Active ammonia transport and excretory nitrogen metabolism in the climbing perch,<i>Anabas testudineus</i>, during 4 days of emersion or 10 minutes of forced exercise on land

SUMMARY Acute ammonia toxicity in vertebrates is thought to be characterized by a cascade of deleterious events resembling those associated with anoxic/ischemic injury in the central nervous system. A key event is the over-stimulation of neuronal N-methyl-D-aspartate (NMDA) receptors, which leads to excitotoxic cell death. The similarity between the responses to acute ammonia toxicity and anoxia suggests that anoxia-tolerant animals such as the goldfish (Carassius auratus Linnaeus) may also be ammonia tolerant. To test this hypothesis, the responses of goldfish were compared with those of the anoxia-sensitive rainbow trout (Oncorhynchus mykiss Walbaum) during exposure to high external ammonia (HEA). Acute toxicity tests revealed that goldfish are ammonia tolerant, with 96 h median lethal concentration (LC50) values of 199 μmol l–1 and 4132 μmol l–1 for NH3 and total ammonia ([TAmm]=[NH3]+[NH4+]), respectively. These values were ∼5–6 times greater than corresponding NH3 and TAmm LC50 values measured in rainbow trout. Further, the goldfish readily coped with chronic exposure to NH4Cl (3–5 mmol l–1) for 5 days, despite 6-fold increases in plasma [T] to ∼1300 μmol l–1 and 3-fold increases in brain [TAmm] to 6700 μmol l–1. Muscle [TAmm] increased by almost 8-fold from ∼900 μmol kg–1 wet mass (WM) to greater than 7000 μmol kg–1 WM by 48 h, and stabilized. Although urea excretion rates (JUrea) increased by 2–3-fold during HEA, the increases were insufficient to offset the inhibition of ammonia excretion that occurred, and increases in urea were not observed in the brain or muscle. There was a marked increase in brain glutamine concentration at HEA, from ∼3000 μmol kg–1 WM to 15,000 μmol kg–1 WM after 48 h, which is consistent with the hypothesis that glutamine production is associated with ammonia detoxification. Injection of the NMDA receptor antagonists MK801 (0.5–8 mg kg–1) or ethanol (1–8 mg kg–1) increased trout survival time by 1.5–2.0-fold during exposure to 2 mmol l–1 ammonia, suggesting that excitotoxic cell death contributes to ammonia toxicity in this species. In contrast, similar doses of MK801 or ethanol had no effect on ammonia-challenged (8–9.5 mmol l–1TAmm) goldfish survival times, suggesting that greater resistance to excitotoxic cell death contributes to the high ammonia-tolerance of the goldfish. Whole-cell recordings measured in isolated brain slices of goldfish telencephalon during in vitro exposure to 5 mmol l–1 or 10 mmol l–1TAmm reversibly potentiated NMDA receptor currents. This observation suggested that goldfish neurons may not be completely resistant to ammonia-induced excitotoxicity. Subsequent western blot and densitometric analyses revealed that NMDA receptor NR1 subunit abundance was 40–60% lower in goldfish exposed to 3–5 mmol l–1TAmm for 5 days, which was followed by a restoration of NR1 subunit abundance after 3 days recovery in ammonia-free water. We conclude that the goldfish brain may be protected from excitotoxicity by downregulating the abundance of functional NMDA receptors during periods when it experiences increased internal ammonia.

Section: Discussion the Anoxia-tolerant Goldfish Is Ammonia Tolerantmentioning

confidence: 99%

The relationship between NMDA receptor function and the high ammonia tolerance of anoxia-tolerant goldfish

Wilkie

Pamenter

Duquette

et al. 2011

“…A very different model would be required to explain NH 3 excretion through the skin into an alkaline boundary layer in these amphibious fish compared with that presented above for freshwater trout gill. Or is it possible that NH 4 + is actively excreted, as has been argued in other amphibious fish (Randall et al, 1999;Tsui et al, 2002;Tay et al, 2006)? Weihrauch et al (Weihrauch et al, 2009 have presented a hypothetical model of how this might occur in K. marmoratus.…”

Section: The Role Of the Skin In Ammonia Excretionmentioning

confidence: 92%

A new paradigm for ammonia excretion in aquatic animals: role of Rhesus(Rh) glycoproteins

Wright

Wood

2009

339

256

SummaryAmmonia 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 NH 3 flux out of the erythrocyte, Rhbg moves it across the basolateral membrane of the branchial ionocyte, and an apical "Na THE JOURNAL OF EXPERIMENTAL BIOLOGY 2304and helps to explain some of the discrepancies in the earlier literature. Retrospection on past controversiesSince the classic divided chamber experiments of Homer Smith (Smith, 1929), it has been known that freshwater fish excrete their nitrogen waste predominantly as ammonia through the gills. August Krogh (Krogh, 1939) presented circumstantial evidence that ammonia excretion is in some way linked to active Na + uptake at the gills of freshwater animals. Jean Maetz (Maetz and GarciaRomeu, 1964) presented experimental evidence for direct Na + /NH 4 + exchange linkage in freshwater fish. Since then, our understanding of how ammonia actually permeates the gills has become less and less clear, as various studies have led to conflicting conclusions. Evidence has been presented to reinforce the predominance of Na + /NH 4 + exchange (Maetz, 1973; Kerstetter and Keeler, 1976;Payan and Girard, 1978;Pressley et al., 1981;McDonald and Prior, 1988;McDonald and Milligan, 1988), whereas others have argued for the dominance of simple NH 3 diffusion down the partial pressure NH 3 gradient maintained by the CO 2 hydration reaction in the gill boundary layer (Cameron and Heisler, 1983;Wilson et al., 1994;Wilkie and Wood, 1994). Intermediate positions have included flexible coupling via diffusion trapping of NH 3 linked to Na + /H + or H + pump/Na + channel mechanisms (Avella and Bornancin, 1989;Heisler, 1990), or mixed mechanisms whereby ammonia moves partly by diffusion and partly by electroneutral exchange (Wright and Wood, 1985;Salama et al., 1999). The overall problem is that ammonia excretion h...

“…Two of the more recently studied examples, the giant mudskipper (Periopthalmodon schlosseri) and the climbing perch (Anabas testudineus) appear to use active NH 4 + excretion to excrete D. Weihrauch, M. P. Wilkie and P. J. Walsh ammonia against massive inwardly directed P NH3 and NH 4 + electrochemical gradients Tay et al, 2006). The details of the process appear well worked out for the giant mudskipper, which is an obligatory air-breathing fish that drowns if denied access to air (Randall et al, 2004).…”

Section: Ammonia Excretion By Amphibious Fishmentioning

confidence: 99%

Ammonia and urea transporters in gills of fish and aquatic crustaceans

Weihrauch

Wilkie

Walsh

2009

246

SUMMARY The diversity of mechanisms of ammonia and urea excretion by the gills and other epithelia of aquatic organisms, especially fish and crustaceans, has been studied for decades. Although the decades-old dogma of `aquatic species excrete ammonia' still explains nitrogenous waste excretion for many species,it is clear that there are many mechanistic variations on this theme. Even within species that are ammonoteles, the process is not purely `passive',often relying on the energizing effects of proton and sodium–potassium ATPases. Within the ammonoteles, Rh (Rhesus) proteins are beginning to emerge as vital ammonia conduits. Many fishes are also known to be capable of substantial synthesis and excretion of urea as a nitrogenous waste. In such species, members of the UT family of urea transporters have been identified as important players in urea transport across the gills. This review attempts to draw together recent information to update the mechanisms of ammonia and urea transport by the gills of aquatic species. Furthermore, we point out several potentially fruitful avenues for further research.