From blood typing to a transport metabolon at a crossroad. Focus on "Ammonium-dependent sodium uptake in mitochondrion-rich cells of medaka (Oryzias latipes) larvae".

THE RH BLOOD GROUP-RELATED PROTEINS have recently been recognized as ammonia transporters acting in erythrocyte and nonerythrocyte membranes. They play an important role in excretion of nitrogenous waste formed mainly from amino acid metabolism. Because of a restricted availability of water, most terrestrial animals detoxify ammonia into urea or uric acid before removal from the body. However, aquatic animals, living in an abundant supply of water, can directly excrete ammonia into the environment immediately as it is produced. This method of ammonia elimination is strategic in avoiding urea synthesis, an anabolic process requiring ATP. In this issue of American Journal of Physiology-Cell Physiology, Wu et al. (28) add a further twist to this adaptive strategy by providing evidence for a coupling of the ammonia excretion with a sodium uptake process, which is essential for living in freshwater conditions. The Rh antigen was first identified about 70 years ago by Landsteiner and Wiener (8), who showed that rabbits and guinea pigs, when immunized with red blood cells of Rhesus monkey, produce an antibody that also agglutinates human red blood cells (Rh for Rhesus). The Rh antigen (Rh), now recognized for its strong immunogenic nature and use for diagnosis of pregnancy at risk for hemolytic disease of the newborn, forms a complex with other subunits including Rh-associated glycoprotein (RhAG), CD47, ICAM-4, and glycophorin B (25). Rh and RhAG are the major components of the complex and belong to the same family of proteins. They were expected to carry some physiologic function based on their well-conserved nature among the species and especially because they have 12 transmembrane-spanning segments characteristic of transporters. A comparative genomic approach led to the following breakthrough discovery that RhAG acts as an ammonia transporter. A low ( 20%) but significant sequence similarity was found by Marini et al. (10) between RhAG and the members of the Mep/Amt family previously identified as ammonia transporters in microorganisms and plants for which ammonia is a preferred source of nitrogen (Mep for methylammonium permease; Amt for ammonium transporter); for the evolution of the Mep/ Amt/Rh family, see Huang and Peng (6). RhAG and its nonerythroid homologues, RhBG and RhCG, were then demonstrated to behave indeed as ammonia transporters (9, 26). The discoveries briefly summarized above have greatly contributed to our understanding of the mechanism of ammonia secretion in fish. Nakada et al. (13) identified four Rh glycoproteins in pufferfish (Rhag, Rhbg, Rhcg1, and Rhcg2; lowercase refers to the proteins of nonhuman species) and determined their locations in the gill, the major site of gas exchange, ionoregulation, and ammonia elimination in adult teleost fish; it is noteworthy that gills are needed for ionoregulation before they are needed for oxygen uptake during development (16). Gill surface area is greatly increased by its lamellar structure. The ammonia transporters Rhag, Rhbg, and Rhcg2 are distributed on these lamellar cells, which is ideal for optimizing ammonia elimination. In contrast, Rhcg1 exhibits a somewhat unexpected localization in mitochondrion-rich cells (MRCs or ionocytes). MRCs are relatively minor among the population of the gill epithelial cells and are specialized to actively absorb or secrete NaCl to maintain body fluid homeostasis. MRCs are present in the skin and yolk sac during early developmental stages before functional gills are present. Since the MRCs found in the skin are easily accessible to experimental analysis, they are frequently studied. Since this report, one or more members of the Rh protein family including those mentioned above (1, 2, 7, 12, 15; for review see Refs. 24 and 27) and Rhp2 (p for primary) (14) have been identified in other fish species including zebrafish, a freshwater fish in which a knockdown experiment can be performed for detailed functional analyses of specific gene products by using antisense morpholino oligonucleotides. In zebrafish, Rhcg1 has been demonstrated to be expressed in the skin and gill MRCs that are rich in vacuolar-type H -ATPase (vH-MRCs) (12, 17), and implicated as being somehow involved in Na uptake (3, 12). Apical localization of Na /H exchanger 3 (NHE3) in vH-MRCs is also observed in zebrafish (29). By integrating these pieces of evidence and other information accumulated by previous molecular physiological studies, a collaborative group of researchers at the McMaster University and the University of Guelph (21, 27) has proposed a “Na /NH4 exchange complex” consisting of the transporters Rhcg, vacuolar-type H -ATPase (vH-ATPase), Na /H exchanger (NHE), and epithelial Na channel (ENaC) working together as a metabolon. Except for the inclusion of ENaC, which is not present in teleost genomes (22), the model is very attractive since the ammonia is not simply discarded; instead, the downhill gradient for ammonia excretion into the water is used to drive uphill Na uptake from the water into the freshwater fish. Wu et al. (28) provided a piece of supporting evidence for the model. After failing to detect significant Na /NH4 exchange in zebrafish, these investigators analyzed another model fish, Japanese medaka (Oryzias latipes). The electrophysiological technique they used warrants a comment. SIET (also known as the self-referencing ion-selective electrode Address for reprint requests and other correspondence: S. Hirose, Dept. of Biological Sciences, Tokyo Institute of Technology, 4259-B-19 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan (e-mail: [email protected]). Am J Physiol Cell Physiol 298: C209–C210, 2010; doi:10.1152/ajpcell.00528.2009. Editorial Focus