Branchial NH4+-dependent acid–base transport mechanisms and energy metabolism of squid (Sepioteuthis lessoniana) affected by seawater acidification
© Hu et al. 2014
Received: 18 March 2014
Accepted: 22 July 2014
Published: 6 August 2014
Cephalopods have evolved strong acid–base regulatory abilities to cope with CO2 induced pH fluctuations in their extracellular compartments to protect gas transport via highly pH sensitive hemocyanins. To date, the mechanistic basis of branchial acid–base regulation in cephalopods is still poorly understood, and associated energetic limitations may represent a critical factor in high power squids during prolonged exposure to seawater acidification.
The present work used adult squid Sepioteuthis lessoniana to investigate the effects of short-term (few hours) to medium-term (up to 168 h) seawater acidification on pelagic squids. Routine metabolic rates, NH4+ excretion, extracellular acid–base balance were monitored during exposure to control (pH 8.1) and acidified conditions of pH 7.7 and 7.3 along a period of 168 h. Metabolic rates were significantly depressed by 40% after exposure to pH 7.3 conditions for 168 h. Animals fully restored extracellular pH accompanied by an increase in blood HCO3− levels within 20 hours. This compensation reaction was accompanied by increased transcript abundance of branchial acid–base transporters including V-type H+-ATPase (VHA), Rhesus protein (RhP), Na+/HCO3− cotransporter (NBC) and cytosolic carbonic anhydrase (CAc). Immunocytochemistry demonstrated the sub-cellular localization of Na+/K+-ATPase (NKA), VHA in basolateral and Na+/H+-exchanger 3 (NHE3) and RhP in apical membranes of the ion-transporting branchial epithelium. Branchial VHA and RhP responded with increased mRNA and protein levels in response to acidified conditions indicating the importance of active NH4+ transport to mediate acid–base balance in cephalopods.
The present work demonstrated that cephalopods have a well developed branchial acid–base regulatory machinery. However, pelagic squids that evolved a lifestyle at the edge of energetic limits are probably more sensitive to prolonged exposure to acidified conditions compared to their more sluggish relatives including cuttlefish and octopods.
KeywordsAcid–base regulation Invertebrate Metabolism Ocean acidification Rh proteins
In teleost fish acid–base regulating epithelia and organs have been extensively studied and the sub-cellular organization of ion transporters localized in mitochondria-rich cells is well described e.g. –. Besides primary active proton extrusion mechanisms via VHA these models suggest an import of HCO3− and export of protons by secondary active transporters such as NHEs and Na+-dependent HCO3− transporters of the SLC4 solute transporter family energized by the NKA located in basolateral membranes. Although a large body of knowledge is available for teleosts, a comparatively small number of studies investigated acid–base and ion regulatory mechanisms in non-model invertebrates like mollusks, echinoderms and crustaceans. For the latter a number of studies exist with osmotic and acid–base regulatory mechanisms recently summarized by Henry et al. , demonstrating the complexity of ion-transport processes in gill epithelia of crustaceans. Although the picture seems more complete for osmoregulatory mechanisms in crustacean gill epithelia the mechanistic basis for acid–base regulation seems largely unexplored as well. Crustaceans were also characterized as strong acid–base regulators that are capable of accumulating high concentrations of HCO3− in their body fluids to buffer an excess of protons. It has been pharmacologically demonstrated that low pH conditions trigger the response of carbonic anhydrase (CA), apical VHA and basolateral Na+/HCO3− exchanger in perfused gills of the euryhaline crab Neohelice (Chasmagnathus) granulata. Furthermore, NHE-dependent acid–base regulation has been suggested in the blue crab that responded with an increase in Na+ uptake when exposed to hypercapnic conditions of 1% CO2. Pharmacological studies suggested the presence of NHEs and VHA in apical membranes of crustacean gills. Another potential model for proton equivalent secretion in crustacean gills has been proposed by Weihrauch and colleagues , suggesting trapping of NH4+ in VHA-rich vesicles and subsequent exocytosis across the apical membrane. The entrance of NH4+ across the basolateral membrane is achieved by the NKA, which may also accept NH4+ as a substrate or by K+/NH4+ channels. In this context a Rhesus protein (RhP) cloned from Dungeness crab Metacarcinus magister is proposed to be substantially involved in branchial NH3/NH4+ regulation during exposure to a high NH4+ environment .
Interestingly, a range of marine invertebrates including bivalves, echinoderms and crustaceans responded with increased NH4+ excretion rates when exposed to acidified conditions –. It has been suggested that this phenomenon may be associated with enhanced protein metabolism to fuel increased acid–base regulatory costs, and/or may support NH4+ based proton equivalent secretion to mediate pH homeostasis. In vertebrates, the excretion of NH4+ mediated by Rhesus C glycoprotein (Rhcg) and Rhesus B glycoprotein (Rhbg) in combination with NHE3 or VHA located in apical membranes has been demonstrated to be connected to net export of protons –. Thus, it can be hypothesized that NH4+-based proton secretion also represents a fundamental pH regulatory pathway, probably connected to a reallocation of energy sources, in marine invertebrates.
The present work aims at identifying and characterizing the acid–base regulatory mechanisms by looking at H+ extrusion and HCO3− import pathways in gill epithelia of adult squid. Additionally, metabolism and excretion are monitored during exposure to acidified conditions, which are important indices for altered energetic features potentially associated with acid–base regulatory efforts. Immunocytochemical techniques in combination with gene expression analyses were applied in order to study the branchial acid–base regulatory machinery. It can be hypothesized that similar to the situation in embryonic epidermal ionocytes, the branchial acid–base regulatory machinery of adult squid involves ion-transporters including NHE3, V-type H+-ATPase, Rh-Protein as well as CAc and NBC, which allows the animal to cope with CO2 induced acid–base disturbances. In this context special attention has been dedicated to the potential role of RhP in mediating pH homeostasis during environmental hypercapnia by supporting proton equivalent transport across membranes. A potential coupling of NH3 and H+ excretion/secretion is proposed, which may represent a fundamental pathway of pH regulation in marine ammonotelic organisms.
Metabolic rates and NH4+ excretion
Extracellular acid–base status
Localization of acid–base relevant transporters in gill epithelia
NKA and VHA activity
Metabolism and excretion
The present work demonstrated that routine metabolic and NH4+ excretion rates in squid Sepioteuthis lessoniana are comparable to those determined for other squid and cuttlefish species ,–. While NH4+ excretion rates were not significantly affected by acidified conditions, metabolic rates were reduced upon prolonged exposure to pH 7.3. To date only a few studies investigated the effects of acidified conditions on metabolic responses in cephalopods. One study using the pelagic squid Dosidicus gigas demonstrated that in response to acute (several minutes) CO2 induced acidified conditions of pH 7.6 (0.1 kPa p CO2) animals respond with depressed metabolic rates accompanied by decreased activity levels . In contrast, routine metabolic rates of the demersal cuttlefish Sepia officinalis remained unaffected by CO2 induced seawater acidification down to pH 7.1 during acute (24 h) exposure . Differential responses observed for the three cephalopod species can be attributed to their very different life styles and abilities to swim and maintain neutral to positive buoyancy in the water column. Migratory pelagic squids like D. gigas swim and maintain positive buoyancy by continuously jetting water through their funnel, whereas S. officinalis has a decoupled swimming mode, by using jetting and fin undulation. Additionally, Sepia spp. have a gas filled cuttlebone to control buoyancy without continuous muscular activity that significantly increases locomotor efficiency . S. lessoniana is a large-finned pelagic squid that has evolved a partially decoupled swimming mode by additionally maintaining buoyancy using their enlarged fins running the full length of its mantle. Powerful pelagic squids that have no decoupled swimming mode need to spend a larger fraction of their energy budget to swim and to maintain neutral buoyancy ,. This higher fraction of energy that is spent for maintaining buoyancy could be a critical factor leading to higher sensitivities. In fact, the energy budget of marine invertebrates has been demonstrated to be compromised by seawater acidification by shifting a larger fraction of energy towards compensatory processes (e.g. acid–base regulation) leading to less energy available for growth and development . The fact that in the present work two individuals died after exposure to pH 7.3 conditions for one week indicates a higher sensitivity towards acidified conditions than cuttlefish S. officinalis that survived with a five-fold increase in body mass during exposure to a seawater pH of 7.1 for 6 weeks . It can be suggested that pelagic squids that evolved a life-style at the edge of energetic limitations, might react more sensitively to seawater acidification due to energetic limitations, compared to less “tuned” cuttlefish and octopus. To test this hypothesis, studies addressing the energetic costs of acid–base regulation in cephalopods will be an important future task.
Acid–base regulation during seawater acidification
The present work demonstrated that squid S. lessoniana can fully compensate for an extracellular acidosis evoked by seawater acidification up to pH 7.3. Stabilization of pHe is accompanied by an increase in blood HCO3− levels, which is a conserved and efficient mechanism to counter a respiratory acidosis found in several taxa, including fish, crustaceans and cephalopods ,,,. The hyperbolic increase in blood HCO3− levels in response to a respiratory acidosis described for other powerful acid–base regulators is in general accordance to the findings for S. lessoniana e.g. ,. Under control conditions venous HCO3− levels of S. lessoniana (2.5 mM) were found to be in the range as described for other cephalopod species including the squid Illex illecebrosus (2.2 mM) and the cuttlefish Sepia officinalis (3.4 mM) ,. An earlier study using the cuttlefish Sepia officinalis demonstrated control blood HCO3− levels of 3.4 mM and a partial compensation of pHe via HCO3− accumulation during exposure to environmental hypercapnia (0.6 kPa p CO2; pH 7.1) . In the same study it was suggested that a partial compensation of 0.2 pH units below control levels is sufficient to achieve sufficient gas transport via the blood pigment hemocyanin under acidified conditions in this less active cephalopod species. However, for the squid S. lessoniana a full compensation of extracellular pH was evident after 20 h during exposure to acidified conditions (pH 7.3). It has been hypothesized that more sluggish cephalopod species like cuttlefish and octopods may not rely on pH dependent oxygen transport to the same extent as more active pelagic squid species ,,. Interestingly, blood HCO3− levels in S. officinalis increased by approximately 7.5 mM within 48 h in response to 0.6 kPa CO2 exposure whereas in this study blood [HCO3−] was only increased by 2 mM when exposed to a similar acidification level. This indicates the presence of differential pH buffering/regulatory mechanisms, including non-bicarbonate buffering and H+ extrusion mechanisms among cephalopods. Non-bicarbonate buffer values determined for squid species ranged between 5 mmol l−1 pH unit−1 (Illex illecebrosus), 5.8 mmol l−1 pH unit−1 (Loligo pealei) and 4.7 mmol l−1 pH unit−1 (S. lessoniana) whereas those determined for cuttlefish, S. officinalis were 10 mmol l−1 pH unit−1,, indicating an even lower HCO3− independent buffering potential in squid species. According to these observations it can be suggested that control of extracellular pH in squids is likely to be attributed to efficient H+ extrusion mechanisms. Earlier studies using fish and crustaceans demonstrated that the compensation of acid–base disturbances elicited by hypercapnia is always associated with significant export of proton equivalents ,,. This feature is particularly important, as HCO3− formation through the hydration of CO2 is always accompanied with the generation of H+. Thus, on the long run organisms that stabilize blood pH via increased HCO3− accumulation require H+ secretion mechanisms as well. These observations are in line with the results of the present work demonstrating that environmental acidification stimulates expression of branchial acid–base transporters involved in HCO3− (NBC, CA) and H+ transport (VHA and RhP). Although an increase of VHA in response to acidified conditions on both the protein and mRNA level has been demonstrated, no significant (p = 0.061) increases in branchial VHA enzyme activities were found. It can be suggested that despite a trend of increased VHA activity during short-term low pH acclimation, statistical analyses failed to prove this effect due to a relatively low experimental “n” (three experimental replicates with six biological replicates) which is always the limitation when working with non-model organisms. Nonetheless, whole animal observations and molecular findings suggest that besides HCO3− buffering H+ secretion pathways across gill epithelia represent probably an even more important mechanism to compensate for acid–base disturbances in active squids. Thus, a special focus of the present work has been dedicated to a better understanding of branchial proton equivalent secretion mechanisms in ammonotelic cephalopods.
Branchial acid–base regulatory machinery
In convergence to fish and crustaceans, cephalopods evolved branchial ion regulatory epithelia, which are equipped with ion transporters including NKA, VHA and NBCe beneficial for coping with acid–base disturbances ,,,,. The present work further demonstrates that gene transcripts coding for Na+/H+ exchanger 3 (NHE3) and Rh protein (RhP), which are essential for proton equivalent transport in vertebrates ,,, are also expressed in the cephalopod gill. NKA-rich cells (NaRs) located in the ion-transporting inner epithelium of the 3 order lamellae of the cephalopod gill showed positive immunoreactivity for VHA (basolateral), NHE3 (apical) and RhP (apical) using antibodies specifically designed for this species. These polyclonal antibodies were designed against conserved regions of the respective protein, and western blot analyses of a previous study  and the present work demonstrated specific immunoreactivity with proteins in the predicted size range. Using in situ hybridization an earlier study demonstrated that an electrogenic Na+/HCO3− cotransporter (NBC) is also highly expressed in the ion-transporting epithelium of the cuttlefish (Sepia officinalis) gill . Together with the results of the present work it can be suggested that this transporter represents an important player in branchial epithelia that mediates extracellular accumulation of HCO3− in cephalopods. Due to the lack of sequence information the existence and role of anion exchangers (e.g. AE1) which were demonstrated to contribute to acid–base homeostasis in teleosts  remains unexplored for cephalopods.
Interestingly, positive VHA immunoractivity was additionally found in pilaster (or pillar) cells spanning through the blood sinus between the inner and the outer epithelium. Little information exists regarding a potential function of pillar cells in ion-regulatory or respiratory processes. Pillar cells in the dogfish (Squalus acanthias) gill were demonstrated to represent an important cell type that may contribute to gas exchange. These pillar cells are characterized by high concentrations of extracellular membrane bound carbonic anhydrase (CA) IV summarized in . This extracellular membrane bound CAIV has been suggested to facilitate the formation of CO2 from HCO3− in concert with basolateral VHA contributing to CO2 excretion across branchial epithelia in dogfish . In Cephalopods carbonic anhydrase has been demonstrated to be associated with the inner ion-transporting epithelium , but information regarding the expression of CA by pilaster cells is not available at present. However, it can be hypothesized that analogous to the situation in dogfish high concentrations of VHA associated with pillar cells may also support gas exchange by providing protons for the formation of CO2.
Branchial NH3/NH4+ transport mechanisms
The dual function of the cephalopod gill in gas exchange and extracellular acid–base regulation is achieved by different epithelia in this organ (depicted in Figures 1 and 9). The thin outer epithelium is believed to be involved in gas exchange by diffusive processes, whereas the inner ion-transporting epithelium is responsible for active transport of acid–base equivalents –. Apical localization of RhP and NHE3 in the inner transporting epithelium supports the hypothesis that the cephalopod gill is a major site of NH4+ excretion. Earlier studies demonstrated that in various cephalopod species the largest fraction of ammonia produced through amino acid metabolism is excreted via branchial epithelia ,. Blood NH4+/NH3 concentrations determined for octopus , cuttlefish  and squid  range from 100 to 500 μmol l−1 and are comparable to those determined for S. lessoniana in the present work (132.11 ± 37.79 μmol l−1; n = 4). Earlier studies hypothesized that ammonia is excreted as ammonia (NH3) accompanied with an excretion of protons to form the ammonium ion (NH4+) ,. This net export of proton equivalents further suggests that NH4+ excretion represents an important mechanism that contributes to acid–base balance in cephalopods. Interestingly, the cephalopod gill shows many morphological and functional similarities to the collecting duct of the mammalian kidney, where NH4+ is transported to the luminal space via Rh glycoproteins and V-type H+-ATPase . In the cephalopod gill, the semi-tubular structure of the 3° gill lamellae creates a luminal space into which NH4+ is secreted by the interplay of RhP and NHE3 (Figure 9). The involvement of NHE3 instead of VHA in this process is thermodynamically favored by the strong Na+ gradient between cytosol (30 mM) and seawater (470 mM). In teleosts Rh proteins including Rhcg and Rhbg were identified as important players in branchial ammonia excretion pathways, as well . The current model denotes the presence of Rhbg in basolateral membranes to facilitate the entry of NH3 into the cell, whereas Rhcg in combination with VHA and NHE2/3 is located in apical membranes. This interplay of H+ and NH3 secretion provides an acid trapping mechanisms for apical NH4+ secretion . Accordingly it can be hypothesized that similar to the situation in teleosts and the mammalian kidney, cephalopods excrete ammonia across gill epithelia by trapping NH4+ in the semi tubular space of the 3° lamellae (Figure 9).
The present work demonstrated that cephalopods have evolved an efficient pH regulatory machinery in branchial epithelia. Acid–base transporters potentially involved in both, HCO3− accumulation and H+ equivalent secretion were identified and localized in gill epithelia suggesting that this represents the major site for acid–base regulatory in cephalopods. Although significant HCO3− buffering capacities to control extracellular pH were only described for few marine species (fish, crustaceans and cephalopods) proton or proton equivalent secretion mechanisms may represent a more direct and ubiquitious pH regulatory pathway. Particularly the coupling of H+ and NH3 secretion can be regarded a fundamental and evolutionary ancient pathway of excretion and acid–base regulation. The present work underlines the importance of NH4+ based proton secretion via RhP that may contribute to well developed acid–base regulatory abilities in cephalopod molluscs.
The rapid compensation of pHe during exposure to acidified conditions is accompanied by a stimulation of branchial acid–base transporters on the protein and mRNA level, suggesting that maintenance of pHe represents a critical and energy consuming process for cephalopods to maintain vital functions. The present work further demonstrated that squids can tolerate short-term exposure without compromising aerobic energy metabolism while medium-term (one week) exposure to acidified conditions evoked decreased metabolic rates and could even lead to mortality. These observations are in accordance to other studies using pelagic squids  but are contrasting to studies conducted on cephalopods that are able to switch to locomotory energy saving modes (e.g. cuttlefish) by burrowing in sediment or maintaining positive buoyancy by using their gas filled cuttlebones and fins . Thus, this study indicates that energetic limitations may represent a critical feature that defines the degree of sensitivity towards seawater acidification. Pelagic squids that evolved a lifestyle at the edge of energetic limits due to high locomotory costs can be expected to be particularly sensitive to prolonged reallocations of energy towards compensatory processes despite their efficient proton equivalent secretion mechanisms. The identification of physiological principles that may lead to differential sensitivities even within one taxa represent an important task for future directions to better predict species sensitivities in times of rapid environmental change.
Seawater physiochemical parameters during the 168 h pH perturbation experiment including pH (NBS scale), CO 2 partial pressure ( p CO 2 ), total CO 2 (TCO 2 ), total alkalinity (TA), salinity (Sal) and temperature (Temp)
8.06 ± 0.002
624.71 ± 11.90
2.27 ± 0.04
2.51 ± 0.05
29.89 ± 0.19
28.63 ± 0.03
7.72 ± 0.019
1585.59 ± 253.20
2.47 ± 0.15
2.56 ± 0.14
29.44 ± 0.19
28.71 ± 0.06
7.34 ± 0.017
4134.16 ± 168.76
2.52 ± 0.12
2.47 ± 0.12
29.56 ± 0.19
28.75 ± 0.08
Metabolic rates and ammonia excretion
Determination of metabolic and ammonia (NH4+) excretion rates were determined at the 20 h and 168 h time points. Sepiotheuthis lessoniana from the pH experiments were starved overnight (12 h) and were gently transferred to glass respiration chambers with a volume of 4 L containing 0.2 μm filtered seawater equilibrated with the appropriate p CO2 level. The digestion process in pelagic squids is finished within 2-6h  and the starvation time of 12 h is sufficient to prevent effects on metabolic rates due to digestion processes . Respiration chambers were closed, and oxygen saturation was measured continuously (once every 30 s) for 20–30 min at 28–29°C using oxygen sensors (PreSens sensor spots, type PSt3) placed in the lid of respiration chambers, connected to an OXY-4 mini multichannel fiber optic oxygen transmitter (PreSens, Regensburg, Germany). The sensors were calibrated according to the manufacturer’s instructions. Preliminary experiments demonstrated that the ventilatory current of the animal could sufficiently mix the water inside the respiration chamber and oxygen concentration decreased linearly. Animal fresh mass was determined on a precision scale after all water was removed from the mantle cavity. When oxygen concentration reached the 75% air saturation level, animals were removed from the respiration chamber. Additionally, a separate glass chamber was incubated without animals to determine background readings of filtered seawater for ammonium excretion and respiration of bacteria. Bacterial respiration was 3.35 ± 3.88 μmol O2 h−1 compared to average oxygen consumption by squids (400.67 ± 161.16 μmol O2 h−1) leading to less than 1% of animal respiration. For calculation of oxygen consumption rates, the linear decrease in oxygen concentration during measuring intervals between 10 min after start and the end of the measurement period was considered. Oxygen consumption rates (MO2) are expressed as μmol O2 gFM−1 h−1.
Ammonium excretion rates were determined from NH4+ concentration measurements prior to and following incubation of squids for respiration measurements. Before and after closing the respiration chambers 10 ml of seawater (stock) were sampled. For NH4+ determinations a 100 μL subsample was taken from the stock and 25 μL of reagent containing orthophthaldialdehyde, sodium sulphite and sodium borate was added . Samples were then incubated for 2 h at room temperature in the dark until fluorescence was determined at an excitation and emission wavelength of 360 and 422 nm, respectively, using a microplate reader (Molecular Device, Spectra Max, M5). Ammonia (NH3) was not measured as NH3 concentrations are negligible at pH values of 8.0–7.1 (0.2–2% of total ammonium/ammonia, . NH4+ concentrations were determined in triplicates and excretion rates were expressed as μmol NH4+ gFM−1 h−1. Blood NH4+ concentrations of four control animals were determined with the same method. This method is suitable for NH4+ determinations in blood samples as it is specific to NH4+ and insensitive to amino acids and proteins .
Extracellular acid–base status
where α (0.039 μmol L−1 Pa) is the solubility coefficient of CO2 in seawater and pK1′ (5.94) the dissociation constant of carbonic acid at a salinity of 30, and a temperature of 29°C .
Immunohistochemistry and western blot analyses
For immunohistochemistry tissues were fixed by direct immersion for 24 h in Bouin’s fixative followed by rinses in 75% ethanol. Samples were fully dehydrated in a graded ethanol series and embedded in Paraplast (Paraplast Plus, Sigma, P3683). Sections of 4 μm were cut on a Leica RM2265 microtome, collected on poly-L-lysine-coated slides. The slides were deparaffinized in Histoclear II® for 10 min and passed through a descending alcohol series (100%, 95%, 90%, 70%, and 50% for 5 min each). Slides were washed in phosphate- buffered saline (PBS), pH 7.3. Subsequently, samples were transferred to a PBS solution containing 5% bovine serum albumin (BSA) for 30 min to block non-specific binding. The primary antibodies, a rabbit polyclonal antibody H-300, raised against the human α subunit of the Na+/K+-ATPase (NKA) (Santa Cruz Biotechnology, INC) and Sepioteuthis lessoniana specific polyclonal antibodies raised against part of the carboxyl-terminal region (IYRVRKVGYDEQFIMSY) of Na+/H+-exchanger3 (NHE3), the subunit A region (SYSKYTRALDEFYDK) of the V-type-H+-ATPase (VHA) for more detail see  and the Rhesus protein (RhP) (antibody designed against the synthetic peptideTRAGYQEFKW) were diluted in PBS (1:50–100) and placed in small droplets of 200 μl onto the sections, and incubated for 12 h at 4°C in a wet chamber. To remove unbound antibodies, the sections were then washed (3 × 5 min) in PBS and incubated for 1 h with small droplets (200 μl) of secondary antibody, anti-mouse Alexa Fluor 488 or anti- rabbit Alexa Fluor 568 (Invitrogen) (dilution 1:250). To allow double-color immunofluorescence staining, one of the polyclonal antibodies was directly labeled with Alexa Fluor dyes using the Zenon antibody labeling kit (Molecular Probes, Eugene, OR, USA). After rinses in PBS (3 × 5 min), sections were examined with a fluorescence microscope (Zeiss imager A1) equipped with an appropriate filter set.
For immunoblotting, 15 μL of crude extracts from gill tissues were used. Proteins were fractionated by SDS-PAGE on 10% polyacrylamide gels, according to Lämmli , and transferred to PVDF membranes (Millipore), using a tank blotting system (Bio-Rad). Blots were pre-incubated 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) blocking reagent (Roche, Mannheim, Germany). Blots were incubated with the primary antibody (see previous section) diluted 1:250–500 at 4°C overnight. After washing with TBS-T, blots were incubated for 2 h with horseradish conjugated goat anti-rabbit IgG antibody (diluted 1:1,000-2,000, at room temperature; Amersham Pharmacia Biotech). Protein signals were visualized by using the enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech) and recorded using Biospectrum 600 imaging system (UVP, Upland, CA, USA). Signal intensity was calculated using the free software “Image J” e.g. .
ATPase activity was measured in crude extracts in a coupled enzyme assay with pyruvate kinase (PK) and lactate dehydrogenase (LDH) by using the method of Schwartz et al. . Crude extracts were obtained by quickly homogenizing the tissue samples using a tissue lyzer (Quiagen) in 10 volumes of ice-cold buffer containing 50 mM imidazole, pH 7.5, 250 mM sucrose, 1 mM EDTA, 5 mM β-mercaptoethanol, 0.1% (w/v) deoxycholate, proteinase inhibitor cocktail from Sigma-Aldrich (catalogue no. P8340). Cell debris was removed by centrifugation for 10 min at 1000 g, 4°C. The supernatant was used as a crude extract. The reaction was started by adding 2 μl of the sample homogenate to the reaction buffer containing 100 mM imidazole, pH 7.5, 80 mM NaCl, 20 mM KCl, 5 mMMgCl2, 5 mM ATP, 0.24 mM Na-(NADH 2 ), 2 mM phosphoenolpyruvate, and about 12 U/ml PK and 17 U/ml LDH in a PK/LDH enzyme mix (Sigma-Aldrich). The oxidation of NADH coupled to the hydrolysis of ATP was followed photometrically at 29°C in a temperature controlled plate reader (Molecular Device, Spectra Max, M5), over a period of 15 min, with the decrease of extinction being measured at λ =339 nm. The fraction of Na+/K+ -ATPase or H+-ATPase activity in total ATPase (TA) activity was determined by the addition of 2 μl ouabain (5 mM final concentration) or bafilomycin (Bafilomycin A1, Sigma-Aldrich) (10 μM final concentration) to the assay, respectively. The concentrations of inhibitors applied were demonstrated to be sufficient to fully inhibit the NKA  and VHA , respectively. Each sample was measured in six replicates (3 with inhibitor dissolved in DMSO and 3 with DMSO). Enzyme activity was calculated by using an extinction coefficient for NADH of ε =6.31 mM−1 · cm−1 and given as micromoles of ATP consumed per gram tissue fresh mass (gFM) per hour.
Preparation of mRNA
Gill tissues (without branchial gland) were homogenized in Trizol reagent (Invitrogen, Carlsbad, CA, USA) using a Tissue lyser (Quiagen). Total RNA was extracted from the aqueous phase after addition of chloroform to Trizol homogenates and purified by addition of isopropanol. DNA contamination was removed with DNase I (Promega, Madison, WI, USA). The mRNA for the RT-PCR was obtained with a QuickPrep Micro mRNA Purification Kit (Amersham Pharmacia, Piscataway, NJ, USA) according to the supplier protocol. The amount of mRNA was determined by spectrophotometry (ND-2000, NanoDrop Technol, Wilmington, DE), and the mRNA quality was checked by running electrophoresis in RNA denatured gels. All mRNA pellets were stored at −80°C.
Real-time quantitative PCR (qPCR)
P rimers used for qRT-PCR
Amplicon size (bp)
Sodium–hydrogen exchanger 3
F 5′- GGCTGTCTTCCAAGAAATGGGTGT -3′
R 5′- AAGAACTTGGCAACACCAAGAGCG -3′
Vacuolar-type H + −ATPase
F 5′- ACGTGAGGGCAGTGTCAGTATTGT -3′
R 5′- TGATCAGCCAGTTGATGGAAGGGA -3′
Na+, K + −ATPase
F 5′- CCGTGCTGAATTTAAGGCAGGTCA -3′
R 5′- GCAAAGCTGATTCAGAAGCGTCAC -3′
Cytosolic Carbonic anhydrase
F 5′- ATGCAGATGGCAGTATTTGCCTGG -3′
R 5′- TTATTGGCTGGGCTGTTTGGGTTC -3′
Statistical analyses were performed using Sigma Stat 3.0 (Systat) software. Statistical differences between pH treatments were analyzed by two-way and one-way ANOVA followed by Tukey’s post-hoc test. Data sets were normally distributed (Kolmogorov-Smirnov test). Equal variance was tested using the Levene median test. The significance level was set to p < 0.05.
This study was financially supported by the grants to Y. C. Tseng from the National Science Council, Taiwan, Republic of China (NSC 102-2321-B-003-002) and an Alexander von Humbold/National Science Council (Taiwan) grant awarded to M. H (NSC 102-2911-I-001-002-2) and M.S. (NSC 103-2911-I-001-506). We gratefully thank Mr. H. T. Lee (assistant of Institute of Cellular and Organismic Biology marine station) for his assistance to maintain experimental systems.
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