Five tropical air-breathing fishes, six different strategies to defend against ammonia toxicity on land

Most tropical fishes are ammonotelic, producing ammonia and excreting it as NH3 by diffusion across the branchial epithelia. Hence, those air-breathing tropical fishes that survive on land briefly or for an extended period would have difficulties in excreting ammonia when out of water. Ammonia is toxic, but some of these air-breathing fishes adopt special biochemical adaptations to ameliorate the toxicity of endogenous ammonia accumulating in the body. The amphibious mudskipper Periophthalmodon schlosseri, which is very active on land, reduces ammonia production by suppressing amino acid catabolism (strategy 1) during aerial exposure. It can also undergo partial amino acid catabolism, leading to the accumulation of alanine (strategy 2) to support locomotory activities on land. In this case, alanine formation is not an ammonia detoxification process but reduces the production of endogenous ammonia. The snakehead Channa asiatica, which exhibits moderate activities on land although not truly amphibious, accumulates both alanine and glutamine in the muscle, with alanine accounting for 80% of the deficit in reduction in ammonia excretion during air exposure. Unlike P. schlosseri, C. asiatica apparently cannot reduce the rates of protein and amino acid catabolism and is incapable of utilizing partial amino acid catabolism to support locomotory activities on land. Unlike alanine formation, glutamine synthesis (strategy 3) represents an ammonia detoxification mechanism that, in effect, removes the accumulating ammonia. The four-eyed sleeper Bostrichyths sinensis, which remains motionless during aerial exposure, detoxifies endogenous ammonia to glutamine for storage. The slender African lungfish Protopterus dolloi, which can aestivate on land on a mucus cocoon, has an active ornithine-urea cycle and converts endogenous ammonia to urea (strategy 4) for both storage and subsequent excretion. Production of urea and glutamine are energetically expensive and appear to be adopted by fishes that remain relatively inactive on land. The Oriental weatherloach Misgurnus anguillicaudatus, which actively burrows into soft mud during drought, manipulates the pH of the body surface to facilitate NH3 volatilization (strategy 5) and develops high ammonia tolerance at the cellular and subcellular levels (strategy 6) during aerial exposure. Hence, with regard to excretory nitrogen metabolism, modern tropical air-breathing fishes exhibit a variety of strategies to survive on land, and they represent a spectrum of specimens through which we may examine various biochemical adaptations that would have facilitated the invasion of the terrestrial habitat by fishes during evolution.     

Branchial ammonia excretion in the Asian weatherloach Misgurnus anguillicaudatus

The weatherloach, Misgurnus anguillicaudatus, is a freshwater, facultative air-breathing fish that lives in streams and rice paddy fields, where it may experience drought and/or high environmental ammonia (HEA) conditions. The aim of this study was to determine what roles branchial Na⁺/K⁺-ATPase, H⁺-ATPase, and Rhcg have in ammonia tolerance and how the weatherloach copes with ammonia loading conditions. The loach's high ammonia tolerance was confirmed as was evident from its high 96 h LC₅₀ value and high tissue tolerance to ammonia. The weatherloach does not appear to make use of Na⁺/NH₄⁺-ATPase facilitated transport to excrete ammonia when exposed to HEA or to high environmental pH since no changes in activity were observed. Using immunofluorescence microscopy, distinct populations of vacuolar (V)-type H⁺-ATPase and Na⁺/K⁺-ATPase immunoreactive cells were identified in branchial epithelia, with apical and basolateral staining patterns, respectively. Rhesus C glycoprotein (Rhcg1), an ammonia transport protein, immunoreactivity was also found in a similar pattern as H⁺-ATPase. Rhcg1 (Slc42a3) mRNA expression also increased significantly during aerial exposure, although not significantly under ammonia loading conditions. The colocalization of H⁺-ATPase and Rhcg1 to the similar non-Na⁺/K⁺-ATPase immunoreactive cell type would support a role for H⁺-ATPase in ammonia excretion via Rhcg by NH₄⁺ trapping. The importance of gill boundary layer acidification in net ammonia excretion was confirmed in this fish; however, it was not associated with an increase in H⁺-ATPase expression, since tissue activity and protein levels did not increase with high environmental pH and/or HEA. However the V-ATPase inhibitor, bafilomycin, did decrease net ammonia flux whereas other ion transport inhibitors (amiloride, SITS) had no effect. H⁺-ATPase inhibition also resulted in a consequent elevation in plasma ammonia levels and a decrease in the net acid flux. In gill, aerial exposure was also associated with a significant increase in membrane fluidity (or increase in permeability) which would presumably enhance NH₃ permeation through the plasma membrane. Taken together, these results indicate the gill of the weatherloach is responsive to aerial conditions that would aid ammonia excretion.     

African sharptooth catfish Clarias gariepinus does not detoxify ammonia to urea or amino acids but actively excretes ammonia during exposure to environmental ammonia

The African sharptooth catfish Clarias gariepinus lives in freshwater, is an obligatory air breather, and exhibits high tolerance of environmental ammonia. This study aimed at elucidating the strategies adopted by C. gariepinus to defend against ammonia toxicity during ammonia exposure. No carbamoyl phosphate synthetase (CPS) I or III activities were detected in the liver or muscle of the adult C. gariepinus. In addition, activities of other ornithine-urea cycle (OUC) enzymes, especially ornithine transcarbamylase, were low in the liver, indicating that adult C. gariepinus does not have a "functional" hepatic OUC. After being exposed to 50 or 100 mM NH4Cl for 5 d, there was no induction of hepatic OUC enzymes and no accumulation of urea in tissues of the experimental animals. In addition, the rate of urea excretion remained low and unchanged. Hence, ammonia exposure did not induce ureogenesis or ureotely in C. gariepinus as suggested elsewhere for another obligatory air-breathing catfish of the same genus, Clarias batrachus, from India. Surprisingly, the local C. batrachus did not possess any detectable CPS I or III activities in the liver or muscle as had been reported for the Indian counterpart. There were no changes in levels of alanine in the muscle, liver, and plasma of C. gariepinus exposed to 50 or 100 mM NH4Cl for 5 d; neither were there any changes in the glutamine levels in these tissues. Yet even after being exposed to 100 mM NH4Cl for 5 d, there was no significant increase in the level of ammonia in the muscle, which constitutes the bulk of the specimen. In addition, the level of ammonia accumulated in the plasma was relatively low compared to other tropical air-breathing fishes. More importantly, for all NH4Cl concentrations tested (10, 50, or 100 mM), the plasma ammonia level was maintained relatively constant (2.2-2.4 mM). These results suggest that C. gariepinus was able to excrete endogenous ammonia and infiltrated exogenous ammonia against a very steep ammonia gradient. When exposed to freshwater (pH 7.0) with or without 10 mM NH4Cl, C. gariepinus was able to excrete ammonia continuously to the external medium for at least 72 h. This was achieved while the plasma NH4+ and NH3 concentrations were significantly lower than those of the external medium. Diffusion trapping of NH3 through boundary layer acidification can be eliminated as the pH of the external medium became more alkaline instead. These results represent the first report on a freshwater fish (C. gariepinus) adopting active excretion of ammonia (probably NH4+) as a major strategy to defend against ammonia toxicity when exposed to environmental ammonia.     

The effect of temperature on post-feeding ammonia excretion and oxygen consumption in the southern catfish

The post-prandial rates of ammonia excretion (TAN) and oxygen consumption in the southern catfish (Silurus meridionalis) were assessed at 2 h intervals post-feeding until the rates returned to those of the fasting rates, at 17.5, 22.5, 27.5, and 32.5°C, respectively. Both fasting TAN and increased with temperature, and were lower than those previously reported for many fish species. The relationship between fasting TAN (mmol NH₃–N kg⁻¹ h⁻¹) and temperature (T, °C) was described as: fasting TAN = 0.144e ^0.0266T (r = 0.526, n = 27, P < 0.05). The magnitude of ammonia excretion and its ratio to total N intake during the specific dynamic action (SDA) tended to increase initially, and then decrease with increasing temperature. The ammonia quotient (AQ), calculated as mol NH₃–N/mol O₂, following feeding decreased as temperature increased. The relationship between AQ during SDA and temperature was described as: AQ_{during SDA} = 0.303e ^−0.0143T (r = 0.739, n = 21, P < 0.05). Our results suggest that ammonia excretion and oxygen consumption post-feeding are operating independently of each other. Furthermore, it appears that the importance of protein as a metabolic substrate in postprandial fish decreases with temperature.     

Role of ureogenesis in tackling problems of ammonia toxicity during exposure to higher ambient ammonia in the air-breathing walking catfish<Emphasis Type="Italic">clarias batrachus</Emphasis>

In the present study, the possible role of ureogenesis to avoid the accumulation of toxic ammonia to a lethal level under hyper-ammonia stress was tested in the air-breathing walking catfishClarias batrachus by exposing the fish at 25 mM NH₄Cl for 7 days. Excretion of ammonia by the NH₄Cl-exposed fish was totally suppressed, which was accompanied by significant accumulation of ammonia in different body tissues. The walking catfish, which is otherwise predominantly ammoniotelic, turned totally towards ureotelism from ammoniotelism with a 5-to 6-fold increase of urea-N excretion during exposure to higher ambient ammonia. Stimulation of ureogenesis was accompanied with significant increase of some of the key urea cycle enzymes such as carbamyl phosphate synthetase (urea cycle-related), argininosuccinate synthetase and argininosuccinate lyase both in hepatic and non-hepatic tissues. Due to this unique physiological strategy of turning towards ureotelism from ammoniotelism via the induced urea cycle, this air-breathing catfish is able to survive in very high ambient ammonia, which they face in certain seasons of the year in the natural habitat.     

氨氮对鱼类毒性的影响因子及气呼吸型鱼类耐氨策略

氨氮广泛存在于养殖水体中,在氨氮过高的养殖环境下可能会导致鱼类的大量死亡。从生态、环境及养殖效益角度来看,研究氨氮对鱼类的毒性以及鱼类应对环境或体内高氨浓度的策略均具有重要意义。某些鱼类具有其特殊的策略来降低氨毒性,使得这些种类能适应极高的环境或体内氨浓度。这些耐氨策略主要为(1)合成谷氨酰胺、(2)合成尿素排出、(3)增强机体排泄、(4)Rh蛋白促进氨解毒、(5)降低周围环境pH、(6)NH_3挥发和体表碱化、(7)降低体内氨生成、(8)特定氨基酸代谢生成丙氨酸、(9)组织高氨耐受性。鱼类的氨耐受策略较多而且是可变的,主要受特定种类的生活习性和栖息环境影响。文章综述了氨氮对鱼类的毒性机理以及鱼类的应对策略,为相关的研究提供基础资料。     

Functional relationship between ammonia and gangliosides in brain

The functional significance of ammonia production in brain under physiological or pathological conditions is not clearly known. NH₄ ⁺ stimulates Na⁺, K⁺ activated ATPase causing stabilization of neuronal membranes of which gangliosides are major structural components. Moreover ammonia is known to inhibit lysosomal enzymes which include enzymes degrading gangliosides. Gangliosides have been shown to stimulate neuritogenesis in neuronal cultures and prevent the damage of the neurons from glutamate toxicity particularly in areas of brain ischemia. Hyperammonemia without any behavioural changes was induced in experimental rats by intraperitoneal administration of either a single dose (0.8 mmol/100 g wt.) or by six hourly doses (0.6 mmol/100 g wt.) of ammonium acetate. An increase in the content of gangliosides along with a rise in the content of GD_1A and GD_1B without any change in -galactosidase and N-acetylhexosaminidase was observed in cerebral cortex, cerebellum, and brain stem, following the administration of single dose of ammonium acetate. Gangliosides, after extraction from the different brain regions, were estimated by the thiobarbituric acid method and expressed in terms of sialic acid. Individual gangliosides were separated and estimated by thin layer chromatography using resorcinol as the staining agent. These results suggest that ammonia production in the neuronal pathways in brain either as a result of repeated stimulation under physiological conditions or as a result of focal ischemia or injury, may likewise cause an increase in the content of gangliosides which may help in neuritic growth (physiological conditions facilitating synaptic plasticity) and may exert a protective effect on the neurons in the ischemic area against glutamate toxicity.Former Professor of Biochemistry, OMC, Hyderabad.     

Ammonia in estuaries and effects on fish

This review aims to explore the biological responses of fish in estuaries to increased levels of environmental ammonia. Results from laboratory and field studies on responses of fish to varying salinity and their responses increased ammonia will be evaluated, although studies which examine responses to ammonia, in relation to varying salinity, pH and temperature together are rare. In a survey of British estuaries the continuous measurement of total ammonia showed values that ranged from background levels increasing up to c. 10 mg N l^?1 although higher values have been noted sporadically. In outer estuaries pH values tended to stabilize towards sea water values (e.g. c. pH 8). Upper reaches of estuaries are influenced by the quality of their fresh waters sources which can show a wide range of pH and water quality values depending on geological, climatic and pollution conditions. In general the ammonia toxicity (96 h LC₅₀) to marine species (e.g. 0·09–3·35 mg l^?1 NH₃) appears to be roughly similar to freshwater species (e.g. 0·068–2·0 mg l^?1 NH₃). Ammonia toxicity is related to differences between species and pH rather than to the comparatively minor influences of salinity and temperature. In the marine environment the toxicity of ionized ammonia should be considered. The water quality standard for freshwater salmonids of 21 μg l^?1 NH₃–N was considered to be protective for most marine fish and estuarine fish although the influence of cyclical changes in pH, salinity and temperature were not considered. During ammonia exposures, whether chronic or episodic, estuarine fish may be most at risk as larvae or juveniles, at elevated temperatures, if salinity is near the seawater value and if the pH value of the water is decreased. They are also likely to be at risk from ammonia intoxication in waters of low salinity, high pH and high ammonia levels. These conditions are likely to promote ammonia transfer from the environment into the fish, both as ionized and unionized ammonia, as well as promoting ammonia retention by the fish. Fish are more likely to be prone to ammonia toxicity if they are not feeding, are stressed and if they are active and swimming. Episodic or cycling exposures should also be considered in relation to the rate at which the animal is able to accumulate and excrete ammonia and the physiological processes involved in the transfer of ammonia. In the complex environment of an estuary, evaluation of ammonia as a pollutant will involve field and laboratory experiments to determine the responses of fish to ammonia as salinity and temperature vary over a period of time. It will also be necessary to evaluate the responses of a variety of species including estuarine residents and migrants.     

Physiological responses of Pinus sylvestris to atmospheric ammonia

Summary Young saplings of Pinus sylvestris were fumigated for 3 months with ammonia in concentrations ranging from 0 to 240 g m^-3. Despite the much higher concentrations than normal in the field, no visible damage occurred. Photosynthesis, dark respiration, transpiration and biomass production were stimulated. At 240 g m^-3 with high irradiance (PAR: 950 mol m^-2 s^-1), net photosynthesis was stimulated by 24% and dark respiration by 76%. Intitial light use efficiency was not significantly affected. Transpiration increased, both in the dark and at 950 mol m^-2 s^-1 by 40% and 57%, respectively. In the presence of ammonia, stomatal control was less efficient. Though growth of roots was not affected by NH₃, that of current year needles was stimulated, resulting in an increased mass ratio of needles to roots. The nitrogen content of the needles increased, but the contents of other mineral components did not change significantly. Due to increased transpiration per unit of needle area and increased mass of needles per tree, water loss per tree was about twice as high in the treatment with 240 g m^-3 as in the control. Towards the end of fumigation, a 10-day period without water supply followed and then the water potential of the shoots was measured as an indicator of water demand. This demand was higher with higher concentrations of NH₃, suggesting a higher risk of injury from drought.     

Diel variation in ammonia excretion,glutamine levels,and hydration status in two species of terrestrial isopods

Terrestrial isopods (suborder Oniscidea) excrete most nitrogen diurnally as volatile ammonia, and ammonia-loaded animals accumulate nonessential amino acids, which may constitute the major nocturnal nitrogen pool. This study explored the relationship between ammonia excretion, glutamine storage/mobilization, and water balance, in two sympatric species Ligidium lapetum (section Diplocheta), a hygric species; and Armadillidium vulgare (Section Crinocheta), a xeric species capable of water-vapor absorption (WVA). Ammonia excretion (12-h), tissue glutamine levels, and water contents were measured following field collection of animals at dusk and dawn. In both species, diurnal ammonia excretion exceeded nocturnal excretion four- to fivefold while glutamine levels increased four- to sevenfold during the night. Most glutamine was accumulated in the somatic tissues (body wall). While data support the role of glutamine in nocturnal nitrogen storage, potential nitrogen mobilization from glutamine breakdown (162 mol g^–1 in A. vulgare) exceeds measured ammonia excretion (2.5 mol g^–1) over 60-fold. This may serve to generate the high hemolymph ammonia concentrations seen during volatilization. The energetic cost of ammonia volatilization is discussed in the light of these findings. Mean water contents were similar at dusk and dawn in both species, indicating that diel cycles of water depletion and replenishment were not occurring.     

The skin of adult rainbow trout is not a significant site of ammonia clearance from the blood

We hypothesized that the skin acts as an extrabranchial route for ammonia excretion in adult rainbow trout (Oncorhynchus mykiss) following high environmental ammonia (HEA) exposure. Trunks of control or HEA-exposed trout were perfused with saline containing 0 or 1 mmol l⁻¹ NH₄⁺. Cutaneous ammonia excretion rates increased 2.5-fold following HEA exposure, however there was no difference in rates between trunks perfused with 0 or 1 mmol l⁻¹ NH₄⁺. The skin is therefore capable of excreting its own ammonia load, but it does not clear circulating ammonia from the plasma.     

Effect of a low-carbohydrate diet on plasma and sweat ammonia concentrations during prolonged nonexhausting exercise

The purpose of this investigation was to examine the effect of low body glycogen stores on plasma ammonia concentration and sweat ammonia excretion during prolonged, nonexhausting exercise of moderate intensity. On two occasions seven healthy untrained men pedalled on a cycle ergometer for 60 min at 50% of their predetermined maximal O₂ uptakes ( _max) firstly, following 3 days on a normal mixed diet (N-diet) (60% carbohydrates, 25% fat and 15% protein) and secondly, following 3 days on a low-carbohydrate diet (LC-diet) (less than 5% carbohydrates, 50% fat and 45% protein) of equal energy content. Blood was collected from the antecubital vein immediately before, at 30th and at 60th min of exercise. Sweat was collected from the hypogastric region using gauze pads. It was shown that plasma ammonia concentrations after the LC-diet were higher than after the N-diet at both the 30th and 60th min of exercise. Sweat ammonia concentration and total ammonia loss through the sweat were also higher after the LC-diet. The higher ammonia concentrations in plasma and sweat after the LC-diet would seem to indicate an increased ammonia production, which may be related to reduced initial carbohydrate stores.     

Benefits to host sea anemones from ammonia contributions of resident anemonefish

Large ectosymbionts (especially fishes and crustaceans) may have major impacts on the physiology of host cnidarians (sea anemones and corals), but these effects have not been well quantified. Here we describe impacts on giant sea anemone hosts (Entacmaea quadricolor) and their endosymbiotic zooxanthellae (Symbiodinium spp.) from the excretion products of anemonefish guests (Amphiprion bicinctus) under laboratory conditions. Starved host anemones were maintained with anemonefish, ammonia supplements (= NH₃ gas and NH₄⁺ ion), or neither for 2 mo. In the presence of external ammonia supplements or resident anemonefish, the zooxanthellae within host anemones increased in abundance (173% and 139% respectively), and provided the hosts with energy that minimized host body size loss. In contrast, anemones cultured with neither ammonia nor anemonefish harbored significantly lower abundances of zooxanthellae (84% of initial abundance) and decreased > 60% in body size. Although they maintained higher zooxanthella abundances, anemones cultured with either ammonia supplements or resident anemonefish exhibited significantly lower ammonia uptake rates (0.065 ± 0.005 µmol g^- 1 h^- 1, and 0.052 ± 0.018 µmol g^- 1 h^- 1 respectively) than did control anemones (0.119 ± 0.009 µmol g^- 1 h^- 1), indicating that their zooxanthellae were more nitrogen sufficient. We conclude that, in this multi-level mutualism, ammonia supplements provide essentially the same level of physiological contribution to host anemones and zooxanthellae as do live resident fish. This nutrient supplement reduces the dependence of the zooxanthellae on host feeding, and allows them to provide abundant photosynthetically-produced energy to the host.     

Evidence from knockout mice against physiologically significant aquaporin 8-facilitated ammonia transport

Aquaporin (AQP)8-facilitated transport of NH₃ has been suggested recently by increased NH₃ permeability in Xenopus oocytes and yeast expressing human or rat AQP8. We tested the proposed roles of AQP8-facilitated NH₃ transport in mammalian physiology by comparative phenotype studies in wild-type vs. AQP8-null mice. AQP8-facilitated NH₃ transport was confirmed in mammalian cell cultures expressing rat or mouse AQP8, in which the fluorescence of a pH-sensing yellow fluorescent protein was measured in response to ammonia (NH₃/NH₄⁺) gradients. Relative AQP8 single-channel NH₃-to-water permeability was 0.03. AQP8-facilitated NH₃ and water permeability in a native tissue was confirmed in membrane vesicles isolated from testes of wild-type vs. AQP8-null mice, in which BCECF was used as an intravesicular pH indicator. A series of in vivo studies were done in mice, including 1) serum ammonia measurements before and after ammonia infusion, 2) renal ammonia clearance, 3) colonic ammonia absorption, and 4) liver ammonia accumulation and renal ammonia excretion after acute and chronic ammonia loading. Except for a small reduction in hepatic ammonia accumulation and increase in ammonia excretion in AQP8-null mice loaded with large amounts of ammonia, there were no significant differences in wild-type vs. AQP8-null mice. Our results support the conclusion that AQP8 can facilitate NH₃ transport but provide evidence against physiologically significant AQP8-facilitated NH₃ transport in mice. water transport; transgenic mouse; liver     

The adaptations of fish to extremely alkaline environments

The Lake Magadi Tilapia (MT; Oreochromis alcalicus grahami, the Lahontan cutthroat trout (LCT; Oncorhynchus clarki henshawi) and the tarek (Ct; Chalcalburnus tarichi) have evolved unique strategies that allow them to overcome problems associated with ammonia excretion (J_Amm) and acid-base regulation in their alkaline environments. In Lake Magadi, Kenya (pH 10), the MT circumvents problems associated with J_Amm by excreting virtually all (>90%) of its waste-nitrogen as urea. Base excretion appears to be facilitated by modified seawater-type gill chloride cells, through apical Cl⁻/HCO₃⁻ exchangers and an outwardly directed OH⁻/HCO₃⁻/CO₃⁼ excretion system. The LCT avoids potentially toxic increases in internal ammonia by permanently lowering ammonia production rates following transfer into alkaline (pH 9.4) Pyramid Lake, Nevada, from its juvenile freshwater (pH 8.4) environment. Greater apical exposure of LCT gill chloride cells, presumably the freshwater variety, probably facilitates base excretion by elevating Cl⁻/HCO₃⁻ exchange capacity. In Lake Van, Turkey (pH 9.8) high ammonia tolerance enables C. tarichi to withstand the high internal ammonia concentrations that it apparently requires for the facilitation of J_Amm. It also excretes unusually high amounts of urea. We conclude that adjustments to nitrogenous waste metabolism and excretion patterns, as well as modifications to gill functional morphology, are necessary adaptations that permit these animals to thrive in environments considered unsuitable for most fishes.     

Cutaneous ion exchange,and renal and extrarenal partitioning of acid and ammonia excretion in the larval tiger salamander,Ambystoma tigrinum,following ingestion of ammonium salts

Summary The skin/gills and the kidneys of aquatic amphibians are potential sites of acid-base regulation. The roles of these organs in acid-base balance were examined in larval Ambystoma tigrinum following gastric infusion of ammonium salts. A single dose of 1.75 mEq NH₄Cl·100 g^-1 produced a mixed acidosis by 1 h after gavage. By 8 h after ingestion, pH and HCO ₃ ^– had increased and PCO₂ had decreased as the animals recovered. A prolonged acidosis was developed in a second group by gavage of an initial dose (1.5 mEq·100 g^-1), followed by periodic maintenance doses (0.25 mEq·100 g^-1) to prolong the disturbance for 8 h. The magnitude of the acidosis during this period was similar to that seen at 1 h after ingestion in the time-course study. A third group of larvae were given NaCl as a control for salt loading, which induced a small but significant respiratory acidosis. Unidirectional fluxes of Na⁺ and Cl^- were examined during these serial ingestions. Salt loading inhibited the influx of the ingested ion. Na⁺ influx increased during the NH₄Cl-induced acidosis. A fourth group of larvae were used to partition acid and ammonia excretion between the skin and the kidneys. These animals were given (NH₄)₂SO₄ to allow re-examination of Cl^- flux rates under non-Cl^--loaded conditions. The ensuing acidosis had a reduced respiratory component and, therefore, pH did not decrease as much. Cl^- influx rates did decrease significantly under these conditions. In both control and acidotic conditions, the majority of the acid efflux was as ammonia and the skin was the primary site of acid excretion. However, both the skin and the kidneys increased total acid excretion, although the efflux across the skin showed a much greater increase. This suggests a primary role for the skin in acid-base regulation in aquatic amphibians.Abbreviations GFR glomerular filtration rate - PO₂ partial pressure of oxygen - PCO₂ partial pressure of carbon dioxide - SITS 4-acetamido-4-isothiocynanatostilbene-2,2-disulfonic acid - TA titratible acidity Present address: Department of Organismal Biology and Anatomy, University of Chicago, 1025 E. 57th St., Chicago, IL 60637, USA     

Physiological and molecular analysis of the interactive effects of feeding and high environmental ammonia on branchial ammonia excretion and Na+ uptake in freshwater rainbow trout

Recently, a “Na⁺/NH₄ ⁺ exchange complex” model has been proposed for ammonia excretion in freshwater fish. The model suggests that ammonia transport occurs via Rhesus (Rh) glycoproteins and is facilitated by gill boundary layer acidification attributable to the hydration of CO₂ and H⁺ efflux by Na⁺/H⁺ exchanger (NHE-2) and H⁺-ATPase. The latter two mechanisms of boundary layer acidification would occur in conjunction with Na⁺ influx (through a Na⁺ channel energized by H⁺-ATPase and directly via NHE-2). Here, we show that natural ammonia loading via feeding increases branchial mRNA expression of Rh genes, NHE-2, and H⁺-ATPase, as well as H⁺-ATPase activity in juvenile trout, similar to previous findings with ammonium salt infusions and high environmental ammonia (HEA) exposure. The associated increase in ammonia excretion occurs in conjunction with a fourfold increase in Na⁺ influx after a meal. When exposed to HEA (1.5 mmol/l NH₄HCO₃ at pH 8.0), both unfed and fed trout showed differential increases in mRNA expression of Rhcg2, NHE-2, and H⁺-ATPase, but H⁺-ATPase activity remained at control levels. Unfed fish exposed to HEA displayed a characteristic reversal of ammonia excretion, initially uptaking ammonia, whereas fed fish (4 h after the meal) did not show this reversal, being able to immediately excrete ammonia against the gradient imposed by HEA. Exposure to HEA also led to a depression of Na⁺ influx, demonstrating that ammonia excretion can be uncoupled from Na⁺ influx. We suggest that the efflux of H⁺, rather than Na⁺ influx itself, is critical to the facilitation of ammonia excretion.     

Influence of sodium bicarbonate ingestion on plasma ammonia accumulation during incremental exercise in man

This investigation evaluated the influence of metabolic alkalosis on plasma ammonia (NH₃) accumulation during incremental exercise. On two occasions separated by at least 6 days, six healthy men cycled at 70, 80, and 90%g of maximum oxygen consumption ( ) for 5 min; each exercise period was followed by 5 min of seated recovery. Exercise was then performed at 100% until exhaustion. Beginning 3 h prior to exercise, subjects ingested 3.6 mmol · kg body mass^– NaHCO₃ (test, T) or 3.0 mmol · kg body mass^–1 CaCO₃ (placebo, P) (both equivalent to 0.3 g · kg^–1) over a 2-h period. Trials were performed after an overnight fast and the order of treatments was randomized. Arterialized venous blood samples for the determination of acid-base status, blood lactate and plasma NH₃ concentrations were obtained at rest before treatment, 15 s prior to each exercise bout (Pre 70%, Pre 80%, Pre 90%, and Pre 100%), and at 0, 5 (5Post), and 10 (10'Post) min after exhaustion. Additional samples for blood lactate and plasma NH₃ determination were obtained immediately after each exercise bout (Post 70%, Post 80%, Post 90%) and at 15 min after exercise (15Post). Time to exhaustion at 100% of was not significantly different between treatments [mean (SE): 173 (42) s and 184 (44) s for T and P respectively]. A significant treatment effect was observed for plasma pH with values being significantly higher on T than on P Pre 70% [7.461 (0.007) vs 7.398 (0.008)], Pre 90% [7.410 (0.010) vs 7.340 (0.016)], and 10'Post [7.317 (0.032) vs 7.242 (0.036)]. The change in plasma pH was significantly greater following the 90%g bout (Pre 100% Pre 90%) for T [–0.09 (0.02)] than for P [–0.06 (0.01)]. Blood base excess and plasma bicarbonate concentrations were significantly higher for T than P before each exercise bout but not at the point of exhaustion. During recovery, base excess was higher for T than P at 5Post and 10Post while the bicarbonate concentration was higher for T than P at 10Post. A significant treatment effect was observed for the blood lactate concentration with T on the average being higher than P [7.0 (1.0) and 6.3 (1.1) mmol · l^–1 for T and P averaged across the 12 sampling times]. Plasma NH₃ accumulation was not different between treatments at any point in time. In addition, no differences were observed between treatments in blood alanine accumulation. The results suggest that under the conditions of the present investigation metabolic alkalosis does not influence plasma NH₃ accumulation or endurance capacity during intense incremental exercise.     

Low assimilation efficiency of photorespiratory ammonia in conifer leaves

Glutamine synthetase (GS) localized in the chloroplasts, GS2, is a key enzyme in the assimilation of ammonia (NH₃) produced from the photorespiration pathway in angiosperms, but it is absent from some coniferous species belonging to Pinaceae such as Pinus. We examined whether the absence of GS2 is common in conifers (Pinidae) and also addressed the question of whether assimilation efficiency of photorespiratory NH₃ differs between conifers that may potentially lack GS2 and angiosperms. Search of the expressed sequence tag database of Cryptomeria japonica, a conifer in Cupressaceae, and immunoblotting analyses of leaf GS proteins of 13 species from all family members in Pinidae revealed that all tested conifers exhibited only GS1 isoforms. We compared leaf NH₃ compensation point (γ_NH3) and the increments in leaf ammonium content per unit photorespiratory activity (NH₃ leakiness), i.e. inverse measures of the assimilation efficiency, between conifers (C. japonica and Pinus densiflora) and angiosperms (Phaseolus vulgaris and two Populus species). Both γ_NH3 and NH₃ leakiness were higher in the two conifers than in the three angiosperms tested. Thus, we concluded that the absence of GS2 is common in conifers, and assimilation efficiency of photorespiratory NH₃ is intrinsically lower in conifer leaves than in angiosperm leaves. These results imply that acquisition of GS2 in land plants is an adaptive mechanism for efficient NH₃ assimilation under photorespiratory environments.