoreceptor, rhodopsin. GC-D and GC-G are localized in olfactory epithelia and serve as sensors at the extracellular domain for external chemical signals such as odorants and pheromones. GC-G also responds to guanylin peptides in the urine, which alters sensitivity to other chemicals. In addition, guanylin peptides that are secreted into the intestinal lumen, a pseudo-external environment, act on the GC-C on the apical membrane for regulation of epithelial transport. In this context, GC-C and GC-G appear to be in transition from exocrine pheromone receptor to endocrine hormone receptor. The pGCs also exist in various deuterostome and protostome invertebrates, and act as receptors for environmental, exocrine and endocrine factors including hormones. Tracing the evolutionary history of pGCs, it appears that pGCs first appeared as a sensor for physicochemical signals in the environment, and then evolved to function as hormone receptors. In this review, the author proposes an evolutionary history of pGCs that highlights the emerging role of the GC/cGMP system for signal transduction in hormone action.

Molecular mechanisms underlying guanylin-induced transcellular Cl- secretion into the intestinal lumen of seawater-acclimated eels.

Guanylin (GN) stimulates Cl- secretion into the intestinal lumen of seawater-acclimated eels, but the molecular mechanisms of transepithelial Cl- transport are still unknown. In Ussing chamber experiments, we confirmed that mucosal application of eel GN reversed intestinal serosa-negative potential difference, indicating Cl- secretion. Serosal application of DNDS or mucosal application of DPC inhibited the GN effect, but serosal application of bumetanide had no effect. Removal of HCO3- from the serosal fluid also inhibited the GN effect. In intestinal sac experiments, mucosal GN stimulated luminal secretion of both Cl- and Na+, which was blocked by serosal DNDS. These results suggest that Cl- is taken up at the serosal side by DNDS-sensitive anion exchanger (AE) coupled with Na+-HCO3- cotransporter (NBC) but not by Na+-K+-2Cl- cotransporter 1 (NKCC1), and Cl- is secreted by unknown DPC-sensitive Cl- channel (ClC) at the mucosal side. The transcriptomic analysis combined with qPCR showed low expression of NKCC1 gene and no upregulation of the gene after seawater transfer, while high expression of ClC2 gene and upregulation after seawater transfer. In addition, SO42- transporters (apical Slc26a3/6 and basolateral Slc26a1) are also candidates for transcellular Cl- secretion in exchange of luminal SO42. Na+ secretion could occur through a paracellular route, as Na+-leaky claudin15 was highly expressed and upregulated after seawater transfer. High local Na+ concentration in the lateral interspace produced by Na+/K+-ATPase (NKA) coupled with K+ channels (Kir5.1b) seems to facilitate the paracellular transport. In situ hybridization confirmed the expression of the candidate genes in the epithelial enterocytes. Together with our previous results, we suggest that GN stimulates basolateral NBCela/AE2 and apical ClC2 to increase transcellular Cl- secretion in seawater eel intestine, which differs from the involvement of apical CFTR and basolateral NKCC1 as suggested in mammals and other teleosts.

Molecular mechanisms for intestinal HCO3- secretion and its regulation by guanylin in seawater-acclimated eels.

The intestine of marine teleosts secretes HCO3- into the lumen and precipitates Ca2+ and Mg2+ in the imbibed seawater as carbonates to decrease luminal fluid osmolality and facilitate water absorption. However, the hormonal regulation of HCO3- secretion is largely unknown. Here, mucosally added guanylin (GN) increased HCO3- secretion, measured by pH-stat, across isolated seawater-acclimated eel intestine bathed in saline at pH 7.4 (5% CO2). The effect of GN on HCO3- secretion was slower than that on the short-circuit current, and the time course of the GN effect was similar to that of bumetanide. Mucosal bumetanide and serosal 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) inhibited the GN effect, suggesting an involvement of apical Na+-K+-2Cl- cotransporter (NKCC2) and basolateral Cl-/HCO3- exchanger (AE)/Na+-HCO3- cotransporter (NBC) in the GN effect. As mucosal DNDS failed to inhibit the GN effect, apical DNDS-sensitive AE may not be involved. To identify molecular species of transporters involved in the GN effect, we performed RNA-seq analyses followed by quantitative real-time PCR after transfer of eels to seawater. Among the genes upregulated after seawater transfer, AE genes (draa, b, and pat1a, c) on the apical membrane, and NBC genes (nbce1a, n1, n2a) and an AE gene (sat-1) on the basolateral membrane were candidates involved in HCO3- secretion. Judging from the slow effect of GN, we suggest that GN inhibits NKCC2b on the apical membrane and decreases cytosolic Cl- and Na+, which then activates apical DNDS-insensitive DRAs and basolateral DNDS-sensitive NBCs to enhance transcellular HCO3- flux across the intestinal epithelia of seawater-acclimated eels.

Preparatory Mechanisms for Salinity Tolerance in Two Congeneric Anuran Species Inhabiting Distinct Osmotic Habitats.

Anurans occupy a wide variety of habitats of diverse salinities, and their osmoregulatory ability is strongly regulated by hormones. In this study, we compared the adaptability and hormonal responses to osmotic stress between two kajika frogs, Buergeria japonica (B.j.) and B. buergeri, (B.b.), which inhabit coastal brackish waters (BW) in the Ryukyu Islands and freshwater (FW) in the Honshu, respectively. Both hematocrit and plasma Na+ concentration were significantly higher in B.j. than in B.b. when both were kept in FW. After transfer to one-third seawater (simulating the natural BW environment), which is slightly hypertonic to their body fluids, their body mass decreased and plasma Na concentration increased significantly in both species. After transfer, plasma Na+ concentration increased significantly in both species. We examined the gene expression of two major osmoregulatory hormones, arginine vasotocin (AVT) and atrial natriuretic peptide (ANP), after partial cloning of their cDNAs. ANP mRNA levels were more than 10-fold higher in B.j. than in B.b. in FW, but no significant difference was observed for AVT mRNA levels due to high variability, although the mean value of B.j. was twice that of B.b. Both AVT and ANP mRNA levels increased significantly after transfer to BW in B.b. but not in B.j., probably because of the high levels in FW. These results suggest that B.j. maintains high plasma Na+ concentration and anp gene expression to prepare for the future encounter of the high salinity. The unique preparatory mechanism may allow B.j. wide distribution in oceanic islands.

Spreading of River Water Guides Migratory Behavior of Homing Chum Salmon Oncorhynchus keta in Otsuchi Bay, a Narrow Inlet with Multiple River Flows.

The Sanriku-ria coast of Japan, a homing area for chum salmon, Oncorhynchus keta, is characterized by a large number of small closed bays into which one or multiple short rivers flow. The present behavioral investigation of chum salmon in this region was designed to gain deeper insight into the migration of chum salmon to their natal rivers. Eighty-three fish caught at the middle part of Otsuchi Bay were tracked using an acoustic transmitter in the narrow inlet into which flow three rivers: the Otsuchi, Koduchi, and Unosumai. The majority of 18 fish that entered the Unosumai River, which flows into the southwest side of the bay, directly approached the river along the southern coast. More than half of fish that entered the Otsuchi and Koduchi Rivers, which flow into the northwest side, also migrated into the inner bay via the southerly route, and then entered these rivers frequently after passing the mouth of the Unosumai River. In the inner bay, the salinity of sea surface water suggested that water from the three rivers circulates in a counterclockwise direction at a depth of less than 1.0 m, flowing eastwardly along the southern coast. The observed migratory paths of homing salmon in Otsuchi Bay thus correspond well with the counterflow of surface river water in the bay. The present results suggest that homing migration of salmon in the Sanriku narrow inlet is guided by natal river flows.

Distribution and co-localization of diversified natriuretic peptides in the eel heart.

Atrial and B-type natriuretic peptides (ANP and BNP) are cardiac hormones important for cardiovascular and body fluid regulation. In some teleost species, an additional member of the natriuretic peptide family, ventricular NP (VNP), has been identified. In this study, we examine tissue distribution of these three NPs in the eel heart. Quantitative real-time PCR showed that anp is almost exclusively expressed in atria, bnp equally in atria and ventricles and vnp three-fold more in ventricles than in atria. The amount of bnp transcript overall in the heart was 1/10 those of anp and vnp. There was no difference in transcript levels between freshwater and seawater-acclimated fishes. Immunohistochemistry using specific antisera and in situ hybridization using gene-specific probes showed that NP signals were detected in most atrial and ventricular myocytes with some regional differences in density. Because of high sequence similarity of the three NPs, each of the three NP antisera individually was pre-incubated with 10-8 M of the other two non-targeted cardiac NPs to increase the specificity. A few atrial myocytes contained all three NPs in the same cell. Immuno-electron microscopy identified many dense-core vesicles containing ANP in atria and VNP in ventricles and some vesicles contained both ANP and VNP as demonstrated using pre-absorbed antisera. Based on these data and those of previous studies, we suggest that in eels ANP is secreted from atria in a regulatory pathway and VNP from ventricles in a constitutive pathway. In addition, VNP, not BNP, is the principal ventricular hormone in eels.

Renoguanylin stimulates apical CFTR translocation and decreases HCO3- secretion through PKA activity in the Gulf toadfish (Opsanus beta).

The guanylin peptides - guanylin, uroguanylin and renoguanylin (RGN) - are endogenously produced hormones in teleost fish enterocytes that are activators of guanylyl cyclase-C (GC-C) and are potent modulators of intestinal physiology, particularly in seawater teleosts. Most notably, they reverse normal net ion-absorbing mechanisms that are vital to water absorption, an important process for seawater teleost survival. The role of guanylin-peptide stimulation of the intestine remains unclear, but it is hypothesized to facilitate the removal of solids from the intestine by providing fluid to enable their removal by peristalsis. The present study used one member of this group of peptides - RGN - to provide evidence for the prominent role that protein kinase A (PKA) plays in mediating the effects of guanylin-peptide stimulation in the posterior intestine of the Gulf toadfish (Opsanus beta). Protein kinase G was found to not mediate the intracellular effects of RGN, despite previous evidence showing that GC-C activation leads to higher cyclic guanosine monophosphate formation. RGN reversed the absorptive short-circuit current and increased conductance in the Gulf toadfish intestine. These effects are correlated to increased trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel to the apical membrane, which is negated by PKA inhibition. Moreover, RGN decreased HCO3- secretion, likely by limiting apical HCO3-/Cl- exchange (possibly by reducing SLC26a6 activity), a reduction that was enhanced by PKA inhibition. RGN seems to alter PKA activity in the posterior intestine to recruit CFTR to the apical membrane and reduce HCO3- secretion.

Molecular and evolutionary perspectives of the renin-angiotensin system from lamprey.

The recent advance and revision on the renin-angiotensin system in lamprey were summarized and we emphasized that presence of two types of angiotensins (Angs) in lamprey. Due to the parasitic nature on fish blood, teleost-type Angs were produced in their buccal gland and secreted into the lamphredin to evade the host immunorejection. A native lamprey angiotensinogen (AGT) was identified in genome and it retains serine-protease inhibitor activity for thrombin that regulates the blood coagulation pathway. The native lamprey angiotensin II (Lp-Ang II) is hypotensive instead of hypertensive, suggesting a functional divergence on cardiovascular regulation from the main vertebrate groups. The renin gene was absent from the lamprey genome so far, and the mutation on the renin-recognition site on lamprey AGT suggested that other proteases may have replaced the role of renin. Lp-Ang II was shown to bind to AT1 receptor and internalized, but the downstream signaling was still unknown. Molecular and phylogenetic evidence on invertebrate ACE-like proteins indicated that they were not homologous to those in vertebrates and could be acting on other native peptides. Although it was generally believed that the RAS was a well-conserved hormone system in vertebrates and invertebrates, revision by molecular data indicated that invertebrates lack homologous RAS components while lamprey possess an almost complete RAS. This suggests that the hormone cascade system was first evolved around cyclostome emergence and invertebrates could have taken up the RAS components from vertebrates through horizontal gene transfer.

Molecular mechanisms underlying active desalination and low water permeability in the esophagus of eels acclimated to seawater.

Marine teleosts can absorb imbibed seawater (SW) to maintain water balance, with esophageal desalination playing an essential role. NaCl absorption from luminal SW was enhanced 10-fold in the esophagus of SW-acclimated eels, and removal of Na+ or Cl- from luminal SW abolished the facilitated absorption, indicating coupled transport. Mucosal/serosal application of various blockers for Na+/Cl- transporters profoundly decreased the absorption. Among the transporter genes expressed in eel esophagus detected by RNA-seq, dimethyl amiloride-sensitive Na+/H+ exchanger (NHE3) and 4,4'-diisothiocyano-2,2'-disulfonic acid-sensitive Cl-/[Formula: see text] exchanger (AE) coupled by the scaffolding protein on the apical membrane of epithelial cells, and ouabain-sensitive Na+-K+-ATPases (NKA1α1c and NKA3α) and diphenylamine-2-carboxylic acid-sensitive Cl- channel (CLCN2) on the basolateral membrane, may be responsible for enhanced transcellular NaCl transport because of their profound upregulation after SW acclimation. Upregulated carbonic anhydrase 2a (CA2a) supplies H+ and [Formula: see text] for activation of the coupled NHE and AE. Apical hydrochlorothiazide-sensitive Na+-Cl- cotransporters and basolateral Na+-[Formula: see text] cotransporter (NBCe1) and AE1 are other possible candidates. Concerning the low water permeability that is typically seen in marine teleost esophagus, downregulated aquaporin genes (aqp1a and aqp3) and upregulated claudin gene (cldn15a) are candidates for transcellular/paracellular route. In situ hybridization showed that these upregulated transporters and tight-junction protein genes were expressed in the absorptive columnar epithelial cells of eel esophagus. These results allow us to provide a full picture of the molecular mechanism of active desalination and low water permeability that are characteristic to marine teleost esophagus and gain deeper insights into the role of gastrointestinal tracts in SW acclimation.

Changes in Plasma and Tissue Long-Chain Polyunsaturated Fatty Acid (LC-PUFA) Content in the Eel Anguilla japonica After External and Internal Osmotic Stress.

We investigated the effect of external and internal osmotic stress on the profile of long-chain polyunsaturated fatty acids (LC-PUFA) in euryhaline eels Anguilla japonica. Freshwater (FW) fish were transferred to seawater (SW) for external osmotic stress or subjected to internal stress through injection with hypertonic saline. FW eels injected with isotonic saline served as controls. Plasma osmolality, Na+ concentration, and gill Na+/K+ -ATPase activity increased, but hematocrit decreased compared with controls in eels exposed to external or internal osmotic stress. The expression of two major transporter genes for SW adaptation, the Na+ -K+ -2Cl - co-transporter 1a (NKCC1a) in the gill and NKCC2b in the intestine, was up-regulated only in SW-transferred eels, suggesting a direct impact of SW on the gill and intestine via SW ingestion. Total LC-PUFA contents and DHA (22:6 n-3) increased in the gill and liver of SW-transferred eels and in the intestine of hypertonic saline-injected eels. However, total LC-PUFA content in plasma decreased after both external and internal osmotic stimuli. In contrast, the gene expression of two key enzymes involved in the LC-PUFA biosynthesis, Δ6 fatty acid desaturase and elongase, did not change in the gill, intestine and liver of osmotically stressed eels. These results indicate that LC-PUFA is possibly involved in osmoregulation and the increased LC-PUFA contents of osmoregulatory organs might be a result of LC-PUFA transport via circulation, rather than through de novo biosynthesis.

The role of the rectum in osmoregulation and the potential effect of renoguanylin on SLC26a6 transport activity in the Gulf toadfish (Opsanus beta).

Teleosts living in seawater continually absorb water across the intestine to compensate for branchial water loss to the environment. The present study reveals that the Gulf toadfish (Opsanus beta) rectum plays a comparable role to the posterior intestine in ion and water absorption. However, the posterior intestine appears to rely more on SLC26a6 (a HCO3 (-)/Cl(-) antiporter) and the rectum appears to rely on NKCC2 (SLC12a1) for the purposes of solute-coupled water absorption. The present study also demonstrates that the rectum responds to renoguanylin (RGN), a member of the guanylin family of peptides that alters the normal osmoregulatory processes of the distal intestine, by inhibited water absorption. RGN decreases rectal water absorption more greatly than in the posterior intestine and leads to net Na(+) and Cl(-) secretion, and a reversal of the absorptive short-circuit current (ISC). It is hypothesized that maintaining a larger fluid volume within the distal segments of intestinal tract facilitates the removal of CaCO3 precipitates and other solids from the intestine. Indeed, the expression of the components of the Cl(-)-secretory response, apical CFTR, and basolateral NKCC1 (SLC12a2), are upregulated in the rectum of the Gulf toadfish after 96 h in 60 ppt, an exposure that increases CaCO3 precipitate formation relative to 35 ppt. Moreover, the downstream intracellular effects of RGN appear to directly inhibit ion absorption by NKCC2 and anion exchange by SLC26a6. Overall, the present findings elucidate key electrophysiological differences between the posterior intestine and rectum of Gulf toadfish and the potent regulatory role renoguanylin plays in osmoregulation.

Development of an animal-borne blood sample collection device and its deployment for the determination of cardiovascular and stress hormones in phocid seals.

An animal-borne blood sampler with data-logging functions was developed for phocid seals, which collected two blood samples for the comparison of endocrinological/biochemical parameters under two different conditions. The sampler can be triggered by preset hydrostatic pressure, acceleration (descending or ascending), temperature, and time, and also manually by light. The sampling was reliable with 39/50 (78%) successful attempts to collect blood samples. Contamination of fluids in the tubing to the next blood sample was <1%, following the prior clearance of the tubing to a waste syringe. In captive harbor seals (Phoca vitulina), the automated blood-sampling method was less stressful than direct blood withdrawal, as evidenced by lower levels of stress hormones (P < 0.05 for ACTH and P = 0.078 for cortisol). HPLC analyses showed that both cortisol and cortisone were circulating in seal blood. Using the sampler, plasma levels of cardiovascular hormones, atrial natriuretic peptide (ANP), AVP, and ANG II were compared in grey seals (Halichoerus grypus), between samples collected when the animals were on land and in the water. HPLC analyses determined that [Met12] ANP (1-28) and various forms of angiotensins (ANG II, III, and IV) were circulating in seal blood. Although water immersion profoundly changes the plasma levels of cardiovascular hormones in terrestrial mammals, there were only tendencies toward an increase in ANP (P = 0.069) and a decrease in AVP (P = 0.074) in the seals. These results suggest that cardiovascular regulation in phocid seals may have undergone adaptation during evolution of the carnivore to a semiaquatic lifestyle.

A second estrogen receptor from Japanese lamprey (Lethenteron japonicum) does not have activities for estrogen binding and transcription.

Estrogens regulate many physiological responses in vertebrates by binding to the estrogen receptor (ER), a ligand-activated transcription factor. To understand the evolution of vertebrate ERs and to investigate how estrogen acts in a jawless vertebrate, we used degenerate primer sets and PCR to isolate DNA fragments encoding two distinct ER subtypes, Esr1a and Esr1b from the Japanese lamprey, Lethenteron japonicum. Phylogenetic analysis indicates that these two ERs are the result of lineage-specific gene duplication within the jawless fishes, different from the previous duplication event of Esr1 (ERα) and Esr2 (ERß) within the jawed vertebrates. Reporter gene assays show that lamprey Esr1a displays both constitutive and estrogen-dependent activation of gene transcription. Domain swapping experiments indicate that constitutive activity resides in the A/B domain of lamprey Esr1a. Unexpectedly, lamprey Esr1b does not bind estradiol and is not stimulated by other estrogens, androgens or corticosteroids. A 3D model of lamprey Esr1b suggests that although estradiol fits into the steroid binding site, some stabilizing contacts between the ligand and side chains that are found in human Esr1 and Esr2 are missing in lamprey Esr1b.

Duplicated CFTR isoforms in eels diverged in regulatory structures and osmoregulatory functions.

Two cystic fibrosis transmembrane conductance regulator (CFTR) isoforms, CFTRa and CFTRb, were cloned in Japanese eel and their structures and functions were studied in different osmoregulatory tissues in freshwater (FW) and seawater (SW) eels. Molecular phylogenetic results suggested that the CFTR duplication in eels occurred independently of the duplication event in salmonid. CFTRa was expressed in the intestine and kidney and downregulated in both tissues in SW eels, while CFTRb was specifically expressed in the gill and greatly upregulated in SW eels. Structurally, the CFTR isoforms are similar in most functional domains except the regulatory R domain, where the R domain of CFTRa is similar to that of human CFTR but the R domain of CFTRb is unique in having high intrinsic negative charges and fewer phosphorylation sites, suggesting divergence of isoforms in terms of gating properties and hormonal regulation. Immunohistochemical results showed that CFTR was localized on the apical regions of SW ionocytes, suggesting a Cl(-) secretory role as in other teleosts. In intestine and kidney, however, immunoreactive CFTR was mostly found in the cytosolic vesicles in FW eels, indicating that Cl(-) channel activity could be low at basal conditions, but could be rapidly increased by membrane insertion of the stored channels. Guanylin (GN), a known hormone that increases CFTR activity in mammalian intestine, failed to redistribute CFTR and to affect its expression in eel intestine. The results suggested that GN-independent CFTR regulation is present in eel intestine and kidney.

Guanylin activates Cl(-) secretion into the lumen of seawater eel intestine via apical Cl(-) channel under simulated in vivo conditions.

Guanylin (GN) action on seawater eel intestine was examined under simulated in vivo conditions, where isotonic luminal fluid has low NaCl and high MgSO4 (MgSO4 Ringer). In Ussing chamber, MgSO4 Ringer induced serosa-negative potential difference (PD) even after bumetanide treatment, which is due to the higher paracellular Na(+) permeability over Cl(-), as confirmed by the replacement by MgCl2 (no Cl(-) gradient) or Na2SO4 Ringer (no Na(+) gradient). Luminal GN reversed serosa-negative PD, probably by enhancing Cl(-) secretion into the lumen, as the GN effect was blocked by apical Cl(-) channel blockers [diphenylamine-2-carboxylic acid (DPC), 5-nitro-2-(3-phenylpropylamino) benzoic acid, glibenclamide but not cystic fibrosis transmembrane regulator (CFTR)inh-172] or replacement of luminal fluid by MgCl2 Ringer. The blockers' effect was undetectable when normal Ringer was on both sides. In the sac preparation, NaCl secretion occurred into the lumen (Na(+) > Cl(-)), and GN further enhanced Cl(-) secretion (Cl(-) > Na(+)), resulting in water secretion. These GN effects were also blocked by DPC. Quantitative analyses showed that isotonic NaCl is absorbed when luminal fluid is normal Ringer, but, when luminal fluid is MgSO4 Ringer, hypertonic NaCl, almost equivalent to seawater, is secreted into the lumen after GN. These results indicate that GN stimulates the secretion of hypertonic NaCl into the lumen of seawater eel intestine, like rectal gland of marine elasmobranchs, to get rid of excess NaCl although marine teleost intestine is thought to have only absorptive-type cells with a unique Na-K-Cl cotransport system. The secreted NaCl may activate the cotransport system and further help absorb water in the final segment of seawater eel intestine.

The differential role of renoguanylin in osmoregulation and apical Cl-/HCO3- exchange activity in the posterior intestine of the Gulf toadfish (Opsanus beta).

The guanylin family of peptides are effective regulators of intestinal physiology in marine teleosts. In the distal intestinal segments, they inhibit or reverse fluid absorption by inhibiting the absorptive short-circuit current (Isc). The present findings demonstrate that mRNA from guanylin and uroguanylin, as well as at least one isoform of the guanylin peptide receptor, apical guanylyl cyclase-C (GC-C), was highly expressed in the intestine and rectum of the Gulf toadfish (Opsanus beta). In the posterior intestine, GC-C, as well as the cystic fibrosis transmembrane conductance regulator and basolateral Na(+)/K(+)/2Cl(-) cotransporter, which comprise a Cl(-)-secretory pathway, were transcriptionally upregulated in 60 parts per thousand (ppt). The present study also shows that, in intestinal tissues from Gulf toadfish held in 35 ppt, renoguanylin (RGN) expectedly causes net Cl(-) secretion, inhibits both the absorptive Isc and fluid absorption, and decreases HCO3(-) secretion. Likewise, in intestinal tissues from Gulf toadfish acclimated to 60 ppt, RGN also inhibits the absorptive Isc and fluid absorption but to an even greater extent, corresponding with the mRNA expression data. In contrast, RGN does not alter Cl(-) flux and, instead, elevates HCO3(-) secretion in the 60-ppt group, suggesting increased apical Cl(-)/HCO3(-) exchange activity by SLC26a6. Overall, these findings reinforce the hypotheses that the guanylin peptide system is important for salinity acclimatization and that the secretory response could facilitate the removal of solids, such as CaCO3 precipitates, from the intestine.

From aquatic to terrestrial life: evolution of the mechanisms for water acquisition.

It is generally accepted that ancient fishes first experienced freshwater (FW), and then variably by lineage moved onto the land or re-entered the seas during evolution. As both land and sea are desiccating environments, animals must change their strategies for body fluid regulation from protecting against overhydration in FW to coping with dehydration in seawater (SW) or on land. The evolution of the mechanisms for acquisition of water surely must have accompanied these dramatic environmental changes. The major route for water acquisition is by oral drinking in terrestrial tetrapods (represented here by mammals) and in SW fishes (represented by teleosts as they are dehydrated in SW), but the regulation is contrasting between the two groups; mechanisms inducing thirst have developed in mammals, whereas inhibitory mechanisms are dominant in marine teleosts as observed in FW teleosts. Thus, the apparent difference was found not between hydrating and dehydrating habitat, but rather between terrestrial and aquatic habitats. This contrast is also reflected in regulatory hormones; dipsogenic hormones such as angiotensin II play pivotal roles in water homeostasis in mammals, whereas antidipsogenic hormones such as atrial natriuretic peptide are essential in teleosts. Imbibed water becomes body fluid only after absorption by the intestine, and there is a distinct difference in the mechanisms for water absorption between mammals and teleosts. Like regulation of drinking, we found that the inhibitory mechanisms are dominant for intestinal water absorption in teleosts. In the initial part of this short review, interesting differences in the body fluid regulation between mammals and teleosts are introduced, particularly with regard to water acquisition (drinking and intestinal absorption). Then an attempt was made to discuss the evolution of the mechanisms from the two perspectives; transitions from aquatic to terrestrial habitats and from hydrating (FW) to dehydrating (land and SW) habitats.