C. Michele Nawata

Rh glycoprotein expression is modulated in pufferfish (Takifugu rubripes) during high environmental ammonia exposure

SUMMARY Rhesus (Rh) protein involvement in ammonia transport processes in freshwater fish has received considerable attention; however, parallel investigations in seawater species are scant. We exposed pufferfish to high environmental ammonia (HEA; 1 and 5 mmol l–1 NH4HCO3) and evaluated the patterns of ammonia excretion and gill Rh mRNA and protein expression. Gill H+-ATPase, NHE1, NHE2, NHE3, Na+/K+-ATPase (NKA), Na+/K+/2Cl– co-transporter (NKCC1) mRNA, H+-ATPase activity, NKA protein and activity, were also quantified. Activation of NKA by NH4+ was demonstrated in vitro. The downregulation of Rhbg mRNA and simultaneous upregulations of Rhcg1, H+-ATPase, NHE3, NKA, NKCC1 mRNA, H+-ATPase activity, and NKA protein and activity levels suggested that during HEA, ammonia excretion was mediated mainly by mitochondria-rich cells (MRCs) driven by NKA with basolateral NH4+ entry via NKA and/or NKCC1, and apical NH3 extrusion via Rhcg1. Reprotonation of NH3 by NHE3 and/or H+-ATPase would minimise back flux through the Rh channels. Downregulated Rhbg and Rhag mRNA observed in the gill during HEA suggests a coordinated protective response to minimise the influx of external ammonia via the pavement cells and pillar cells, respectively, while routing ammonia excretion through the MRCs. Exposure to hypercapnia (1% CO2 in air) resulted in downregulated gill and erythrocyte Rhag mRNA. Surprisingly, Rhag, Rhbg, Rhcg1 and Rhcg2 proteins responded to both hypercapnia and HEA with changes in their apparent molecular masses. A dual NH3/CO2 transport function of the pufferfish Rh proteins is therefore suggested. The results support and extend an earlier proposed model of pufferfish gill ammonia excretion that was based on immunolocalisation of the Rh proteins. Passive processes and/or Rhbg and Rhcg2 in the pavement cells may maintain basal levels of plasma ammonia but elevated levels may require active excretion via NKA and Rhcg1 in the MRCs.

The effects of CO2 and external buffering on ammonia excretion and Rhesus glycoprotein mRNA expression in rainbow trout.

SUMMARY Rhesus (Rh) proteins were recently characterized as ammonia gas (NH3) channels. Studies indicate, however, that Rh proteins also facilitate CO2 transport in a green alga and in human erythrocytes. Previously, we reported that Rh mRNA expression in various rainbow trout tissues responded to high environmental ammonia. To determine whether or not Rh proteins may also be involved in CO2 transport in rainbow trout, we examined the effects of a 12 h exposure to external hypercapnia (1% CO2 in air) on Rh mRNA expression in the gill, skin and erythrocytes. External hypercapnic conditions lowered the water pH and facilitated ammonia excretion; therefore, we also studied the effects of hypercapnia and normocapnia in the presence of 10 mmol l–1 Hepes-buffered water. Hepes treatment prevented water acidification, but resulted in elevated plasma ammonia levels and reduced ammonia excretion rates. Hypercapnia exposure without buffering did not elicit changes in Rh mRNA expression in the gill or skin. However, Rhcg2 mRNA expression was downregulated in the gills and upregulated in the skin of both normocapnia- and hypercapnia-exposed fish in Hepes-buffered water. mRNA expression of a newly cloned Rhbg2 cDNA was downregulated in the skin of fish exposed to buffered water, and Rhag mRNA expression in erythrocytes was decreased with exposure to normocapnia in buffered water but not with hypercapnia exposure in either buffered or unbuffered water. With the aid of Hepes buffering, we were able to observe the effects of both CO2 and ammonia on Rh mRNA expression. Overall, we conclude that high CO2 did not directly elicit changes in Rh mRNA transcription levels in the gill and skin, and that the changes observed probably reflect responses to high plasma ammonia, mirroring those in trout exposed to high environmental ammonia. Therefore a dual function for gill and skin Rh proteins in CO2 and ammonia transport is not evident from these results. Rhag expression, however, responded differentially to high CO2 and high ammonia, suggesting a possible dual role in the erythrocytes.

A nose-to-nose comparison of the physiological and molecular responses of rainbow trout to high environmental ammonia in seawater versus freshwater

SUMMARY Steelhead rainbow trout acclimated to either freshwater (FW) or seawater (SW) were exposed to high environmental ammonia (HEA, 1000 μmol l–1 NH4HCO3, pH 7.8–8.0) for 24 h. SW trout restored ammonia excretion more rapidly (3–6 h versus 9–12 h in FW), despite higher production rates and lower plasma pH. Plasma total ammonia levels stabilized at comparable levels below the external HEA concentration, and blood acid–base disturbances were small at both salinities. The electrochemical gradients for NH4+ entry (FNH4+) were the same in the two salinities, but only because FW trout allowed their transepithelial potential to rise by ∼15 mV during HEA exposure. Elevation of plasma [cortisol] during HEA exposure was more prolonged in SW fish. Plasma [glucose] increased in SW, but decreased in FW trout. Plasma [urea-N] also decreased in FW, in concert with elevated urea transporter (UT) mRNA expression in the gills. Of 13 branchial transporters, baseline mRNA expression levels were higher for Rhcg1, NHE2, NKCC1a and UT, and lower for NBC1 and NKA-α1a in SW trout, whereas NKA-α1b, NHE3, CA2, H+-ATPase, Rhag, Rhbg and Rhcg2 did not differ. Of the Rh glycoprotein mRNAs responding to HEA, Rhcg2 was greatly upregulated in both FW and SW, Rhag decreased only in SW and Rhcg1 decreased only in FW. H+-ATPase mRNA increased in FW whereas NHE2 mRNA increased in SW; NHE3 did not respond, and V-type H+-ATPase activity declined in SW during HEA exposure. Branchial Na+,K+-ATPase activity was much higher in SW gills, but could not be activated by NH4+. Overall, the more effective response of SW trout was explained by differences in physical chemistry between SW and FW, which greatly reduced the plasma NH3 tension gradient for NH3 entry, as well as by the higher [Na+] in SW, which favoured Na+-coupled excretion mechanisms. At a molecular level, responses in SW trout showed subtle differences from those in FW trout, but were very different than in the SW pufferfish. Upregulation of Rhcg2 appears to play a key role in the response to HEA in both FW and SW trout, and NH4+ does not appear to move through Na+,K+-ATPase.

Modulation of Rh glycoproteins, ammonia excretion and Na+ fluxes in three freshwater teleosts when exposed chronically to high environmental ammonia

SUMMARY We investigated relationships among branchial unidirectional Na+ fluxes, ammonia excretion, urea excretion, plasma ammonia, plasma cortisol, and gill transporter expression and function in three freshwater fish differing in their sensitivity to high environmental ammonia (HEA). The highly ammonia-sensitive salmonid Oncorhynchus mykiss (rainbow trout), the less ammonia-sensitive cyprinid Cyprinus carpio (common carp) and the highly ammonia-resistant cyprinid Carassius auratus (goldfish) were exposed chronically (12–168 h) to 1 mmol l−1 ammonia (as NH4HCO3; pH 7.9). During HEA exposure, carp and goldfish elevated ammonia excretion (JAmm) and Na+ influx rates () while trout experienced higher plasma ammonia (TAmm) and were only able to restore control rates of JAmm and . All three species exhibited increases in Na+ efflux rate (). At the molecular level, there was evidence for activation of a ‘Na+/NH4+ exchange metabolon’ probably in response to elevated plasma cortisol and TAmm, though surprisingly, some compensatory responses preceded molecular responses in all three species. Expression of Rhbg, Rhcg (Rhcg-a and Rhcg-b), H+-ATPase (V-type, B-subunit) and Na+/K+-ATPase (NKA) mRNA was upregulated in goldfish, Rhcg-a and NKA in carp, and Rhcg2, NHE-2 (Na+/H+ exchanger) and H+-ATPase in trout. Branchial H+-ATPase activity was elevated in goldfish and trout, and NKA activity in goldfish and carp, but NKA did not appear to function preferentially as a Na+/NH4+-ATPase in any species. Goldfish alone increased urea excretion rate during HEA, in concert with elevated urea transporter mRNA expression in gills. Overall, goldfish showed more effective compensatory responses towards HEA than carp, while trout were least effective.

Rhesus Glycoprotein and Urea Transporter Genes Are Expressed in Early Stages of Development of Rainbow Trout (Oncorhynchus mykiss)

The objective of this study was to determine if the genes for the putative ammonia transporters, Rhesus glycoproteins (Rh) and the facilitated urea transporter (UT) were expressed during early development of rainbow trout, Oncorhynchus mykiss Walbaum. We predicted that the Rh isoforms Rhbg, Rhcg1 and Rhcg2 would be expressed shortly after fertilization but UT expression would be delayed based on the ontogenic pattern of nitrogen excretion. Embryos were collected 3, 14 and 21 days postfertilization (dpf), whereas yolk sac larvae were sampled at 31 dpf and juveniles at 60 dpf (complete yolk absorption). mRNA levels were quantified using quantitative polymerase chain reaction and expressed relative to the control gene, elongation factor 1alpha. All four genes (Rhbg, Rhcg1, Rhcg2, UT) were detected before hatching (25-30 dpf). As predicted, the mRNA levels of the Rh genes, especially Rhcg2, were relatively high early in embryonic development (14 and 21 dpf), but UT mRNA levels remained low until after hatching (31 and 60 dpf). These findings are consistent with the pattern of nitrogen excretion in early stages of trout development. We propose that early expression of Rh genes is critical for the elimination of potentially toxic ammonia from the encapsulated embryo, whereas retention of the comparatively benign urea molecule until after hatch is less problematic for developing tissues and organ systems.

Differential responses in ammonia excretion, sodium fluxes and gill permeability explain different sensitivities to acute high environmental ammonia in three freshwater teleosts.

We examined the acute physiological responses to high environmental ammonia (HEA), particularly the linkages between branchial ammonia fluxes and unidirectional Na(+) fluxes, as well as urea excretion, cortisol, and indicators of gill permeability in three freshwater teleosts differing in their sensitivities to ammonia; the highly sensitive salmonid Oncorhynchus mykiss (rainbow trout), the less sensitive cyprinid Cyprinus carpio (common carp) and the highly resistant cyprinid Carassius auratus (goldfish). Fish were acutely exposed to two sub-lethal ammonia concentrations (as NH(4)HCO(3)) at pH 7.9: 1 mM for a period of 12 h, identical for all species, and 5 mM for the cyprinids and 1.4 mM for the trout for 3 h. Elevation of plasma cortisol at both levels of HEA was apparent in all species. At 1 mM, ammonia excretion (J(amm)) was inhibited to a greater extent in trout than cyprinids and concurrently a significantly higher plasma ammonia level was evident in trout. However J(amm) was reversed in all species at 5 or 1.4 mM. Goldfish showed a significant increase in urea excretion rate (J(urea)) during HEA exposure. In carp and trout, neither level of HEA elevated J(urea) but urea production was increased as evidenced by a considerable elevation of plasma urea. At 1mM HEA, Na(+) imbalance became progressively more severe in trout and carp due to a stimulation of unidirectional Na(+) efflux (J(out)(Na)) without a concomitant increase in unidirectional Na(+) influx (J(in)(Na)). Additionally, a transient reduction of J(in)(Na) was evident in trout. Goldfish showed an opposite trend for J(out)(Na) with reduced efflux rates and a positive Na(+) balance during the first few hours of HEA. However, after 12 h of exposure, both J(in)(Na) and J(out)(Na) were also increased in both carp and goldfish, whereas only J(out)(Na) was increased in trout, leading to a net Na(+) loss. Na(+) homeostasis was entirely disrupted in all three species when subjected to the 5 or 1.4 mM ammonia for 3 h: J(in)(Na) was significantly inhibited while considerable activation of J(out)(Na) was observed. Diffusive water efflux rates and net K(+) loss rates across the gills were enhanced during HEA only in trout, indicating an increment in gill transcellular permeability. Transepithelial potential was increased in all the species during ammonia exposure, but to the least extent in goldfish. Overall, for several different physiological systems, trout were most disturbed, and goldfish were least disturbed by HEA, helping to explain the differential ammonia tolerance of the three species.

Rh Proteins and NH4+ –activated Na+-ATPase in the Magadi Tilapia (Alcolapia grahami), a 100% Ureotelic Teleost Fish.

SUMMARY The small cichlid fish Alcolapia grahami lives in Lake Magadi, Kenya, one of the most extreme aquatic environments on Earth (pH ~10, carbonate alkalinity ~300 mequiv l−1). The Magadi tilapia is the only 100% ureotelic teleost; it normally excretes no ammonia. This is interpreted as an evolutionary adaptation to overcome the near impossibility of sustaining an NH3 diffusion gradient across the gills against the high external pH. In standard ammoniotelic teleosts, branchial ammonia excretion is facilitated by Rh glycoproteins, and cortisol plays a role in upregulating these carriers, together with other components of a transport metabolon, so as to actively excrete ammonia during high environmental ammonia (HEA) exposure. In Magadi tilapia, we show that at least three Rh proteins (Rhag, Rhbg and Rhcg2) are expressed at the mRNA level in various tissues, and are recognized in the gills by specific antibodies. During HEA exposure, plasma ammonia levels and urea excretion rates increase markedly, and mRNA expression for the branchial urea transporter mtUT is elevated. Plasma cortisol increases and branchial mRNAs for Rhbg, Rhcg2 and Na+,K+-ATPase are all upregulated. Enzymatic activity of the latter is activated preferentially by NH4+ (versus K+), suggesting it can function as an NH4+-transporter. Model calculations suggest that active ammonia excretion against the gradient may become possible through a combination of Rh protein and NH4+-activated Na+-ATPase function.

Transepithelial water and urea permeabilities of isolated perfused Munich-Wistar rat inner medullary thin limbs of Henle's loop

To better understand the role that water and urea fluxes play in the urine concentrating mechanism, we determined transepithelial osmotic water permeability (Pf) and urea permeability (Purea) in isolated perfused Munich-Wistar rat long-loop descending thin limbs (DTLs) and ascending thin limbs (ATLs). Thin limbs were isolated either from 0.5 to 2.5 mm below the outer medulla (upper inner medulla) or from the terminal 2.5 mm of the inner medulla. Segment types were characterized on the basis of structural features and gene expression levels of the water channel aquaporin 1, which was high in the upper DTL (DTLupper), absent in the lower DTL (DTLlower), and absent in ATLs, and the Cl-(1) channel ClCK1, which was absent in DTLs and high in ATLs. DTLupper Pf was high (3,204.5 ± 450.3 μm/s), whereas DTLlower showed very little or no osmotic Pf (207.8 ± 241.3 μm/s). Munich-Wistar rat ATLs have previously been shown to exhibit no Pf. DTLupper Purea was 40.0 ± 7.3 × 10(-5) cm/s and much higher in DTLlower (203.8 ± 30.3 × 10(-5) cm/s), upper ATL (203.8 ± 35.7 × 10(-5) cm/s), and lower ATL (265.1 ± 49.8 × 10(-5) cm/s). Phloretin (0.25 mM) did not reduce DTLupper Purea, suggesting that Purea is not due to urea transporter UT-A2, which is expressed in short-loop DTLs and short portions of some inner medullary DTLs close to the outer medulla. In summary, Purea is similar in all segments having no osmotic Pf but is significantly lower in DTLupper, a segment having high osmotic Pf. These data are inconsistent with the passive mechanism as originally proposed.

Sensitivity of ventilation and brain metabolism to ammonia exposure in rainbow trout, Oncorhynchus mykiss.

SUMMARY Ammonia has been documented as a respiratory gas that stimulates ventilation, and is sensed by peripheral neuroepithelial cells (NECs) in the gills in ammoniotelic rainbow trout. However, the hyperventilatory response is abolished in trout chronically exposed (1+ months) to high environmental ammonia [HEA; 250 μmol l−1 (NH4)2SO4]. This study investigates whether the brain is involved in the acute sensitivity of ventilation to ammonia, and whether changes in brain metabolism are related to the loss of hyperventilatory responses in trout chronically exposed to HEA (‘HEA trout’). Hyperventilation (via increased ventilatory amplitude rather than rate) and increased total ammonia concentration ([TAmm]) in brain tissue were induced in parallel by acute HEA exposure in control trout in a concentration-series experiment [500, 750 and 1000 μmol l−1 (NH4)2SO4], but these inductions were abolished in HEA trout. Ventilation was correlated more closely to [TAmm] in brain rather than to [TAmm] in plasma or cerebrospinal fluid. The close correlation of hyperventilation and increased brain [TAmm] also occurred in control trout acutely exposed to HEA in a time-series analysis [500 μmol l−1 (NH4)2SO4; 15, 30, 45 and 60 min], as well as in a methionine sulfoxamine (MSOX) pre-injection experiment [to inhibit glutamine synthetase (GSase)]. These correlations consistently suggest that brain [TAmm] is involved in the hyperventilatory responses to ammonia in trout. The MSOX treatments, together with measurements of GSase activity, TAmm, glutamine and glutamate concentrations in brain tissue, were conducted in both the control and HEA trout. These experiments revealed that GSase plays an important role in transferring ammonia to glutamate to make glutamine in trout brain, thereby attenuating the elevation of brain [TAmm] following HEA exposure, and that glutamate concentration is reduced in HEA trout. The mRNAs for the ammonia channel proteins Rhbg, Rhcg1 and Rhcg2 were expressed in trout brain, and the expression of Rhbg and Rhcg2 increased in HEA trout, potentially as a mechanism to facilitate the efflux of ammonia. In summary, the brain appears to be involved in the sensitivity of ventilation to ammonia, and brain ammonia levels are regulated metabolically in trout.

Alternative channels for urea in the inner medulla of the rat kidney

The ascending thin limbs (ATLs) and lower descending thin limbs (DTLs) of Henles loop in the inner medulla of the rat are highly permeable to urea, and yet no urea transporters have been identified in these sections. We hypothesized that novel, yet-unidentified transporters in these tubule segments could explain the high urea permeability. cDNAs encoding for Na(+)-glucose transporter 1a (SGLT1a), Na(+)-glucose transporter 1 (NaGLT1), urea transporter (UT)-A2c, and UT-A2d were isolated and cloned from the Munich-Wistar rat inner medulla. SGLT1a is a novel NH2-terminal truncated variant of SGLT1. NaGLT1 is a Na(+)-dependent glucose transporter primarily located in the proximal tubules and not previously described in the thin limbs. UT-A2c and UT-A2d are novel variants of UT-A2. UT-A2c is truncated at the COOH terminus, and UT-A2d has one exon skipped. When rats underwent water restriction for 72 h, mRNA levels of SGLT1a increased in ATLs, NaGLT1 levels increased in both ATLs and DTLs, and UT-A2c increased in ATLs. [(14)C]urea uptake assays performed on Xenopus oocytes heterologously expressing these proteins revealed that despite having structural differences from their full-length versions, SGLT1a, UT-A2c, and UT-A2d enhanced urea uptake. NaGLT1 also facilitated urea uptake. Uptakes were Na(+) independent and inhibitable by phloretin and/or phloridzin. Our data indicate that there are several alternative channels for urea in the rat inner medulla that could potentially contribute to the high urea permeabilities in thin limb segments.