Ammonia excretion in rainbow trout (Oncorhynchus mykiss): evidence for Rh glycoprotein and H+-ATPase involvement

2007 ◽  
Vol 31 (3) ◽  
pp. 463-474 ◽  
Author(s):  
C. Michele Nawata ◽  
Carrie C. Y. Hung ◽  
Tommy K. N. Tsui ◽  
Jonathan M. Wilson ◽  
Patricia A. Wright ◽  
...  
Keyword(s):  
Boundary Layer ◽  
Rainbow Trout ◽  
Ammonia Excretion ◽  
Pavement Cell ◽  
New Members ◽  
Pavement Cells ◽  
Cdna Sequences ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Ammonia Transport ◽  
Genes Encoding

Branchial ammonia transport in freshwater teleosts is not well understood. Most studies conclude that NH3 diffuses out of the gill and becomes protonated to NH4+ in an acidified gill boundary layer. Rhesus (Rh) proteins are new members of the ammonia transporter superfamily and rainbow trout possess genes encoding for Rh30-like1 and Rhcg2. We identified seven additional full-length trout Rh cDNA sequences: one Rhag and two each of Rhbg, Rhcg1, and Rh30-like. The mRNA expression of Rhbg, Rhcg1, and Rhcg2 was examined in trout tissues (blood, brain, eye, gill, heart, intestine, kidney, liver, muscle, skin, spleen) exposed to high external ammonia (HEA; 1.5 mmol/l NH4HCO3, pH 7.95, 15°C). Rhbg was expressed in all tissues, Rhcg1 was expressed in brain, gill, liver, and skin, and Rhcg2 was expressed in gill and skin. Brain Rhbg and Rhcg1 were downregulated, blood Rh30-like and Rhag were downregulated, and skin Rhbg and Rhcg2 were upregulated with HEA. After an initial uptake of ammonia into the fish during HEA, excretion was reestablished, coinciding with upregulations of gill Rh mRNA in the pavement cell fraction: Rhcg2 at 12 and 48 h, and Rhbg at 48 h. NHE2 expression remained unchanged, but upregulated H+-ATPase (V-type, B-subunit) and downregulated carbonic anhydrase (CA2) expression and activity were noted in the gill and again expression changes occurred in pavement cells, and not in mitochondria-rich cells. Together, these results indicate Rh glycoprotein involvement in ammonia transport and excretion in the rainbow trout while underscoring the significance of gill boundary layer acidification by H+-ATPase.

The linkage between Na+ uptake and ammonia excretion in rainbow trout: kinetic analysis, the effects of (NH4)2SO4 and NH4HCO3 infusion and the influence of gill boundary layer pH

1999 ◽  
Vol 202 (6) ◽  
pp. 697-709 ◽  
Author(s):  
A. Salama ◽  
I.J. Morgan ◽  
C.M. Wood
Keyword(s):  
Boundary Layer ◽  
Rainbow Trout ◽  
Kinetic Analysis ◽  
Dominant Role ◽  
Ammonia Excretion ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Total Ammonia ◽  
Stimulation Of

The nature of the linkage between between branchial ammonia excretion (JAmm) and unidirectional Na+ influx (JNain) was studied in the freshwater rainbow trout (Oncorhynchus mykiss). Arterial plasma total [ammonia], PNH3 and JAmm were all elevated approximately threefold by intravascular infusion for 24 h with either 70 mmol l-1 (NH4)2SO4 or 140 mmol l-1 NH4HCO3 at a rate of approximately 400 micromol kg-1 h-1. Both treatments markedly stimulated JNain. NH4HCO3 induced metabolic alkalosis in the blood plasma, whereas (NH4)2SO4 caused a slight metabolic acidosis. Experiments with Hepes-buffered water (5 mmol l-1) under control conditions demonstrated that increases in gill boundary layer pH were associated with decreases in both JNain and JAmm. Thus, the stimulation of JNain caused by ammonium loading was not simply a consequence of a Na+-coupled H+ extrusion mechanism activated by internal acidosis or by alkalosis in the gill boundary layer. Indeed, there was no stimulation of net acidic equivalent excretion accompanying NH4HCO3 infusion. Michaelis-Menten kinetic analysis by acute variation of water [Na+] demonstrated that both infusions caused an almost twofold increase in JNamax but no significant change in Km, indicative of an increase in transporter number or internal counterion availability without an alteration in transporter affinity for external Na+. The increase in JNain was larger with (NH4)2SO4 than with NH4HCO3 infusion and in both cases lower than the increase in JAmm. Additional evidence of quantitative uncoupling was seen in the kinetics experiments, in which acute changes in JNain of up to threefold had negligible effects on JAmm under either control or ammonium-loaded conditions. In vitro measurements of branchial Na+/K+-ATPase activity demonstrated no effect of NH4+ concentration over the concentration range observed in vivo in infused fish. Overall, these results are consistent with a dominant role for NH3 diffusion as the normal mechanism of ammonia excretion, but indicate that ammonium loading directly stimulates JNain, perhaps by activation of a non-obligatory Na+/NH4+ exchange rather than by an indirect effect (e.g. Na+-coupled H+ excretion) mediated by altered internal or external acid-base status.

Shortening of Branchial Tight Junction Acid-Exposed Rainbow Trout (Oncorhynchus mykiss)

1991 ◽  
Vol 48 (10) ◽  
pp. 2028-2033 ◽  
Author(s):  
J. Freda ◽  
D. A. Sanchez ◽  
H. L. Bergman
Keyword(s):  
Rainbow Trout ◽  
Oncorhynchus Mykiss ◽  
Tight Junctions ◽  
Relative Magnitude ◽  
Chloride Cells ◽  
Pavement Cells ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Plasma Electrolytes ◽  
Gill Structure ◽  
Electrolyte Loss

The objective of this study was to investigate possible sites for Na+ loss in fish exposed to low environmental pH. In rainbow trout (Oncorhynchus mykiss) exposed to pH 4.0 for 1 h, a net loss of Na+ was stimulated, and changes in gill structure occurred. In addition to epithelial lifting and necrosis in the gills of acid-exposed fish, tight junctions between pavement epithelial cells and chloride cells decreased in length by 25% whereas tight junctions between adjacent pavement cells did not significantly change. In a second experiment where fish were moved from pH 4.0 or 3.5 water to pH 6.5 water, we observed that Na+ loss declined immediately and approached control levels. The reversible nature of the stimulation of Na+ loss indicates that the site of Na+ loss in the fish gill can be reversibly opened and closed, which is consistent with the known properties of tight junctions. We hypothesize that the opening of tight junctions contributes to the loss of plasma electrolytes at low environmental pH. However, the relative magnitude of electrolyte loss through the tight junctions remains unknown.

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.

Excretion and distribution of ammonia and the influence of boundary layer acidification in embryonic rainbow trout (Oncorhynchus mykiss)

1996 ◽  
Vol 199 (12) ◽  
pp. 2713-2723 ◽  
Author(s):  
E Rahaman-Noronha ◽  
M Donnell ◽  
C Pilley ◽  
P Wright
Keyword(s):  
Rainbow Trout ◽  
Oncorhynchus Mykiss ◽  
Electrical Potential ◽  
Ammonia Excretion ◽  
Free Water ◽  
Bulk Water ◽  
Water Addition ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
External Water ◽  
Excretion Rates

This study examined ammonia excretion by embryos of the rainbow trout (Oncorhynchus mykiss). The distribution of ammonia in relation to the H+ distribution and electrical potential was determined. The influence of the pH of the unstirred layer (USL) of water next to the external surface of the embryo was also assessed. Eyed-up embryos (35­40 days post-fertilization) were exposed to various external water conditions [pH 6.0, pH 10.0, 1.6 mmol l-1 NaCl, 0.0 mmol l-1 NaCl, 0.2 mmol l-1 NH4Cl, 2.5 mmol l-1 borax buffer (Na2B4O7.10H2O), 2.5 mmol l-1 Hepes, 0.1 mmol l-1 amiloride] for 30 min and ammonia excretion rates, ammonia concentration in the perivitelline fluid (PVF) and yolk, and the pH of the PVF, yolk and USL were measured. The rate of ammonia excretion was dependent, in part, on the partial pressure gradient of NH3 ( PNH3) from the PVF to the USL. Exposure to water of pH 6 increased, whereas NH4Cl or pH 10 exposure decreased, ammonia excretion rates. Elevated external Na+ levels also influenced the rate of ammonia excretion, but neither Na+-free water nor amiloride had any effect. The distribution of ammonia between the PVF and USL was dependent on the H+ distribution, but ammonia was distributed according to the electrical potential between the PVF and yolk. The USL was 0.32 pH units more acidic than the bulk water. Addition of buffer to the external water eliminated the acid USL and decreased ammonia excretion rates. We conclude that rainbow trout embryos excrete ammonia primarily as NH3, but when external Na+ levels are elevated, ammonia excretion may be independent of the PNH3 gradient. The acidic USL next to the chorion probably facilitates NH3 diffusion by maintaining the PNH3 through the conversion of NH3 to NH4+ upon entry into the USL.

A respirometric analysis of fuel use during aerobic swimming at different temperatures in rainbow trout (Oncorhynchus mykiss)

1998 ◽  
Vol 201 (22) ◽  
pp. 3123-3133 ◽  
Author(s):  
JD Kieffer ◽  
D Alsop ◽  
CM Wood
Keyword(s):  
Rainbow Trout ◽  
Oncorhynchus Mykiss ◽  
Swimming Speed ◽  
Ammonia Excretion ◽  
Whole Body ◽  
Acclimation Temperature ◽  
Fish Swimming ◽  
Fuel Use ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Absolute Basis

Instantaneous fuel usage at 5 degreesC or 15 degreesC was assessed by measurement of rates of O2 consumption (O2), CO2 excretion (CO2) and nitrogenous waste excretion (nitrogen =ammonia-N + urea-N) in juvenile rainbow trout (Oncorhynchus mykiss) at rest and during swimming at 45 % and 75 % of aerobic capacity (Ucrit). After 2 weeks of training at approximately 1 body length s-1 (BL s-1), critical swimming speeds (approximately 3.0 BL s-1) and whole-body energy stores (total protein, lipids and carbohydrates) were identical in fish acclimated to 5 degreesC or 15 degreesC. O2 and CO2 increased with swimming speed at both temperatures and were higher at 15 degreesC than at 5 degreesC at all speeds, but the overall Q10 values (1.23-1.48) were low in these long-term (6 weeks) acclimated fish. The respiratory quotient (CO2/O2, approximately 0.85) was independent of both temperature and swimming speed. In contrast to O2 and CO2, the rate of ammonia excretion was independent of swimming speed, but more strongly influenced by temperature (Q10 1. 4-2.8). Urea excretion accounted for 15-20 % of nitrogen, was unaffected by swimming speed and showed a tendency (P<0.07) to be positively influenced by temperature at one speed only (45 % Ucrit). Nitrogen quotients (NQ nitrogen/O2) were generally higher in warm-acclimated fish, remaining independent of swimming speed at 15 degreesC (0.08), but decreased from about 0.08 at rest to 0.04 during swimming at 5 degreesC. Instantaneous aerobic fuel use calculations based on standard respirometric theory showed that both acclimation temperature and swimming speed markedly influenced the relative and absolute use of carbohydrates, lipids and proteins by trout. At rest, cold-acclimated trout used similar proportions of carbohydrates and lipids and only 27 % protein. During swimming, protein use decreased to 15 % at both speeds while the relative contributions of both lipid and carbohydrate increased (to more than 40 %). On an absolute basis, carbohydrate was the most important fuel for fish swimming at 5 degreesC. In contrast, resting fish acclimated to 15 degreesC utilized 55 % lipid, 30 % protein and only 15 % carbohydrate. However, as swimming speed increased, the relative contribution of carbohydrate increased to 25 %, while the protein contribution remained unchanged at approximately 30 %, and lipid use decreased slightly (to 45 %). On an absolute basis, lipid remained the most important fuel in fish swimming at 15 degreesC. These results support the concept that lipids are a major fuel of aerobic exercise in fish, but demonstrate that the contribution of protein oxidation is much smaller than commonly believed, while that of carbohydrate oxidation is much larger, especially at higher swimming speeds and colder temperature.

Oxygen concentration in the water boundary layer next to rainbow trout (Oncorhynchus mykiss) embryos is influenced by hypoxia exposure time, metabolic rate, and water flow

2008 ◽  
Vol 65 (10) ◽  
pp. 2170-2177 ◽  
Author(s):  
Silvana C. Miller ◽  
Shannon E. Reeb ◽  
Patricia A. Wright ◽  
Todd E. Gillis
Keyword(s):  
Boundary Layer ◽  
Rainbow Trout ◽  
Oncorhynchus Mykiss ◽  
Metabolic Rate ◽  
Water Flow ◽  
Exposure Time ◽  
Chronic Hypoxia ◽  
Acute Hypoxia ◽  
Hypoxia Exposure ◽  
Rainbow Trout Oncorhynchus Mykiss

The objective of this study was to examine the influence of hypoxia exposure time, metabolic rate, and water flow rate on the O2 concentration in the boundary layer outside and inside the chorion of rainbow trout ( Oncorhynchus mykiss ) embryos. Oxygen consumption, growth, and dissolved O2 in the boundary layer were measured at 15, 22, and 29 days post fertilization (dpf) from embryos reared in normoxia (O2 concentration at 100% saturation) and chronic hypoxia (>24 h, 50% saturation) and exposed to acute hypoxia (30 min, 50% saturation). Chronic and acute hypoxia exposure decreased the dissolved O2 in the boundary layer to the same extent at 15 and 22 dpf; however, at 29 dpf, O2 levels were significantly lower in acute relative to chronic hypoxia. At 29 dpf, O2 uptake per individual was significantly lower in embryos exposed to chronic relative to acute hypoxia. In addition, mass-specific O2 uptake in chronic hypoxia-exposed embryos was ~40% less than that of controls but the same as that of acutely exposed embryos. This correlates with reduced growth in embryos exposed to chronic hypoxia. We conclude that boundary layer O2 is lower after 30 min of hypoxia compared with 2 weeks of hypoxia simply because embryos exposed to chronic hypoxia grow slower and consume less O2.

Sign in / Sign up

Export Citation Format

Share Document