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Am J Physiol Regul Integr Comp Physiol 295: R1660-R1670, 2008. First published September 17, 2008; doi:10.1152/ajpregu.90403.2008
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ENVIRONMENTAL PHYSIOLOGY

Acclimation of ion regulatory capacities in gills of marine fish under environmental hypercapnia

Katrin Deigweiher, Nils Koschnick, Hans-Otto Pörtner, and Magnus Lucassen

Alfred Wegener Institute for Polar and Marine Research, Marine Animal Physiology, Bremerhaven, Germany

Submitted 5 May 2008 ; accepted in final form 9 September 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The preservation of ion balance and pH despite environmental fluctuations is essential for the maintenance of vital cellular functions. While several ion transporters contribute to acid-base regulation in fish, the involvement and expression of key transporters under hypercapnia remain to be established. Here, two members of the HCO3– transporter family (Na+/HCO3– cotransporter NBC1 and Cl–/HCO3– exchanger AE1) were described for the first time in gills of marine fish. Benthic eelpout Zoarces viviparus were acclimated to 10,000 ppm CO2. Hypercapnia did not affect whole animal oxygen consumption over a period of 4 days. During a time series of 6 wk NBC1 mRNA levels first decreased by about 40% (8 to 24 h) but finally increased about threefold over control. mRNA expression of AE1 decreased transiently by 50% at day 4 but recovered to control levels only. Reduced mRNA levels were also found for two Na+/H+ exchangers (NHE1A, NHE1B) during the first days (by 50–60% at days 1 and 2), followed by restoration of control levels. This pattern was mirrored in a slight decrease of NHE1 protein contents and its subsequent recovery. In contrast, Na+-K+-ATPase mRNA and protein contents, as well as maximum activity, rose steadily from the onset of hypercapnia, and reached up to twofold control levels at the end. These results indicate shifting acclimation patterns between short- and long-term CO2 exposures. Overall, ion gradient-dependent transporter mRNA levels were transiently downregulated in the beginning of the disturbance. Upregulation of NBC1 on long timescales stresses the importance of this transporter in the hypercapnia response of marine teleosts. Long-term rearrangements include Na+-K+-ATPase at higher densities and capacities, indicating a shift to elevated rates of ion and acid-base regulation under environmental hypercapnia.

Zoarces viviparus; Na+-K+-ATPase; Na+/HCO3– cotransporter; Cl–/HCO3– anion exchanger; Na+/H+ exchanger; real-time polymerase chain reaction

VARIOUS ASPECTS OF ORGANISMAL acid-base regulation across fish gill epithelia have been studied extensively in the past decades, and studies of the patterns of ion and pH regulation in response to environmental stimuli have a long history (16, 19). Ion transport mechanisms have been under investigation since the discovery of the role of fish gill Na+-K+-ATPase in adaptation to seawater by Epstein et al. (15). A large variety of ion transporters in fish gills has been described (for reviews, see Refs. 16, 32, 40). With rising concern about the impact of anthropogenic CO2 emissions on ocean physicochemistry and marine organisms (reviewed by Ref. 43), the response of the ion transport machinery to ambient hypercapnia (increased CO2 partial pressure) and ocean acidification has attracted renewed interest. CO2 concentrations in atmosphere and ocean surface waters are expected to increase from current levels of 380 ppm to a maximum of 1,130 ppm until the end of the century (23a). Deep-sea carbon dioxide sequestration ventures as proposed more than 30 years ago (30) would expose marine animals to even higher CO2 partial pressures. Identification of the mechanisms affected by elevated CO2 levels in marine animals is crucial in understanding ecosystem level effects of ocean acidification.

In fish, acid-base regulation under elevated CO2 levels is achieved by a direct or indirect net accumulation of bicarbonate, accompanied by an equimolar loss of anions, mostly Cl–. An involvement of the bicarbonate transporter family seems likely (19, 27, 50). So far, two kinds of bicarbonate transporters have been described for gills of freshwater fish, the Cl–/HCO3– exchanger (named AE for anion exchanger) and the Na+/HCO3– cotransporter (NBC1), both from the solute carrier (SLC) 4 gene family (16, 44). AE1 has been localized in the apical membrane of gill epithelial cells (53), mediating Cl– import correlated with HCO3– secretion. Thus, HCO3–/Cl– exchange may play an important role during alkalosis but might also be operative during hypercapnia-induced acidosis as proposed earlier (7, 19). In the cyprinid Osorezan dace, inhabiting an extremely acidic lake, NBC1 has been localized in the basolateral membrane, mediating export of Na+ and HCO3– to the extracellular space (22). It remains to be established whether these transporters are functionally expressed in seawater fish.

Direct secretion of protons may contribute to reduce the acid load under hypercapnic conditions. Again, the mechanisms involved may differ between freshwater and seawater fish. Studies in freshwater fish have proposed active export of protons by an apical V-type H+-ATPase, coupled to an import of Na+ ions via an ENaC (epithelial Na+-channel)-like channel (10, 28). In seawater fish, use of the Na+/H+ exchanger and the existing Na+ gradient is energetically cheaper. Both the proton ATPase and sodium/proton exchange exist in seawater, as well as in freshwater fish gills but may participate in different proportions. The required extrusion of Na+ ions is mediated by the basolateral Na+-K+-ATPase, transporting Na+ against K+ into the extracellular space. Together with the secretion of Cl– ions via apical Cl–-channels, a positive transepithelial potential is generated, which is thought to drive the diffusion of Na+ ions through leaky tight junctions into the surrounding water. This process may be paralleled by the activity of a basolateral Na+/K+/2Cl– cotransporter, which mediates the import of these ions from the extra- into the intracellular space. Recycling of K+ ions is achieved by basolateral K+-channels (for reviews, see Refs. 7 and 16).

Previous studies have examined the responses of freshwater and marine fish to hypercapnia for up to 20 days, focusing on general patterns and mechanisms involved in acid-base regulation (8, 9, 27, 33, 37, 50). In the present study, we identified essential transport proteins involved in the hypercapnia response of the marine eelpout Zoarces viviparus. The viviparous eelpout with its low dispersal range has become a model organism to monitor and study the response to climate change and associated environmental factors (42). We studied transcriptional and translational patterns, as well as the functional capacity of Na+-K+-ATPase as a key enzyme driving most energy-dependent ion transport processes, including those involved in acid-base regulation over a period of 6 wk. Furthermore, we characterized two members of the bicarbonate transporter family, namely NBC1 and AE1, for the first time in marine fish gills. Together with Na+/H+ exchanger (NHE1), the expression of these ion gradient-dependent transporters was assessed. As in previous investigations 10,000 ppm of CO2 has been used in the present study to elucidate essential and responsive molecular processes under more extreme levels of hypercapnia. Future studies will need to elaborate the role of such mechanisms under CO2 concentrations as predicted by climate scenarios (23a).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and experimental procedures. Common eelpout (Z. viviparus) were obtained from the Nordseeaquarium Büsum (Germany) in May 2005 and kept in a flow-through aquarium system at 10°C under a 12:12-h day-night cycle. They were fed twice a week with North Sea shrimps ad libitum prior and during the acclimation period but not during the last 5 days before sampling. For incubations under hypercapnia, the animals were transferred to another aquarium system containing seawater equilibrated with 10,000 ppm CO2, resulting in a drop in water pH from control levels of 8.1 to 6.9. The gas mixture was provided by a mass flow controller (MKS Instruments Deutschland, München, Germany) through mixing of 1% CO2 and 99% air. pH measurements were performed with a pH-Meter (340i, Wissenschaftlich-Technische Werkstätten, Weilheim, Germany) equipped with a SenTix 81 electrode (WTW) calibrated between pH 7 and 10, according to the National Bureau of Standards. In order to take possible effects of handling into account, the control group was also transferred to another tank 24 h before sampling. Groups of 8 or 9 fishes were sampled at every designated time point (0 h control, 8 h, 24 h, 48 h, 4 days, 7 days, 14 days, 42 days). Animals were caught consecutively and stunned by a blow to the head. After weight and length determinations (overall means 40.4 ± 21.6 g, 20.2 ± 3.0 cm), the fish were killed by cutting their spine as close to the cranium as possible. From this cut, the cranium was opened and the brain was dissected first. Thereafter, gill and other tissue samples were dissected quickly, frozen in liquid nitrogen and stored at –80°C until further analysis. Tissues were not perfused before freezing, because the contribution of blood to whole gill expression levels has been previously shown to be negligible (38, 46). Furthermore, other possibly blood-contaminated tissues like liver did not show any mRNA expression of NHE1a or NHE1b in Z. viviparus at all (M. Lucassen, N. Koschnick, E. Sokolov, H. Pörtner, unpublished data). Handling and killing of the fish were conducted in conformity with the recommendations of the American Veterinary Medical Association. An approval of the work was issued by competent German authority (Freie Hansestadt Bremen, reference number 522-27-11/2-0; date: 2002-11-28).

mRNA expression of ion transporters. Total RNA from gill tissue was isolated according to the RNeasy Mini Kit (Qiagen, Hilden, Germany), and complete removal of DNA was ensured by DNase digestion with the DNA-free kit (Applied Biosystems, Darmstadt, Germany). Integrity of the RNA was controlled with a RNA 6000 Nano LabChip assay (Agilent Technologies, Waldbronn, Germany), and the concentration was determined spectrophotometrically (BioPhotometer Eppendorf, Hamburg, Germany).

For expression studies, cDNA sequences of Na+-K+-ATPase (ATN-A1) of two isoforms of the Na+/H+ exchanger (NHE1A, NHE1B) and of β-actin (26) as endogenous control were used from earlier studies (M. Lucassen, N. Koschnick, E. Sokolov, H. Pörtner, unpublished data; accession numbers: ATN-A1: EU810373, NHE1A: EU810375, NHE1B: EU810376) For the NBC1, the entire cDNA sequence was isolated from Z. viviparus using a combined approach of reverse transcriptase-linked PCR with conserved primers and rapid amplification of cDNA ends (RACE), as described earlier (31). In the same way, a partial cDNA sequence from zoarcid Cl–/HCO3– exchanger (AE1) was obtained (for primers, see Supplemental Table 1 in the online version of this article). PCR reactions were performed in a gradient cycler (TGradient, Biometra, Göttingen, Germany) as follows: 1 cycle of 94°C for 4 min, followed by 33 cycles of 94°C for 40 s, 53.1–62.9°C for 1 min, and 72°C for 1 min (final elongation at 72°C for 8 min). Separation, cloning, and analyses of PCR fragments were performed as described by Mark et al. (31). The sequences have been submitted to GenBank and can be obtained under the following accession numbers: NBC1: EU552532 and AE1: EU552533.


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  		Table 1. Primer and probe sequences used for qRT-PCR

 
Total RNA from all specimens was transcribed into cDNA with the high-capacity cDNA archive kit (Applied Biosystems, Darmstadt, Germany) and used as a template for quantitative real-time PCR performed on an ABI 7500 Real Time PCR System (Applied Biosystems). Primers and Taqman probes for real-time PCR were designed by using the Primer Express software (ver. 3.0, Applied Biosystems; see Table 1).

PCR reactions contained 0.2 to 1 ng of cDNA, 900 nM of each primer, 200 nM of each probe and 2x TaqMan PCR Master Mix (Applied Biosystems). Real-time PCR reactions were performed, according to the standardized protocol. All samples were run in triplicate, after each PCR condition had been optimized, with cDNA concentrations ranging about five orders of magnitude according to the manufacturer's instructions. To ensure that background or genomic DNA contaminations were negligible, no-template controls and no-reverse-transcribed-RNA controls were run, respectively. mRNA expression values of the different transporters were normalized against the "housekeeping" gene β-actin. Recent studies on the suitability of "housekeeping" genes emphasized the necessity of testing the expression stability of these genes (21, 35). Depending on experimental conditions, animal species and tissue type, not every "housekeeping" gene can serve as an applicable endogenous control. In case of Z. viviparus gill tissue, expression of β-actin did not change significantly over time to the extent that the genes under study did. For the calculation of relative expession levels under condition "n" compared to condition "control" (0), the comparative Ct method was used: {formula: 2^[(Cttransporter,n – Ctβ-actin,n) – (Cttransporter,0 – Ctβ-actin,0)]}, where Ct corresponds to the threshold cycle number.

Protein quantification. Frozen gill tissue samples were quickly homogenized with a conical glass tissue grinder in 10 volumes of ice-cold buffer [50 mM imidazole, pH 7.4, 250 mM sucrose, 1 mM EDTA, 5 mM β-mercaptoethanol, 0.1% (wt/vol) deoxycholate, protease inhibitor cocktail from Sigma-Aldrich, Taufkirchen, Germany; Cat. No. P 8340] followed by Ultra Turrax treatment (3 x 10 s). Cell debris was removed by centrifugation for 10 min at 1,000 g and 0°C. One-half of the supernatant was used as a crude extract for Na+-K+-ATPase activity measurements and protein quantification, and the other half was used for membrane preparation by centrifugation for 1 h at 20,000 g and 0°C. The sedimented membrane fraction was resuspended in 2 volumes of extraction buffer and was used for NHE1 protein quantification. Total protein contents of crude and membrane extracts were determined according to the method of Bradford (2), using BSA as the standard.

For immunoblotting, 8 µl of crude extracts for Na+-K+-ATPase and 20 µl of membrane extracts for Na+/H+ exchanger were used. Proteins were fractionated by SDS-PAGE on 10% polyacrylamide gels, according to Laemmli (26), and transferred to PVDF membranes (Bio-Rad, Munich, Germany), using a tank blotting system (Bio-Rad). Blots were preincubated for 1 h at room temperature in TBS-Tween buffer [TBS-T, 50 mM Tris·HCl, pH 7.4, 0.9% (wt/vol) NaCl, 0.1% (vol/vol) Tween20] containing 5% (wt/vol) nonfat skimmed milk powder. As primary antibody for the Na+-K+-ATPase {alpha}-subunit, the {alpha}5 monoclonal antibody (culture supernatant: 1:100), originally developed against the chicken {alpha}-subunit by D. M. Fambrough (John Hopkins University, Baltimore, MD), was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Development, maintained by the University of Iowa, Department of Biological Sciences, Iowa City, Iowa. For NHE1 mouse monoclonal antibody 4E9 raised against porcine NHE1 (45), (Chemicon, Temecula, CA) was used in the same buffer (2 µg in 5 ml). Blots were incubated with primary antibodies at 4°C overnight. After washing with TBS-T, blots were incubated for 1 h with goat anti-mouse IgG antibody (horseradish peroxidase conjugated; Pierce, Rockford, IL) diluted 1:2,000 in TBS-T containing 5% nonfat skimmed milk powder. Protein signals were visualized by using the ECL Western blotting detection reagents (GE Healthcare, Munich, Germany) and recorded by a LAS-1000 charge-coupled device camera (Fuji, Tokyo, Japan). Signal intensity was calculated using the AIDA Image Analyzer software (ver. 3.52, Raytest, Straubenhardt, Germany), and results were expressed as values normalized to the 0 h control. One randomly chosen sample was used on every gel for normalization of different immunoblots. Membrane preparations were used to determine the optimal concentration ratio for antigen over primary and secondary antibody. For quantification, an intermediate protein concentration was used in a range, where the signal changed linearly with antibody binding.

Na+-K+-ATPase activity. Na+-K+-ATPase activity was measured in gill crude extracts (see Protein quantification) in a coupled enzyme assay with pyruvate kinase (PK) and lactate dehydrogenase (LDH) using the method of Allen and Schwarz (1). The reaction was started by adding the sample homogenate to the reaction buffer containing 100 mM imidazole, pH 7.4, 80 mM NaCl, 20 mM KCl, 5 mM MgCl2, 5 mM ATP, 0.24 mM Na-(NADH2), 2 mM phosphoenolpyruvate, about 12 U/ml PK and 17 U/ml LDH, using a PK/LDH enzyme mix (Sigma-Aldrich). The oxidation of NADH coupled to the hydrolysis of ATP was followed photometrically at 10°C in a DU7400i spectrophotometer (Beckman Coulter, Krefeld, Germany) over a period of 10 min, measuring the decrease of extinction at {lambda} = 339 nm. The fraction of Na+-K+-ATPase activity in total ATPase (TA) activity was determined by the addition of 5 mM ouabain to the assay. Enzyme activity was calculated using an extinction coefficient for NADH of {varepsilon} = 6.31 mM–1·cm–1 and given as micromoles consumed ATP per gram tissue fresh weight (fwt) per hour.

Standard metabolic rate. Animals were starved for 2 days prior to and during the experiments. Standard metabolic rates (SMR) were determined using intermittent closed respirometry. Briefly, animals weighing 37.7 ± 6.2 g (n = 11) were incubated in cylindrical perspex chambers (diameter = 3 cm, length = 25 cm) for a period of 10 days. For control measurements and during prehypercapnia and posthypercapnia treatments, chambers were perfused with freshly aerated (100% air) seawater from a reservoir, using a peristaltic pump (ISM 404B, Ismatec, Wertheim-Mondfeld, Germany) and gas-tight tubing (Tygon T4406-23, Saint-Gobain Performance Plastics, Charny, France). For hypercapnia a gas mixture of 1% CO2 and 99% air was provided by a gas mixing pump (Wösthoff, Bochum, Germany). Water flow rates of 60 to 66 ml/min ensured chamber oxygen partial pressures of approximately 18 to 20 kPa between measurements. Temperature was maintained at 10°C (± 0.2°C) by placing the four replicate chambers in a temperature-controlled water bath. Respirometry runs were performed twice a day (11:00 AM, 4:00 PM), by creating a closed-loop circulation. Oxygen partial pressures were measured using a fiber-optic oxygen sensing system (Oxy-4 micro, PreSens, Regensburg, Germany) with oxygen optodes (needle-type microsensors, PreSens) incorporated into the closed loop. Within 10 to 12 min, animals usually had reduced chamber oxygen levels to 14–16 kPa. Oxygen consumption rates were calculated from linear declines in chamber oxygen partial pressure with time using the following formula: M(O2) = {Delta}P(O2) β(O2) V w–1, where M(O2) is the oxygen consumption rate [µmol(O2)·g–1·h–1], {Delta}P(O2) is the gradient of oxygen decrease over time in the chamber [kPa], β(O2) is the oxygen capacity of water [µmol(O2)·l–1·kPa–1], V is the volume of the closed chamber loop (liters), and w is the animal weight (g).

Statistics. Values were expressed as means ± SE. Because of the limited amount of gill tissue available from some animals, two or four among the total amount of samples for each treatment were pooled from two individual fish of same size and gender. The pooled sample was treated as n = 1 in the statistical analysis. Outliers were identified at the 95% significance level using Nalimov's test and were removed. Statistical significance was tested with the remaining samples at the P < 0.05 level using one-factor ANOVA and the post hoc Dunnett's test for comparing the samples from different time points of hypercapnia incubation to the 0 h control sample. Where indicated, additional ANOVA tests were performed to identify significant changes during the early or long-term phase of hypercapnia exposures. For SMR measurements, two-factor ANOVA and the post hoc Student-Newman-Keuls' test for all pairwise multiple comparisons have been used to test for significant influences of the factor time and the factor hypercapnia on whole animal oxygen consumption.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
All molecular and functional results were obtained from measurements in gill tissue samples taken during the hypercapnia (10,000 ppm CO2) trial at 0 h (control), 8 h, 24 h, 48 h, 4 days, 7 days, 14 days, and 42 days.

Ion transporter mRNA expression. The mRNA expression levels of the Na+/H+ exchanger first decreased at the onset of hypercapnia but recovered to control levels thereafter and remained unchanged until the end of the incubation period (fig. 1A). The dominant isoform NHE1A, which was generally found expressed at 10-fold higher levels than isoform 1B, decreased by about 60% during the first 2 days and recovered slowly thereafter, while isoform 1B showed an initial 50% decrease and was already fully recovered after 2 days (Fig. 1A). The Cl–/HCO3– anion exchanger AE1 showed an expression pattern with minimal expression after day 4 (50% below control levels, albeit marginally and only significant when testing 4 days against control and 42 days) followed by full recovery to control values (Fig. 1B). After an initial, slight decline within the first 2 days (40%), Na+/HCO3–-cotransporter mRNA was stabilized until week 2 but was upregulated to about 300% after 6 wk of acclimation (Fig. 1C). In contrast to gradient-dependent ion transporters, mRNA expression of Na+-K+-ATPase increased significantly during the first 24 h after the onset of the CO2 treatment and was maintained—with the exception of day 4—at a significantly higher level of about 200% for the remaining time period (Fig. 1D).


Figure 1
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  		Fig. 1. Gill ion transporter gene expression during 6 wk of exposure to 10,000 ppm hypercapnia. A: Na+/H+ exchanger (NHE1) isoforms A and B. B: Cl–/HCO3– anion exchanger (AE1), C: Na+/HCO3– cotransporter (NBC1), D: Na+-K+-ATPase (ATN-A1). Expression is given relative to the expression of β-actin and is normalized to the respective 0 h controls. Significant differences compared to the 0 h control group (P < 0.05) are indicated by asterisks (one-factor ANOVA). Asterisks in parentheses indicate significant differences to controls according to one-factor ANOVA for the acute (24 h) or long-term phase (up to 6 wk) of acclimation. The acute phase is depicted by gray shading. Values are expressed as means ± SE (n = 5–7).

 
Ion transporter protein levels. Na+-K+-ATPase and Na+/H+ exchanger protein levels were determined by immunoblotting (fig. 2). After cell fractionation, both antibodies detected a single, clear band depicting the expected size in the membrane fraction; no signals were detectable in the cytosol (data not shown). For Na+-K+-ATPase, in some samples, degradation products became visible due to the sensitivity of the detection system, which account for up to 5% of the main signal (not included into the quantification). Because of the high abundance of Na+-K+-ATPase, the respective antibody reaction was quantified in crude extracts, while NHE1 was determined in isolated membrane fractions. Since antibody NHE 4E9 was raised against porcine NHE1, it is reasonable to assume that this antibody did not differentiate between the two zoarcid NHEs, which are both closely related to mammalian isoform 1. Protein levels tended to follow the patterns of mRNA levels and were slightly decreased within the first 2 days, albeit only significantly for day 1 when using ANOVA for this early time frame (Fig. 3A). Thereafter, NHE protein levels returned back to control values. Na+-K+-ATPase protein levels were found significantly increased after 2 days of hypercapnia and were maintained about 25 to 35% above control levels until the end of the trial (Fig. 3B).


Figure 2
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  		Fig. 2. Quantification of Na+-K+-ATPase (A) and Na+/H+ exchanger (B) protein levels in control animals (n = 6) or after 6 wk of hypercapnia (10,000 ppm) (n = 6). Distinct immunoreactive bands were observed for {alpha}–ATN-A1 and NHE1 with monoclonal antibodies corresponding to molecular masses of about 105 kDa and about 90 kDa, respectively. Sample 3 (6 wk) showed significant degradation of the Na+-K+-ATPase protein and was removed according to the outlier test. A reference sample (#) was used to equalize signal intensities from different blots.

 

Figure 3
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  		Fig. 3. Gill ion transporter protein abundance under 10,000 ppm hypercapnia. Expression is normalized to the respective 0 h controls. A: Na+/H+ exchanger (NHE1), B: Na+-K+-ATPase {alpha}-subunit (ATN-A1). Significant differences compared to the 0 h control group (P < 0.05) are indicated by asterisks (one-factor ANOVA). Asterisks in parentheses indicate significant differences according to one-factor ANOVA for the early phase of acclimation, which is depicted by gray shading. Values are expressed as means ± SE (n = 5–7).

 
Na+-K+-ATPase activity. Functional capacities of Na+-K+-ATPase rose steadily during the hypercapnia trial. The maximum activity of Na+-K+-ATPase was determined at 114.6 ± 16.2 µmol ATP·gfwt–1·h–1. From day 2 onward, levels were significantly increased and reached a 60% higher activity level at the end of the 6-wk trial (Fig. 4).


Figure 4
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  		Fig. 4. Specific gill Na+-K+-ATPase activity under hypercapnia treatment (10,000 ppm). Activity is given in micromoles ATP per gram fresh weight (fwt) per hour. Significant differences compared to the 0 h control group (P < 0.05) are indicated by asterisks (one-factor ANOVA). Values are means ± SE (n = 5–7).

 
Standard metabolic rate. Oxygen consumption rates of Z. viviparus (n = 7) during acute hypercapnia are presented in Fig. 5. Starting from significant elevated values of about 2.5 µmol (O2)·g–1·h–1, oxygen consumption stabilized after 2.5 days at about 1.5 µmol (O2)·g–1·h–1. The onset of hypercapnia, indicated by the pH drop from 8.1 to 6.9, had no influence on SMR, which remained unchanged for 4 days, and also during 3 days of recovery under normocapnia at 1.54 ± 0.21 µmol (O2)·g–1·h–1 (means ± SD). In two control animals, the same stabilization pattern during the first 3 days became apparent, with a constant respiration rate of 1.55 ± 0.24 µmol (O2)·g–1·h–1 for the following 8 days (means ± SD). Generally, eelpouts are sluggish fish and an excellent model for SMR measurements, since they prefer to hide in small caves or tubes without moving during normal maintenance, as also reported earlier (52). In the present study, the observed activity levels were minimal during the whole experimentation.


Figure 5
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  		Fig. 5. Standard metabolic rate (SMR) of Zoarces viviparus under short-term hypercapnia (10,000 ppm). The left y-axis shows SMR as micromoles O2 consumed per gram fish weight per hour. The right y-axis shows the change in water pH during the hypercapnia trial. After 3 days of normocapnia, CO2 concentrations were increased for 4 days (indicated by the pH drop from 8.1 to 6.9; gray circles). Thereafter, recovery under normocapnia was monitored for 3 days. Values are expressed as means ± SE (n = 7; diamonds). Respiration rates of two control animals (triangles: median ± SD) were measured over a period of 11 days under normocapnia. Two-way ANOVA with the factors time and hypercapnia revealed significantly elevated oxygen consumption levels for the first two time points compared to the remaining time period, but there was no significant influence of hypercapnia exposure.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Acid-base and associated ion regulation in fish gills comprise about 90% of the organism's total acid-base regulation (7, 16, 18, 19, 41). Studies of the genetic basis and molecular structure of essential ion transporters have focused on the involvement of specific ion transporters in acid-secretion such as Na+/H+ exchanger, Na+/HCO3– exchanger, V-type H+-ATPase, and a putative H+/K+-ATPase, or on the contrasting role of Cl–/HCO3– exchangers for base secretion (5, 6, 13, 39, 49, 51).

In marine teleosts (Perciformes, Salmoniformes, Anguilliformes), hypercapnia levels such as those applied in the present study lead to a transient drop of extracellular and intracellular pH values (27, 33, 37, 50). Blood pH is usually fully restored to control levels within 10–24 h [with the exception of Sparus aurata, in which a small, but significant decrease in pHe was still visible at day 3 (33)]. pH compensation may involve net proton release but ultimately leads to the net accumulation of bicarbonate in all investigated species (27, 33, 37, 50).

Responses of the Na+-K+-ATPase. Na+/K+-ATPase is expected to be the key enzyme providing the driving force for many gradient-dependent transport processes in the membranes of fish gills. Although the molecular regulation of Na+-K+-ATPase has been studied in fish with regard to osmoregulation, temperature, and development (reviewed in Ref. 32), only a few studies have examined its response to hypercapnia. Seidelin et al. (47) found Na+-K+-ATPase mRNA levels of developing smolts (Atlantic salmon) reduced under short-term hypercapnia (4 days, 20,000 ppm CO2), while its enzymatic activity remained unaffected. In contrast, Ishimatsu et al. (24) reported a significant increase of Na+-K+-ATPase activity in Japanese flounder after exposure to 1% and 5% hypercapnia (unpublished data cited in Ref. 24).

In the gills of Z. viviparus mRNA expression, protein abundance and functional capacities of Na+-K+-ATPase were upregulated during 6 wk of hypercapnia. The present data on the control Na+-K+-ATPase capacities are identical to earlier measurements in this species [120.4 ± 9.2 µmol ATP·gfwt–1·h–1 (n = 8); K. Deigweiher and M. Lucassen, unpublished data], sampled 3 mo earlier. Thus, the Na+-K+-ATPase capacities are stable over time, if no further treatment is applied, and the observed responses in the present study can be clearly attributed to the hypercapnia stress. The transcriptional response of Na+-K+-ATPase took place within 2 days after the onset of hypercapnia. While mRNA increased immediately, the protein level seemed to initially decrease and respond to the mRNA increments with a delay of about 24 h. At day 4, mRNA had returned to control levels, which was apparently mirrored in a decreasing protein level at day 7. Thereafter, mRNA and protein levels increased to 200 and 140% of control levels, respectively, and remained elevated until the end of the 6-wk acclimation period.

Similar regulatory patterns involving a delay between the transcriptional and functional upregulation of Na+-K+-ATPase activity in fish gills have been seen after salinity transfer experiments. In the gills of Atlantic salmon smolts, there was a direct increase of Na+-K+-ATPase mRNA expression after 24 h, followed by an increase in activity after 11 days, when mRNA levels had transiently returned to control levels. Values started to increase again at the end of the trial after 25 days (11). During short-term hyposmotic shock in the milkfish Chanos chanos, Lin et al. (29) found within an "adjustive phase" significantly increased Na+-K+-ATPase activity (after 3 h) and elevated protein contents (after 12 h). After return of these values to control levels within 24 h, mRNA expression increased only within the "regulatory phase" (48–96 h), followed by another increase of protein abundance and activity.

Overall, higher functional capacities of Na+-K+-ATPase support ion- and acid-base status under environmental hypercapnia. This increment is apparently achieved by progressively higher transcription and translation levels of the respective gene, involving continuous upregulation during the long-term treatment (6 wk). The regulatory stimulus for upregulation remains unclear. The similarity of the patterns observed in the present study to those seen under osmotic stress indicates that gene expression may respond to changes in demand rather than, for example, transient hypercapnia-associated acidosis. Na+-K+-ATPase provides an ion gradient, which directly drives transporters involved in proton equivalent ion exchange. The elevated demand on steady-state acid-base regulation may thus trigger the upregulation of Na+-K+-ATPase.

Implications of hypercapnia for gradient-dependent ion transporters. Gene expression of gradient-dependent ion transporters was regulated differently from that of Na+-K+-ATPase. In general, a short-term repression in the beginning was followed by a restoration until the end of the hypercapnia trial, with the exception of a long-term accumulation of Na+/HCO3– cotransporter transcripts after 6 wk. Our findings are in accordance with the mechanisms generally found for marine fish under environmental hypercapnia. During the initial stage of acclimation, the pH drop in response to CO2 accumulation is compensated for by nonbicarbonate buffering, which leads to a limited rise in plasma bicarbonate levels. Further pH compensation is probably achieved by net importing bicarbonate from the environment via epithelial ion transporters, since higher environmental bicarbonate concentrations support the recovery rate (19). In Scyliorhinus stellaris, environmental HCO3– concentration was even revealed as the limiting factor for acid-base relevant ion transfer (19, 20). Additionally, higher proton export rates would support higher serum levels of bicarbonate originated from endogenous sources. Increments in extracellular bicarbonate levels usually involve a decrease of Cl– levels (9, 27). Therefore, existence of a Cl–/HCO3– exchange mechanism at the apical membrane was postulated (19). A Na+/HCO3– cotransporter in the basolateral membrane may support net bicarbonate transport from epithelial cells into the blood. However, Cl–/HCO3– exchanger (49, 53) and Na+/HCO3– cotransporter (22, 39) have only been characterized in the gills of freshwater fish so far.

The role of the Na+/HCO3– cotransporter in acid-base regulation has been explored in limnic fish. In the Osorezan dace (Tribolodon hakonensis), which live in a naturally extremely acidic lake, the protein has been localized by immunostaining at the basolateral membrane of gill chloride cells using a primary antibody against a C-terminal fraction of NBC1 (22). Also, mRNA expression increased within 5 days after transfer to low-pH water (pH 3). Similarly, NBC1 mRNA expression was increased under acute hypercapnia (10,000 ppm, 6 h) in gills of the rainbow trout O. mykiss (39). In marine Z. viviparus, however, NBC1 mRNA level was 50% downregulated during the first 24 h of hypercapnia (Fig. 1C). After the onset of hypercapnia, nonbicarbonate buffering and the accumulation of metabolically produced bicarbonate may reduce the need for HCO3– transport into the plasma. Furthermore, if bicarbonate accumulates in the epithelial cells, the hydration of CO2 and dissociation to protons and bicarbonate are downregulated. This would minimize the production of H+ in the cells, which supports the capacity for pH recovery (see Fig. 6).


Figure 6
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  		Fig. 6. Preliminary model of acute/short-term (A) and long-term (B) adjustments to environmental hypercapnia in the gills of seawater teleosts [modified after Claiborne et al. (7) and Evans et al. (16)]. CO2 is hydrated by carbonic anhydrase (CA) after diffusive entry resulting in HCO3– and H+. A: Acute pH compensation is achieved by nonbicarbonate buffering together with net H+ extrusion supported by transitional downregulation (–) of the basolateral Na+/H+ exchanger (NHE1) and Na+/HCO3– cotransporter (NBC1). Na+-K+-ATPase (NKA) expression is maintained and then upregulated (=/+). A delayed downregulation (=/–) of apical Cl–/HCO3– anion exchanger (AE1) supports the maintenance of higher bicarbonate levels in the cell and plasma when extracellular pH is already restored. During long-term compensation (B), net accumulation of extracellular HCO3– is supported by an increase (+) in the abundance of basolateral NBC1 and the maintenance (=) of AE1 and NHE1 levels compared to controls. Net Cl– decrease in blood may be mediated by a basolateral Na+/K+/2Cl– cotransporter (NKCC) and apical Cl– channels. NHE1 is operating at control levels under long-term steady state conditions. Net proton extrusion is possibly achieved by apical NHE2/3. The driving force for the new steady state is provided by elevated Na+-K+-ATPase levels in the basolateral membrane. Excess Na+ can diffuse via leaky tight junctions into the surrounding water.

 
Extended hypercapnia exposure led to the restoration of NBC1 mRNA within 2 wk and to a threefold higher expression at the end of the trial, indicating a differentiated response to acute vs. long-term hypercapnic conditions. As with Na+-K+-ATPase and NHE1, the change in message likely results in changing protein levels. In case of the Na+/HCO3– cotransporter, elevated protein contents would support maintenance of elevated bicarbonate levels (Fig. 6B), as described for several fishes (9, 27, 33, 50). The threefold increment of NBC1 mRNA at the end of the trial emphasizes an important role of this transporter in steady-state acid-base regulation under long-term hypercapnia.

Since HCO3– accumulation under hypercapnia is usually accompanied by a decrease of Cl– concentration, a role for gill Cl–/HCO3– exchange has been postulated (see introduction). However, AE1 mRNA expression in Z. viviparus was only slightly affected, with a trend to decrease initially under hypercapnia, reaching a 50% decrease at day 4, after which time it recovered slowly to control levels until the end of the trial (Fig. 1B). This pattern is not in line with a rising importance of Cl–/HCO3–-exchange under hypercapnia. As AE1 is abundantly expressed in fish erythrocytes (17, 23), a contribution of blood mRNA to total tissue mRNA expression level might be possible. For NHE1A or NHE1B, blood contamination of the patterns seen in gills in the present study is likely negligible (see MATERIALS AND METHODS). Furthermore, Perry et al. (38) found no obvious contribution of blood mRNA to total tissue mRNA expression levels of H+-ATPase, although also being expressed in blood cells. The existence and location of AE1 have been identified at least in freshwater fish gills (53). Thus, a functional role of AE1 in marine fish gills seems likely, but the localization and thus the transepithelial transport direction might be completely different. If AE1 in seawater fish gills is located apically, our findings would be in line with a role for the exchanger in base release. As base release is likely reduced during the transient acidosis, the putative decrement of AE1 content would cause lower Cl– influx and support net bicarbonate uptake by reducing HCO3– loss (Fig. 6A). Alternatively, AE1 might be localized basolaterally in marine fish. Although this localization could support higher plasma bicarbonate and lower chloride levels under hypercapnia, the expression of this transporter remained at control levels after 6 wk. Therefore, a role of AE1 for direct basolateral Cl–/HCO3– exchange under hypercapnia seems unlikely, and the regulation pattern under acute and long-term hypercapnia would be most consistent with a role for apical AE1.

Two mechanisms have emerged in recent years for the net release of protons from the organism via the gills: ATP-consuming proton extrusion via a V-type H+-ATPase, electrochemically coupled to a Na+ channel, and electroneutral Na+/H+ exchange via a gradient-driven ion transporter. The latter might be more important in marine fish, because the intrusion of Na+ would be favored by the inward gradient of Na+. In both cases the extra Na+ load would have to be compensated for by Na+-K+-ATPase (for reviews, see Refs. 7, 16, 41). For Z. viviparus, two isoforms of the Na+/H+ exchanger have been isolated with degenerated primers for NHE1. Both isoforms differ by about 20% (comparable to the interspecies differences within teleosts) and clearly belong to the NHE1 isoform family when compared with NHE2 or NHE3. No mRNA expression of NHE1A or NHE1B could be detected in liver or white muscle of Z. viviparus (M. Lucassen, N. Koschnick, E. Sokolov, H. Pörtner, unpublished data). Thus, further isoform differentiation may have taken place in the teleost branch, possibly accounting for the specific requirements within the marine environment. For both isoforms, mRNA expression was decreased by about 50% during the first days of hypercapnia, followed by a recovery to control values until the end of the trial. Isoform 1A was expressed 10-fold higher than isoform 1B, and the recovery of the former took about 2 days longer. The protein abundance of NHE1 was measured by immunoblotting with an antibody that has been successfully used in several other studies to specifically identify NHE1 in marine and euryhaline teleosts and elasmobranchs (4, 6, 14). The decrease after 2 days (probably representing the more abundant isoform 1A) followed by restoration to control levels thus parallels the mRNA data.

The functional role of different NHE isoforms may be relevant to understand how downregulation of the Na+/H+ exchanger genes and of their protein expression in the early phase could help to overcome hypercapnic acidosis. So far eight distinct isoforms of the SLC9 gene family have been found in mammals (reviewed in Ref. 36). In marine fish, the existence of at least three isoforms (NHE1, NHE2, and NHE3) has been demonstrated by use of antibodies and/or sequencing (reviewed in Refs. 7 and 16). Recently, Edwards et al. (14) studied NHE isoform expression in the gills of freshwater (FW) and seawater (SW) adapted euryhaline fish Fundulus heteroclitus after 1 h of hypercapnia treatment (10,000 ppm CO2) and found an increase in the expression of NHE2 in FW fish, while in SW fish, both NHE1 and NHE3 were increased. These findings are not in line with our study, which is rather in line with an earlier study by the same group, in which a decrease in NHE1 after metabolic acidosis was found in the gills of marine sculpin. The authors postulated that NHE1 is localized at the basolateral membrane (6). Downregulation of NHE1 in the beginning would then favor net acid excretion, thereby counteracting the acute effects of hypercapnia, while isoform NHE2/3 at the apical membrane is maintained or even upregulated (Fig. 6A). Indeed, NHE2 has been localized in the apical membrane, and protein abundance remained constant after acute acidosis over 8 h in gills of the marine teleost Myoxocephalus octodecemspinosus (3). Further isoforms at the apical membrane might thus be involved in H+ secretion. However, the existence of such isoforms remains unconfirmed for Z. viviparus despite analysis by means of different antibodies or by cDNA approaches.

Proposed working model of gill ion transporter regulation under hypercapnia. The present study provides insight into shifting roles of transporters in acid-base regulation during acclimation to environmental hypercapnia. Even though changes in protein levels might be less pronounced than responses of mRNA expression to hypercapnia, the shifts in mRNA levels found here for AE1 and NBC1 over time likely reflect the respective changes in protein levels. In line with available literature, we propose a biphasic acclimation mechanism to hypercapnia for Z. viviparus. The acute response within the gill, when the pH drop is being compensated for through nonbicarbonate buffering and stimulated ion exchange by the existing transporter inventory, occurs within minutes to hours. Within this recovery phase (usually completed within 10–24 h), a first response at the mRNA level takes place by downregulation of basolateral NHE1 (after 24 h) and NBC1 (after 8 h). Within the epithelial cells, this may support higher bicarbonate levels and subsequent inhibition of carbonic anhydrase on the one hand (NBC1) and reduced proton export into the plasma on the other (NHE1). Net H+ extrusion by further transporters (e.g. NHE2 and NHE3) has to be postulated to explain the recovery of pH (Fig. 6A). After this initial compensation, the protein inventory is rearranged long term to meet the requirements of the new ion composition. During this acclimatory phase, when pH compensation is most probably completed, short-term downregulation of AE1 (day 4) may support the maintenance of the now required higher bicarbonate concentration (Fig. 6A). Accordingly, Na+-K+-ATPase as the driving force of ion and pH regulation is maintained from the beginning and then progressively upregulated under long-term hypercapnia up to 6 wk and beyond (Fig. 6, A and B). For Na+-K+-ATPase, a feedback regulation of mRNA and protein levels in the early acclimation phase became visible, as previously postulated under hyposmotic exposure (29). For maintaining the new steady-state of acid-base regulation, with elevated HCO3– and lower chloride levels in the serum, net proton release is likely reduced, and ion transport capacities are rearranged and adapted to the new requirements, with restoration of Cl–/HCO3– exchanger AE1 and Na+/H+ exchanger NHE1 and upregulation of the Na+-HCO3– cotransporter NBC1 (Fig. 6B).

Consequences of hypercapnia for whole animal performance? Whole animal oxygen consumption (Fig. 5), which was similar to values reported for Z. viviparus earlier (52), was not affected by short-term hypercapnia. Thus, severe impairments of the marine fish under hypercapnic conditions (10,000 ppm) were not detectable. Nonetheless, the expression patterns indicate rearrangements of the protein inventory during the early phase of hypercapnic exposure. It is assumed that the energy demand of ion and pH regulation is elevated if the main ion transporter—the Na+-K+-ATPase—operates at an elevated activity level. In isolated gill tissue and perfused gills, respectively, the energy demand of the Na+-K+-ATPase accounted for up to 30% of total oxygen consumption (34, 48). Theoretical calculations of the contribution of epithelial ion transport to whole animal oxygen consumption range from 0.5 to 15% in seawater fish (12, 25). Thus, the increased Na+-K+-ATPase capacities found in the present study appear to be too small to be detectable within SMR measurements. Furthermore, an increased energy demand of ion regulation might be compensated for through decreased rates of protein turnover, anabolism, or activity levels. As the observed levels of motor activity were minimal in these sluggish benthic fish under all experimental conditions applied, a response at that functional level can be excluded as unlikely. Energy allocation in hypercapnia-exposed animals would have to be determined to assess relative shifts in energy budget associated with hypercapnia. Negative effects on whole animal performance like those on growth are conceivable, as Na+-K+-ATPase is operating at a higher rate under hypercapnia.

Perspectives and Significance

Investigation of further transporters and other isoforms, as well as differentiation between cell types, will be required in future analyses to deepen the understanding of the complex interaction of ion transport mechanisms. Furthermore, the regulatory signals eliciting the shift in gene expression of ion transport mechanisms and their response to rates and steady-state parameters of ion and acid-base status require elaboration. The present study already points to NBC1 and Na+-K+-ATPase as key transporters supporting long-term ion and acid-base regulation under hypercapnia. The analyses of these transporters under the predicted CO2 scenarios, especially in combination with proposed temperature increments may serve as sensitive markers for the detection of long-term effects on the animals' resilience.


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The study was supported by a student grant from the University of Bremen.


   ACKNOWLEDGMENTS
 
The authors would like to thank Boris Klein for aquarium maintenance and the acquisition of animals, and Dr. Lars G. Eckerle for support during fish sampling. Magdalena Gutowska and Dr. Frank Melzner are thankfully acknowledged for their support with the respiration measurements and helpful discussions.

A contribution to the MARCOPOLI research program of the Alfred Wegener Institute (POL4: Response of higher marine life to change).


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Lucassen, Alfred Wegener Institute for Polar and Marine Research, Marine Animal Physiology, Am Handelshafen 12, D-27570 Bremerhaven, Germany (e-mail: Magnus.Lucassen{at}awi.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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