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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Department of Life Science, National Taiwan Normal University, Taipei, Taiwan, Republic of China; and 2Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan, Republic of China
Submitted 14 May 2008 ; accepted in final form 22 September 2008
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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H+-adenosine 5'-triphosphatase; ionocytes; Rhesus glycoprotein; ammonium; knockdown; ion-selective electrode
The dominant model indicates that branchial ammonia is excreted from freshwater fish mainly down favorable blood-to-water (transbranchial) ammonia gradients (25). Passive NH3 diffusion is based on evidence that ammonia flow requires a suitable blood-to-water gradient. Ammonia flow is inhibited when the gradient is reduced by high ambient ammonia (1, 29). Wright et al. (33) suggested that maintenance of the transbranchial ammonia gradient relies on acidification of unstirred layers of the apical surface of the branchial epithelium. As it is acidified, this layer creates a microenvironment for a favorable transbranchial NH3 gradient that facilitates ammonia excretion. This is especially advantageous during exposure to high ammonia (HA), which reverses the normally positive ammonia gradient. Abolishing this layer with HEPES buffer during HA reduced the flux of total ammonia (29). It was also suggested that the H+ arising from CO2 hydration traps NH3 as NH4+ as it enters the unstirred layers on the surfaces of gills (29, 33).
In addition to this "acid-trapping" model, early studies proposed the possibility of apical Na+/NH4+ exchange in freshwater teleosts (26). In this model, Na+ uptake across the apical side of the gill epithelium is linked to NH4+ extrusion, which replaces H+ on the Na+/H+ exchanger (NHE). Although, the NHE has been identified in gills of several freshwater species including rainbow trout (Oncorhynchus mykiss) (27), tilapia (Oreochromis mossambicus) (27), Osorezan dace (Tribolodon hakonensis) (8), and zebrafish (Danio rerio) (34), the contribution of the apical NHE to ammonia transport has been debated (19, 29).
Recent evidence has shown that the branchial surface might also be acidified by H+ extruded from an apical vacuolar H+-ATPase. Accumulating data using pharmacological, physiological, immunocytochemical, and molecular approaches have demonstrated the role of V-type H+-ATPase in the apical Na+ uptake mechanism of freshwater teleosts (3, 11). Recently, Nawata et al. (19) examined the mRNA expression of Rhesus (Rh) protein (a putative ammonia transporter/channel), and H+-ATPase with a real-time PCR in rainbow trout exposed to HA. They found that Rhcg2, Rhbg, and H+-ATPase (the B-subunit) mRNA expressions were upregulated in pavement cell fractions during HA, and they suggested the contribution of Rh proteins and H+-ATPase to ammonia transport across gill epithelium. However, direct evidence by a loss-of-function or gain-of-function study has not yet reported. Several isoforms of the Rh glycoprotein (Rhag, Rhbg, Rhcg1, and Rhcg2) have been identified in the gill epithelium of pufferfish (Takifugu rubripes) (18), killifish (Kryptolebias marmoratus) (10), and zebrafish (17); however, their exact functions are still unclear.
The zebrafish (D. rerio) has been used as a good model for studying ion and acid-base regulation in freshwater teleosts (11). Lin et al. (16) identified two subtypes of ionocytes, Na+ pump-rich cells and H+ pump-rich (HR) cells in zebrafish larva, by molecular and electrophysiological approaches. Knockdown of H+-ATPase translation with specific morpholino oligonucleotides (MOs) (9) and bafilomycin inhibition (16) demonstrated that H+-ATPase contributes to acid secretion in the skin of zebrafish larvae. Recently, Nakada et al. (17) reported that Rhcg1 was located in the apical membrane of HR cells in zebrafish. Therefore, we hypothesized that the HR cells are the main locations for ammonia excretion in zebrafish. We attempted to measure NH4+ excretion in specific skin cells with scanning ion-selective electrode technique (SIET), and we provide direct evidence for the contributions of H+-ATPase and Rhcg1 in ammonia excretion.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Scanning ion-selective electrode technique. To measure NH4+ and H+ activities and fluxes at the surface of zebrafish larvae, NH4+- and H+-selective microelectrodes were constructed. Glass capillary tubes (no. TW 150-4, World Precision Instruments, Sarasota, FL) were pulled on a Sutter P-97 Flaming Brown pipette puller (Sutter Instruments, San Rafael, CA) into micropipettes with tip diameters of 3–4 µm. These glass micropipettes were baked at 120°C overnight and then vapor-silanized with dimethyl chlorosilane (Fluka, Buchs, Switzerland) for 30 min. Before use, the micropipettes were backfilled with 1 cm of a column of 100 mM NH4Cl or KH2PO4 to respectively create an NH4+ or H+ microelectrode. Then the micropipettes were frontloaded with a 20- to 30-µm column of liquid ion exchanger cocktail (NH4+ ionophore I cocktail A or H+ ionophore I cocktail B; Fluka). The details of the system were described in a previous report (16). The Nernstian property of each microelectrode was measured by placing the microelectrode in a series of standard solutions (0.1, 1, and 10 mM NH4Cl; pH 6, 7, and 8). By plotting the voltage output of the probe against the log NH4+ and H+ concentrations, a linear regression yielded a Nernstian slope of 57.8 ± 0.6 (n = 10) for NH4+ and 57.5 ± 2.5 (n = 10) for H+.
Measurement of surface NH4+ and H+ gradients.
SIET was performed at room temperature (26–28°C) in a small plastic recording chamber filled with 1 ml "recording medium" that contained NW, 300 µM MOPS buffer (Sigma, St. Louis, MO), and 0.1 mg/l ethyl 3-aminobenzoate (Tricaine, Sigma; pH 7.0). The selectivity coefficients of the Fluka NH4+ ionophore I (cocktail A) is only four times more selective for NH4+ than for K+. Therefore "K+-free recording medium" (in which KH2PO4 and K2HPO4 were replaced by NaH2PO4 and Na2HPO4) was used to probe for NH4+. Before the measurement, an anesthetized embryo was positioned in the center of the chamber with its lateral side contacting the base of the chamber. To record the NH4+ or H+ activities at the surface of a larva, the microelectrode was moved to the target position 10–20 µm away from the skin. After recording a target point, the microelectrode was then moved away (
10 mm) to record the background. In this study, 
[NH4+] and [H+] were respectively used to represent the measured NH4+ and H+ gradients between the point of interest (skin surface) and background.
Measurement of NH4+ flux in ionocytes and keratinocytes.
The anesthetized larva was laid laterally in the chamber for SIET measurements. Under a differential interference contrast (DIC) microscope, the apical membrane of ionocytes could be identified in its skin. To record the local NH4+ flux at specific cell surface sites, the microelectrode was moved to a position 2–3 µm above the surface of interest. In addition to "single-spot" recordings, a "line scan" was made by probing a series of spots along a line (9 spots along 40 µm) across the surface of ionocytes and adjacent keratinocytes. At every spot, the voltage difference in microvolts was measured by probing orthogonally to the surface at 10-µm intervals. The calculation of NH4+ flux was shown in previous reports (4, 6). Briefly, voltage gradients obtained from the ASET software were converted into concentration (activity) gradient using the following equation:
 | (1) |
C is the concentration gradient between the two points measured in µmol·l–1·cm–3; Cb is the background ion concentration, calculated as the average of the concentration at each point measured in µmol/l; V is the voltage gradient obtained from ASET in µV; and S is the Nernst slope of the electrode. The concentration gradient was subsequently converted into (extracellular) ion flux using Fick's law of diffusion in the following equation:
 | (2) |
C is the concentration gradient in pmol/cm3, and X is the distance between the two points measured in centimeters. Concanavalin A labeling. Concanavalin A (Con-A), a kind of lectin, was found to bind with the apical membrane of HR cells and was used as a vital marker for HR cells (16). Live larvae were incubated in NW containing 0.5 mg/ml Alexa 488-conjugated Con-A (Molecular Probes, Eugene, OR) for 10 min. After staining, the larvae were washed with NW for 3 min.
Treatment with amiloride, 5-ethylisopropyl amiloride, and bafilomycin A1. The amiloride or 5-ethylisopropyl amiloride (EIPA; Sigma) stock solutions were prepared by dissolving amiloride or EIPA in dimethyl sulfoxide (DMSO, Sigma). A final concentration of 0.1 mM or 1 mM was applied to the larvae for 10 min. After treatment, the larvae were immediately measured with SIET. The bafilomycin A1 (Sigma) stock solution was dissolved in DMSO and then added to the media to a final concentration of 10 µM. The larvae were incubated in 10 µM bafilomycin A1 for 1 h and then measured with SIET. Effect of DMSO has been tested in a preliminary experiment. DMSO did not affect H+ or NH4+ gradient, thus we used NW as control in the inhibitor experiments.
Morpholino design and microinjection. MOs were obtained from Gene Tools (Philomath, OR). The detailed method was previously reported (9). The MOs against H+-ATPase (atp6v1a) and Rhcg 1 were atp6v1a (5'-ATCCATCTTGTGTGTTAGAAAACTG-3') and rhcg1 (5'-CAGTTGCCCATGTCTACAGCTTGAG-3'), which covers the start codon of Rhcg1. A standard control MO (5'-CCTCTTACCTCAGTTACAATTTATA-3') was used as the control. The standard control oligo provided by Gene Tools has no target and no significant biological activity. The MO solution was prepared with sterile water and contained 0.1% phenol red as a visualizing indicator. The MO at 8 ng was microinjected into the embryo at the 1- to 4-cell stage with an IM-300 microinjector system (Narishigi Scientific Instrument Laboratory, Tokyo, Japan). In the preliminary test and our previous study (9), embryos injected with 8 ng of control MO showed no significant difference in survival rate, morphology, or H+ or NH4+ secretion (measured with SIET) compared with wild-type embryos, indicating that an injection of 8 ng control MO caused little or no specific effect.
Immunohistochemistry. Zebrafish larvae were anesthetized on ice and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 h at 4°C. After being rinsed with PBS, the larvae were postfixed and permeabilized with 90% ethanol at –20°C for 10 min. After being washed with PBS, samples were incubated with 3% bovine serum albumin and 5% normal goat serum for 30 min to block nonspecific binding. The larvae were then incubated overnight at 4°C with polyclonal antibody against zebrafish Rhcg1 (diluted 1:1,000 with PBS). The antibody was provided by Dr. S. Hirose (17). After being rinsed with PBS for 20 min, the larvae were further incubated in goat anti-rabbit IgG conjugated with FITC (diluted 1:1,000; Jackson Immunoresearch Laboratories, West Grove, PA), for 2 h at room temperature (26–28°C).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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[NH4+] at the six spots was determined by calculating the difference between the target spot and background (10 mm away from the larva). Figure 1B shows [NH4+] values measured at the six spots from four individuals. [NH4+] at the yolk sac was higher than at the other locations, indicating that NH4+ was highly secreted in the yolk sac area. When the microelectrode was moved step by step away from the yolk sac surface, [NH4+] gradually declined with distance (data not shown).
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[NH4+] at 10-µm intervals. Under a microscope, ionocytes and keratinocytes can easily be identified by their morphology (oval and polygonal, respectively; Fig. 2A). The measured ionocytes were located mainly in the border between the yolk-extension and trunk of larvae. Figure 2B shows a serial probing (line scan) over the surface of an ionocyte, which was further identified as an H+ pump-rich cell (HRC) by fluorescent Con-A, which is a specific and vital marker for HRCs (16; Fig. 2C). SIET detected weaker outward flux of NH4+ in keratinocytes, but this gradually increased when probing toward the apical surface of an HRC (Fig. 2B). In contrast, no surge in the signal was found when probing over the surface of Con-A-negative cells (other types of ionocytes). Figure 2D shows a comparison of NH4+ flux in HRCs (Con-A+), other ionocytes (Con-A–), and keratinocytes (KCs). The flux from HRCs was about threefold higher than that from other cell types.
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[NH4+] was measured in 5-dpf larvae that had been preacclimated to various concentrations of NH4+ (0, 0.5, 5, and 10 mM) for 5 days (Fig. 3A). During acclimation, no significant increase in mortality was found even in 10 mM HA, indicating that zebrafish can tolerate HA. When the larvae in the recording medium (containing 0.1 mM NH4Cl) were measured, NH4+ gradients showed a dose-dependent increase with the amount of treated [NH4+] (Fig. 3A). Moreover, the NH4+gradients of larvae also increased with development from 3 to 7 dpf (Fig. 3B). At 7 dpf, [NH4+] was measured at the surface of gills instead of yolk sac, which was absorbed. In these stages, the NH4+ gradient was significantly higher in larvae exposed to 5 mM HA than larvae in NW (Fig. 3B).
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[H+] value was positive and gradually increased with development in NW larvae (Fig. 4). However, the [H+] value of HA larvae was significantly lower than that of NW larvae, particularly in larvae exposed to HA for 5 days (the 5-pdf larvae). At that time, the [H+] of HA decreased to 10% of control.
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[NH4+] was significantly higher in pH 6 medium than in pH 7 or pH 8 medium (Fig. 5A). When larvae in the medium with strong buffer (5 mM MOPS) were measured, both [NH4+] and [H+] decreased to 64% and 54%, respectively (Fig. 5, B and C; NW contained only 300 µM MOPS).
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[NH4+] and [H+] remarkably decreased in atp6v1a morphants (larvae injected with MOs). The inhibited levels of ammonium and proton gradients were both close to 70%. This inhibitory effect was also confirmed by bafilomycin A1 treatment (10 µM for 1 h), which significantly decreased both proton and ammonium gradient at larval skin (Fig. 7, A and B). In larvae injected with the rhcg1 morpholino, [NH4+] decreased by 36%; however, [H+] remained at the same level (Fig. 6, C and D). To verify the effect of rhcg1 knockdown, immunostaining was done in the larvae (Fig. 8). Rhcg1-specific antibody reacted with a group of ionocytes (it has been reported to be HRCs; 17) in the yolk sac skin of wild-type (Fig. 8A) and control larvae (Fig. 8D). However, the Rhcg1 signal totally disappeared in the larvae injected with 8 ng rhcg1 morpholino (Fig. 8C).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In previous reports on fish ammonia transport, the net flux of total ammonia (including NH3 and NH4+) was determined by calculating changes in total ammonia in the water during the experiment (29, 32). However, that method provides the ammonia flux from the entire animal but not specific organs or cell types. In the present study, SIET was used to measure the NH4+ activity and flux at specific locations on the surface of larval skin. SIET has been used to detect various ions, including H+, K+, Na+, Ca2+, Cl–, etc. (2, 15, 21, 22, 23) of samples ranging from plants to animals. Unlike traditional ion-selective microelectrodes, which are used to measure intra- or extracellular ion activities, SIET detects extracellular ion activity and ion flux at specific locations. However, it should be noted that SIET measures diffusion flux of ion (calculated with Fick's law) outside of the skin or cell, instead of transepithelial (transmembrane) ion flux. Although, the extracellular ion gradient or flux is caused mainly by transmembrane ion flux, several factors may affect the measured values. In the case of H+ probing, H+ activity at skin surface is related to direct H+ efflux, H+ generated by the CO2 hydration and loss of H+ via titration of buffers in water and NH3 excreted (the main point of this study). To discriminate specific source, we used inhibitors such as bafilomycin in this study to block H+-ATPase-dependent H+ secretion. Thus we can conclude that the measured H+ gradient reflects transmembrane H+ transport through H+-ATPase to some degree.
The application of SIET to probing NH4+ has been reported in studies of plant root absorption (7) and mosquito NH4+ excretion (4). For the first time, the present study used SIET to detect cellular NH4+ transport in an intact vertebrate model, the zebrafish. In this study, the ammonia secreted by larval skin was presented as [NH4+] calculated from the difference in NH4+ activities between the background and the location of interest. To calibrate the NH4+ activity and flux, a linear Nernstian regression was done using a series of known concentrations of NH4Cl. The slope of the concentrations above 0.1 mM was fitted to a linear Nernstian slope of 58 but gradually decayed at concentrations below 0.1 mM (data not shown). However, the NH4+ concentration in NW was lower than 0.01 mM and was thus beyond the linear range of the calibrating line. For a practical and precise calibration, the NH4+ concentration in the medium for SIET measurement was raised by adding 0.1 mM NH4Cl.
In previous studies, SIET was used to determine H+ transport in the skin of zebrafish larvae and was shown to be a good approach for examining ion transport in a zebrafish model (9, 16). The present study further used SIET to examine ammonia transport in zebrafish and provides direct evidence for the cellular location and molecular mechanism of ammonia transport in zebrafish larvae. In a study on the air-breathing mudskipper (Periophthalmodon schlosseri), mitochondria-rich cells (MRCs) were suggested to be the location of branchial ammonia transport (28). In zebrafish, vH-MRCs (which refer to HRCs in our study) were also suggested to be involved in ammonia transport based on evidence of Rhcg1 in the apical membrane (17). Furthermore, pavement cells were also suggested to be involved in ammonia transport of zebrafish (17), pufferfish (18), and rainbow trout (19). However, those studies provide molecular evidence for the localization of the ammonia transporter, and functional evidence on these cells has not been reported. In this study, SIET detected outward NH4+ fluxes of about threefold higher in HRCs than in adjacent keratinocytes or other ionocytes. This finding coincides with our previous finding that HRCs are acid-secreting cells (16) and consequently drive a significant amount of NH3 transport by the mechanism of "acid-trapping." Although the NH4+ flux in HRCs is relatively high, keratinocytes should greatly contribute to overall NH4+ transport due to their large number and surface area.
The unstirred acid layer facilitating branchial ammonia transport (by the acid-trapping mechanism) has been proposed and may be the dominant mechanism for ammonia excretion in freshwater teleosts (25). This mechanism of skin ammonia transport was demonstrated in larval zebrafish by the present data. A high external pH (pH 8) and strong MOPS buffer (5 mM) diminished the formation of the surface acid layer (acid gradient) and consequently ammonia transport. In addition, the acid gradient at the skin surface was greatly diminished in larvae acclimated to 5 mM HA (Fig. 4). These data can be interpreted as increased NH3 secretion from HA larvae combined (consumed) with the secreted H+ at the skin surface thus generated a higher NH4+ gradient. If a larva secretes total ammonia mainly in the form of NH4+, we would expect to see an increase in the surface acid gradient instead of a decrease. Our data apparently do not support this point. However, we cannot completely rule out a minor portion of NH4+ excretion.
More importantly, we suggest that H+-ATPase in the apical membrane of HRCs contributes >70% to skin acid secretion and the accompanying ammonia transport. Both bafilomycin inhibition and atp6v1a gene knockdown directly demonstrated the contribution of H+-ATPase to NH4+ secretion. In contrast, CO2 diffusion and H+ transport by NHE contributed relatively little to acid and ammonia secretion. In gills of the mudskipper (P. schlosseri), NHE was suggested to play a critical role in active ammonium transport (Na+/NH4+ exchange) (20); however, recent studies do not support this mechanism in freshwater fish (19). Our data show that amiloride or EIPA inhibited both H+ and NH4+ secretion by 20%, and the blocked NH4+ secretion was more likely due to a decrease in acid trapping than to Na+/NH4+ exchange. Na+/NH4+ exchange by NHE was first reported in a mammalian study in 1981 (14), in which functional assay of NHE was done with rabbit renal microvillus membrane vesicles. However, we cannot find other functional studies that convincingly demonstrate Na+/NH4+ exchange by NHE with in vitro expression systems. In mammalian studies, NH4+ is thought to be transported by Na+-K+(NH4+)-ATPase and Na+-K+ (NH4+)-2Cl– cotransporter because of the similar property and size of K+ and NH4+ (24). However, the size of H+ and NH4+ is quite different. If NHE can carry NH4+ needs to be further studied with expression system such as Xenopus oocytes.
Rh glycoproteins (members of the Amt/MEP/Rh superfamily) are generally thought to function as NH3/NH4+ transporters in a broad range of species (30). Recent studies suggested that Amt/MEP/Rh proteins are more like channel proteins that deprotonate NH4+ and then conduct NH3 (12, 13). Four isoforms of the Rh protein (Rhag, Rhbg, Rhcg1, and Rhcg2) have been identified in the gill epithelium of zebrafish (17). Among them, Rhcg1 expression has been located in the apical membrane of HRCs in the yolk sac and gills (17). In this study, we further conducted a loss-of-function study (rhcg1 gene knockdown) to demonstrate the function of Rhcg1 in ammonia transport. In Rhcg1 morphants, about a 40% decrease in ammonia secretion but no significant change in acid secretion was revealed. This suggests that other Rh isoforms might also be involved in ammonia transport, and thus the effect of rhcg1 knockdown alone was not as high as H+-ATPase knockdown or bafilomycin treatment. Taking this evidence together, we suggest a model that H+-ATPase in HRCs generates an extracellular H+ gradient to drive facilitative NH3 diffusion through Rhcg1 in HRCs and possibly through other Rh glycoproteins in keratinocytes. Further study is needed to investigate the function and regulation of other isoforms in zebrafish.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
|---|
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|---|
2. Breton S, Smith PJ, Lui B, Brown D. Acidification of the male reproductive tract by a proton pumping (H+)-ATPase. Nat Med 2: 470–472, 1996.[CrossRef][Web of Science][Medline]
3. Claiborne JB, Edwards SL, Morrison-Shetlar AI. Acid-base regulation in fishes: cellular and molecular mechanisms. J Exp Zool 293: 302–319, 2002.[CrossRef][Web of Science][Medline]
4. Donini A, O'Donnell MJ. Analysis of Na+, Cl–, K+, H+ and NH4+ concentration gradients adjacent to the surface of anal papillae of the mosquito Aedes aegypti: application of self-referencing ion-selective microelectrodes. J Exp Biol 208: 603–610, 2005.
5. Evans DH, Piermarini PM, Choe KP. The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste. Physiol Rev 85: 97–177, 2005.
6. Faszewski EE, Kunkel JG. Covariance of ion flux measurements allows new interpretation of Xenopus laevis oocyte physiology. J Exp Zool 290: 652–661, 2001.[CrossRef][Web of Science][Medline]
7. Henriksen GH, Bloom AJ, Spanswick RM. Measurement of net fluxes of ammonium and nitrate at the surface of barley roots using ion-selective microelectrodes. Plant Physiol 93: 271–280, 1990.
8. Hirata T, Kaneko T, Ono T, Nakazato T, Furukawa N, Hasegawa S, Wakabayashi S, Shigekawa M, Chang MH, Romero MF, Hirose S. Mechanism of acid adaptation of a fish living in a pH 3.5 lake. Am J Physiol Regul Integr Comp Physiol 284: R1199–R1212, 2003.
9. Horng JL, Lin LY, Huang CJ, Katoh F, Kaneko T, Hwang PP. Knockdown of V-ATPase subunit A (atp6v1a) impairs acid secretion and ion balance in zebrafish (Danio rerio). Am J Physiol Regul Integr Comp Physiol 292: R2068–R2076, 2007.
10. Hung CY, Tsui KN, Wilson JM, Nawata CM, Wood CM, Wright PA. Rhesus glycoprotein gene expression in the mangrove killifish Kryptolebias marmoratus exposed to elevated environmental ammonia levels and air. J Exp Biol 210: 2419–2429, 2007.
11. Hwang PP, Lee TH. New insights into fish ion regulation and mitochondrion-rich cells. Comp Biochem Physiol A Mol Integr Physiol 148: 479–497, 2007.[CrossRef][Medline]
12. Javelle A, Lupo D, Ripoche P, Fulford T, Merrick M, Winkler FK. Substrate binding, deprotonation, and selectivity at the periplasmic entrance of the Escherichia coli ammonia channel AmtB. Proc Natl Acad Sci USA 105: 5040–5045, 2008.
13. Khademi S, O'Connell J, 3rd Remis J, Robles-Colmenares Y, Miercke LJ, Stroud RM. Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 A. Science 305: 1587–1594, 2004.
14. Kinsella JL, Aronson PS. Interaction of NH4+ and Li+ with the renal microvillus membrane Na+-H+ exchanger. Am J Physiol Cell Physiol 241: C220–C226, 1981.
15. Land SC, Sanger RH, Smith PJ. O2 availability modulates transmembrane Ca2+ flux via second-messenger pathways in anoxia-tolerant hepatocytes. J Appl Physiol 82: 776–783, 1997.
16. Lin LY, Horng JL, Kunkel JG, Hwang PP. Proton pump-rich cell secretes acid in skin of zebrafish larvae. Am J Physiol Cell Physiol 290: C371–C378, 2006.
17. Nakada T, Hoshijima K, Esaki M, Nagayoshi S, Kawakami K, Hirose S. Localization of ammonia transporter Rhcg1 in mitochondrion-rich cells of yolk sac, gill, and kidney of zebrafish and its ionic strength-dependent expression. Am J Physiol Regul Integr Comp Physiol 293: R1743–R1753, 2007.
18. Nakada T, Westhoff CM, Kato A, Hirose S. Ammonia secretion from fish gill depends on a set of Rh glycoproteins. FASEB J 21: 1067–1074, 2007.
19. Nawata CM, Hung CC, Tsui TK, Wilson JM, Wright PA, Wood CM. Ammonia excretion in rainbow trout (Oncorhynchus mykiss): evidence for Rh glycoprotein and H+-ATPase involvement. Physiol Genomics 31: 463–474, 2007.
20. Randall DJ, Wilson JM, Peng KW, Kok TW, Kuah SS, Chew SF, Lam TJ, Ip YK. The mudskipper, Periophthalmodon schlosseri, actively transports NH4+ against a concentration gradient. Am J Physiol Regul Integr Comp Physiol 277: R1562–R1567, 1999.
21. Shirihai O, Smith P, Hammar K, Dagan D. Microglia generate external proton and potassium ion gradients utilizing a member of the H/K ATPase family. Glia 23: 339–348, 1998.[CrossRef][Web of Science][Medline]
22. Smith PJ, Sanger RH, Jaffe LF. The vibrating Ca2+ electrode: a new technique for detecting plasma membrane regions of Ca2+ influx and efflux. Methods Cell Biol 40: 115–134, 1994.[Web of Science][Medline]
23. Smith PJ, Trimarchi J. Noninvasive measurement of hydrogen and potassium ion flux from single cells and epithelial structures. Am J Physiol Cell Physiol 280: C1–C11, 2001.
24. Weiner ID, Hamm LL. Molecular mechanisms of renal ammonia transport. Annu Rev Physiol 69: 317–340, 2007.[CrossRef][Web of Science][Medline]
25. Wilkie MP. Ammonia excretion and urea handling by fish gills: present understanding and future research challenges. J Exp Zool 293: 284–301, 2002.[CrossRef][Web of Science][Medline]
26. Wilkie MP. Mechanisms of ammonia excretion across fish gills. Comp Biochem Physiol A Mol Integr Physiol 118: 39–50, 1997.
27. Wilson JM, Laurent P, Tufts BL, Benos DJ, Donowitz M, Vogl AW, Randall DJ. NaCl uptake by the branchial epithelium in freshwater teleost fish: an immunological approach to ion-transport protein localization. J Exp Biol 203: 2279–2296, 2000.[Abstract]
28. Wilson JM, Randall DJ, Donowitz M, Vogl AW, Ip AK. Immunolocalization of ion-transport proteins to branchial epithelium mitochondria-rich cells in the mudskipper (Periophthalmodon schlosseri). J Exp Biol 203: 2297–2310, 2000.[Abstract]
29. Wilson RW, Wright PM, Munger RS, Wood CM. Ammonia excretion in fresh water rainbow trout (Oncorhynchus mykiss) and the importance of gill boundary layer acidification: lack of evidence for Na+/NH4+ exchange. J Exp Biol 191: 37–58, 1994.[Abstract]
30. Winkler FK. Amt/MEP/Rh proteins conduct ammonia. Pflügers Arch 451: 701–707, 2006.[CrossRef][Web of Science][Medline]
31. Wood CM. Ammonia and urea metabolism and excretion. In: The Physiology of Fishes, edited by Evans DH. Boca Raton, FL: CRC, 1993, p. 379–425.
32. Wright PA. Nitrogen excretion and enzyme pathways for ureagenesis in fresh water tilapia (Oreochromis niloticus). Physiol Zool 66: 881–901, 1993.
33. Wright PA, Randall DJ, Perry SF. Fish gill water boundary layer: a site of linkage between carbon dioxide and ammonia excretion. J Comp Physiol 158: 627–635, 1989.[CrossRef]
34. Yan JJ, Chou MY, Kaneko T, Hwang PP. Gene expression of Na+/H+ exchanger in zebrafish H+-ATPase-rich cells during acclimation to low-Na+ and acidic environments. Am J Physiol Cell Physiol 293: C1814–C1823, 2007.
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 T. Nakada, C. M. Westhoff, Y. Yamaguchi, S. Hyodo, X. Li, T. Muro, A. Kato, N. Nakamura, and S. Hirose Rhesus Glycoprotein P2 (Rhp2) Is a Novel Member of the Rh Family of Ammonia Transporters Highly Expressed in Shark Kidney J. Biol. Chem., January 22, 2010; 285(4): 2653 - 2664. [Abstract] [Full Text] [PDF]
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 M. H. Braun, S. L. Steele, and S. F. Perry The responses of zebrafish (Danio rerio) to high external ammonia and urea transporter inhibition: nitrogen excretion and expression of rhesus glycoproteins and urea transporter proteins J. Exp. Biol., December 1, 2009; 212(23): 3846 - 3856. [Abstract] [Full Text] [PDF]
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J.-L. Horng, P.-P. Hwang, T.-H. Shih, Z.-H. Wen, C.-S. Lin, and L.-Y. Lin Chloride transport in mitochondrion-rich cells of euryhaline tilapia (Oreochromis mossambicus) larvae Am J Physiol Cell Physiol, October 1, 2009; 297(4): C845 - C854. [Abstract] [Full Text] [PDF]
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D. Weihrauch, M. P. Wilkie, and P. J. Walsh Ammonia and urea transporters in gills of fish and aquatic crustaceans J. Exp. Biol., June 1, 2009; 212(11): 1716 - 1730. [Abstract] [Full Text] [PDF]
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 M. H. Braun, S. L. Steele, M. Ekker, and S. F. Perry Nitrogen excretion in developing zebrafish (Danio rerio): a role for Rh proteins and urea transporters Am J Physiol Renal Physiol, May 1, 2009; 296(5): F994 - F1005. [Abstract] [Full Text] [PDF]
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J.-L. Horng, L.-Y. Lin, and P.-P. Hwang Functional regulation of H+-ATPase-rich cells in zebrafish embryos acclimated to an acidic environment Am J Physiol Cell Physiol, April 1, 2009; 296(4): C682 - C692. [Abstract] [Full Text] [PDF]
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