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Patrick J Walsh - One of the best experts on this subject based on the ideXlab platform.
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physiological and molecular responses of the spiny dogfish shark squalus acanthias to high environmental ammonia scavenging for nitrogen
The Journal of Experimental Biology, 2015Co-Authors: C. Michele Nawata, Patrick J Walsh, Chris M WoodAbstract:In teleosts, a branchial metabolon links ammonia excretion to Na+ uptake via Rh glycoproteins and other transporters. Ureotelic elasmobranchs are thought to have low branchial ammonia permeability, and little is known about Rh function in this ancient group. We cloned Rh cDNAs (Rhag, Rhbg and Rhp2) and evaluated gill ammonia handling in Squalus acanthias. Control ammonia excretion was
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Evolution of urea transporters in vertebrates: adaptation to urea's multiple roles and metabolic sources.
The Journal of experimental biology, 2015Co-Authors: Christophe M R Lemoine, Patrick J WalshAbstract:In the two decades since the first cloning of the mammalian kidney urea transporter (UT-A), UT genes have been identified in a plethora of organisms, ranging from single-celled bacteria to metazoans. In this review, focusing mainly on vertebrates, we first reiterate the multiple catabolic and anabolic pathways that produce urea, then we reconstruct the phylogenetic history of UTs, and finally we examine the tissue distribution of UTs in selected vertebrate species. Our analysis reveals that from an ancestral UT, three homologues evolved in piscine lineages (UT-A, UT-C and UT-D), followed by a subsequent reduction to a single UT-A in lobe-finned fish and amphibians. A later internal tandem duplication of UT-A occurred in the amniote lineage (UT-A1), followed by a second tandem duplication in mammals to give rise to UT-B. While the expected UT expression is evident in excretory and osmoregulatory tissues in Ureotelic taxa, UTs are also expressed ubiquitously in non-Ureotelic taxa, and in tissues without a complete ornithine-urea cycle (OUC). We posit that non-OUC production of urea from arginine by arginase, an important pathway to generate ornithine for synthesis of molecules such as polyamines for highly proliferative tissues (e.g. testis, embryos), and neurotransmitters such as glutamate for neural tissues, is an important evolutionary driving force for the expression of UTs in these taxa and tissues.
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Interactions between cortisol and Rhesus glycoprotein expression in ureogenic toadfish, Opsanus beta.
The Journal of experimental biology, 2012Co-Authors: Tamara M Rodela, Patrick J Walsh, M. Danielle Mcdonald, Kathleen M. GilmourAbstract:In their native environment, gulf toadfish excrete equal quantities of ammonia and urea. However, upon exposure to stressful conditions in the laboratory (i.e. crowding, confinement or air exposure), toadfish decrease branchial ammonia excretion and become Ureotelic. The objective of this study was to determine the influences of cortisol and ammonia on ammonia excretion relative to expression of Rhesus (Rh) glycoproteins and the ammonia-fixing enzyme, glutamine synthetase (GS). In vivo infusions and/or injections were used to manipulate corticosteroid activity and plasma ammonia concentrations in Ureotelic toadfish. Metyrapone treatment to lower circulating cortisol levels resulted in a 3.5-fold elevation of ammonia excretion rates, enhanced mRNA expression of two of the toadfish Rh isoforms (Rhcg1 and Rhcg2), and decreased branchial and hepatic GS activity. Correspondingly, cortisol infusion decreased ammonia excretion 2.5-fold, a change that was accompanied by reduced branchial expression of all toadfish Rh isoforms (Rhag, Rhbg, Rhcg1 and Rhcg2) and a twofold increase in hepatic GS activity. In contrast, maintenance of high circulating ammonia levels by ammonia infusion enhanced ammonia excretion and Rh expression (Rhag, Rhbg and Rhcg2). Toadfish treated with cortisol showed an attenuated response to ammonia infusion with no change in Rh mRNA expression or GS activity. In summary, the evidence suggests that ammonia excretion in toadfish is modulated by cortisol-induced changes in both Rh glycoprotein expression and GS activity.
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Dogmas and controversies in the handling of nitrogenous wastes: ureotely and ammonia tolerance in early life stages of the gulf toadfish, Opsanus beta.
Journal of Experimental Biology, 2004Co-Authors: John F. Barimo, Shelby L. Steele, Patricia A. Wright, Patrick J WalshAbstract:The marine gulf toadfish ( Opsanus beta ) is an unusual teleost fish as it is able to switch between ammoniotelism and ureotelism in response to a variety of laboratory conditions. The present study integrates field work conducted in Biscayne and Florida Bays, USA with laboratory studies to examine ureotelism during the early life history stages of O. beta . Adult toadfish voluntarily nested in artificial shelters placed amongst seagrass beds and were found to be predominantly Ureotelic under natural conditions as the internal shelter water had mean urea and ammonia concentrations ( N =51) of 14.2±1.6 μmol N l–1 and 8.9±0.9 μmol N l–1, respectively. Toadfish successfully spawned in shelters, providing eggs, larvae and juvenile toadfish for laboratory study. In the lab, juvenile toadfish were also Ureotelic and urea was excreted in pulsatile events that accounted for 62.0±5.9% of total urea-N excreted. Excretion rates of urea-N and ammonia-N were 1.018±0.084 μmol N h–1 g–1 and 0.235±0.095 μmol N h–1 g–1, respectively. Field-collected eggs, larvae and juveniles expressed significant levels of the ornithine–urea cycle enzymes carbamoyl-phosphate synthetase III, ornithine transcarbamylase and arginase and the accessory enzyme glutamine synthetase, all of which increased in activity as toadfish developed through early life stages. In juveniles, the ammonia 96-h LC50 value was 875 μmol N l–1 and there was a 3-fold increase in ornithine transcarbamylase activity in the 1000 μmol N l–1 NH4Cl treatment. The results are discussed in the context of the causal factor(s) for ureotelism in toadfish. Furthermore, the results of this study suggest it is unlikely that the adaptive significance of ureotelism in toadfish is a means to prevent fouling nests with ammonia and in turn poisoning offspring; however, additional study is warranted.
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branchial and renal excretion of urea and urea analogues in the plainfin midshipman porichthys notatus
Journal of Comparative Physiology B-biochemical Systemic and Environmental Physiology, 2002Co-Authors: M D Mcdonald, Patrick J Walsh, Chris M WoodAbstract:This study investigated whether urea transport mechanisms were present in the gills of the ammoniotelic plainfin midshipman (Porichthysnotatus), similar to those recently documented in its Ureotelic relative (family Batrachoididae), the gulf toadfish (Opsanusbeta). Midshipmen were fitted with internal urinary and caudal artery catheters for repetitive sampling of urine and blood in experiments and radiolabeled urea analogues ([14C]-thiourea and [14C]-acetamide) were used to evaluate the handling of these substances. Isosmotically balanced infusions of urea were used to raise plasma and urine urea concentrations to levels surpassing physiological levels by 8.5-fold and 6.4-fold, respectively. Despite these high urea levels, there was no observable transport maximum in either renal or branchial urea excretion rate, a result mirrored by the total uptake of fish exposed to a range of environmental urea concentrations. Permeability to urea appeared to be symmetrical in the two directions. At comparable plasma concentrations the branchial clearance rate of acetamide was 74% that of urea while branchial clearance rate of thiourea was 55% that of urea. For influx, the comparable values were 60% and 36%, indicating the same pattern. In contrast, the secretion clearance rate of acetamide by the kidney was 56% that of urea while the rate of thiourea secretion clearance was 137% greater than that of urea, with both urea and thiourea being more concentrated in the urine than in the plasma. In addition, the secretion clearance rates of thiourea and urea were significantly greater than those of water and Cl–, whereas acetamide, water and Cl– were found equally in the plasma and urine, appearing to passively equilibrate between the two fluids. Based on our findings, there appear to be two distinct transport mechanisms involved in urea excretion in the plainfin midshipmen, one in the gill (a facilitated diffusion type transporter) and one in the kidney (an active transport mechanism), each of which does not saturate even at plasma urea concentrations that greatly exceed physiological levels. These transporters appear to be similar to those in the midshipman's Ureotelic relative, the gulf toadfish.
Chris M Wood - One of the best experts on this subject based on the ideXlab platform.
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Nitrogen handling in the elasmobranch gut: a role for microbial urease.
The Journal of experimental biology, 2019Co-Authors: Chris M Wood, Hon Jung Liew, Gudrun De Boeck, J Lisa Hoogenboom, W Gary AndersonAbstract:Ureotelic elasmobranchs require nitrogen for both protein growth and urea-based osmoregulation, and therefore are probably nitrogen-limited in nature. Mechanisms exist for retaining and/or scavenging nitrogen in the gills, kidney, rectal gland and gut, but as yet, the latter are not well characterized. Intestinal sac preparations of the Pacific spiny dogfish shark (Squalus acanthias suckleyi) incubated in vitro strongly reabsorbed urea from the lumen after feeding, but mucosal fluid ammonia concentrations increased with incubation time. Phloretin (0.25 mmol l-1, which blocked urea reabsorption) greatly increased the rate of ammonia accumulation in the lumen. A sensitive [14C]urea-based assay was developed to examine the potential role of microbial urease in this ammonia production. Urease activity was detected in chyme/intestinal fluid and intestinal epithelial tissue of both fed and fasted sharks. Urease was not present in gall-bladder bile. Urease activities were highly variable among animals, but generally greater in chyme than in epithelia, and greater in fed than in fasted sharks. Comparable urease activities were found in chyme and epithelia of the Pacific spotted ratfish (Hydrolagus colliei), a Ureotelic holocephalan, but were much lower in ammonotelic teleosts. Urease activity in dogfish chyme was inhibited by acetohydroxamic acid (1 mmol l-1) and by boiling. Treatment of dogfish gut sac preparations with acetohydroxamic acid blocked ammonia production, changing net ammonia accumulation into net ammonia absorption. We propose that microbial urease plays an important role in nitrogen handling in the elasmobranch intestine, allowing some urea-N to be converted to ammonia, which is then reabsorbed for amino acid synthesis or reconversion to urea.
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physiological and molecular responses of the spiny dogfish shark squalus acanthias to high environmental ammonia scavenging for nitrogen
The Journal of Experimental Biology, 2015Co-Authors: C. Michele Nawata, Patrick J Walsh, Chris M WoodAbstract:In teleosts, a branchial metabolon links ammonia excretion to Na+ uptake via Rh glycoproteins and other transporters. Ureotelic elasmobranchs are thought to have low branchial ammonia permeability, and little is known about Rh function in this ancient group. We cloned Rh cDNAs (Rhag, Rhbg and Rhp2) and evaluated gill ammonia handling in Squalus acanthias. Control ammonia excretion was
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Rh proteins and NH4(+)-activated Na+-ATPase in the Magadi tilapia (Alcolapia grahami), a 100% Ureotelic teleost fish.
The Journal of experimental biology, 2013Co-Authors: Chris M Wood, C. Michele Nawata, Pierre Laurent, Claudine Chevalier, Harold L. Bergman, Jonathan M Wilson, Adalto Bianchini, John N Maina, Ora E Johannsson, Lucas F BianchiniAbstract:The small cichlid fish Alcolapia grahami lives in Lake Magadi, Kenya, one of the most extreme aquatic environments on Earth (pH ~10, carbonate alkalinity ~300 mequiv l(-1)). The Magadi tilapia is the only 100% Ureotelic teleost; it normally excretes no ammonia. This is interpreted as an evolutionary adaptation to overcome the near impossibility of sustaining an NH3 diffusion gradient across the gills against the high external pH. In standard ammoniotelic teleosts, branchial ammonia excretion is facilitated by Rh glycoproteins, and cortisol plays a role in upregulating these carriers, together with other components of a transport metabolon, so as to actively excrete ammonia during high environmental ammonia (HEA) exposure. In Magadi tilapia, we show that at least three Rh proteins (Rhag, Rhbg and Rhcg2) are expressed at the mRNA level in various tissues, and are recognized in the gills by specific antibodies. During HEA exposure, plasma ammonia levels and urea excretion rates increase markedly, and mRNA expression for the branchial urea transporter mtUT is elevated. Plasma cortisol increases and branchial mRNAs for Rhbg, Rhcg2 and Na(+),K(+)-ATPase are all upregulated. Enzymatic activity of the latter is activated preferentially by NH4(+) (versus K(+)), suggesting it can function as an NH4(+)-transporter. Model calculations suggest that active ammonia excretion against the gradient may become possible through a combination of Rh protein and NH4(+)-activated Na(+)-ATPase function.
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Rh proteins and NH4+-activated Na+-ATPase in the Magadi tilapia (Alcolapia grahami), a 100% Ureotelic teleost fish
Journal of Experimental Biology, 2013Co-Authors: Chris M Wood, C. Michele Nawata, Pierre Laurent, Claudine Chevalier, Harold L. Bergman, Jonathan M Wilson, Adalto Bianchini, John N Maina, Ora E Johannsson, Lucas F BianchiniAbstract:SUMMARY The small cichlid fish Alcolapia grahami lives in Lake Magadi, Kenya, one of the most extreme aquatic environments on Earth (pH ~10, carbonate alkalinity ~300 mequiv l −1 ). The Magadi tilapia is the only 100% Ureotelic teleost; it normally excretes no ammonia. This is interpreted as an evolutionary adaptation to overcome the near impossibility of sustaining an NH 3 diffusion gradient across the gills against the high external pH. In standard ammoniotelic teleosts, branchial ammonia excretion is facilitated by Rh glycoproteins, and cortisol plays a role in upregulating these carriers, together with other components of a transport metabolon, so as to actively excrete ammonia during high environmental ammonia (HEA) exposure. In Magadi tilapia, we show that at least three Rh proteins ( Rhag , Rhbg and Rhcg2 ) are expressed at the mRNA level in various tissues, and are recognized in the gills by specific antibodies. During HEA exposure, plasma ammonia levels and urea excretion rates increase markedly, and mRNA expression for the branchial urea transporter mtUT is elevated. Plasma cortisol increases and branchial mRNAs for Rhbg , Rhcg2 and Na + ,K + -ATPase are all upregulated. Enzymatic activity of the latter is activated preferentially by NH 4 + ( versus K + ), suggesting it can function as an NH 4 + -transporter. Model calculations suggest that active ammonia excretion against the gradient may become possible through a combination of Rh protein and NH 4 + -activated Na + -ATPase function.
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rh proteins and nh4 activated na atpase in the magadi tilapia alcolapia grahami a 100 Ureotelic teleost fish
The Journal of Experimental Biology, 2013Co-Authors: Chris M Wood, Pierre Laurent, Claudine Chevalier, Harold L. Bergman, Jonathan M Wilson, Adalto Bianchini, John N Maina, Michele C Nawata, Ora E JohannssonAbstract:SUMMARY The small cichlid fish Alcolapia grahami lives in Lake Magadi, Kenya, one of the most extreme aquatic environments on Earth (pH ~10, carbonate alkalinity ~300 mequiv l −1 ). The Magadi tilapia is the only 100% Ureotelic teleost; it normally excretes no ammonia. This is interpreted as an evolutionary adaptation to overcome the near impossibility of sustaining an NH 3 diffusion gradient across the gills against the high external pH. In standard ammoniotelic teleosts, branchial ammonia excretion is facilitated by Rh glycoproteins, and cortisol plays a role in upregulating these carriers, together with other components of a transport metabolon, so as to actively excrete ammonia during high environmental ammonia (HEA) exposure. In Magadi tilapia, we show that at least three Rh proteins ( Rhag , Rhbg and Rhcg2 ) are expressed at the mRNA level in various tissues, and are recognized in the gills by specific antibodies. During HEA exposure, plasma ammonia levels and urea excretion rates increase markedly, and mRNA expression for the branchial urea transporter mtUT is elevated. Plasma cortisol increases and branchial mRNAs for Rhbg , Rhcg2 and Na + ,K + -ATPase are all upregulated. Enzymatic activity of the latter is activated preferentially by NH 4 + ( versus K + ), suggesting it can function as an NH 4 + -transporter. Model calculations suggest that active ammonia excretion against the gradient may become possible through a combination of Rh protein and NH 4 + -activated Na + -ATPase function.
Paul M. Anderson - One of the best experts on this subject based on the ideXlab platform.
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N-Acetyl-l-glutamate and the Urea Cycle in Gulf Toadfish (Opsanus beta) and Other Fish
Archives of biochemistry and biophysics, 1998Co-Authors: Eric A. Julsrud, Patrick J Walsh, Paul M. AndersonAbstract:Carbamoyl phosphate synthetase I (CPSase I) catalyzes the first reaction of the urea cycle in mammalian Ureotelic species. The positive allosteric cofactor N-acetyl-L-glutamate (AGA) is required for CPSase I activity and is important for regulation of the urea cycle. A similar enzyme, CPSase III, catalyzes this reaction in fish; CPSase III differs from CPSase I in that it utilizes glutamine as the nitrogen-donating substrate instead of ammonia. AGA also stimulates the CPSase III-catalyzed reaction, but is not absolutely required for activity if the glutamine concentration is high. There has been no report of the presence or function of AGA in fish. Here we report that AGA is present in those species and tissues of fish that have significant levels of CPSase III and urea cycle activity; the levels of AGA were higher in liver of adult gulf toadfish (Opsanus beta) and spiny dogfish shark (Squalus acanthias), both of which have high CPSase III activity, than in bass (Micropterus salmoides) or trout (Oncorhynchus mykiss), which have much lower or no CPSase III activity, respectively. In the toadfish the levels of AGA in liver and muscle tissue were considerably higher in the fed than in the fasting state, as is observed in mammalian species; in liver, but not in muscle, the level of AGA increased when the toadfish were confined (stressed), which has been shown to induce a Ureotelic response. Toadfish muscle had CPSase III and ornithine carbamoyltransferase activities; the increase in AGA concentration in muscle when fed suggests that the presence of these first two enzymes of the urea cycle in muscle may be physiologically significant. The results indicate that the fish investigated have physiologically significant levels of AGA and that the levels correlate with parameters related to urea cycle activity.
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Carbamyl Phosphate Synthetases in an Air-Breathing Teleost, Heteropneustes fossilis
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 1997Co-Authors: Nirmalendu Saha, Paul M. Anderson, Jacqueline Dkhar, Braja K RathaAbstract:Abstract The Indian air-breathing teleost fish Heteropneustes fossilis has been shown to have a functional urea cycle and to be able to switch from ammoniotelic to Ureotelic nitrogen metabolism when exposed to high levels of ammonia or air. The objective of this study was to identify the type of carbamyl phosphate synthetase (CPS) catalyzing the first step of the urea cycle in H. fossilis . Mitochondrial CPS III [glutamine- and N-acetyl-L-glutamate (NAG)-dependent] and cytosolic CPS II (glutamine-dependent) activities were found to be present in liver, analogous to that described for two other teleosts that have CPS III activity. The same activities and subcellar localization were found in kidney. Unexpectedly, a CPS I-like activity (ammonia- and NAG-dependent) was found to be present at levels higher than the CPS III activity in the mitochondrial fraction of both liver and kidney. The urea cycle-related CPS III found in invertebrates and fish is considered to be the evolutionary precursor of the urea cycle-related CPS I in Ureotelic mammalian and amphibian species. Whether or not this CPS I-like activity 1) is due to the presence of a separate CPS I gene in addition to a CPS III gene or 2) represents an adapted CPS III activity in H. fossilis , these results suggest that the presence of both CPS I-like and CPS III activities may play an important physiological adaptive role in the tolerance of these fish to high concentrations of external ammonia.
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3 Urea Cycle in Fish: Molecular and Mitochondrial Studies
Fish Physiology, 1995Co-Authors: Paul M. AndersonAbstract:Publisher Summary This chapter discusses the molecular and mitochondrial studies of the urea cycle in fish. A significant proportion of energy production in fish involves the catabolism and oxidation of proteins and amino acids. Consistent with their water habitat, the major end product of nitrogen metabolism in most fish is ammonia. Carbamoyl phosphate is the precursor for two major metabolic pathways: the urea cycle and pyrimidine nucleotide biosynthesis. The first step of the urea cycle in mammalian and amphibian Ureotelic species is catalyzed by carbamoyl-phosphate synthetase I. The function of CPSase II is to catalyze carbamoyl phosphate formation as the first step in pyrimidine nucleotide biosynthesis. The utilization of the amide group of glutamine for the biosynthesis of carbamoyl phosphate in the glutamine-dependent CPSases involves the reaction of glutamine with a cysteine SH group on the enzyme to form a γ-glutamyl thioester intermediate, releasing ammonia, which reacts with an activated intermediate common to all CPSase. The unique co-functioning of glutamine synthetase and CPSase III in ammonia assimilation in the mitochondrial matrix probably reflects the adaptation of urea synthesis for the dual role of ureoosmotic and Ureotelic functions.
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Glutamine-dependent urea synthesis in elasmobranch fishes.
Biochemistry and cell biology = Biochimie et biologie cellulaire, 1991Co-Authors: Paul M. AndersonAbstract:Marine elasmobranchs (sharks, skates, and rays) synthesize and retain in their tissues high concentrations of urea (up to 0.4 M) and certain amines, such as trimethylamine oxide (up to 0.2 M), for the purpose of osmoregulation. Although the pathway of urea synthesis in ureosmotic elasmobranchs is similar to the classical urea cycle in Ureotelic species, there are several properties that are unique. Indeed, this might be anticipated, since the primary function of urea synthesis in elasmobranchs is osmoregulation, rather than ammonia detoxification. The existence in different species of a major biosynthetic pathway resulting in the same end product, but which serves quite different physiological purposes in each species, can provide an opportunity for comparative investigations that contribute to our understanding of the regulatory properties and evolutionary development of the pathway. The purpose of this commentary is to consider the unique properties of urea synthesis in elasmobranchs and to comment on the comparative relationships to teleost fishes and mammalian species; most studies discussed here have been carried out with spiny dogfish shark, Squalus acanthias, a representative elasmobranch. Like Ureotelic species, hepatic urea synthesis in ureosmotic elasmobranchs requires both mitochondrial and cytosolic enzymes, but there are notable differences (Fig. 1). The most significant difference is that the initial step of ammonia assimilation for urea synthesis in hepatic mitochondria of elasmobranchs is the formation of glutamine, which is subsequently utilized as the substrate for carbamoyl-phosphate synthesis (Anderson and Casey, J. Biol. Chem., 259, 456-462, 1984). This is due to the presence of high levels of glutamine synthetase (GSase) and a unique acetylglutamateand glutamine-dependent carbamoyl-phosphate synthetase (CPSase 111), both localized exclusively in the mitochondrial matrix (Casey and Anderson, J. Biol. Chem., 257,8449-8453, 1982, and Comp. Biochem. Physiol., 82B, 307-315,1985). GSase is a cytosolic enzyme in liver of mammalian species and in some, but not all, teleost fishes. In mammalian and amphibian species the first step of ammonia assimilation for urea synthesis in liver is direct conversion of ammonia to carbamoyl phosphate catalyzed by a corresponding mitochondrial acetylglutamateand ammoniadependent CPSase I (which cannot utilize glutamine as a substrate). The properties of elasmobranch mitochondrial CPSase I11 are similar to the mammalian mitochondrial CPSase I, except for the fact that glutamine rather than ammonia serves as the nitrogen-donating substrate (Anderson, J. Biol. Chem., 256, 12 228 12 238, 1981).
C. Michele Nawata - One of the best experts on this subject based on the ideXlab platform.
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physiological and molecular responses of the spiny dogfish shark squalus acanthias to high environmental ammonia scavenging for nitrogen
The Journal of Experimental Biology, 2015Co-Authors: C. Michele Nawata, Patrick J Walsh, Chris M WoodAbstract:In teleosts, a branchial metabolon links ammonia excretion to Na+ uptake via Rh glycoproteins and other transporters. Ureotelic elasmobranchs are thought to have low branchial ammonia permeability, and little is known about Rh function in this ancient group. We cloned Rh cDNAs (Rhag, Rhbg and Rhp2) and evaluated gill ammonia handling in Squalus acanthias. Control ammonia excretion was
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Rh proteins and NH4(+)-activated Na+-ATPase in the Magadi tilapia (Alcolapia grahami), a 100% Ureotelic teleost fish.
The Journal of experimental biology, 2013Co-Authors: Chris M Wood, C. Michele Nawata, Pierre Laurent, Claudine Chevalier, Harold L. Bergman, Jonathan M Wilson, Adalto Bianchini, John N Maina, Ora E Johannsson, Lucas F BianchiniAbstract:The small cichlid fish Alcolapia grahami lives in Lake Magadi, Kenya, one of the most extreme aquatic environments on Earth (pH ~10, carbonate alkalinity ~300 mequiv l(-1)). The Magadi tilapia is the only 100% Ureotelic teleost; it normally excretes no ammonia. This is interpreted as an evolutionary adaptation to overcome the near impossibility of sustaining an NH3 diffusion gradient across the gills against the high external pH. In standard ammoniotelic teleosts, branchial ammonia excretion is facilitated by Rh glycoproteins, and cortisol plays a role in upregulating these carriers, together with other components of a transport metabolon, so as to actively excrete ammonia during high environmental ammonia (HEA) exposure. In Magadi tilapia, we show that at least three Rh proteins (Rhag, Rhbg and Rhcg2) are expressed at the mRNA level in various tissues, and are recognized in the gills by specific antibodies. During HEA exposure, plasma ammonia levels and urea excretion rates increase markedly, and mRNA expression for the branchial urea transporter mtUT is elevated. Plasma cortisol increases and branchial mRNAs for Rhbg, Rhcg2 and Na(+),K(+)-ATPase are all upregulated. Enzymatic activity of the latter is activated preferentially by NH4(+) (versus K(+)), suggesting it can function as an NH4(+)-transporter. Model calculations suggest that active ammonia excretion against the gradient may become possible through a combination of Rh protein and NH4(+)-activated Na(+)-ATPase function.
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Rh proteins and NH4+-activated Na+-ATPase in the Magadi tilapia (Alcolapia grahami), a 100% Ureotelic teleost fish
Journal of Experimental Biology, 2013Co-Authors: Chris M Wood, C. Michele Nawata, Pierre Laurent, Claudine Chevalier, Harold L. Bergman, Jonathan M Wilson, Adalto Bianchini, John N Maina, Ora E Johannsson, Lucas F BianchiniAbstract:SUMMARY The small cichlid fish Alcolapia grahami lives in Lake Magadi, Kenya, one of the most extreme aquatic environments on Earth (pH ~10, carbonate alkalinity ~300 mequiv l −1 ). The Magadi tilapia is the only 100% Ureotelic teleost; it normally excretes no ammonia. This is interpreted as an evolutionary adaptation to overcome the near impossibility of sustaining an NH 3 diffusion gradient across the gills against the high external pH. In standard ammoniotelic teleosts, branchial ammonia excretion is facilitated by Rh glycoproteins, and cortisol plays a role in upregulating these carriers, together with other components of a transport metabolon, so as to actively excrete ammonia during high environmental ammonia (HEA) exposure. In Magadi tilapia, we show that at least three Rh proteins ( Rhag , Rhbg and Rhcg2 ) are expressed at the mRNA level in various tissues, and are recognized in the gills by specific antibodies. During HEA exposure, plasma ammonia levels and urea excretion rates increase markedly, and mRNA expression for the branchial urea transporter mtUT is elevated. Plasma cortisol increases and branchial mRNAs for Rhbg , Rhcg2 and Na + ,K + -ATPase are all upregulated. Enzymatic activity of the latter is activated preferentially by NH 4 + ( versus K + ), suggesting it can function as an NH 4 + -transporter. Model calculations suggest that active ammonia excretion against the gradient may become possible through a combination of Rh protein and NH 4 + -activated Na + -ATPase function.
B.k. Ratha - One of the best experts on this subject based on the ideXlab platform.
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unique hepatic cytosolic arginase evolved independently in ureogenic freshwater air breathing teleost heteropneustes fossilis
PLOS ONE, 2013Co-Authors: Shilpee Srivastava, B.k. RathaAbstract:Hepatic cytosolic arginase (ARG I), an enzyme of the urea cycle operating in the liver of Ureotelic animals, is reported to be present in an ammoniotelic freshwater air-breathing teleost, Heteropneustes fossilis which has ureogenic potential. Antibodies available against mammalian ARG I showed no cross reactivity with the H. fossilis ARG I. We purified unique ARG I from H. fossilis liver. Purified ARG I is a homotrimer with molecular mass 75 kDa and subunit molecular mass of 24 kDa. The pI value of the enzyme was 8.5. It showed maximum activity at pH 10.5 and 55°C. The Km of purified enzyme for L-arginine was 2.65±0.39 mM. L-ornithine and Nω-hydroxy-L-arginine showed inhibition of the ARG I activity, with Ki values 0.52±0.02mM and 0.08±0.006mM, respectively. Antibody raised against the purified fish liver ARG I showed exclusive specificity, and has no cross reactivity against fish liver ARG II and mammalian liver ARG I and ARG II. We found another isoform of arginase bound to the outer membrane of the mitochondria which was released by 150–200 mM KCl in the extraction medium. This isoform was immunologically different from the soluble cytosolic and mitochondrial arginase. The results of present study support that hepatic cytosolic arginase evolved in this ureogenic freshwater teleost, H. fossilis. Phylogenetic analysis confirms an independent evolution event that occurred much after the evolution of the cytosolic arginase of Ureotelic vertebrates.
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Unique Hepatic Cytosolic Arginase Evolved Independently in Ureogenic Freshwater Air-Breathing
2013Co-Authors: Teleost Heteropneustes Fossilis, Shilpee Srivastava, B.k. RathaAbstract:Hepatic cytosolic arginase (ARG I), an enzyme of the urea cycle operating in the liver of Ureotelic animals, is reported to be present in an ammoniotelic freshwater air-breathing teleost, Heteropneustes fossilis which has ureogenic potential. Antibodies available against mammalian ARG I showed no cross reactivity with the H. fossilis ARG I. We purified unique ARG I from H. fossilis liver. Purified ARG I is a homotrimer with molecular mass 75 kDa and subunit molecular mass of 24 kDa. The pI value of the enzyme was 8.5. It showed maximum activity at pH 10.5 and 55uC. The Km of purified enzyme for L-arginine was 2.6560.39 mM. L-ornithine and Nv-hydroxy-L-arginine showed inhibition of the ARG I activity, with Ki values 0.5260.02 mM and 0.0860.006 mM, respectively. Antibody raised against the purified fish liver ARG I showed exclusive specificity, and has no cross reactivity against fish liver ARG II and mammalian liver ARG I and ARG II. We found another isoform of arginase bound to the outer membrane of the mitochondria which was released by 150–200 mM KCl in the extraction medium. This isoform was immunologically different from the soluble cytosolic and mitochondrial arginase. The results of present study support that hepatic cytosolic arginase evolved in this ureogenic freshwater teleost, H. fossilis. Phylogenetic analysis confirms an independent evolution event that occurred much after the evolution of the cytosolic arginase of Ureotelic vertebrates
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Does fish represent an intermediate stage in the evolution of Ureotelic cytosolic arginase I
Biochemical and biophysical research communications, 2009Co-Authors: Shilpee Srivastava, B.k. RathaAbstract:Arginase catalyses the last step of the urea cycle. At least two isoenzymes of arginase are known; cytosolic ARG I and mitochondrial ARG II. ARG I is predominantly expressed in liver cytosol, as a part of urea cycle in Ureotelic animals. The second isoform ARG II is primarily responsible for non-ureogenic functions, expressed in mitochondria of both hepatic and non-hepatic tissues in most vertebrates. Most micro-organisms and invertebrates are known to have only one type of arginase, whose function is unrelated to ornithine-urea cycle (OUC). However, in ureo-osmotic marine elasmobranchs arginase is localized in liver mitochondria as a part of OUC to synthesize urea for osmoregulation. An evolutionary transition occurred in arginase enzyme in terrestrial Ureotelic vertebrates, with the evolution of ARG I from a pre-existing ancestral mitochondrial ARG II. This cytosolic ARG I activity is supposed to have first appeared in lung fishes, but the 40% and 60% distribution of arginase I and II activity in liver and kidney tissue of Heteropneustes fossilis indicates reconsideration of the above fact.
