Ammonia excretion by the skin of zebrafish (Danio rerio) larvae

2008 ◽  
Vol 295 (6) ◽  
pp. C1625-C1632 ◽  
Author(s):  
Tin-Han Shih ◽  
Jiun-Lin Horng ◽  
Pung-Pung Hwang ◽  
Li-Yih Lin
Keyword(s):  
Direct Evidence ◽  
Ammonia Excretion ◽  
Bafilomycin A1 ◽  
Selective Electrode ◽  
Zebrafish Larvae ◽  
Trapping Mechanism ◽  
Morpholino Oligonucleotides ◽  
Electrode Technique ◽  
External Ph ◽  
Freshwater Teleosts

The mechanism of ammonia excretion in freshwater teleosts is not well understood. In this study, scanning ion-selective electrode technique was applied to measure H+ and NH4+ fluxes in specific cells on the skin of zebrafish larvae. NH4+ extrusion was relatively high in H+ pump-rich cells, which were identified as the H+-secreting ionocyte in zebrafish. Minor NH4+ extrusion was also detected in keratinocytes and other types of ionocytes in larval skin. NH4+ extrusion from the skin was tightly linked to acid secretion. Increases in the external pH and buffer concentration (5 mM MOPS) diminished H+ and NH4+ gradients at the larval surface. Moreover, coupled decreases in NH4+ and H+ extrusion were found in larvae treated with an H+-pump inhibitor (bafilomycin A1) or H+-pump gene ( atp6v1a) knockdown. Knockdown of Rhcg1 with morpholino-oligonucleotides also decreased NH4+ excretion. This study demonstrates ammonia excretion in epithelial cells of larval skin through an acid-trapping mechanism, and it provides direct evidence for the involvement of the H+ pump and an Rh glycoprotein (Rhcg1) in ammonia excretion.

Rhcg1 and NHE3b are involved in ammonium-dependent sodium uptake by zebrafish larvae acclimated to low-sodium water

2012 ◽  
Vol 302 (1) ◽  
pp. R84-R93 ◽  
Author(s):  
Tin-Han Shih ◽  
Jiun-Lin Horng ◽  
Sian-Tai Liu ◽  
Pung-Pung Hwang ◽  
Li-Yih Lin
Keyword(s):  
Yolk Sac ◽  
Ion Selective Electrode ◽  
Loss Of Function ◽  
Selective Electrode ◽  
Uptake Mechanism ◽  
Sodium Uptake ◽  
Zebrafish Larvae ◽  
Low Sodium ◽  
Morpholino Oligonucleotides ◽  
Electrode Technique

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-of-function evidence for the involvement of Rhcg1 and NHE3b in the ammonium-dependent Na+ uptake mechanism in zebrafish larvae subjected to low-Na+ water.

Ammonium-dependent sodium uptake in mitochondrion-rich cells of medaka (Oryzias latipes) larvae

2010 ◽  
Vol 298 (2) ◽  
pp. C237-C250 ◽  
Author(s):  
Shu-Chen Wu ◽  
Jiun-Lin Horng ◽  
Sian-Tai Liu ◽  
Pung-Pung Hwang ◽  
Zhi-Hong Wen ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Real Time Pcr ◽  
Skin Surface ◽  
Selective Electrode ◽  
Trapping Mechanism ◽  
Water Ph ◽  
Acid Secreting ◽  
Apical Membranes ◽  
Electrode Technique

In this study, a scanning ion-selective electrode technique (SIET) was applied to measure H+, Na+, and NH4+ gradients and apparent fluxes at specific cells on the skin of medaka larvae. Na+ uptake and NH3/NH4+ excretion were detected at most mitochondrion-rich cells (MRCs). H+ probing at MRCs revealed two group of MRCs, i.e., acid-secreting and base-secreting MRCs. Treatment with EIPA (100 μM) blocked 35% of the NH3/NH4+ secretion and 54% of the Na+ uptake, suggesting that the Na+/H+ exchanger (NHE) is involved in Na+ and NH3/NH4+ transport. Low-Na+ water (<0.001 mM) or high-NH4+ (5 mM) acclimation simultaneously increased Na+ uptake and NH3/NH4+ excretion but decreased or even reversed the H+ gradient at the skin and MRCs. The correlation between NH4+ production and H+ consumption at the skin surface suggests that MRCs excrete nonionic NH3 (base) by an acid-trapping mechanism. Raising the external NH4+ significantly blocked NH3/NH4+ excretion and Na+ uptake. In contrast, raising the acidity of the water (pH 7 to pH 6) enhanced NH3/NH4+ excretion and Na+ uptake by MRCs. In situ hybridization and real-time PCR showed that the mRNAs of the Na+/H+ exchanger ( slc9a3) and Rhesus glycoproteins ( Rhcg1 and Rhbg) were colocalized in MRCs of medaka, and their expressions were induced by low-Na+ acclimation. This study suggests a novel Na+/NH4+ 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.

Rhcg1 and Rhbg mediate ammonia excretion by ionocytes and keratinocytes in the skin of zebrafish larvae: H+-ATPase-linked active ammonia excretion by ionocytes

2013 ◽  
Vol 304 (12) ◽  
pp. R1130-R1138 ◽  
Author(s):  
Tin-Han Shih ◽  
Jiun-Lin Horng ◽  
Yi-Ting Lai ◽  
Li-Yih Lin
Keyword(s):  
Ammonia Excretion ◽  
Apical Surface ◽  
Concentration Gradients ◽  
Skin Cells ◽  
Zebrafish Larvae ◽  
Skin Keratinocytes ◽  
Morpholino Oligonucleotides ◽  
Apical Membranes

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.

Proton-facilitated ammonia excretion by ionocytes of medaka (Oryzias latipes) acclimated to seawater

2013 ◽  
Vol 305 (3) ◽  
pp. R242-R251 ◽  
Author(s):  
Sian-Tai Liu ◽  
Lin Tsung ◽  
Jiun-Lin Horng ◽  
Li-Yih Lin
Keyword(s):  
Boundary Layer ◽  
Ammonia Excretion ◽  
Oryzias Latipes ◽  
Yolk Sac ◽  
Pcr Analysis ◽  
Selective Electrode ◽  
High Ammonia ◽  
Rh Glycoproteins ◽  
Electrode Technique

The proton-facilitated ammonia excretion is critical for a fish's ability to excrete ammonia in freshwater. However, it remains unclear whether that mechanism is also critical for ammonia excretion in seawater (SW). Using a scanning ion-selective electrode technique (SIET) to measure H+ gradients, an acidic boundary layer was detected at the yolk-sac surface of SW-acclimated medaka ( Oryzias latipes) larvae. The H+ gradient detected at the surface of ionocytes was higher than that of keratinocytes in the yolk sac. Treatment with Tricine buffer or EIPA (a NHE inhibitor) reduced the H+ gradient and ammonia excretion of larvae. In situ hybridization and immunochemistry showed that slc9a2 (NHE2) and slc9a3 (NHE3) were expressed in the same SW-type ionocytes. A real-time PCR analysis showed that transfer to SW downregulated branchial mRNA expressions of slc9a3 and Rhesus glycoproteins ( rhcg1, rhcg2, and rhbg) but upregulated that of slc9a2. However, slc9a3, rhcg1, rhcg2, and rhbg expressions were induced by high ammonia in SW. This study suggests that SW-type ionocytes play a role in acid and ammonia excretion and that the Na+/H+ exchanger and Rh glycoproteins are involved in the proton-facilitated ammonia excretion mechanism.

Visualization in zebrafish larvae of Na+ uptake in mitochondria-rich cells whose differentiation is dependent on foxi3a

2007 ◽  
Vol 292 (1) ◽  
pp. R470-R480 ◽  
Author(s):  
Masahiro Esaki ◽  
Kazuyuki Hoshijima ◽  
Sayako Kobayashi ◽  
Hidekazu Fukuda ◽  
Koichi Kawakami ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Differentiation ◽  
Direct Evidence ◽  
Zebrafish Embryos ◽  
Morpholino Oligonucleotide ◽  
Gastrula Stage ◽  
Zebrafish Larvae ◽  
Transport Inhibitors ◽  
Antisense Morpholino ◽  
Na Uptake

Uptake of Na+ from the environment is an indispensable strategy for the survival of freshwater fish, as they easily lose Na+ from the plasma to a diluted environment. Nevertheless, the location of and molecules involved in Na+ uptake remain poorly understood. In this study, we utilized Sodium Green, a Na+-dependent fluorescent reagent, to provide direct evidence that Na+ absorption takes place in a subset of the mitochondria-rich (MR) cells on the yolk sac surface of zebrafish larvae. Combined with immunohistochemistry, we revealed that the Na+-absorbing MR cells were exceptionally rich in vacuolar-type H+-ATPase (H+-ATPase) but moderately rich in Na+-K+-ATPase. We also addressed the function of foxi3a, a transcription factor that is specifically expressed in the H+-ATPase-rich MR cells. When foxi3a was depleted from zebrafish embryos by antisense morpholino oligonucleotide injection, differentiation of the MR cells was completely blocked and Na+ influx was severely reduced, indicating that MR cells are the primary sites for Na+ absorption. Additionally, foxi3a expression is initiated at the gastrula stage in the presumptive ectoderm; thus, we propose that foxi3a is a key gene in the control of MR cell differentiation. We also utilized a set of ion transport inhibitors to assess the molecules involved in the process and discuss the observations.

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