Patricia A. Wright

A new paradigm for ammonia excretion in aquatic animals: Role of rhesus (RH) glycoproteins

SUMMARY Ammonia excretion at the gills of fish has been studied for 80 years, but the mechanism(s) involved remain controversial. The relatively recent discovery of the ammonia-transporting function of the Rhesus (Rh) proteins, a family related to the Mep/Amt family of methyl ammonia and ammonia transporters in bacteria, yeast and plants, and the occurrence of these genes and glycosylated proteins in fish gills has opened a new paradigm. We provide background on the evolution and function of the Rh proteins, and review recent studies employing molecular physiology which demonstrate their important contribution to branchial ammonia efflux. Rhag occurs in red blood cells, whereas several isoforms of both Rhbg and Rhcg occur in many tissues. In the branchial epithelium, Rhcg appears to be localized in apical membranes and Rhbg in basolateral membranes. Their gene expression is upregulated during exposure to high environmental ammonia or internal ammonia infusion, and may be sensitive to synergistic stimulation by ammonia and cortisol. Rhcg in particular appears to be coupled to H+ excretion and Na+ uptake mechanisms. We propose a new model for ammonia excretion in freshwater fish and its variable linkage to Na+ uptake and acid excretion. In this model, Rhag facilitates NH3 flux out of the erythrocyte, Rhbg moves it across the basolateral membrane of the branchial ionocyte, and an apical “Na+/NH +4 exchange complex” consisting of several membrane transporters (Rhcg, V-type H+-ATPase, Na+/H+ exchanger NHE-2 and/or NHE-3, Na+ channel) working together as a metabolon provides an acid trapping mechanism for apical excretion. Intracellular carbonic anhydrase (CA-2) and basolateral Na+/HCO –3 cotransporter (NBC-1) and Na+/K+-ATPase play indirect roles. These mechanisms are normally superimposed on a substantial outward movement of NH3 by simple diffusion, which is probably dependent on acid trapping in boundary layer water by H+ ions created by the catalysed or non-catalysed hydration of expired metabolic CO2. Profitable areas for future investigation of Rh proteins in fish are highlighted: their involvement in the mechanism of ammonia excretion across the gills in seawater fish, their possible importance in ammonia excretion across the skin, their potential dual role as CO2 transporters, their responses to feeding, and their roles in early life stages prior to the full development of gills.

Rhesus glycoprotein gene expression in the mangrove killifish Kryptolebias marmoratus exposed to elevated environmental ammonia levels and air

SUMMARY The mechanism(s) of ammonia excretion in the presence of elevated external ammonia are not well understood in fish. Recent studies in other organisms have revealed a new class of ammonia transporters, Rhesus glycoprotein genes (Rh genes), which may also play a role in ammonia excretion in fish. The first objective of this study was to clone and characterize Rh genes in a fish species with a relatively high tolerance to environmental ammonia, the mangrove killifish Kryptolebias marmoratus (formerly Rivulus marmoratus). We obtained full-length cDNAs of three Rh genes in K. marmoratus: RhBG (1736 bp), RhCG1 (1920 bp) and RhCG2 (2021 bp), which are highly homologous with other known Rh gene sequences. Hydropathy analysis revealed that all three Rh genes encode membrane proteins with 10–12 predicted transmembrane domains. RhBG, RhCG1 and RhCG2 are highly expressed in gill tissue, with RhBG also present in skin of K. marmoratus. Exposure to elevated environmental ammonia (2 mmol l–1 NH4HCO3) for 5 days resulted in a modest (+37%) increase in whole-body ammonia levels, whereas gill RhCG2 and skin RhCG1 mRNA levels were upregulated by 5.8- and 7.7-fold, respectively. RhBG mRNA levels were also increased in various tissues, with 3- to 7-fold increases in the liver and skeletal muscle. In a separate group of killifish exposed to air for 24 h, RhCG1 and RhCG2 mRNA levels were elevated by 4- to 6-fold in the skin. Thus, the multifold induction of Rh mRNA levels in excretory tissues (gills and skin) and internal tissues in response to conditions that perturb normal ammonia excretion suggests that RhBG, RhCG1 and RhCG2 may be involved in facilitating ammonia transport in this species. Furthermore, the findings support earlier studies demonstrating that the skin is an important site of ammonia excretion in K. marmoratus.

Molecular characterization of an elasmobranch urea transporter

Marine elasmobranch fishes retain relatively high levels of urea to balance the osmotic stress of living in seawater. To maintain osmotic balance and reduce the energetic costs of making urea, it is important for these animals to minimize urea excretion to the environment. We have isolated a novel 2.2-kb cDNA from Squalus acanthias (spiny dogfish shark) kidney encoding a 380-amino acid hydrophobic protein (ShUT) with 66% identity to the rat facilitated urea transporter protein UT-A2. Injection of ShUT cRNA into Xenopusoocytes induced a 10-fold increase in14C-labeled urea uptake, inhibitable by phloretin (0.35 mM). ShUT mRNA is expressed in kidney and brain. Related mRNA species are found in liver, blood, kidney, gill, intestine, muscle, and rectal gland. This is the first facilitated urea transporter to be identified in a marine fish. We propose that the ShUT protein is involved in urea reabsorption by the renal tubules of the dogfish shark, which in turn minimizes urea loss in the urine.

Urea and Water Permeability in Dogfish (Squalus acanthias) Gills

We used a perfused gill preparation from dogfish to investigate the origin of low branchial permeability to urea. Urea permeability (14C-urea) was measured simultaneously with diffusional water permeability (3H2O). Permeability coefficients for urea and ammonia in the perfused preparation were almost identical to in vivo values. The permeability coefficient of urea was 0.032 x 10(-6) cm/sec and of 3H2O 6.55 x 10(-6) cm/sec. Adrenalin (1 x 10(-6) M) increased water and ammonia effluxes by a factor of 1.5 and urea efflux by a factor of 3.1. Urea efflux was almost independent of the urea concentration in the perfusion medium. The urea analogue thiourea in the perfusate had no effect on urea efflux, whereas the non-competitive inhibitor of urea transport, phloretin, increased efflux markedly. The basolateral membrane is approximately 14 times more permeable to urea than the apical membrane. We conclude that the dogfish apical membrane is extremely tight to urea, but the low apparent branchial permeability may also relate to the presence of an active urea transporter on the basolateral membrane that returns urea to the blood and hence reduces the apical urea gradient.

Expression and Activity of Carbamoyl Phosphate Synthetase III and Ornithine Urea Cycle Enzymes in Various Tissues of Four Fish Species

Abstract Previous studies have reported low activities of carbamoyl phosphate synthetase (CPSase) and other ornithine urea cycle enzymes in the liver of many teleost fishes, but it was not established if the CPSase was the urea cycle-related CPSase III or pyrimidine pathway-related CPSase II. The purpose of this study was to investigate the expression of the urea cycle-related CPSase III and other ornithine urea cycle enzymes in the liver, muscle, intestine, and kidney of three adult teleost fish (common carp, Cyprinus carpio ; goldfish, Carassius auratus ; channel catfish, Ictalurus punctatus ) and a holostean fish (bowfin, Amia calva ). In contrast to previous literature reports, CPSase III activity was absent from the liver of all fish studied. Surprisingly, CPSase III was present in the muscle of the common carp and bowfin and in the intestine of bowfin. Variable levels of the remaining urea cycle enzymes, as well as the pyrimidine nucleotide pathway-related CPSase II were expressed by all tissues and fish examined. We conclude that hepatic CPSase III expression may be relatively rare in teleost and holostean fishes, but expression in extrahepatic tissues, particularly muscle, may be more typical.

Expression of Carbamoyl-phosphate Synthetase III mRNA during the Early Stages of Development and in Muscle of Adult Rainbow Trout (Oncorhynchus mykiss)

It has been reported that the activities of the urea cycle-related enzymes ornithine carbamoyltransferase and carbamoyl-phosphate synthetase III (CPSase III) are induced during early life stages of ammonotelic rainbow trout (Oncorhynchus mykiss), suggesting that the urea cycle may play a physiological role in early development in teleost fish (Wright, P. A., Felskie, A., and Anderson, P. M. (1995) J. Exp. Biol. 198, 127-135). CPSase III cDNA prepared from embryo mRNA was sequenced, confirming the existence of the CPSase III gene in trout and its expression. The deduced amino acid sequence of the CPSase III is homologous to other CPSases. Supporting evidence for the expression of CPSase III activity in trout embryos was obtained by demonstrating expression of CPSase III mRNA as early as day 3 post-fertilization, reaching a maximum at 10-14 days, declining to a minimum at day 70, and then increasing to a relatively constant level from days 90 to 110 (relative to total RNA). Unexpectedly, in tissues of adult and fingerling trout, CPSase III mRNA was found to be present in muscle but not in other tissues, including liver. This finding was confirmed by assay of extracts, which showed CPSase III and ornithine carbamoyltransferase activity in muscle but not in other tissues. The pyrimidine nucleotide pathway-related CPSase II mRNA was expressed in all tissues.

Seven things fish know about ammonia and we don’t ☆

In this review we pose the following seven questions related to ammonia and fish that represent gaps in our knowledge. 1. How is ammonia excretion linked to sodium uptake in freshwater fish? 2. How much does branchial ammonia excretion in seawater teleosts depend on Rhesus (Rh) glycoprotein-mediated NH(3) diffusion? 3. How do fish maintain ammonia excretion rates if branchial surface area is reduced or compromised? 4. Why does high environmental ammonia change the transepithelial potential across the gills? 5. Does high environmental ammonia increase gill surface area in ammonia tolerant fish but decrease gill surface area in ammonia intolerant fish? 6. How does ammonia contribute to ventilatory control? 7. What do Rh proteins do when they are not transporting ammonia? Mini reviews on each topic, which are able to present only partial answers to each question at present, are followed by further questions and/or suggestions for research approaches targeted to uncover answers.

UREA PRODUCTION AND TRANSPORT IN TELEOST FISHES

Teleosts appear to have retained the genes for the urea cycle enzymes. A few species express the full complement of enzymes and are ureotelic (e.g., Lake Magadi tilapia) or ammoniotelic (e.g., largemouth bass), whereas most species have low or non-detectable enzyme activities in liver tissue and excrete little urea (e.g., adult rainbow trout). It was surprising, therefore, to find the expression of four urea cycle enzymes during early life stages of rainbow trout. The urea cycle may play a role in ammonia detoxification during a critical time of development. Exposure to alkaline water (pH 9.0-9.5) or NH4Cl (0.2 mmol/l) increased urea excretion by several-fold in trout embryos, free embryos and alevin. Urea transport is either by passive simple diffusion or via carried-mediated transport proteins. Molecular studies have revealed that a specialised urea transport protein is present in kidney tissue of elasmobranchs, similar to the facilitated urea transporter found in the mammalian inner medulla of the kidney.

Nitrogen excretion and enzyme pathways for ureagenesis in freshwater tilapia (Oreochromis niloticus)

The tilapia of Lake Magadi (pH 10), Oreochromis alcalicus grahami, excrete all nitrogen wastes as urea and possess the ornithine urea cycle (OUC), unlike the majority of teleost fish. It was proposed that liver OUC enzymes could be induced in the freshwater ammoniotelic tilapia Oreochromis niloticus in response to environmental or dietary manipulations that stimulate urea excretion in other animals. Elevated water ammonia (1 mM NH₄Cl) inhibited ammonia excretion over the first 12 h and increased urea excretion rates three- to fivefold over 7 d. The OUC was not functional in control fish, as carbamoyl phosphate synthetase III activity was not detectable and ornithine carbamoyltransferase activity was relatively low. No changes in OUC enzyme activities were detected in NH₄Cl-exposed fish, but the activity of the uricolytic enzyme allantoicase was twofold higher. Exposure to pH 10 water had only a transient effect on nitrogen excretion, and OUC enzymes and allantoicase activities were unchanged. The rate of urea and ammonia excretion increased with an increase in the dietary protein content, but OUC or uricolytic enzymes were not induced. These results demonstrate that O. niloticus, unlike Lake Magadi tilapia, do not recruit the hepatic OUC, even under environmental or dietary conditions that enhance urea excretion. Rather, uricolysis and argininolysis appear to be the major routes for ureagenesis.

Neuroepithelial cells and the hypoxia emersion response in the amphibious fish Kryptolebias marmoratus

SUMMARY Teleost fish have oxygen-sensitive neuroepithelial cells (NECs) in the gills that appear to mediate physiological responses to hypoxia, but little is known about oxygen sensing in amphibious fish. The mangrove rivulus, Kryptolebias marmoratus, is an amphibious fish that respires via the gills and/or the skin. First, we hypothesized that both the skin and gills are sites of oxygen sensing in K. marmoratus. Serotonin-positive NECs were abundant in both gills and skin, as determined by immunohistochemical labelling and fluorescence microscopy. NECs retained synaptic vesicles and were found near nerve fibres labelled with the neuronal marker zn-12. Skin NECs were 42% larger than those of the gill, as estimated by measurement of projection area, and 45% greater in number. Moreover, for both skin and gill NECs, NEC area increased significantly (30–60%) following 7 days of exposure to hypoxia (1.5 mg l–1 dissolved oxygen). Another population of cells containing vesicular acetylcholine transporter (VAChT) proteins were also observed in the skin and gills. The second hypothesis we tested was that K. marmoratus emerse in order to breathe air cutaneously when challenged with severe aquatic hypoxia, and this response will be modulated by neurochemicals associated chemoreceptor activity. Acute exposure to hypoxia induced fish to emerse at 0.2 mg l–1. When K. marmoratus were pre-exposed to serotonin or acetylcholine, they emersed at a significantly higher concentration of oxygen than untreated fish. Pre-exposure to receptor antagonists (ketanserin and hexamethonium) predictably resulted in fish emersing at a lower concentration of oxygen. Taken together, these results suggest that oxygen sensing occurs at the branchial and/or cutaneous surfaces in K. marmoratus and that serotonin and acetylcholine mediate, in part, the emersion response.