Download From blood typing to a transport metabolon at a crossroad. Focus on

Am J Physiol Cell Physiol 298: C209 –C210, 2010;
doi:10.1152/ajpcell.00528.2009.
Editorial Focus
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”
Shigehisa Hirose1 and Tsutomu Nakada2
1
Department of Biological Sciences, Tokyo Institute of Technology, Yokohama, Japan; and 2Department of Molecular
Pharmacology, Shinshu University School of Medicine, Nagano, Japan
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]).
http://www.ajpcell.org
proteins 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⫹/NH⫹
4 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⫹/NH⫹
4 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
0363-6143/10 $8.00 Copyright © 2010 the American Physiological Society
C209
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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 glyco-
Editorial Focus
C210
DISCLOSURES
No conflicts of interest are declared by the author(s).
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298 • FEBRUARY 2010 •
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technique) is a noninvasive method for measuring minute
extracellular ion gradients created within the unstirred layer on
the surface of ion-transporting epithelial cells (18, 19). SIET uses
a microelectrode having a tip diameter of ⬃5 ␮m and a thin layer
(⬃250 ␮m) of an ion-selective ionophore cocktail in its tip. By a
modulation technique using a vibrating electrode, which is termed
self-referencing, the drift components inherent to electrodes can
be removed and the electrochemical detection of signals is greatly
improved. The principle is simple: A microelectrode is placed
within the unstirred boundary layer of a cell; a second measurement is then made, with the same electrode attached to a vibrator,
at a known distance away, and the two compared; the process is
repeated with the help of a scanning device. By using fast
responding electrodes, this self-referencing method is now being
improved so as to characterize rapid events such as a singlechannel activity under native conditions (11). Using SIET, Wu et
al. (28) first demonstrated that MRCs on the skin of medaka larvae
⫹
actually secrete NH⫹
4 and absorb Na and then showed colocalization of Rhcg1 and NHE3 (slc9a3) in MRCs by in situ hybridization. Tight coupling of ammonia secretion and sodium uptake
was also suggested by inhibition experiments and by altering
compositions of bathing media. It is interesting that the rate of
ammonia secretion by individual MRCs is more than 10 times
higher in medaka than in zebrafish (17). Future studies should aim
to provide direct evidence for the physical coupling of Rhcg1 and
NHE3 (i.e., a transport metabolon). The recently developed proximity ligation assay (4, 20) may prove useful in this model.
Concerning the mechanism for Na⫹ uptake by MRCs of freshwater fish, the role of the apical Na⫹-Cl⫺ cotransporter (NCC)
should be considered because it has been demonstrated in another
teleost fish, tilapia (5), while in zebrafish MRCs’ the NCC has
been shown to be mainly involved in Cl⫺ uptake and play only an
indirect or minor role in Na⫹ uptake (23).