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* Department of Biological Sciences, Tokyo Institute of Technology, Yokohama, Japan;
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA; and
American Red Cross, Philadelphia, Pennsylvania, USA
1Correspondence: Department of Biological Sciences, Tokyo Institute of Technology, 4259-B-19 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. E-mail: shirose{at}bio.titech.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: RhAG • Rhesus glycoprotein • ammonium transporter • mitochondria-rich cell • teleost
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4)
.
Fish gills consist of a large number of filaments arranged along the gill arches. The surfaces of the filaments are greatly enlarged by a series of plate-like lamellae. Each lamella is composed of two sheets of epithelia separated by a thin space through which blood circulates to allow the exchange of respiratory gases. The epithelial sheets consist of thin squamous pavement cells. The separation between the epithelial sheets is maintained by pillar cells and basal lamina. Pillar cells are spool-shaped cells connecting two epithelial sheets of the respiratory lamella in the gills. In the basal region of the lamella, mitochondrion-rich cells (MRCs) are rich in mitochondria and sodium/potassium ATPase (Na+,K+-ATPase), reflecting their extraordinary power of active ion transport. Thus, gill epithelium is composed of several distinct cell types, but it is controversial from which cells, and by which mechanism, ammonia is excreted (1
, 5)
.
Members of the ammonia transporter/methylammonium permease/Rh glycoprotein (Amt/MEP/Rh) family are involved in ammonia transport in a broad range of organisms (6
7
8)
. Rh-associated glycoprotein (RhAG), a member of the Rh family, is associated with the major blood group antigens on the surface of red blood cells in humans (9)
. Rh type B glycoproteins (RhBG) and Rh type C glycoproteins (RhCG) have been localized to the basolateral and apical membranes, respectively, in the collecting segment and collecting duct in the kidney, which is a major site of transepithelial ammonia transport (10
11
12)
. The Rh glycoproteins were shown to have ammonia transport activity when expressed in Saccharomyces cerevisiae and Xenopus oocyte heterologous systems (6
, 13
14
15
16)
. These findings lead us to hypothesize that Rh glycoprotein homologs in fish are involved in ammonia excretion, and specifically in the fish gill, where passive diffusion through the plasma membrane is generally thought to be the mechanism of transport.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RNA isolation
Total RNA was isolated from the tissues by acid guanidinium thiocyanate-phenol-chloroform extraction with Isogen (Nippon Gene, Tokyo, Japan) as described previously (17)
. Briefly, tissues were homogenized in Isogen (1 g of tissue per 10 ml of Isogen) using a Polytron tissue homogenizer followed by chloroform extraction, isopropanol precipitation, and 75% (v/v) ethanol washing of precipitated RNA. The RNA was dissolved in diethyl pyrocarbonate (DEPC) -treated water and the concentration was measured spectrophotometrically at 260 nm.
Cloning of fugu Rh glycoproteins
Fragments of fugu Rh cDNAs were isolated by RT-polymerase chain reaction (RT-PCR) from gill or kidney RNA with primers that were designed based on the fugu genomic database (http://genome.jgi-psf.org/fugu6/fugu6.home.html). The PCR products were subcloned into pBluescript II SK(–) (Stratagene, La Jolla, CA, USA) or pZErO-2 (Invitrogen, Carlsbad, CA, USA) and sequenced. These clones were used as a probe for Northern blot analysis. Full-length cDNAs were obtained by 5'-RACE, 3'-RACE, and RT-PCR as described previously (17)
. All primers used are listed in Table 1
.
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Northern blot analysis
Total RNA (20 µg/lane) from the tissues of pufferfish was electrophoresed on formaldehyde-agarose (1%) denaturing gels in MOPS running buffer (20 mM MOPS, pH 7.0, 8 mM acetate, 1 mM EDTA), then transferred onto Hybond-N+ nylon membranes (GE Healthcare Bioscience, Piscataway, NJ, USA) by capillary blotting. After transfer, membranes were baked for 2 h at 80°C and prehybridized for 2 h at 65°C in PerfectHyb hybridization solution (Toyobo, Osaka, Japan). The probes were labeled with [
-32P]dCTP (3000 Ci/mmol) using a Ready-To-Go DNA labeling kit (GE Healthcare Bioscience) and the unincorporated nucleotides were removed by passage through a Sephadex G-50 column (GE Healthcare Bioscience). The membranes were then hybridized with each 32P-labeled probe in the same buffer at 68°C for 16 h. The blots were subsequently washed with increasingly stringent conditions (final wash: 1x saline-sodium citrate and 0.1% SDS for 30 min at 60°C). Membranes were exposed to imaging plates (Fuji Film, Tokyo, Japan) in a cassette overnight. Results were analyzed using a Fuji BAS2000 Bioimage analyzer (Fuji Film). A ß-actin probe (18)
was used as a control to verify loading and RNA integrity. A ß-globin probe was isolated by RT-PCR from blood total RNA with primers based on the fugu genomic database.
In situ hybridization
Gills from anesthetized pufferfish, perfusion-fixed with 10% buffered neutral formalin (Muto Pure Chemicals, Tokyo, Japan) were harvested, embedded in paraffin, and sectioned (4 µm). The following DNA templates were used to prepare digoxigenin (DIG) -labeled riboprobes: a 371 bp fragment of fRhag cDNA (nucleotides 1112–1482), a 359 bp fragment of fRhbg cDNA (nucleotides 1158–1516), a 159 bp fragment of fRhcg1 cDNA (nucleotide 1471–1629), and a 219 bp fragment of fRhcg2 cDNA (nucleotide 1372–1590). The DIG RNA Labeling Mix (Roche Diagnostics, Mannheim, Germany) was used for synthesizing DIG-labeled sense and antisense probes. Alkaline phosphatase-conjugated anti-DIG antibody and NBT/BCIP substrates were used to visualize the signal, followed by counterstaining with Kernechtrot (Muto Pure Chemicals).
Antibody production
cDNA fragments encoding a part of the COOH terminus of fRhag (amino acid residues 386–441), fRhbg (407–458), fRhcg1 (426–485), and fRhcg2 (420–481) were subcloned into the BamHI/EcoRI sites of the bacterial expression vector pHAT (Clontech, Palo Alto, CA, USA) or pRSET (Invitrogen). The recombinant proteins were purified with Talon metal affinity resins (Clontech) following the manufacturer’s instructions. Briefly, BL21 cells transformed with the expression vectors were used to inoculate 1.5 L of LB broth containing 100 µg/ml ampicillin. The cultures were grown to an A600 of 0.5 at 37°C, and protein expression was induced by adding isopropyl-1-thio-D-galactopyranoside to a final concentration of 1 mM for 3 h at 37°C. The cells were harvested from the cultures by centrifugation and resuspended in 20 ml of Extraction/Wash buffer, then disrupted by freezing-thawing and sonication. After centrifugation (10,000 g at 4°C), supernatants were saved and purified with Talon metal affinity resin. After purification, recombinant proteins were dialyzed against saline at 4°C. Polyclonal antibodies were prepared in Japanese white rabbits by injecting 
200 µg of purified recombinant proteins, emulsified with the adjuvant TiterMax Gold (CytRx) (1:1), intramuscularly at multiple sites. The rabbits were injected three times at 1 month intervals and bled 7 days after the third immunization.
Immunohistochemistry
Gills from pufferfish were fixed in 0.1 M phosphate buffer, pH 7.4, containing 4% (w/v) paraformaldehyde for 1 h at 4°C. After incubation in 0.1 M phosphate buffer, pH 7.4, containing 20% (w/v) sucrose for 16 h at 4°C, specimens were frozen in Tissue Tek OCT Compound on a cryostat holder. Sections (6 µm) were prepared in –20°C cryostat, mounted on 3'-amino propyltriethoxy silane-coated glass slides, and air dried for 1 h. After washing with PBS, sections were first incubated in PBS with 0.1% (v/v) Triton X-100 for 10 min, then incubated for 2 h at room temperature with 2.5% (v/v) normal goat serum. After blocking, sections were reacted with anti-fRhag antiserum (1:1000), anti-fRhbg antiserum (1:1000), anti-fRhcg1 antiserum (1:1000), anti-fRhcg2 antiserum (1:1000), and each preimmune serum (1:1000) overnight at 4°C. Sections were then washed with PBS and treated with Alexa Fluor 488-conjugated anti-rabbit IgG (1:2000, Invitrogen) and Hoechst 33342 (100 ng/ml, Invitrogen) for 1 h at room temperature. When treated with fRhag, fRhbg, and fRhcg2 antisera, TRITC-phalloidin (0.1 µg/ml, Sigma, St. Louis, MO, USA) was also added to the secondary antibody solutions. The rat anti-Na+,K+-ATPase antiserum (1:1000) (16)
and Cy3-conjugated anti-rat IgG (1:2000, Jackson ImmunoResearch, West Grove, PA, USA) were used to stain MRCs. Fluorescence images were acquired using Axiovert 200M epifluorescence microscope (Carl Zeiss, Thornwood, NY, USA) equipped with an ApoTome optical sectioning device (Carl Zeiss).
In vitro transcription/translation and Western blot analysis
The pufferfish recombinant Rh glycoproteins were synthesized by in vitro transcription and translation with TNT Quick Coupled Reticulocyte Lysate system (Promega, Madison, WI, USA) and each Rh plasmid in the presence of [35S]methionine (>1000 Ci mmol–1; BD Biosciences Clontech) according to the manufacturer’s instructions. Two microliters of the reaction mix were separated by 10% SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. Nonspecific binding was blocked with 5% nonfat skim milk in TBST (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature. The membranes were incubated with anti-fRhag (1:3000), anti-fRhbg (1:1000), antifRhcg1 (1:10,000), or anti-fRhcg2 (1:10,000) antiserum overnight at 4°C. After washing with TBST, membranes were then reacted with horseradish peroxidase-conjugated donkey anti-rabbit IgG (1:10,000; Jackson ImmunoResearch) for 1 h at room temperature. The bound secondary antibody was visualized by enhanced chemiluminescence detection using ECL-Plus reagents (Amersham Bioscience) according to the manufacturer’s instructions. After chemiluminescence detection, 35S-labeled proteins were visualized by autoradiography.
Oocyte injection and [14C]methylammonium uptake assay
Stage V and VI defolliculated oocytes were injected with 34 nl (1 ng/nl) of cRNA, or water for controls, and placed in individual wells in 96-well plates with 200 µl of SOS containing in mM (100 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, pH 7.6, 200 mosM) with 2.5 mM sodium pyruvate and 100 µg/ml gentamicin at 16°C. Radiolabeled methylammonium ([14C]CH3NH3+) (ICN, Irvine, CA, USA) uptake was measured 3 days postinjection. Experiments were performed at room temperature by placing groups of six oocytes in 200 µl of low K+ (0.2 mM) SOS uptake buffer containing 1 µCi/ml [14C]methylammonium (MA) and unlabeled MA to a final concentration of 1.5 mM. For all experiments, radiotracer uptake was terminated by washing the oocytes several times with 1.2 ml of ice-cold unlabeled uptake buffer. Oocytes were solubilized in 200 µl of 5% SDS and analyzed for radioactivity in 5 ml of CytoScint (ICN) by liquid scintillation counting. Water-injected control oocytes were evaluated in parallel in all assays, and control uptake values were subtracted from experimental values for cRNA-injected oocytes.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). The pufferfish has one RhAG ortholog (fRhag), one Rh blood group CED antigen ortholog (fRhced), one RhBG ortholog (fRhbg), and two RhCG orthologs (fRhcg1 and fRhcg2). Two others—fRhag-like1 and fRhag-like2—belong to an independent branch (Fig. 1A
).
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To localize tissue expression, we isolated cDNA fragments of the pufferfish RH orthologs by RT-PCR and carried out Northern blot analysis. The blots were also probed with a ß-globin probe to control for the presence of red blood cell contamination in the tissues. The erythroid homologue fRhag mRNA was found in the spleen and kidney, in the blood, and in the gill (Fig. 1B
). However, the fRhag mRNA signal in the kidney and spleen, which are hematopoietic organs in teleosts, was the result of red blood cells as shown by the equivalent ß-globin mRNA signal. The fRhced mRNA was confined to expression in blood cells, as in the case of the mammalian ortholog. It important to note that the positive fRhag hybridization signal in the gill was not due to contamination with red blood cells, as verified by the undetectable level of ß-globin mRNA in the gill (Fig. 1B
). fRhbg, fRhcg1, and fRhcg2 mRNAs were also specifically expressed in the gill but were not found in the kidney (Fig. 1B
). These results suggest that all four pufferfish Rh glycoprotein genes (fRhag, fRhbg, fRhcg1, and fRhcg2)are likely to be involved in the elimination of ammonia in the gill. No expression of fRhag-like1 and fRhag-like2 was observed in the tissues sampled.
To clarify the cellular location of each gene product in the gill and to characterize the potential transport function, the complete coding sequences of the four pufferfish Rh glycoproteins were obtained by 5'- and 3'-RACE and RT-PCR. The amino acid sequences deduced from the isolated cDNAs showed 60–70% identity with the corresponding human orthologs and are predicted to have 12 putative transmembrane spans (Fig. 2
).
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Localization of fRhag, fRhbg, fRhcg1, and fRhcg2 in the gill
To determine the cellular localization of the mRNAs in the gill, we performed in situ hybridization (Fig. 3
A). Clear labeling was detected in pillar cells with the fRhag probe. Pavement cells were stained with both fRhbg and fRhcg2 probes. A population of cells in the basal regions of the lamellae was stained with the fRhcg1 probe, whose distribution is reminiscent of MRCs.
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We carried out immunohistochemical analysis to determine the subcellular localization of the pufferfish Rh glycoproteins. Antibodies against the COOH terminus of each protein were raised in rabbits using recombinant protein fragments as antigens. Concanavalin A (Con A) and an antibody to the Na+,K+-ATPase were used to visualize the basal lamina and basolateral membranes of MRCs (18
, 19)
, respectively (Fig. 3B
). The fRhag antibody revealed that fRhag protein was localized in the apical and basolateral membranes of pillar cells. The fRhcg2 and fRhbg proteins were localized in the apical and basolateral membranes of the pavement cells, respectively. In contrast, only the fRhcg1 protein was localized in the apical membrane of the MRCs (Fig. 3B
), and no evidence for Rh glycoprotein expression was observed on the basolateral membranes of MRCs.
Since the amino acid sequences of some pufferfish Rh glycoproteins are quite similar, the cross-reactivity of the antisera was assessed by Western blot. Recombinant Rh glycoproteins were synthesized by in vitro transcription/translation. Each antiserum showed a specific signal with the appropriate translation product with the predicted molecular weight (Fig. 3C
). There was no cross-reactivity between the Rh glycoproteins.
Functional analysis of fugu Rh glycoproteins
To determine whether the pufferfish Rh glycoprotein homologs also function to mediate ammonia transport, heterologous expression studies in Xenopus oocytes were conducted. The radioactive analog tracer MA [14C]CH3NH3+ was used as a measure of ammonia transport as described (15)
. As Xenopus oocytes have an endogenous ammonium uptake system, water-injected control oocytes were evaluated in parallel. Expression of fRhag, fRhbg, fRhcg1, or fRhcg2 enhanced the rate of [14C]MA uptake compared with controls (Fig. 4
). Uptake mediated by the pufferfish proteins was 2- to 5-fold greater at 15–30 min and was still 4- to 5-fold greater at 1 h compared with the linear rate of uptake in the controls. Methylammonium uptake was competitively inhibited with ammonium chloride, confirming the specificity of the transport (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The ammonia concentration gradient between the vascular space and environmental water may be the driving force for this lamellar excretion system. Apical expression of fRhcg1 in MRCs suggests that MRCs may also excrete ammonia. Passage of ammonia across the basolateral membrane of MRCs may potentially be mediated by ion pumps or transporters that have a high affinity for ammonium ions. For example, the Na+,K+-ATPase in the thick ascending limb of rabbit kidney transports NH4+ in place of K+ (20
, 21)
. In the mudskipper Periophthalmondon schlosseri, active ammonia excretion has been shown to be sensitive to ouabain, a Na+,K+-ATPase inhibitor (5)
. As MRCs contain abundant Na+,K+-ATPase in the basolateral membrane, it is possible that this basolateral pump may cooperate with the apical fRhcg1 in active excretion of ammonia in MRCs to assure complete elimination of ammonia. In contrast, the lamellar fRhag/fRhbg/fRhcg system may function to remove ammonia when the plasma concentration is high. It is therefore tempting to speculate that ammonia levels are initially lowered mainly by the lamellar transporters, and these levels are further reduced to nontoxic levels by the MRC system, but this remains to be proved.
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Pufferfish Rh glycoproteins are ideally located in the pillar cells, pavement cells, and MRCs for ammonia excretion (Fig. 5)
; they transport the ammonia analog MA when expressed in Xenopus oocytes. Currently there are several models of ammonia excretion from the gill, and simple NH3 diffusion is thought to be the most significant (1
, 2)
. A protein-mediated pathway would be more efficient than simple diffusion of NH3.
Numerous cDNA fragments identified as Rhag, Rhbg, Rhcg1, and Rhcg2 orthologs are present in the expression sequence tag database from the gill of the three-spined stickleback, Gasterosteus aculeatus. The Zebrafish Information Network shows that an Rhbg ortholog is expressed in the gill of 5-day-old zebrafish embryos (http://zfin.org/; Jan 2006, ZFIN ID: ZDB-FIG-050630–10384). These observations strongly suggest that the ammonia excretion system described here is present in other ammonotelic teleosts.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication July 26, 2006. Accepted for publication November 9, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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