Shigehisa Hirose

Lung surfactant is a complex mixture of lipids and proteins, which is secreted from the alveolar type II epithelial cell and coats the surface of alveoli as a thin layer. It plays a crucial role in the prevention of alveolar collapse through its ability to reduce surface tension. Under normal conditions, surfactant homeostasis is maintained by balancing its release and the uptake by the type II cell for recycling and the internalization by alveolar macrophages for degradation. Little is known about how the surfactant pool is monitored and regulated. Here we show, by an analysis of gene-targeted mice exhibiting massive accumulation of surfactant, that Ig-Hepta/GPR116, an orphan receptor, is expressed on the type II cell and sensing the amount of surfactant by monitoring one of its protein components, surfactant protein D, and its deletion results in a pulmonary alveolar proteinosis and emphysema-like pathology. By a coexpression experiment with Sp-D and the extracellular region of Ig-Hepta/GPR116 followed by immunoprecipitation, we identified Sp-D as the ligand of Ig-Hepta/GPR116. Analyses of surfactant metabolism in Ig-Hepta^+/+ and Ig-Hepta^−/− mice by using radioactive tracers indicated that the Ig-Hepta/GPR116 signaling system exerts attenuating effects on (i) balanced synthesis of surfactant lipids and proteins and (ii) surfactant secretion, and (iii) a stimulating effect on recycling (uptake) in response to elevated levels of Sp-D in alveolar space.

Secretion of HCO₃⁻ at the apical side of the epithelial cells of the choroid plexus is an essential step in the formation of cerebrospinal fluid. Anion conductance with a high degree of HCO₃⁻ permeability has been observed and suggested to be the major pathway for HCO₃⁻ transport across the apical membrane. Recently, it was found that NBC (Na⁺/HCO₃⁻ co-transporter) 4, an electrogenic member of the NBC family, was expressed in the choroid plexus. We found that a novel variant of the NBC4 [NBC4g/Slc4a5 (solute carrier family 4, sodium bicarbonate co-transporter, member 5)] is almost exclusively expressed in the apical membrane of rat choroid plexus epithelium at exceptionally high levels. RNA interference-mediated knockdown allowed the functional demonstration that NBC4g is the major player in the HCO₃⁻ transport across the apical membrane of the choroid plexus epithelium. When combined with a recent observation that in choroid plexus epithelial cells electrogenic NBC operates with a stoichiometry of 3:1, the results of the present study suggest that NBC4g mediates the efflux of HCO₃⁻ and contributes to cerebrospinal fluid production.

Freshwater fishes actively absorb salt from their environment to tolerate low salinities. We previously reported that vacuolar-type H+-ATPase/mitochondrion-rich cells (H-MRCs) on the skin epithelium of zebrafish larvae (Danio rerio) are primary sites for Na+ uptake. In this study, in an attempt to clarify the mechanism for the Na+ uptake, we performed a systematic analysis of gene expression patterns of zebrafish carbonic anhydrase (CA) isoforms and found that, of 12 CA isoforms, CA2a and CA15a are highly expressed in H-MRCs at larval stages. The ca2a and ca15a mRNA expression were salinity-dependent; they were up-regulated in 0.03 mM Na+ water whereas ca15a but not ca2a was down-regulated in 70 mM Na+ water. Immunohistochemistry demonstrated cytoplasmic distribution of CA2a and apical membrane localization of CA15a. Furthermore, cell-surface immunofluorescence staining revealed external surface localization of CA15a. Depletion of either CA2a or CA15a expression by Morphorino antisense oligonucleotides resulted in a significant decrease in Na+ accumulation in H-MRCs. An in situ proximity ligation assay demonstrated a very close association of CA2a, CA15a, Na+/H+ exchanger 3b (Nhe3b), and Rhcg1 ammonia transporter in H-MRC. Our findings suggest that CA2a, CA15a, and Rhcg1 play a key role in Na+ uptake under freshwater conditions by forming a transport metabolon with Nhe3b.

The process of NaCl reabsorption in the distal nephron allows freshwater fishes to excrete hypotonic urine and seawater fishes to excrete urine containing high concentrations of divalent ions; the relevant transporters, however, have not yet been identified. In the mammalian distal nephron, NaCl absorption is mediated by Na⁺-K⁺-Cl⁻cotransporter 2 (NKCC2, Slc12a1) in the thick ascending limb, Na⁺-Cl⁻cotransporter (NCC, Slc12a3) in the distal convoluted tubule, and epithelial sodium channel (ENaC) in the collecting duct. In this study, we compared the expression profiles of these proteins in the kidneys of euryhaline and seawater pufferfishes. Mining the fugu genome identified one NKCC2 gene and one NCC gene, but no ENaC gene. RT-PCR and in situ hybridization analyses demonstrated that NKCC2 was highly expressed in the distal tubules and NCC was highly expressed in the collecting ducts of euryhaline pufferfish (mefugu, Takifugu obscurus ). On the other hand, the kidney of seawater pufferfish (torafugu, Takifugu rubripes ), which lacked distal tubules, expressed very low levels of NCC, and, in the collecting ducts, high levels of NKCC2. Acclimation of mefugu to seawater resulted in a 2.7× decrease in NCC expression, whereas NKCC2 expression was not markedly affected. Additionally, internalization of NCC from the apical surface of the collecting ducts was observed. These results suggest that NaCl reabsorption in the distal nephron of the fish kidney is mediated by NCC and NKCC2 in freshwater and by NKCC2 in seawater.

FXYD proteins, small single-transmembrane proteins, have been proposed to be auxiliary regulatory subunits of Na⁺–K⁺-ATPase and have recently been implied in ion osmoregulation of teleost fish. In freshwater (FW) fish, numerous ions are actively taken up through mitochondrion-rich cells (MRCs) of the gill and skin epithelia, using the Na⁺ electrochemical gradient generated by Na⁺–K⁺-ATPase. In the present study, to understand the molecular mechanism for the regulation of Na⁺–K⁺-ATPase in MRCs of FW fish, we sought to identify FXYD proteins expressed in MRCs of zebrafish. Reverse-transcriptase PCR studies of adult zebrafish tissues revealed that, out of eight fxyd genes found in zebrafish database, only zebrafish fxyd11 (zfxyd11) mRNA exhibited a gill-specific expression. Double immunofluorescence staining showed that zFxyd11 is abundantly expressed in MRCs rich in Na⁺–K⁺-ATPase (NaK-MRCs) but not in those rich in vacuolar-type H⁺-transporting ATPase. An in situ proximity ligation assay demonstrated its close association with Na⁺–K⁺-ATPase in NaK-MRCs. The zfxyd11 mRNA expression was detectable at 1 day postfertilization, and its expression levels in the whole larvae and adult gills were regulated in response to changes in environmental ionic concentrations. Furthermore, knockdown of zFxyd11 resulted in a significant increase in the number of Na⁺–K⁺-ATPase–positive cells in the larval skin. These results suggest that zFxyd11 may regulate the transport ability of NaK-MRCs by modulating Na⁺–K⁺-ATPase activity, and may be involved in the regulation of body fluid and electrolyte homeostasis.

The rh blood group-related proteins have recently been recognized as ammonia transporters acting in erythrocyte and nonerythrocyte membranes. They play an important role in excretion of nitrogenous waste formed mainly from amino acid metabolism. Because of a restricted availability of water, most terrestrial animals detoxify ammonia into urea or uric acid before removal from the body. However, aquatic animals, living in an abundant supply of water, can directly excrete ammonia into the environment immediately as it is produced. This method of ammonia elimination is strategic in avoiding urea synthesis, an anabolic process requiring ATP. In this issue of American Journal of Physiology-Cell Physiology , Wu et al. ([28][1]) add a further twist to this adaptive strategy by providing evidence for a coupling of the ammonia excretion with a sodium uptake process, which is essential for living in freshwater conditions. The Rh antigen was first identified about 70 years ago by Landsteiner and Wiener ([8][2]), who showed that rabbits and guinea pigs, when immunized with red blood cells of Rhesus monkey, produce an antibody that also agglutinates human red blood cells (Rh for Rhesus). The Rh antigen (Rh), now recognized for its strong immunogenic nature and use for diagnosis of pregnancy at risk for hemolytic disease of the newborn, forms a complex with other subunits including Rh-associated glycoprotein (RhAG), CD47, ICAM-4, and glycophorin B ([25][3]). Rh and RhAG are the major components of the complex and belong to the same family of proteins. They were expected to carry some physiologic function based on their well-conserved nature among the species and especially because they have 12 transmembrane-spanning segments characteristic of transporters. A comparative genomic approach led to the following breakthrough discovery that RhAG acts as an ammonia transporter. A low (∼20%) but significant sequence similarity was found by Marini et al. ([10][4]) between RhAG and the members of the Mep/Amt family previously identified as ammonia transporters in microorganisms and plants for which ammonia is a preferred source of nitrogen (Mep for methylammonium permease; Amt for ammonium transporter); for the evolution of the Mep/Amt/Rh family, see Huang and Peng ([6][5]). RhAG and its nonerythroid homologues, RhBG and RhCG, were then demonstrated to behave indeed as ammonia transporters ([9][6], [26][7]). The discoveries briefly summarized above have greatly contributed to our understanding of the mechanism of ammonia secretion in fish. Nakada et al. ([13][8]) identified four Rh glycoproteins in pufferfish (Rhag, Rhbg, Rhcg1, and Rhcg2; lowercase refers to the proteins of nonhuman species) and determined their locations in the gill, the major site of gas exchange, ionoregulation, and ammonia elimination in adult teleost fish; it is noteworthy that gills are needed for ionoregulation before they are needed for oxygen uptake during development ([16][9]). Gill surface area is greatly increased by its lamellar structure. The ammonia transporters Rhag, Rhbg, and Rhcg2 are distributed on these lamellar cells, which is ideal for optimizing ammonia elimination. In contrast, Rhcg1 exhibits a somewhat unexpected localization in mitochondrion-rich cells (MRCs or ionocytes). MRCs are relatively minor among the population of the gill epithelial cells and are specialized to actively absorb or secrete NaCl to maintain body fluid homeostasis. MRCs are present in the skin and yolk sac during early developmental stages before functional gills are present. Since the MRCs found in the skin are easily accessible to experimental analysis, they are frequently studied. Since this report, one or more members of the Rh protein family including those mentioned above ([1][10], [2][11], [7][12], [12][13], [15][14]; for review see Refs. [24][15] and [27][16]) and Rhp2 (p for primary) ([14][17]) have been identified in other fish species including zebrafish, a freshwater fish in which a knockdown experiment can be performed for detailed functional analyses of specific gene products by using antisense morpholino oligonucleotides. In zebrafish, Rhcg1 has been demonstrated to be expressed in the skin and gill MRCs that are rich in vacuolar-type H+-ATPase (vH-MRCs) ([12][13], [17][18]), and implicated as being somehow involved in Na+ uptake ([3][19], [12][13]). Apical localization of Na+/H+ exchanger 3 (NHE3) in vH-MRCs is also observed in zebrafish ([29][20]). By integrating these pieces of evidence and other information accumulated by previous molecular physiological studies, a collaborative group of researchers at the McMaster University and the University of Guelph ([21][21], [27][16]) has proposed a “Na+/NH4+ exchange complex” consisting of the transporters Rhcg, vacuolar-type H+-ATPase (vH-ATPase), Na+/H+ exchanger (NHE), and epithelial Na+ channel (ENaC) working together as a metabolon. Except for the inclusion of ENaC, which is not present in teleost genomes ([22][22]), the model is very attractive since the ammonia is not simply discarded; instead, the downhill gradient for ammonia excretion into the water is used to drive uphill Na+ uptake from the water into the freshwater fish. Wu et al. ([28][1]) provided a piece of supporting evidence for the model. After failing to detect significant Na+/NH4+ exchange in zebrafish, these investigators analyzed another model fish, Japanese medaka ( Oryzias latipes ). The electrophysiological technique they used warrants a comment. SIET (also known as the self-referencing ion-selective electrode technique) is a noninvasive method for measuring minute extracellular ion gradients created within the unstirred layer on the surface of ion-transporting epithelial cells ([18][23], [19][24]). SIET uses a microelectrode having a tip diameter of ∼5 μm and a thin layer (∼250 μm) of an ion-selective ionophore cocktail in its tip. By a modulation technique using a...

Trappin is a multifunctional host-defense peptide that has antiproteolytic, antiinflammatory, and antimicrobial activities. The numbers and compositions of trappin paralogs vary among mammalian species: human and sheep have a single trappin-2 gene; mouse and rat have no trappin gene; pig and cow have multiple trappin genes; and guinea pig has a trappin gene and two other derivativegenes. Independent duplications of trappin genes in pig and cow were observed recently after the species were separated. To determine whether these trappin gene duplications are restricted only to certain mammalian lineages, we analyzed recently-developed genome databases for the presence of duplicate trappin genes. The database analyses revealed that: 1) duplicated trappin multigenes were found recently in the nine-banded armadillo; 2) duplicated two trappin genes had been found in the Afrotherian species (elephant, tenrec, and hyrax) since ancient days; 3) a single trappin-2 gene was found in various eutherians species; and 4) no typical trappin gene has been found in chicken, zebra finch, and opossum. Bayesian analysis estimated the date of the duplication of trappin genes in the Afrotheria, guinea pig, armadillo, cow, and pig to be 244, 35, 11, 13, and 3 million-years ago, respectively. The coding regions of trappin multigenes of almadillo, bovine, and pig evolved much faster than the noncoding exons, introns, and the flanking regions, showing that these genes have undergone accelerated evolution, and positive Darwinian selection was observed in pig-specific trappin paralogs. These results suggest that trappin is an eutherian-specific molecule and eutherian genomes have the potential to form trappin multigenes.