Ammonia excretion via Rhcg1 facilitates Na+ uptake in larval zebrafish, Danio rerio, in acidic water

2011 ◽  
Vol 301 (5) ◽  
pp. R1517-R1528 ◽  
Author(s):  
Yusuke Kumai ◽  
Steve F. Perry
Keyword(s):  
Freshwater Fish ◽  
Ammonia Excretion ◽  
Acidic Water ◽  
Chemical Gradients ◽  
Larval Zebrafish ◽  
Zebrafish Larvae ◽  
Pharmacological Data ◽  
Water Ph ◽  
Rh Proteins ◽  
Na Uptake

The involvement of a Na+/H+ exchanger (NHE) in mediating Na+ uptake by freshwater fish is currently debated. Although supported indirectly by empirical molecular and pharmacological data, theoretically its operation should be constrained thermodynamically, owing to unfavorable chemical gradients. Recently, there has been an increasing focus on ammonia channels (Rh proteins) as potentially contributing to Na+ uptake across the freshwater fish gill. In this study, we tested the hypothesis that Rhcg1, a specific apical isoform of Rh protein, is critically important in facilitating Na+ uptake in zebrafish larvae via its interaction with NHE. Treating larvae (4 days postfertilization) with 5-( N-ethyl- N-isopropyl) amiloride (EIPA), an inhibitor of NHE, caused a significant reduction in Na+ uptake in fish reared in acidic water (pH ∼ 4.0). A role for NHE in Na+ uptake was further confirmed by translational knockdown of NHE3b, an isoform of NHE thought to be responsible for Na+/H+ exchange in zebrafish larvae. Exposing the larvae reared in acidic water to 5 mM external ammonium sulfate or increasing the buffering capacity of the water with 10 mM HEPES caused concurrent reductions in ammonia excretion and Na+ uptake. Furthermore, translational knockdown of Rhcg1 significantly reduced ammonia excretion and Na+ uptake in larvae chronically (4 days) or acutely (24 h) exposed to acidic water. Unlike in sham-injected larvae, EIPA did not affect Na+ uptake in fish experiencing Rhcg1 knockdown. Additionally, exposure of larvae to bafilomycin A1 (an inhibitor of H+-ATPase) significantly reduced Na+ uptake in fish reared in acidic water. These observations suggest the existence of multiple mechanisms of Na+ uptake in larval zebrafish in acidic water: one in which Na+ uptake via NHE3b is linked to ammonia excretion via Rhcg1, and another facilitated by H+-ATPase.

scholarly journals A role for sodium-chloride cotransporters in the rapid regulation of ion uptake following acute environmental acidosis: new insights from the zebrafish model

2016 ◽  
Vol 311 (6) ◽  
pp. C931-C941 ◽  
Author(s):  
Raymond W. M. Kwong ◽  
Steve F. Perry
Keyword(s):  
Acid Exposure ◽  
Whole Body ◽  
Cell Type ◽  
Acidic Water ◽  
Zebrafish Model ◽  
Larval Zebrafish ◽  
Water Ph ◽  
Control Water ◽  
Compensatory Regulation

The effects of acute exposure to acidic water on Na+ and Cl− homeostasis, and the mechanisms underlying their compensatory regulation, were investigated in the larval zebrafish Danio rerio. Exposure to acidic water (pH 4.0; control pH 7.6) for 2 h significantly reduced Na+ uptake and whole body Na+ content. Nevertheless, the capacity for Na+ uptake was substantially increased in fish preexposed to acidic water but measured in control water. Based on the accumulation of the Na+-selective dye, Sodium Green, two ionocyte subtypes exhibited intracellular Na+ enrichment after preexposure to acidic water: H+-ATPase rich (HR) cells, which coexpress the Na+/H+ exchanger isoform 3b (NHE3b), and a non-HR cell population. In fish experiencing Na+-Cl− cotransporter (NCC) knockdown, we observed no Sodium Green accumulation in the latter cell type, suggesting the non-HR cells were NCC cells. Elimination of NHE3b-expressing HR cells did not prevent the increased Na+ uptake following acid exposure. On the other hand, the increased Na+ uptake was abolished when the acidic water was enriched with Na+ and Cl−, but not with Na+ only, indicating that the elevated Na+ uptake after acid exposure was associated with the compensatory regulation of Cl−. Further examinations demonstrated that acute acid exposure also reduced whole body Cl− levels and increased the capacity for Cl− uptake. Moreover, knockdown of NCC prevented the increased uptake of both Na+ and Cl− after exposure to acidic water. Together, the results of the present study revealed a novel role of NCC in the compensatory regulation of Na+ and Cl− uptake following acute acidosis.

scholarly journals The role of cAMP-mediated intracellular signaling in regulating Na+ uptake in zebrafish larvae

2014 ◽  
Vol 306 (1) ◽  
pp. R51-R60 ◽  
Author(s):  
Yusuke Kumai ◽  
Raymond W. M. Kwong ◽  
Steve F. Perry
Keyword(s):  
Protein Trafficking ◽  
Intracellular Signaling ◽  
Whole Body ◽  
Larval Zebrafish ◽  
Zebrafish Larvae ◽  
Β Adrenergic Receptors ◽  
Camp Levels ◽  
Na Uptake ◽  
Stimulation Of

In the current study, the role of cAMP in stimulating Na+ uptake in larval zebrafish was investigated. Treating larvae at 4 days postfertilization (dpf) with 10 μM forskolin or 1 μM 8-bromo cAMP significantly increased Na+ uptake by three-fold and twofold, respectively. The cAMP-dependent stimulation of Na+ uptake was probably unrelated to protein trafficking via microtubules because pretreatment with 200 μM colchicine or 30 μM nocodazole did not attenuate the magnitude of the response. Na+ uptake was stimulated markedly following acute (2 h) exposure to acidic water. The acid-induced increase in Na+ uptake was accompanied by a twofold elevation in whole body cAMP levels and attenuated by inhibiting PKA with 10 μM H-89. Knockdown of Na+-H+ exchanger 3b (NHE3b) attenuated, but did not abolish, the stimulation of Na+ uptake during forskolin treatment. In glial cell missing 2 morphants, in which the role of NHE3b in Na+ uptake is diminished and the Na+-Cl− cotransporter (NCC) becomes the predominant route of Na+ entry, forskolin treatment continued to increase Na+ uptake. These data suggest that at least NHE3b and NCC are targeted by cAMP in zebrafish larvae. Staining of larvae with fluorescent forskolin and propranolol revealed the presence of transmembrane adenylyl cyclase within multiple subtypes of ionocytes expressing β-adrenergic receptors. Taken together, results of the present study demonstrate that cAMP-mediated intracellular signaling may regulate multiple Na+ transporters and plays an important role in regulating Na+ uptake in zebrafish larvae during acute exposure to an acidic environment.

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.

scholarly journals Fumonisin B1 Production by Fusarium Species and Mycotoxigenic Effect on Larval Zebrafish

2020 ◽  
Vol 31 (3) ◽  
pp. 91-107 ◽  
Author(s):  
Najihah Azman ◽  
Nur Ain Izzati Mohd Zainudin ◽  
Wan Norhamidah Wan Ibrahim
Keyword(s):  
Liquid Chromatography ◽  
Danio Rerio ◽  
Morphological Changes ◽  
Fusarium Species ◽  
Fumonisin B1 ◽  
Larval Zebrafish ◽  
Zebrafish Larvae ◽  
Significant Difference ◽  
Successful Amplification ◽  
Fast Liquid Chromatography

Fumonisin B1 (FB1) is a common mycotoxin produced by Fusarium species particularly F. proliferatum and F. verticillioides. The toxin produced can cause adverse effects on humans and animals. The objectives of this study were to detect the production of FB1 based on the amplification of FUM1 gene, to quantify FB1 produced by the isolates using Ultra-fast Liquid Chromatography (UFLC) analysis, to examine the embryotoxicity effect of FB1 and to determine EC50 toward the larvae of zebrafish (Danio rerio). Fifty isolates of Fusarium species were isolated from different hosts throughout Malaysia. Successful amplification of the FUM1 gene showed the presence of this gene (800 bp) in the genome of 48 out of 50 isolates. The highest level of FB1 produced by F. proliferatum isolate B2433 was 6677.32 ppm meanwhile F. verticillioides isolate J1363 was 954.01 ppm. From the assessment of embryotoxicity test of FB1 on larvae of zebrafish, five concentrations of FB1 (0.43 ppm, 0.58 ppm, 0.72 ppm, 0.87 ppm and 1.00 ppm) were tested. Morphological changes of the FB1 exposed-larvae were observed at 24 to 168 hpf. The mortality rate and abnormality of zebrafish larvae were significantly increased at 144 hpf exposure. Meanwhile, the spontaneous tail coiling showed a significant difference. There were no significant differences in the heartbeat rate. As a conclusion, the presence of FUM1 in every isolate can be detected by FUM1 gene analysis and both of the species produced different concentrations of FB1. This is the first report of FB1 produced by Fusarium species gave a significant effect on zebrafish development.

Muscle metabolism of freshwater fish, Tilapia mossambica (Peters), during acute exposure and acclimation to sublethal acidic water

1981 ◽  
Vol 59 (10) ◽  
pp. 1909-1915 ◽  
Author(s):  
V. Krishna Murthy ◽  
P. Reddanna ◽  
M. Bhaskar ◽  
S. Govindappa
Keyword(s):  
Freshwater Fish ◽  
White Muscle ◽  
Glycogen Content ◽  
Acid Stress ◽  
Muscle Metabolism ◽  
Acute Exposure ◽  
Tilapia Mossambica ◽  
Red Muscle ◽  
Water Ph ◽  
Red And White Muscles

Freshwater fish, Tilapia mossambica (Peters), were subjected to acute exposure and acclimation to sublethal acid water (pH 4.0), and the muscle metabolism was investigated. Differential patterns of carbohydrate metabolism were witnessed in the red and white muscles in response to both acute exposure and acclimation. The glycogen content of red muscle was elevated whereas that of white muscle was depleted on acute exposure. But on acclimation, both the muscles had elevated glycogen content. The red muscle seems to mobilize carbohydrates into both hexose mono- and di-phosphate pathways, but white muscle does so only into the hexose monophosphate pathway on acclimation. In general, both the muscles exhibited suppressed glycolysis and elevated oxidative phase leading to elevated glycogen level. The muscle metabolism was oriented towards conservation of carbohydrates and lesser production of organic acids on acclimation, as a possible metabolic adaptive mechanism of the fish, enabling them to counteract the imposed acid stress.

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.

The effect of highly alkaline water (pH 9.5) on the morphology and morphometry of chloride cells and pavement cells in the gills of the freshwater rainbow trout: relationship to ionic transport and ammonia excretion

2000 ◽  
Vol 78 (2) ◽  
pp. 307-319 ◽  
Author(s):  
Pierre Laurent ◽  
Michael P Wilkie ◽  
Claudine Chevalier ◽  
Chris M Wood
Keyword(s):  
Rainbow Trout ◽  
Surface Area ◽  
Ammonia Excretion ◽  
Chloride Cells ◽  
Apical Surface ◽  
Influx Rate ◽  
Pavement Cells ◽  
Alkaline Water ◽  
Partial Restoration ◽  
Water Ph

Exposure of rainbow trout (Oncorhynchus mykiss) to alkaline water (pH 9.5) impairs ammonia excretion (JAmm) and gill-mediated ion-exchange processes, as characterized by decreased Cl- (JC1in) and Na+ influx (JNain) across the gill. Scanning electron microscopy suggested that the depression of JC1in was concomitant with an early decrease in the population of the most active chloride cells (CCs), partly compensated for by an increasing number of immature CCs. However, within 72 h after the onset of exposure to alkaline water, there was a 2-fold increase in the fractional apical surface area of CCs that paralleled complete recovery of the maximal Cl- influx rate (JC1max). These results suggest that recovery of JC1max was associated with greater CC surface area, resulting in more transport sites on the gill epithelium. Morphometric analysis of the outermost layer of pavement cells on the lamellar epithelium showed a greater density of microvilli during exposure to alkaline water, which may have contributed to partial restoration of the number of Na+ transport sites (JNamax). Finally, the blood-to-water gill-diffusion distance decreased by 27% after 72 h at pH 9.5, and likely contributed to progressive restoration of ammonia excretion in alkaline water.

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