Rainbow Trout Gill Epithelial Cells Research Articles

Single and combined effects of titanium (TiO2) and zinc (ZnO) oxide nanoparticles in the rainbow trout gill cell line RTgill-W1.

Understanding the environmental impact of nanoparticle (NP) mixtures is essential to accurately assess the risk they represent for aquatic ecosystems. However, although the toxicity of individual NPs has been extensively studied, information regarding the toxicity of combined NPs is still comparatively rather scarce. Hence, this research aimed to investigate the individual and combined toxicity mechanisms of two widely consumed nanoparticles, zinc oxide (ZnO NPs) and titanium dioxide (TiO2 NPs), using an in vitro model, the RTgill-W1 rainbow trout gill epithelial cell line. Sublethal concentrations of ZnO NPs (0.1µgmL-1) and TiO2 (30µgmL-1) and a lethal concentration of ZnO NPs causing 10% mortality (EC10, 3µgmL-1) were selected based on cytotoxicity assays. Cells were then exposed to the NPs at the selected concentrations alone and to their combination. Cytotoxicity assays, oxidative stress markers, and targeted gene expression analyses were employed to assess the NP cellular toxicity mechanisms and their effects on the gill cells. The cytotoxicity of the mixture was identical to the one of ZnO NPs alone. Enzymatic and gene expression (nrf2, gpx, sod) analyses suggest that none of the tested conditions induced a strong redox imbalance. Metal detoxification mechanisms (mtb) and zinc transportation (znt1) were affected only in cells exposed to ZnO NPs, while tight junction proteins (zo1 and cldn1), and apoptosis protein p53 were overexpressed only in cells exposed to the mixture. Osmoregulation (Na + /K + ATPase gene expression) was not affected by the tested conditions. The overall results suggest that the toxic effects of ZnO and TiO2 NPs in the mixture were significantly enhanced and could result in the disruption of the gill epithelium integrity. This study provides new insights into the combined effects of commonly used nanoparticles, emphasizing the importance of further investigating how their toxicity may be influenced in mixtures.

The flame retardant triphenyl phosphate alters the epigenome of embryonic cells in an aquatic in vitro model.

Triphenyl phosphate (TPhP) is an organophosphate flame retardant and plasticizer that is added to a wide variety of consumer and industrial products. It is also a ubiquitous environmental pollutant. Exposure to TPhP has been shown to alter gene expression in metabolic and estrogenic signaling pathways in in vitro and in vivo models of a variety of species, and as such, is considered to be an endocrine disrupting chemical. Exposure to endocrine disrupting chemicals is increasingly being associated with changes to the epigenome, especially during embryonic development. The aim of this study was to evaluate whether TPhP exposure in aquatic ecosystems has the ability to alter the epigenome in two immortal cell lines derived from trout (Oncorhynchus mykiss). This study assessed whether 24 h exposure to TPhP resulted in changes to histone modification and DNA methylation profiles in steelhead trout embryonic cells and rainbow trout gill epithelial cells. Results show that several epigenetic modifications on histone H3 and DNA methylation are altered in the embryonic cells following TPhP exposure, but not in the gill epithelial cells. Specifically, histone H3 acetylation, histone H3 mono-methylation and global DNA methylation were found to be reduced. The alterations of these epigenetic modification profiles in the embryonic cells suggest that exposure to TPhP during fetal development may alter gene expression in the developing embryo, likely in metabolic and estrogenic pathways. The impacts to the epigenome determined in this study may even carry multigenerational detrimental effects on human and ecosystem health, which requires further investigation.

Innate response of rainbow trout gill epithelial (RTgill-W1) cell line to ultraviolet-inactivated VHSV and FliC and rhabdovirus infection

Gills reportedly play a crucial role in induction of an antiviral immune response in fish. We investigated the expression of innate response genes in the rainbow trout gill epithelial cell line RTgill-W1 36 h after pretreatment with ultraviolet-inactivated viral hemorrhagic septicemia virus (UV-VHSV), flagellin C protein from Edwardsiella tarda (FliC), VHSV and SVCV using an Agilent 4 × 44k cGRASP salmonid microarray. RTgill-W1 cells pretreated with UV-VHSV, triggered an independent gene expression profile from those treated with a recombinant flagellin C protein from Edwardsiella tarda. In addition, exposure of RTgill-W1 cells to live viruses spring viremia of carp virus and viral hemorrhagic septicemia virus induced a less robust transcriptional change of 24 and 22 gene probes, respectively, when compared to 123 genes for UV-VHSV. Further the pretreatment of RTgill-W1 cells with (UV-VHSV) significantly reduced VHSV genome copy number at 6 d post infection (dpi) relative to the FliC-treated and untreated control. A quantitative PCR was used to study the transcriptional modulation of a set of 25 innate immune-related genes highlighted by the microarray data and a panel of 7 established antiviral genes in the protected cells. Notably, the expression of ifn1, ifn2, mx1 and mx3 were expressed more in untreated cells than in UV-VHSV-treated cells where virus replication was inhibited. The results from this study shed light on the mechanisms and pathways used by teleost gill epithelium innate immunity in combating viral and bacterial infection.

Impact of lithiated cobalt oxide and phosphate nanoparticles on rainbow trout gill epithelial cells

Metal oxide and phosphate nanoparticles (NPs) are ubiquitous in emerging applications, ranging from energy storage to catalysis. Cobalt-containing NPs are particularly important, where their widespread use raises questions about the relationship between composition, structure, and potential for environmental impacts. To address this gap, we investigated the effects of lithiated metal oxide and phosphate NPs on rainbow trout gill epithelial cells, a model for environmental exposure. Lithium cobalt oxide (LCO) NPs significantly reduced cell viability at10 µg/mL, while a 10-fold higher concentration of lithiated cobalt hydroxyphosphate (LCP) NPs was required to significantly reduce viability. Exposure to Li+ and Co2+ alone, at concentrations relevant to ion released from the NPs, did not reduce cell viability and minimally impacted reactive oxygen species (ROS) levels. Both LCO- and LCP-NPs were found within membrane-bound organelles. However, only LCP-NPs underwent rapid and complete dissolution in artificial lysosomal fluid. Unlike LCP-NPs, LCO-NPs significantly increased intracellular ROS, could be found within abnormal multilamellar bodies, and induced formation of intracellular vacuoles. Increased p53 gene expression, measured in individual cells, was observed at sub-toxic concentrations of both LCO- and LCP-NPs, implicating both in inductions of cellular damage and stress at concentrations approaching predicted environmental levels. Our results implicate the intact NP, not the dissolved ions, in the observed adverse effects and show that LCO-NPs significantly impact cell viability accompanied by increase in intracellular ROS and formation of organelles indicative of cell stress, while LCP-NPs have minimal adverse effects, possibly due to their rapid dissolution in acidic organelles.

Temporary protection of rainbow trout gill epithelial cells from infection with viral haemorrhagic septicaemia virus IVb.

The branchial epithelium is not only a primary route of entry for viral pathogens, but is also a site of viral replication and subsequent shedding may also occur from the gill epithelium. This study investigated the potential of agents known to stimulate innate immunity to protect rainbow trout epithelial cells (RTgill-W1) from infection with VHSV IVb. RTgill-W1 cells were pretreated with poly I:C, FuGENE(®) HD+poly I:C, lipopolysaccharide (LPS), LPS+poly I:C or heat-killed VHSV IVb and then infected with VHSV IVb 4 days later. Cytopathic effect (CPE) was determined at 2, 3, 4, 7 and 11days post-infection. Virus in cells and supernatant was detected using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). All of the treatments delayed the onset of CPE (per cent of monolayer destruction), compared with untreated controls; however, killed VHSV or poly I:C combined with LPS was the most effective. Similarly, the detection of viral RNA in the supernatant was delayed, and the quantity was significantly (P<0.05) reduced by all treatments with the exception of LPS alone (4 days). Unlike many of the other treatments, pretreatment of RTgill-W1 with heat-killed VHSV did not upregulate interferon 1, 2 or MX 1 gene expression.

The p53 inhibitor, pifithrin-α, disrupts microtubule organization, arrests growth, and induces polyploidy in the rainbow trout gill cell line, RTgill-W1

Pifithrin-α (PFT-α) blocks p53-dependent transcription and is an example of the many drugs being developed to target the p53 pathway in humans that could be released into the environment with potential impacts on aquatic animals if they were to become successful pharmaceuticals. In order to understand how p53 drugs might act on fish, the effects of PFT-α on rainbow trout gill epithelial cell line, RTgill-W1, were studied. PFT-α was not cytotoxic to RTgill-W1 in cultures with or without fetal bovine serum (FBS), but at 5.25μg/ml, PFT-α completely arrested proliferation. When FBS was present, PFT-α increased the number of polyploid cells over 12days. Those results suggest that like in mammals, p53 appears to regulate ploidy in fish. However, several effects were seen that have not been observed with mammalian cells. PFT-α caused a transient rise in the mitotic index and a disruption in cytoskeletal microtubules. These results suggest that in fish cells PFT-α affects microtubules either directly through an off-target action on tubulin or indirectly through an on-target action on p53-regulated transcription.

Cortisol 유발 세포독성에 대한 아연 관련 항산화 유전자 발현 증가에 의한 세포보호 효과

The protective effect of zinc against cortisol-induced cell injury was examined in rainbow trout gill epithelial cells. Cells exposed to cortisol for 24 h showed increased leakage of lactate dehydrogenase (LDH) as well as decreased cell viability in a dose-dependent manner. Treatment with zinc (100 μM ZnSO₄) reduced the severity of both LDH release and cell death as well as protected cells against cortisol-induced caspase-3 activation, indicating reduction of apoptosis. Cortisol-induced cell death, leakage of LDH, and caspase-3 activation were blocked by the glucocorticoid receptor antagonist Mifepristone (RU-486), suggesting that cell injury was cortisol-dependent. In addition, we studied the effect of zinc on the expression of antioxidant genes such as metallothionein A (MTA), metallothionein B (MTB), glutathione-S-transferase (GST), and glucose-6-phosphate dehydrogenase (G6PD) during cortisol-induced cell injury. MTA, MTB, GST, and G6PD mRNA levels increased after treatment with zinc or cortisol, separately or in combination. Higher mRNA levels of MTA, MTB, GST, and G6PD were detected when cells were treated with 100 μM ZnSO₄ and 1 μM cortisol in combination at the same time compared to treatment with zinc or cortisol separately. Cells treated with zinc showed increased intracellular free zinc concentrations, and this response was significantly enhanced in cells treated with cortisol and zinc. In conclusion, zinc treatment inhibited cortisol-induced cytotoxicity and apoptosis through indirect antioxidant action.

Development of the microsporidian parasite, Loma salmonae, in a rainbow trout gill epithelial cell line (RTG-1): evidence of xenoma development in vitro.

Growth and propagation of fish-infecting microsporidians within cell culture has been more difficult to achieve than for insect- and human-infecting microsporidians. Fish microsporidia tend to elicit xenoma development rather than diffuse growth in vivo, and this process likely increases host specificity. We present evidence that the fish microsporidian, Loma salmonae, has the capacity to develop xenomas within a rainbow trout gill epithelial cell line (RTG-1). Spore numbers increased over a 4 weeks period within cell culture flasks. Xenoma-like structures were observed using phase contrast microscopy, and then confirmed using transmission electron microscopy. Optimization of the L. salmonae-RTG-1 cell model has important implications in elucidating the process of xenoma development induced by microsporidian parasites.

Long-term storage and impedance-based water toxicity testing capabilities of fluidic biochips seeded with RTgill-W1 cells

Rainbow trout gill epithelial cells (RTgill-W1) are used in a cell-based biosensor that can respond within one hour to toxic chemicals that have the potential to contaminate drinking water supplies. RTgill-W1 cells seeded on enclosed fluidic biochips and monitored using electric cell-substrate impedance sensing (ECIS) technology responded to 18 out of the 18 toxic chemicals tested within one hour of exposure. Nine of these chemical responses were within established concentration ranges specified by the U.S. Army for comparison of toxicity sensors for field application. The RTgill-W1 cells remain viable on the biochips at ambient carbon dioxide levels at 6°C for 78weeks without media changes. RTgill-W1 biochips stored in this manner were challenged with 9.4μM sodium pentachlorophenate (PCP), a benchmark toxicant, and impedance responses were significant (p<0.001) for all storage times tested. This poikilothermic cell line has toxicant sensitivity comparable to a mammalian cell line (bovine lung microvessel endothelial cells (BLMVECs)) that was tested on fluidic biochips with the same chemicals. In order to remain viable, the BLMVEC biochips required media replenishments 3 times per week while being maintained at 37°C. The ability of RTgill-W1 biochips to maintain monolayer integrity without media replenishments for 78weeks, combined with their chemical sensitivity and rapid response time, make them excellent candidates for use in low cost, maintenance-free field-portable biosensors.

Flow cytometric analysis of BDE 47 mediated injury to rainbow trout gill epithelial cells

The polybrominated diphenyl ethers (PBDEs) are ubiquitous environmental contaminants whose residues are increasing in fish, wildlife and human tissues. However, relatively little is known regarding the mechanisms of cell injury caused by PBDE congeners in fish. In the present study, we employed flow cytometry-based analyses to understand the onset and mechanisms of cell injury in rainbow trout gill cells (RTgill-W1 cells) exposed to 2,2′,4,4′-tetrabromodiphenyl ether (BDE 47). Substantial optimization and validation for flow cytometry protocols were required during assay development for the trout gill cell line. Exposure to micromolar concentrations of BDE 47 elicited a significant loss in RTgill-W1 cell viability that was accompanied by a decrease in NAD(P)H autofluorescence, a marker associated with disruption of cellular redox status. This loss in NAD(P)H content was accompanied by a decrease in nonyl acridine orange fluorescence, indicating mitochondrial membrane lipid peroxidation. Furthermore, low doses of BDE 47 altered cellular forward angle light scatter (FS, a measure of cell diameter or size) and side light scatter properties (SS, a measure of cellular internal complexity), consistent with the early stages of apoptosis. These changes were more pronounced at higher BDE 47 concentrations, which led to an increase in the percentage of cells undergoing frank apoptosis as evidenced by sub-G1 DNA content. Apoptosis was also observed at a relatively low dose (3.2 μM) of BDE 47 if cells were exposed for an extended period of time (24 h). Collectively, the results of these studies indicate that exposure of rainbow trout gill cells to BDE47 is associated with the induction of apoptosis likely originating from disruption of cellular redox status and mitochondrial oxidative injury. The current report extends observations in other species demonstrating that oxidative stress is an important mechanism of BDE 47 mediated cellular toxicity, and supports the use of oxidative stress-associated biomarkers in assessing the sublethal effects of PBDEs and their replacements in fish. The application of flow cytometry endpoints using fish cell lines should facilitate study of the mechanisms of chemical injury in aquatic species.

Cortisol stimulates the zinc signaling pathway and expression of metallothioneins and ZnT1 in rainbow trout gill epithelial cells

Intracellular zinc signaling is important in the control of a number of cellular processes. Hormonal factors that regulate cellular zinc influx and initiate zinc signals are poorly understood. The present study investigates the possibility for cross talk between the glucocorticoid and zinc signaling pathways in cultured rainbow trout gill epithelial cells. The rainbow trout metallothionein A (MTA) gene possesses a putative glucocorticoid response element and multiple metal response elements 1042 base pairs upstream of the start codon, whereas metallothionein B (MTB) and zinc transporter-1 (ZnT1) have multiple metal response elements but no glucocorticoid response elements in this region. Cortisol increased MTA, MTB, and ZnT1 gene expression, and this stimulation was enhanced if cells were treated with cortisol together with zinc. Cells treated with zinc showed increased zinc accumulation, transepithelial zinc influx (apical to basolateral), and intracellular labile zinc concentrations. These responses were also significantly enhanced in cells pretreated with cortisol and zinc. The cortisol-mediated effects were blocked by the glucocorticoid receptor (GR) antagonist RU-486, indicating mediation via a GR. In reporter gene assays, zinc stimulated MTA promoter activity, whereas cortisol did not. Furthermore, cortisol significantly reduced zinc-stimulated MTA promoter activity in cells expressing exogenous rainbow trout GR. These results demonstrate that cortisol enhances cellular zinc uptake, which in turn stimulates expression of MTA, MTB, and ZnT1 genes.

Localization and characterization of phenamil-sensitive Na+influx in isolated rainbow trout gill epithelial cells

Percoll density-gradient separation, combined with peanut lectin agglutinin (PNA) binding and magnetic bead separation, was used to separate dispersed fish gill cells into sub-populations. Functional characterization of each of the sub-populations was performed to determine which displayed acid-activated phenamil- and bafilomycin-sensitive Na(+) uptake. Analysis of the mechanism(s) of (22)Na(+) influx was performed in control and acid-activated (addition of 10 mmoll(-1) proprionic acid) cells using a variety of Na(+) transport inhibitors (ouabain, phenamil, HOE-694 and bumetanide) and a V-type ATPase inhibitor (bafilomycin). We found that cells migrating to a 1.03-1.05 g ml(-1) Percoll interface [pavement cells (PVCs)] possessed the lowest rates of Na(+) uptake and that influx was unchanged during either bafilomycin (10 nmoll(-1)) treatment or internal acidification with addition of proprionic acid (10 mmoll(-1)). Mitochondria-rich (MR) cells that migrated to the 1.05-1.09 g ml(-1) interface of the Percoll gradient demonstrated acidification-activated bafilomycin and phenamil-sensitive Na(+) influx. Further separation of the MR fraction into PNA(+) and PNA(-) fractions using magnetic separation demonstrated that only the PNA(-) cells (alpha-MR cells) demonstrated phenamil-and bafilomycin-sensitive acid-activated (22)Na(+) uptake. We confirm the coupling of a V-type H(+)-ATPase with phenamil-sensitive Na(+) uptake activity and conclude that high-density alpha-MR cells function in branchial Na(+) uptake in freshwater fish.

Minimising aerobic respiratory demands could form the basis to sub-lethal copper tolerance by rainbow trout gill epithelial cells in vitro

Mechanisms of Cu tolerance were investigated in respiratory epithelial cell cultures, from rainbow trout gills, by studying O2 consumption and protein synthesis rates, intracellular Na concentration and TER. The lowest concentration found to reduce O2 consumption was 25 μM Cu. This did not affect either protein synthesis rate or intracellular Na concentration and was interpreted in terms of copper tolerance; i.e., how these two energetically demanding processes are maintained despite a reduction in aerobic ATP supply. The relationship between protein synthesis rate and synthesis cost is exponential and the cost of protein synthesis in gill cells was found to be minimal (i.e., this cell occupies a position on the asymptotic section of the protein synthesis rate/synthesis cost model) and unaffected by 25 μM Cu. Thus protein synthesis rates could be maintained since any reduction would represent an insignificant energy saving. Intracellular Na concentrations and O2 consumption rates were linearly correlated suggesting reducing intracellular maintenance costs would have a greater significance in terms of overall energetic conservation. Intracellular Na maintenance costs, calculated from O2 consumption rates and intracellular Na concentrations, were found to decline after exposure to 25 μM Cu. Since TER was unaffected this implied the reduced costs arose from membrane `channel arrest'. Thus the Na/K ATPase energy demands, associated with maintaining intracellular Na concentration, could be reduced by decoupling metabolic demand and membrane function. Therefore this study may demonstrate how the flexibility of cellular energetics enables gill epithelial cells to tolerate sub-lethal Cu.

Glutathione S-transferase and UDP-glucuronyltransferase activity in primary cultures of rainbow trout gill epithelial cells.

The objective of this study was to characterize rainbow trout (Oncorhynchus mykiss) gill epithelial cells in primary culture by evaluating their ability to maintain glutathione and glucuronide conjugating enzymes. The activity and inducibility of the phase II enzymes was investigated as a function of culture time. Glutathione S-transferase (GST) and UDP-glucuronyltransferase (UDPGT) enzyme activities were measured in freshly isolated cells and in cells cultured for 7 and 12 days. GST activity, determined with 1-chloro-2,4-dinitrobenzene, decreased gradually to 72% after 7 days and to 38% after 12 days in culture compared with freshly isolated cells. There was no significant difference between UDPGT activities in freshly isolated cells compared with cells cultured up to 12 days although a transient decrease in activity was observed at day 7. In vitro induction of the enzymes was studied using beta-naphtoflavone (BNF) and 3-methylcholanthrene (3-MC) as inducers. GST activity increased 2-fold after exposure to BNF and 1.5-fold after 3-MC exposure for 48 h in 7 days old cultures. No induction was observed in 12 days old cultures. UDPGT activity was not induced either at day 7 or 12.

Xenobiotic and steroid biotransformation activities in rainbow trout gill epithelial cells in culture

The biotransformation of xenobiotics and steroids was investigated in cultured respiratory epithelial cells from rainbow trout ( Oncorhynchus mykiss) gills. As a first approach, ethoxyresorufin- O-deethylase (EROD), chosen as a marker of CYP1A activity, was measured in monolayers of adherent cells. The induction of this enzyme was studied in cells exposed to β-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in concentrations ranging from 10 −6 to 10 −12 M. After 24 h, TCDD showed a maximal induction at a concentration of 10 −9 M while BNF showed a maximal induction at a concentration of 10 −7 M. Concurrently, a variety of substrates involved in cytochrome P450-dependent metabolism as well as phase II reactions, namely ethoxycoumarin, aniline and testosterone were incubated with cultured gill cells for 2 or 8 h and with freshly isolated hepatocytes for comparison. Our results revealed a significant cytochrome P450-dependent activity in gill cells with ethoxycoumarin and aniline, but no hydroxylation was observed with testosterone as substrate. No trace of sulfate conjugate was detected. With 2.5 μM aniline as substrate, 2-hydroxyaniline accounted for 32.1% of the radioactivity after 2 h incubation whereas acetanilide amounted to 6.4%. Significant differences were found between gill cells and isolated hepatocytes in the capacity of these systems to conduct oxidative and conjugating metabolic pathways. Qualitatively, the main difference was observed for testosterone which is hydroxylated in position 6β and 16β and conjugated to glucuronic acid in liver cells, whereas reductive biotransformation giving rise to dihydrotestosterone and androstanediol and traces of androstenedione were observed in gill cells. Quantitatively, the biotransformation activity in gill epithelial cells, expressed as pmol/h per mg protein, was between 1.5 and 14% of the activity level observed in isolated hepatocytes, depending on the substrate.

Corrigendum — Rainbow trout gill epithelial cells in primary culture communicate through gap junctions as demonstrated by dye-coupling

Communication across gap junctions between cells in various tissues is considered an important mechanism for control of cellular growth, differentiation and function. Although cell-cell coupling in the gill epithelium is likely for functional reasons, the common view is that gap junctions are not present between cells in the gill epithelium of teleostean fish. Gap junction mediated cell-cell communication was analysed in the present study in primary and secondary cultures of rainbow trout gill epithelial cells by microinjecting the fluorescent dye Lucifer yellow CH. In 4–14 day-old primary and secondary cell cultures, 58% of the cells injected with Lucifer yellow were coupled to at least one other cell. To exclude the possibility that intercellular dye transfer occurred through cytoplasmic bridges instead of gap junctions, we also microinjected FITC-dextran, which because of its molecular size does not pass through gap junctions. None of the cells injected with FITC-dextran showed spreading of the dye. The number of cells spreading Lucifer yellow was decreased in a dose-dependent way when the cells were treated with dioctanoylglycerol, a synthetic diacylglycerol known to affect the open state of gap junctions, which further supports that the cells are coupled through functional gap junctions.

Localization of mRNA for the proton pump (H+-ATPase) and exchanger in the rainbow trout gill

In situ hybridization was performed on sections of rainbow trout (Oncorhynchus mykiss) gill tissue using oligonucleotide probes complementary to the mRNA of the 31-kilodalton subunit of the bovine renal V-type H+-ATPase or rat kidney Band 3 anion exchanger ([Formula: see text] exchanger). This was conducted in conjunction with measurements of whole-body net acid fluxes and blood acid–base status during imposed conditions of respiratory acidosis (external hypercapnia) or metabolic alkalosis (NaHCO3infusion). A positive hybridization signal for the H+-ATPase mRNA was localized predominantly in lamellar epithelial cells and was less apparent in cells associated with the filament or interlamellar regions. The H+-ATPase hybridization signal was enhanced during hypercapnic acidosis concurrently with a marked increase in whole-body net acid excretion. A positive hybridization signal for the [Formula: see text] exchanger mRNA was observed in epithelial cells on both the filament and lamella. During metabolic alkalosis induced by intra-arterial infusion of NaHCO3, there was a marked increase in the [Formula: see text] exchanger mRNA hybridization signal in cells on both the filament and lamella that occurred concurrently with a decrease in net acid excretion. The results of this study support the existence of a V-type H+-ATPase and a [Formula: see text] exchanger in rainbow trout gill epithelial cells and demonstrate that alterations in gene expression for the pump–exchanger may be a significant mechanism underlying the altered rates of net acid equivalent excretion during acid – base disturbances.