Hirose, Shigehisa

AbstractMarine teleosts ingest large amounts of seawater containing various ions, including 0.4 mM boric acid, which can accumulate at toxic levels in the body. However, the molecular mechanisms by which marine teleosts absorb and excrete boric acid are not well understood. Aquaporins (Aqps) are homologous to the nodulin‐like intrinsic protein (NIP) family of plant boric acid channels. To investigate the potential roles of Aqps on boric acid transport across the plasma membrane in marine teleosts, we analyzed the function of Aqps of Japanese pufferfish (Takifugu rubripes) expressed in Xenopus laevis oocytes. Takifugu genome database contains 16 genes encoding the aquaporin family members (aqp0a, aqp0b, aqp1aa, aqp1ab, aqp3a, aqp4a, aqp7, aqp8bb, aqp9a, aqp9b, aqp10aa, aqp10bb, aqp11a, aqp11b, aqp12, and aqp14). When T. rubripes Aqps (TrAqps) were expressed in X. laevis oocytes, a swelling assay showed that boric acid permeability was significantly increased in oocytes expressing TrAqp3a, 7, 8bb, 9a, and 9b. The influx of boric acid into these oocytes was also confirmed by elemental quantification. Electrophysiological analysis using a pH microelectrode showed that these TrAqps increase B(OH)3 permeability. These results indicate that TrAqp3a, 7, 8bb, 9a, and 9b act as boric acid transport systems, likely as channels, in marine teleosts.

Marine teleost fish precipitate divalent cations as carbonate deposits in their intestine to minimize the potential for excessive calcium entry and to stimulate water absorption by removing osmolyte ions. This carbonate deposit formation therefore plays an important role in osmoregulation in the seawater environment and also requires controlled secretion of HCO 3 − to match the amount of calcium entering the intestinal lumen. Despite this physiological importance, the process of carbonate precipitate formation has not been characterized at the molecular level. We first isolated 24 candidate cDNA clones representing all possible membrane proteins capable of transporting HCO 3 − from the euryhaline pufferfish, mefugu Takifugu obscurus based on the DNA sequence information on the T. rubripes genome, then performed Northern blot analyses to select clones highly expressed in the intestine of seawater mefugu, and finally determined the cellular locations by immunohistochemistry. Based on these observations, we proposed a model for HCO 3 − excretion across the intestinal epithelium of marine teleost fish that includes a basolateral NBCe1 (Na + /bicarbonate cotransporter) and apical Slc26a6 (Cl − /HCO 3 − exchangers).

Euryhaline pufferfish ( Takifugu obscurus, mefugu) can live in freshwater (FW) with ion concentration less than 1/100 of its body fluid or acclimate to seawater (SW) which has more than 3 times the ion concentration of its body fluid. However, the body‐fluid ion concentrations are maintained stable by the function of epithelial cells in the gill, intestine, and kidney which transport ions directionally and selectively. To understand the mechanisms of the fish body‐fluid homeostasis at the molecular level, there are several essentials: identification of transporters expressed in the epithelia, determination of transporter localization, and characterization of transporter activities. Analyses of various transporter mRNA levels in mefugu tissues identified increases for Slc26a6A and Slc26a6B in the intestine and the kidney of SW‐acclimated mefugu. To study their functions for SW acclimation, we subcloned the cDNAs into a Xenopus expression vector and assayed their functions using microelectrodes in oocytes. The electrophysiological analyses of the oocytes revealed that (a) Slc26a6A and Slc26a6B are electrogenic Cl − / n HCO 3 − exchangers; (b) Slc26a6A and Slc26a6B are also exchangers of Cl − /formate, Cl − /oxalate, and Cl − /SO 4 2− ; and (c) the slope of I–V relationship of Slc26a6A in the presence of SO 4 2− is 70 times higher than that of Slc26a6B. These results suggest significant roles for Slc26a6A and Slc26a6B in intestinal HCO 3 − secretion and a role for Slc26a6A in renal SO 4 2− excretion during SW acclimation. This work was supported by MEXT Japan, NIH (EY017732) and CFF grant (Romero‐06G0).

The gas in the swimbladder of teleost fish is composed primarily of O 2 , and the swimbladder volume is regulated by O 2 transfer between the luminal space of the swimbladder and the blood. In the swimbladder, lactic acid generation by anaerobic glycolysis in the gas gland epithelial cells and its recycling through the rete mirabile bundles of countercurrent capillaries are essential for local blood acidification and oxygen liberation from hemoglobin by the “Root effect.” To clarify molecules that are involved in the blood acidification and visualize the route of lactic acid movement, we analyzed the expression of 16 members of the H + /monocarboxylate transporter (MCT) family in the fugu genome and found that only MCT1b and MCT4b are highly expressed in the fugu swimbladder. Immunohistochemistry demonstrated that (i) MCT4b expresses in gas gland cells together with the glycolytic enzyme GAPDH at high level and mediate lactic acid secretion by gas gland cells, and (ii) MCT1b expresses in arterial, but not venous, capillary endothelial cells in rete mirabile and mediates recycling of lactic acid in the rete mirabile by solute‐specific transcellular transport. Electrophysiological analyses demonstrated that MCT1b is a high‐affinity lactate transporter whereas MCT4b is a low‐affinity/high‐conductance lactate transporter. These results clarified the mechanism of the blood acidification in the swimbladder by spatially organized two lactic acid transporters MCT4b and MCT1b.

Boron is a vital micronutrient and is toxic at high concentrations, however, little is known about whole‐body boric acid homeostasis in animals. Slc4a11 was reported to function as a Na + ‐coupled borate transporter in mammals, presumably for borate absorption. However, seawater (SW) contains 0.4 mM boric acid, and the bladder urine of a euryhaline pufferfish mefugu ( Takifugu obscures ) in SW contains ~20 mM boric acid. Therefore, mefugu kidney is a good model to study a borate efflux system. In the mefugu kidney, a paralog of Slc4a11 (Slc4a11A) was markedly induced after transfer to SW and localized to the apical membrane of renal tubules. When Xenopus oocytes expressing Slc4a11A were voltage‐clamped at a holding potential of −60 mV and exposed medium containing borate, intracellular pH was increased and an outward current (anion influx) was observed. The borate current was not altered when Na + was replaced with other cations such as choline and Li + , but was eliminated when Na + was replaced with borate chelating agent NMDG. The borate (boron) influx was confirmed by elemental analysis of the oocytes. These results indicate that Slc4a11A is a Na + ‐independent B(OH) 4 − channel which is suitable for borate secretion, and clarify at first the mechanism of the borate efflux system in animal that prevent marine fishes from the toxic effects of borate.

The solute carrier 26 (Slc26) transporters are anion transporters with diverse substrate specificity. They are expressed mainly in epithelia and play a central role in secretion and absorption of anions such as chloride, oxalate (ox 2− ), sulfate (SO 4 2− ), bicarbonate (HCO 3 − ), iodide, formate and hydroxyl. In mammals the Slc26a6 protein is expressed in kidney and gut epithelia, and mediates electrogenic Cl − /ox 2− , Cl − / n HCO 3 − , Cl − /SO 4 2− exchange. Recently, chicken and zebrafish prestin (Slc26a5) were shown to mediate electrogenic Cl − /ox 2− and Cl − /SO 4 2− exchange rather than being an auditory sensor. We have cloned and characterized the Slc26a5 (fPrestin) transporter from the euryhaline pufferfish Takifugu obscurus (mefugu). This fPrestin mRNA is expressed ubiquitously unlike fSlc26a6A‐C which is found in kidney and gut. While the fSlc26a6A‐C mRNAs are increased in response to a freshwater to saltwater transition, fPrestin expression is unchanged. We next expressed fSlc26a5 in Xenopus oocytes and evaluated transport using microelectrodes. These experiments reveal that fPrestin mediates electrogenic Cl − /ox 2− , and Cl − /SO 4 2− as previously shown for chicken and zebrafish Slc26a5. Furthermore, fPrestin also can mediate electrogenic Cl − / n HCO 3 − exchange. These transport features are quite similar to mammalian Slc26a6 which has been implicated in renal Ca‐oxalate, stones.

The kidney of marine teleosts is the major site of Mg2+excretion and produces urine with a high Mg2+concentration. However, the transporters involved in Mg2+excretion are poorly understood. The cyclin M (Cnnm; also known as ancient conserved domain protein) family comprises membrane proteins homologous to the bacterial Mg2+and Co2+efflux protein, CorC. To understand the molecular mechanism of Mg2+homeostasis in marine teleosts, we analyzed the expression of the C nnm family genes in the seawater (SW) pufferfish, torafugu ( Takifugu rubripes), and the closely related euryhaline species, mefugu ( Takifugu obscurus). Database mining and phylogenetic analysis indicated that the Takifugu genome contains six members of the Cnnm family: two orthologs of Cnnm1, one of Cnnm2, one of Cnnm3, and two of Cnnm4. RT-PCR analyses indicated that Cnnm2, Cnnm3, and Cnnm4a are expressed in the kidney, whereas other members are mainly expressed in the brain. Renal expression of Cnnm3 was upregulated in SW mefugu, whereas renal expression of Cnnm2 was upregulated in freshwater (FW) mefugu. No significant difference was observed in renal expression of Cnnm4a between SW and FW mefugu. In situ hybridization and immunohistochemical analyses of the SW mefugu kidney revealed that Cnnm3 is expressed in the proximal tubule, and its product localizes to the lateral membrane. When Cnnm3 was expressed in Xenopus laevis oocytes, whole cellular Mg2+content and free intracellular Mg2+activity significantly decreased. These results suggest that Cnnm3 is involved in body fluid Mg2+homeostasis in marine teleosts.

Zebrafish Na+/H+exchanger 3b (zNHE3b) is highly expressed in the apical membrane of ionocytes where Na+is absorbed from ion-poor fresh water against a concentration gradient. Much in vivo data indicated that zNHE3b is involved in Na+absorption but not leakage. However, zNHE3b-mediated Na+absorption has not been thermodynamically explained, and zNHE3b activity has not been measured. To address this issue, we overexpressed zNHE3b in Xenopus oocytes and characterized its activity by electrophysiology. Exposure of zNHE3b oocytes to Na+-free media resulted in significant decrease in intracellular pH (pHi) and intracellular Na+activity ( aNai). aNaiincreased significantly when the cytoplasm was acidified by media containing CO2-HCO3−or butyrate. Activity of zNHE3b was inhibited by amiloride or 5-ethylisopropyl amiloride (EIPA). Although the activity was accompanied by a large hyperpolarization of ∼50 mV, voltage-clamp experiments showed that Na+/H+exchange activity of zNHE3b is electroneutral. Exposure of zNHE3b oocytes to medium containing NH3/NH4+resulted in significant decreases in pHiand aNaiand significant increase in intracellular NH4+activity, indicating that zNHE3b mediates the Na+/NH4+exchange. In low-Na+(0.5 mM) media, zNHE3b oocytes maintained aNaiof 1.3 mM, and Na+-influx was observed when pHiwas decreased by media containing CO2-HCO3−or butyrate. These results provide thermodynamic evidence that zNHE3b mediates Na+absorption from ion-poor fresh water by its Na+/H+and Na+/NH4+exchange activities.

The second most abundant cation in seawater (SW), Mg2+, is present at concentrations of ∼53 mM. Marine teleosts maintain plasma Mg2+concentration at 1–2 mM by excreting Mg2+into the urine. Urine Mg2+concentrations of SW teleosts exceed 70 mM, most of which is secreted by the renal tubular epithelial cells. However, molecular mechanisms of the Mg2+secretion have yet to be clarified. To identify transporters involved in Mg2+secretion, we analyzed the expression of fish homologs of the Slc41 Mg2+transporter family in various tissues of SW pufferfish torafugu ( Takifugu rubripes) and its closely related euryhaline species mefugu ( Takifugu obscurus). Takifugu genome contained five members of Slc41 genes, and only Slc41a1 was highly expressed in the kidney. Renal expression of Slc41a1 was markedly elevated when mefugu were transferred from fresh water (FW) to SW. In situ hybridization analysis and immunohistochemistry at the light and electron microscopic levels revealed that Slc41a1 is localized to vacuoles in the apical cytoplasm of the proximal tubules. These results suggest that pufferfish Slc41a1 is a Mg2+transporter involved in renal tubular transepithelial Mg2+secretion by mediating Mg2+transport from the cytosol to the vacuolar lumen, and support the hypothesis that Mg2+secretion is mediated by exocytosis of Mg2+-rich vacuoles to the lumen.

Na+/H+exchanger 3 (NHE3) provides one of the major Na+absorptive pathways of the intestine and kidney in mammals, and recent studies of aquatic vertebrates (teleosts and elasmobranchs) have demonstrated that NHE3 is expressed in the gill and plays important roles in ion and acid-base regulation. To understand the role of NHE3 in elasmobranch osmoregulatory organs, we analyzed renal and intestinal expressions and localizations of NHE3 in a marine elasmobranch, Japanese banded houndshark ( Triakis scyllium). mRNA for Triakis NHE3 was most highly expressed in the gill, kidney, spiral intestine, and rectum. The kidney and intestine expressed a transcriptional isoform of NHE3 (NHE3k/i), which has a different amino terminus compared with that of NHE3 isolated from the gill (NHE3g), suggesting that NHE3k/i and NHE3g arise from a single gene by alternative promoter usage. Immunohistochemical analyses of the Triakis kidney demonstrated that NHE3k/i is expressed in the apical membrane of a part of the proximal and late distal tubules in the sinus zone. In the bundle zone of the kidney, NHE3k/i was expressed in the apical membrane of the early distal tubules known as the diluting segment. In the spiral intestine and rectum, NHE3k/i was localized toward the apical membrane of the epithelial cells. The transcriptional levels of NHE3k/i were increased in the kidney when Triakis was acclimated in 130% seawater, whereas those in the spiral intestine were increased in fish acclimated in diluted seawater. These results suggest that NHE3 is involved in renal Na+reabsorption, urine acidification, and intestinal Na+absorption in elasmobranchs.