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Zebrafish lateral-line hair cells are an in vivo model for studying hair cell development, function, and ototoxicity. However, the molecular identification and properties of the mechanotransducer (MET) channel in hair cells are still controversial. In this study, a noninvasive electrophysiological method, the scanning ion-electrode technique (SIET), was applied for the first time to investigate properties of MET channels in intact zebrafish embryos. With the use of a Ca2+-selective microelectrode to deflect hair bundles and simultaneously record the Ca2+ flux, the inward Ca2+ flux was detected at stereocilia of hair cells in 2- to ~4-day postfertilization embryos. Ca2+ influx was blocked by MET channel blockers (BAPTA, La3+, Gd3+, and curare). In addition, 10 μM aminoglycoside antibiotics (neomycin and gentamicin) were found to effectively block Ca2+ influx within 10 min. Elevating the external Ca2+ level (0.2-2 mM) neutralized the effects of neomycin and gentamicin. However, elevating the Mg2+ level up to 5 mM neutralized blockade by gentamicin but not by neomycin. This study demonstrated MET channel-mediated Ca2+ entry at hair cells and showed that the SIET to be a sensitive approach for functionally assaying MET channels in zebrafish. [ABSTRACT FROM AUTHOR]

In zebrafish, Rhcg1 was found in apical membranes of skin ionocytes [H+-ATPase-rich (HR) cells], which are similar to α-type intercalated cells in mammalian collecting ducts. However, the cellular distribution and role of Rhbg in zebrafish larvae have not been well investigated. In addition, HR cells were hypothesized to excrete ammonia against concentration gradients. In this study, we attempted to compare the roles of Rhbg and Rhcg1 in ammonia excretion by larval skin and compare the capability of skin cells to excrete ammonia against concentration gradients. Using in situ hybridization and immunohistochemistry, Rhbg was localized to both apical and basolateral membranes of skin keratinocytes. A scanning ion-selective electrode technique (SIET) was applied to measure the NH4 + flux at the apical surface of keratinocytes and HR cells. Knockdown of Rhbg with morpholino oligonucleotides suppressed ammonia excretion by keratinocytes and induced compensatory ammonia excretion by HR cells. To compare the capability of cells to excrete ammonia against gradients, NH4 + flux of cells was determined in larvae exposed to serial concentrations of external NH4 +. Results showed that HR cells excreted NH4 + against higher NH4 + concentration than did keratinocytes. Knockdown of the expression of either Rhcg1 or H+-ATPase in HR cells suppressed the capability of HR cells. [ABSTRACT FROM AUTHOR]

In the present study, medaka embryos were exposed to acidified freshwater (pH 5) to investigate the mechanism of acid secretion by mitochondrion-rich (MR) cells in embryonic skin. With double or triple in situ hybridization/ immunocytochemistry, the Na+/H+ exchanger 3 (NHE3) and H+-ATPase were localized in two distinct subtypes of MR cells. NHE3 was expressed in apical membranes of a major proportion of MR cells, whereas H+-ATPase was expressed in basolateral membranes of a much smaller proportion of MR cells. Gill mRNA levels of NHE3 and H+-ATPase and the two subtypes of MR cells in yolk sac skin were increased by acid acclimation, however, the mRNA level of NHE3 was remarkably higher than that of H+-ATPase. A scanning ion-selective electrode technique was used to measure H+, Na+, and NH4+ transport by individual MR cells in larval skin. Results showed that Na+ uptake and NH4+ excretion by MR cells increased after acid acclimation. These findings suggested that the NHE3/Rh glycoprotein-mediated Na+uptake/NH4+ excretion mechanism plays a critical role in acidic equivalent (H+/NH4+) excretion by MR cells of the freshwater medaka. [ABSTRACT FROM AUTHOR]

To investigate whether Na+ uptake by zebrafish is dependent on NH4+ excretion, a scanning ion-selective electrode technique was applied to measure Na+ and NH4+ gradients at the yolk-sac surface of zebrafish larvae. Low-Na+ acclimation induced an inward Na+ gradient (uptake), and a combination of low Na+ and high NH4+ induced a larger inward Na+ gradient. When measuring the ionic gradients, raising the external NH4+ level (5 mM) blocked NH4+ excretion and Na+ uptake; in contrast, raising the external Na+ level (10 mM) simultaneously enhanced Na+ uptake and NH4+ excretion. The addition of MOPS buffer (5 mM), which is known to block NH4+ excretion, also suppressed Na+ uptake. These results showed that Na+ uptake and NH4+ excretion by larval skin are associated when ambient Na+ level is low. Knockdown of Rhcg1 translation with morpholino-oligonucleotides decreased both NH4+ excretion and Na+ uptake by the skin and Na+ content of whole larvae. Knockdown of nhe3b translation or inhibitor (5-ethylisopropyl amiloride) treatment also decreased both the NH4+ excretion and Na+ uptake. This study provides loss-offunction evidence for the involvement of Rhcg1 and NHE3b in the ammonium-dependent Na+ uptake mechanism in zebrafish larvae subjected to low-Na+ water. [ABSTRACT FROM AUTHOR]

A noninvasive technique, the scanning ion-selective electrode technique (SIET) was applied to measure Na+ and Cl- transport by the yolk-sac skin and individual mitochondrion-rich cells (MRCs) in intact medaka larvae (Oryzias latipes). In seawater (SW)-acclimated larvae, significant outward Na+ and Cl- gradients were measured at the yolk-sac surface, indicating secretions of Na+ and Cl- from the yolk-sac skin. With Na+ pump immunostaining and microscopic observation, two groups of MRCs were identified on the yolk-sac skin of SW-larvae. These were single MRCs (s-MRCs), which do not have an accompanying accessory cell (AC), and multicellular complex MRCs (mc-MRCs), which usually consist of an MRC and an accompanying AC. The percentage of mc-MRC was ∼60% in 30 parts per thousand of SW, and it decreased with the decrease of external salinity. By serial SIET probing over the surface of the MRCs and adjacent keratinocytes (KCs), significant outward fluxes of Na+ and Cl- were detected at the apical opening (membrane) of mc-MRCs, whereas only outward Cl- flux, but not Na+ flux, was detected at s-MRCs. Treatment with 100 μM ouabain or bumetanide effectively blocked the Na+ and Cl- secretion. Following freshwater (FW) to SW transfer, Na+ and Cl- secretions by the yolk-sac skin were fully developed in 5 h and 2 h, respectively. In contrast, both Na+ and Cl- secretions downregulated rapidly after SW to FW transfer. Sequential probing at individual MRCs found that Na+ and Cl- secretions declined dramatically after SW to FW transfer and Na+/Cl- uptake was detected at the same s-MRCs and mc-MRCs after 5 h. This study provides evidence demonstrating that ACs are required for Na+ excretion and MRCs possess a functional plasticity in changing from a Na+/Cl--secreting cell to a Na+/Cl--absorbing cell. [ABSTRACT FROM AUTHOR]

Similar to mammalian proximal tubular cells, H+-ATPase rich (HR) cells in zebrafish skin and gills are also responsible for Na+ uptake and acid secretion functions. However, the basolateral transport pathways in HR cells are still unclear. In the present study, we tested the hypothesis if there are specific slc4 members involved in basolateral ion transport pathways in HR cells. Fourteen isoforms were identified in the zebrafish(z) slc4 family, and the full-length cDNAs of two novel isoforms, zslc4a1b (anion exchanger, zAE1b) and zslc4a4b (Na+/HCO3- cotransporter, zNBCe1b), were sequenced. mRNA signals of zslc4a1b and zslc4a4b were mainly detected in certain groups of ionocytes in zebrafish skin/gills. Further double immunocytochemistry or in situ hybridization demonstrated that zAE1b, but not zNBCe1b, was localized to basolateral membranes of HR cells. Acclimation to low-Na+ or acidic environments stimulated the mRNA expression of zslc4a1b in zebrafish gills, and loss-of-function of zslc4a1b with specific morpholinos caused significant decreases in both the whole body Na+ content and the skin H+ activity in the morphants. On the basis of these results, it was concluded that zAE1b, but not zNBCe1b, is involved in the basolateral transport pathways in Na+ uptake/acid secretion mechanisms in zebrafish HR cells. [ABSTRACT FROM AUTHOR]

In this study, a scanning ion-selective electrode technique (SIET) was applied to measure Ht Na+, and NH+ gradients and apparent fluxes at specific cells on the skin of medaka larvae. Na+ uptake and NH3/NH+4 excretion were detected at most niitochondrion-rich cells (MRCs). H+ probing at MRCs revealed two group of MRCs, i.e., acid-secreting and base-secreting MRCs. Treatment with EIPA (100 p.M) blocked 35% of the NH3/ NH+ secretion and 54% of the Na+ uptake, suggesting that the Na+/H+ exchanger (NHE) is involved in Na+ and NH3/NH+4transport. Low-Na+ water (<0.001 mM) or high-NH+ (5 mM) acclimation simultaneously increased Na+ uptake and NH3/NH+ excretion but decreased or even reversed the H+ gradient at the skin and MRCs. The correlation between NH+ production and H+ consumption at the skin surface suggests that MRCs excrete nonionic NH3 (base) by an acid-trapping mechanism. Raising the external NH+ significantly blocked NH3/NH+ excretion and Na+ uptake. In contrast, raising the acidity of the water (pH 7 to pH 6) enhanced NH3/NH+ excretion and Na+ uptake by MRCs. In situ hybridization and real-time PCR showed that the mRNAs of the Na4/H+ exchanger (slc9a3) and Rhesus glycoproteins (Rhcgl and Rhbg) were colocalized in MRCs of medaka, and their expressions were induced by low-Na+ acclimation. This study suggests a novel Na+/NH+ exchange pathway in apical membranes of MRCs, in which a coupled NHE and Rh glycoprotein is involved and the Rh glycoprotein may drive the NHE by generating H+ gradients across apical membranes of MRCs. [ABSTRACT FROM AUTHOR]

A noninvasive scanning ion-selective electrode technique (SIET) was applied to measure Cl- transport at individual mitochondrion-rich cells (MRCs) in the skin of euryhaline tilapia (Oreochromis mossambicus) larvae. In seawater (SW)-acclimated larvae, outward C1 gradients (20∼80 mM higher than the background) were measured at the surface, indicating a secretion of Cl- from the skin. By serial probing over the surface of MRCs and adjacent keratinocytes (KCs), a significant outward flux of Cl- was detected at the apical opening (membrane) of MRCs. Treatment with 100 μM ouabain or bumetanide inhibited the Cl- secretion by ∼75%. In freshwater (FW)acclimated larvae, a lower level of outward Cl- gradients (0.2∼1 mM) was measured at the skin surface. Low-Cl- water (<0.005 mM) acclimation increased the apical Na+-Cl- cotransporter (NCC) immunoreactivity of MRCs in the larval skin. An inward flux of Cl- was detected when probing the exterior surface of a group of MRCs (convex-MRCs) that express the NCC. An NCC inhibitor (100 μM metolazone) reduced the flux by ∼90%. This study provides direct and convincing evidence for Cl- transport by MRCs of SW- and FW-acclimated euryhaline tilapia and the involvement of an apical NCC in Cluptake of MRCs of FW-acclimated fish. [ABSTRACT FROM AUTHOR]

H+-ATPase-rich (HR) cells in zebrafish are known to be involved in acid secretion and Na+ uptake mechanisms in zebrafish gills/skin: however, little is known about how HR cells are functionally regulated. In the present work, we studied the roles of Drosophila gilal cell missing (gem), a cell fate-related transcription factor, in the differentiation and functional regulation of zebrafish HR cells. Zebmfish gem2 (zgcm2) was found to begin expression in zebrafish embryos at 10 h postfertilization (hpf), and to be extensively expressed in gills but only mildly so in eyes, heart, muscles, and testes. By whole mount in situ hybridization, zgcm2 mRNA signals were found in a group of cells on the zebrafish yolk sac surface initially in the tail bud stage (10 hpf); they had disappeared at 36 hpf and thereafter appeared again in the gill region from 48 hpf. Double fluorescence in situ hybridization further demonstrated specific colocalizatian of zgcm2 mRNA in HR. cells in zebrafish embryos. Knockdown of zgcm2 with a specific morpholino oligonucleotide caused the complete disappearance of HR cells with a concomitant decrease in H+ activity at the apical surface of HR cells, but it did not affect the occurrence of Na+-K+-ATPase-rich cells. A decrease in the H+-ATPase subunit A (zatp6vla) expression and no change in zgcm2 expression in zebrafish gills were seen from 12 h to 3 days after transfer to acidic fresh water, but a compensatory stimulation in the expressions of both genes appeared 4 days posttransfer. In conclusion, functional regulation of HR cells is probably achieved by enhancing cell differentiation via zGCM2 activation. [ABSTRACT FROM AUTHOR]

H~-ATPase-rich (HR) cells in zebrafish gills/skin were found to carry out Na~ uptake and acid-base regulation through a mechanism similar to that which occurs in mammalian proximal tubular cells. However, the roles of carbonic anhydrases (CAs) in this mechanism in zebrafish HR cells are still unclear. The present study used a functional genomic ap- proach to identify 20 CA isoforms in zebrafish. By screening with whole mount in situ hybridization, only zca2-like a and zcal5a were found to be expressed in specific groups of cells in zebrafish gills/skin, and further analyses by triple in situ hybridization and immunocyto- chemistry demonstrated specific colocalizations of the two zca iso- forms in HR cells. Knockdown of zca2 -like a caused no change in and knockdown of zca]5a caused an increase in H~ activity at the apical surface of HR cells at 24 h postfertilization (hpf). Later, at 96 hpf, both the zca2 -like a and zcal5a morphants showed decreased H~ activity and increased Na~ uptake, with concomitant upregulation of znhe3b and downregulation of zatp6vla (H~-ATPase A-subunit) expressions. Accli- mation to both acidic and low-Na~ fresh water caused upregulation of zcal5a expression but did not change the zca2 -like a mRNA level in zebrafish gills. These results provide molecular physiological evidence to support the roles of these two zCA isoforms in Na~ uptake and acid-base regulation mechanisms in zebrafish HR cells. [ABSTRACT FROM AUTHOR]

In the skin of zebrafish embryo, the vacuolar H+-ATPase (V-ATPase, H+ pump) distributed mainly in the apical membrane of H+-pump-rich cells, which pump internal acid out of the embryo and function similarly to acid-secreting intercalated cells in mammalian kidney. In addition to acid excretion, the electrogenic H+ efflux via the H+-ATPases in the gill apical membrane of freshwater fish was proposed to act as a driving force for Na+ entry through the apical Na+ channels. However, convincing molecular physiological evidence in vivo for this model is still lacking. In this study, we used morpholino-modified antisense oligonucleotides to knockdown the gene product of H+-ATPase subunit A (atp6v1a) and examined the phenotype of the mutants. The H+-ATPase knockdown embryos revealed several abnormalities, including suppression of acid-secretion from skin, growth retardation, trunk deformation, and loss of internal Ca2+ and Nat This finding reveals the critical role of H+-ATPase in embryonic acid-secretion and ion balance, as well. [ABSTRACT FROM AUTHOR]

The mammalian kidney excretes its metabolic acid load through the proton-transporting cells, intercalated cells, in the distal nephron and collecting duct. Fish excrete acid through external organs, gill, or skin; however, the cellular function is still controversial. Ill this study, molecular and electrophysiological approaches were used to identify a novel cell type secreting acid in skin of zebrafish (Danio rerio) larvae. Among keratinocytes covering the larval surface, novel proton-secreting ionocytes, proton pump (H+-ATPase)-rich cells, were identified to generate strong outward H+ flux. The present work demonstrates for the first time, with a noninvasive technique, H+-secreting cells in an intact animal model, the zebrafish, showing it to be a suitable model in which to study the functions of vertebrate transporting epithelia in vivo. [ABSTRACT FROM AUTHOR]