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

Abstract

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...

Genomics of Adaptation to Multiple Concurrent Stresses: Insights from Comparative Transcriptomics of a Cichlid Fish from One of Earth’s Most Extreme Environments, the Hypersaline Soda Lake Magadi in Kenya, East Africa

“…By expressing the complete complement of the OUC pathway genes, the gill becomes another unusual tissue known to express urea pathway genes in A. grahami, following an earlier equally surprising finding of the complete OUC enzyme repertoire in the muscles (Randall et al 1989). The liver is the usual site for urea enzyme activity in terrestrial ureotelic vertebrates and some teleosts (Wright and Wood 2009). Although it is generally agreed that OUC pathway genes may actually be present in the early life stages of most teleost species, most adult teleost fish are ammoniotelic (Wright and Fyhn 2001).…”

Recent advances in understanding trans-epithelial acid-base regulation and excretion mechanisms in cephalopods

“…54,105 In vertebrate systems Rh glycoproteins including Rhbg and Rhcg were identified as central players facilitating the export of ammonia (NH 3 ) in concert with proton secretion mechanisms allowing a NH 4 C trapping mechanism in the acidified boundary layer of excretory cells. [106][107][108][109] Just lately Rh proteins were also found and characterized in a range of invertebrates including crustaceans and insects. [110][111][112] Also in cephalopods one Rh-protein (RhP) has been identified, which has the highest degree of identity to RhPs cloned from the decapod crustaceans Carcinus maenas (50%) and Metacarcinus magister (56 %).…”

“…Moreover, co-localization of NHE3 and RhP strongly suggests an acid-trapping mechanism of NH 4 C similar to that demonstrated for diverse vertebrate excretory organs including skin, gill and kidneys. 107,114,115 For example in acid-secreting intercalated cells of the mammalian kidney acid-secretion is coupled to the export of ammonium ions that are trapped in the acidified urine. 106 Here, the basolateral localization of Rhbg and Rhcg is believed to facilitate the entry of the de-protonized ammonia (NH 3 ) from the blood into the cell where the majority of NH 3 is protonized to the ammonium ion at a physiological pH of approximately 7.2.…”

Features of nitrogen metabolism in fishes

“…Ammonia output from the apical pole of the gill epithelium, rich in mitochondria (Evans DH et al 2005;Wright and Wood Ch 2009). In the water, adding free protons of the ammonia molecule, on the one hand, lose the ability to back diffusion through the lipophilic environment of biological membranes, on the other hand, the conversion of ammonia into less toxic substance-ammonium cation maintains the concentration gradient of ammonia on the interface between the external and internal environment.…”

“…The results of recent research underline the importance of the physiological role of the described mechanism, directly conjugated with the basic homeostatic functions of the gill apparatus, ensuring the stability of the parameters of gas exchange, acid-base and osmotic balance of internal environment (Evans et al 2005). However, some authors ascertain the relevance of further research, whose aim is, firstly, the creation of adequate models of clearance of ammonia, which is closely associated with the work of the ion-transporting systems of gill epithelium (Wright and Wood Ch 2009), in-Second, the analysis of physiological and evolutionary aspects of the ways renal clearance of ammonia in fish (Nakada et al 2007), as well as to study the features of neuro-humoral control systems of homeostatic functions of the gill apparatus of fish (Hoshijima and Hirose Sh 2007;Tse et al 2007). …”