13C tracer analysis reveals the landscape of metabolic checkpoints in human CD8+ T cell differentiation and exhaustion.

Introduction: Naïve T cells remain in an actively maintained state of quiescence until activation by antigenic signals, upon which they start to proliferate and generate effector cells to initiate a functional immune response. Metabolic reprogramming is essential to meet the biosynthetic demands of the differentiation process, and failure to do so can promote the development of hypofunctional exhausted T cells. Methods: Here we used 13C metabolomics and transcriptomics to study the metabolism of CD8+ T cells in their complete course of differentiation from naïve over stem-like memory to effector cells and in exhaustion-inducing conditions. Results: The quiescence of naïve T cells was evident in a profound suppression of glucose oxidation and a decreased expression of ENO1, downstream of which no glycolytic flux was detectable. Moreover, TCA cycle activity was low in naïve T cells and associated with a downregulation of SDH subunits. Upon stimulation and exit from quiescence, the initiation of cell growth and proliferation was accompanied by differential expression of metabolic enzymes and metabolic reprogramming towards aerobic glycolysis with high rates of nutrient uptake, respiration and lactate production. High flux in anabolic pathways imposed a strain on NADH homeostasis, which coincided with engagement of the proline cycle for mitochondrial redox shuttling. With acquisition of effector functions, cells increasingly relied on glycolysis as opposed to oxidative phosphorylation, which was, however, not linked to changes in mitochondrial abundance. In exhaustion, decreased effector function concurred with a reduction in mitochondrial metabolism, glycolysis and amino acid import, and an upregulation of quiescence-associated genes, TXNIP and KLF2, and the T cell suppressive metabolites succinate and itaconate. Discussion: Overall, these results identify multiple metabolic features that regulate quiescence, proliferation and effector function, but also exhaustion of CD8+ T cells during differentiation. Thus, targeting these metabolic checkpoints may be a promising therapeutic strategy for both prevention of exhaustion and promotion of stemness of anti-tumor T cells.

Quantum based effects of therapeutic nuclear magnetic resonance persistently reduce glycolysis.

Electromagnetic fields are known to induce the clock protein cryptochrome to modulate intracellular reactive oxygen species (ROS) via the quantum based radical pair mechanism (RPM) in mammalian cells. Recently, therapeutic Nuclear Magnetic Resonance (tNMR) was shown to alter protein levels of the circadian clock associated Hypoxia Inducible Factor-1α (HIF-1α) in a nonlinear dose response relationship. Using synchronized NIH3T3 cells, we show that tNMR under normoxia and hypoxia persistently modifies cellular metabolism. After normoxic tNMR treatment, glycolysis is reduced, as are lactate production, extracellular acidification rate, the ratio of ADP/ATP and cytosolic ROS, whereas mitochondrial and extracellular ROS, as well as cellular proliferation are increased. Remarkably, these effects are even more pronounced after hypoxic tNMR treatment, driving cellular metabolism to a reduced glycolysis while mitochondrial respiration is kept constant even during reoxygenation. Hence, we propose tNMR as a potential therapeutic tool in ischemia driven diseases like inflammation, infarct, stroke and cancer.

Oxygen consumption and acid secretion in isolated gas gland cells of the European eel Anguilla anguilla.

Swimbladder gas gland cells are known to produce lactic acid required for the acidification of swimbladder blood and decreasing the oxygen carrying capacity of swimbladder blood, i.e., the onset of the Root effect. Gas gland cells have also been shown to metabolize glucose via the pentose phosphate shunt, but the role of the pentose phosphate shunt for acid secretion has not yet been evaluated. Similarly, aerobic metabolism of gas gland cells has been largely neglected so far. In the present study, we therefore simultaneously assessed the role of glycolysis and of the pentose phosphate shunt for acid secretion and recorded oxygen consumption of isolated swimbladder gas gland cells of the European eel. Presence of glucose was essential for acid secretion, and at glucose concentrations of about 1.5 mmol l-1 acid secretion of gas gland cells reached a maximum, indicating that glucose concentrations in swimbladder blood should not be limiting acid production and secretion under physiological conditions. The data revealed that most of the acid was produced in the glycolytic pathway, but a significant fraction was also contributed by the pentose phosphate shunt. Addition of glucose to gas gland cells incubated in a glucose-free medium resulted in a reduction of oxygen uptake. Inhibition of mitochondrial respiration significantly reduced oxygen consumption, but a fraction of mitochondria-independent respiration remained in presence of rotenone and antimycin A. In the presence of glucose, application of either iodo-acetate inhibiting glycolysis or 6-AN inhibiting the pentose phosphate shunt did not significantly affect oxygen uptake, indicating an independent regulation of oxidative phosphorylation and of acid production. Inhibition of the muscarinic acetylcholine receptor caused a slight elevation in acid secretion, while forskolin caused a concentration-dependent reduction in acid secretion, indicating muscarinic and c-AMP-dependent control of acid secretion in gas gland cells.

Aging Cell Culture - Genetic and Metabolic Effects of Passage Number on Zebrafish Z3 Cells.

BACKGROUND/AIMS: Since cell lines are cultured and extensively used in a variety of different research disciplines, we determined the effects of passage numbers on a commonly used embryonic zebrafish cell line (Z3). METHODS: Senescence markers, DNA damage, the redox state, gene expression, and metabolic parameters have been investigated in young (passage 5) up to very old (passage 40 and higher) cells. RESULTS: Besides increasing DNA damage, we also found elevated metabolic capacity and a shift to a more reduced cellular redox state in the cells. Interestingly, several parameters showed a non-linear course regarding the passage number or cell age, so that for example young and mid-aged cells appeared to cluster with very old rather than with old cells. CONCLUSION: This study illustrates the importance of passage number and suggests pre-testing specific parameters to assure the generation of accurate and reproducible data.

Demystifying biotrophs: FISHing for mRNAs to decipher plant and algal pathogen-host interaction at the single cell level.

Plant-pathogen interactions follow spatial and temporal developmental dynamics where gene expression in pathogen and host undergo crucial changes. Therefore, it is of great interest to detect, quantify and localise where and when key genes are active to understand these processes. Many pathosystems are not accessible for genetic amendments or other spatially-resolved gene expression monitoring methods. Here, we adapt single molecule FISH techniques to demonstrate the presence and activity of mRNAs at the single-cell level using phytomyxids in their plant and algal host in lab and field material. This allowed us to monitor and quantify the expression of genes from the clubroot pathogen Plasmodiophora brassicae, several species of its Brassica hosts, and of several brown algae, including the genome model Ectocarpus siliculosus, infected with the phytomyxid Maullinia ectocarpii. We show that mRNAs are localised along a spatiotemporal gradient, thus providing a proof-of-concept of the usefulness of single-molecule FISH to increase knowledge about the interactions between plants, algae and phytomyxids. The methods used are easily applicable to any interaction between microbes and their algal or plant host, and have therefore the potential to rapidly increase our understanding of key, spatially- and temporally-resolved processes underpinning complex plant-microbe interactions.

Assessment of the Retina of Plp-α-Syn Mice as a Model for Studying Synuclein-Dependent Diseases.

Purpose: Synucleinopathies such as multiple system atrophy (MSA) and Parkinson's disease are associated with a variety of visual symptoms. Functional and morphological retinal aberrations are therefore supposed to be valuable biomarkers for these neurodegenerative diseases. This study examined the retinal morphology and functionality resulting from human α-synuclein (α-Syn) overexpression in the transgenic Plp-α-Syn mouse model. Methods: Immunohistochemistry on retinal sections and whole-mounts was performed on 8- to 11-week-old and 12-month-old Plp-α-Syn mice and C57BL/6N controls. Quantitative RT-PCR experiments were performed to study the expression of endogenous and human α-Syn and tyrosine hydroxylase (TH). We confirmed the presence of human α-Syn in the retina in western blot analyses. Multi-electrode array (MEA) analyses from light-stimulated whole-mounted retinas were used to investigate their functionality. Results: Biochemical and immunohistochemical analyses showed human α-Syn in the retina of Plp-α-Syn mice. We found distinct staining in different retinal cell layers, most abundantly in rod bipolar cells of the peripheral retina. In the periphery, we also observed a trend toward a decline in the number of retinal ganglion cells. The number of TH+ neurons was unaffected in this human α-Syn overexpression model. MEA recordings showed that Plp-α-Syn retinas were functional but exhibited mild alterations in dim light conditions. Conclusions: Together, these findings implicate an impairment of retinal neurons in the Plp-α-Syn mouse. The phenotype partly relates to retinal deficits reported in MSA patients. We further propose the suitability of the Plp-α-Syn retina as a biological model to study synuclein-mediated mechanisms.

Metabolic interaction of hydrogen peroxide and hypoxia in zebrafish fibroblasts.

Cells require oxygen for aerobic metabolism, which may also result in the production of reactive oxygen species (ROS) as a by-product. Under low oxygen conditions, ROS formation has been reported to either increase or decrease. We addressed this physiological response for the first time in zebrafish embryonic fibroblasts (Z3) and used a hydrogen peroxide (H2O2)-specific fluorescent protein (roGFP2-Orp1) either targeted to the mitochondria or expressed in the cytosol. Microfluidic live-cell imaging measurements showed that oxygen deprivation in Z3 cells results in decreased or stable H2O2 levels within the mitochondria or the cytosol, respectively, and that the reductive shift recorded in the mitochondrial matrix is directly dependent on oxygen concentration. The response was accompanied by a transient increase in extracellular acidification rate (ECAR) and a lower cellular reducing potential as assessed by the viability stain alamarBlue. Complex I and III inhibition with Rotenone and Antimycin A led to H2O2 production under normoxia but these inhibitors were not able to avert the reductive shift under hypoxia. Only by system-wide inhibition of flavin-containing oxidases with Diphenyleneiodonium (DPI) were we able to decrease the reductive shift, while selective inhibition of NADPH oxidases with the inhibitor Apocynin had no effect on the hypoxia response. Since DPI also led to a strong increase in ECAR we found that, in order to keep the cytosolic H2O2 levels stable, glycolytic metabolism was of fundamental importance. According to our experiments with the glucose-6-phosphate dehydrogenase inhibitor 6-Aminonicotinamide, this was attributable to the pentose phosphate pathway producing reducing equivalents required for ROS degradation.

Metabolic Plasticity Enables Circadian Adaptation to Acute Hypoxia in Zebrafish Cells.

BACKGROUND/AIMS: Reduced oxygen availability, hypoxia, is frequently encountered by organisms, tissues and cells, in aquatic environments as well as in high altitude or under pathological conditions such as infarct, stroke or cancer. The hypoxic signaling pathway was found to be mutually intertwined with circadian timekeeping in vertebrates and, as reported recently, also in mammals. However, the impact of hypoxia on intracellular metabolic oscillations is still unknown. METHODS: For determination of metabolites we used Multilabel Reader based fluorescence and luminescence assays, circadian levels of Hypoxia Inducible Factor 1 alpha and oxidized peroxiredoxins were semi quantified by Western blotting and ratiometric quantification of cytosolic and mitochondrial H2O2 was achieved with stable transfections of a redox sensitive green fluorescent protein sensor into zebrafish fibroblasts. Circadian oscillations of core clock gene mRNA´s were assessed using realtime qPCR with subsequent cosine wave fit analysis. RESULTS: Here we show that under normoxia primary metabolic activity of cells predominately occurs during day time and that after acute hypoxia of two hours, administrated immediately before each sampling point, steady state concentrations of glycolytic key metabolites such as glucose and lactate reveal to be highly rhythmic, following a circadian pattern with highest levels during the night periods and reflecting the circadian variation of the cellular response to hypoxia. Remarkably, rhythms in glycolysis are transferred to cellular energy states under normoxic conditions, so that ADP/ATP ratios oscillate as well, which is the first evidence for cycling ADP/ATP pools in a metazoan cell line to our knowledge. Furthermore, the hypoxia induced alterations in rhythms of glycolysis lead to the alignment of three major cellular redox systems, namely the circadian oscillations of NAD+/NADH and NADP+/NADPH ratios and of increased nocturnal levels of oxidized peroxiredoxins, resulting in a highly oxidized nocturnal cellular environment. Of note, circadian rhythms of cytosolic H2O2 remain unaltered, while the transcriptional clock is already attenuated, as it is known to occur also under chronic hypoxia. CONCLUSION: We therefor propose that the realignment of metabolic redox oscillations might initiate the observed hypoxia induced attenuation of the transcriptional clock, based on the reduced binding affinity of the CLOCK/BMAL complex to the DNA in an oxidized environment.

Cadmium Protection Strategies--A Hidden Trade-Off?

Cadmium (Cd) is a non-essential transition metal which is introduced into the biosphere by various anthropogenic activities. Environmental pollution with Cd poses a major health risk and Cd toxicity has been extensively researched over the past decades. This review aims at changing the perspective by discussing protection mechanisms available to counteract a Cd insult. Antioxidants, induction of antioxidant enzymes, and complexation of Cd to glutathione (GSH) and metallothionein (MT) are the most potent protective measures to cope with Cd-induced oxidative stress. Furthermore, protection mechanisms include prevention of endoplasmic reticulum (ER) stress, mitophagy and metabolic stress, as well as expression of chaperones. Pre-exposure to Cd itself, or co-exposure to other metals or trace elements can improve viability under Cd exposure and cells have means to reduce Cd uptake and improve Cd removal. Finally, environmental factors have negative or positive effects on Cd toxicity. Most protection mechanisms aim at preventing cellular damage. However, this might not be possible without trade-offs like an increased risk of carcinogenesis.

Chronodisruption increases cardiovascular risk in zebrafish via reduced clearance of senescent erythrocytes.

The circadian clock and the hypoxic signaling pathway play critical roles in physiological homeostasis as well as in pathogenesis. The bi-directionality of the interaction between both pathways has been shown on physiological and only recently also on molecular level. But the consequences of a disturbed circadian rhythm for the hypoxic response and the cardiovascular system have never been addressed in any organism. Here we show that the hypoxic response of animals subjected to chronodisruption is reduced by approximately 30%, as reflected by decreased expression levels of hypoxia inducible factor 1 and its down-stream target genes erythropoietin, responsible for the generation of red blood cells (RBC) and vascular endothelial growth factor, which is essential for proper vascularization. Beside malformations of their vascular beds, chronodisrupted animals surprisingly revealed elevated numbers of senescent erythrocytes under normoxic conditions, due to a reduced clearance rate via apoptosis. Over-aged erythrocytes in turn are characterized by decreased oxygen transport capacities and an increased tendency for aggregation, explaining the higher mortality of chronodisrupted animals observed in our study. The present study shows for the first time that chronodisruption strongly interferes with the hypoxic signalling cascade, increasing the cardiovascular risk in zebrafish due to elevated proportions of senescent erythrocytes. The results might shed new light on the etiology of the increased cardiovascular risk observed among shiftworkers.

Finding a robust strain for biomethanation: anaerobic fungi (Neocallimastigomycota) from the Alpine ibex (Capra ibex) and their associated methanogens.

Anaerobic fungi occupy the rumen and digestive tract of herbivores, where they play an important role in enzymatic digestion of lignocellulosic and cellulosic substrates, i.e. organic material that their hosts are unable to decompose on their own. In this study we isolated anaerobic fungi from a typical alpine herbivore, the Alpine ibex (C. ibex). Three fungal strains, either as pure culture (ST2) or syntrophic co-culture with methanogens (ST3, ST4) were successfully obtained and morphologically characterised by different microscopy- and staining-techniques and by rDNA ITS gene sequencing. The isolated fungi were identified as Neocallimastix frontalis (ST2) and Caecomyces communis (ST3 and ST4). We introduce a novel field of application for lactofuchsin-staining, combined with confocal laser scanning microscopy. This approach proved as an effective method to visualize fungal structures, especially in the presence of plant biomass, generally exhibiting high autofluorescence. Moreover, we could demonstrate that fungal morphology is subject to changes depending on the carbon source used for cultivation. Oxygen tolerance was confirmed for both, C. communis-cultures for up to three, and for the N. frontalis-isolate for up to 12 h, respectively. With PCR, FISH and an oligonucleotide microarray we found associated methanogens (mainly Methanobacteriales) for C. communis, but not for N. frontalis.

A method to evaluate the efficiency of transfection reagents in an adherent zebrafish cell line.

We present a simple and robust method to evaluate the transfection efficiency of commercially available transfection reagents intended to be established for use in nonmammalian cell lines. To illustrate the method, we compare the ability of four different reagents to transfect the embryonic zebrafish cell line Z3. Z3 cells were seeded in a 96-well plate and simultaneously transfected in several variations by using minimum volumes of transfection reagent and a vector DNA encoding an amplified version of green fluorescent protein (GFP). After 24 and 48 h, transfection efficiency was determined by a dual fluorescence plate reader measurement of GFP and Hoechst 33342 fluorescence, an indicator of cell density. Of the four different reagents tested, certain variations of JetPrime(™) reagent and X-tremeGene(™) HP reagent produced the highest fluorescence signal per cell after 24- and 48-h incubation, respectively. The simultaneous multivariate setup enables comparing different reagent/DNA combinations at different time points well, independent of cell growth variability or seeding density.

Cortisol affects tight junction morphology between pavement cells of rainbow trout gills in single-seeded insert culture.

A primary culture system of rainbow trout gill pavement cells grown on permeable support (single-seeded insert, SSI) was used to examine histological and physiological changes induced by the addition of the corticosteroid hormone cortisol. Pavement cell epithelia were cultured under symmetrical conditions (L15 apical/L15 basolateral) and developed a high transepithelial resistance (TER, 6.84 ± 1.99 kΩ cm(2), mean ± SEM) with a low phenol red diffusion rate (PRD, 0.15 ± 0.03 µmol l(-1)/day). Addition of cortisol to the basolateral compartment increased TER twofold and reduced PRD threefold over a 5-day period. A similar increase in TER could be seen after 24 h apical freshwater (FW) in control cultures. In cortisol-treated cultures FW exposure did not change TER, but PRD increased significantly. Histochemical staining of the cytoskeleton of cells in SSI culture revealed a morphological partitioning into a single mucosal layer of polarized, polygonal cells featuring cortical F-actin rings which were comparable to F-actin rings of epithelial cells on the lamellar and filamental surface, and several unorganized serosal layers of cells with F-actin stress fibers. Addition of cortisol increased cell density by 18% and in the mucosal layer it led to smaller, less polygonal cells with increased height and increased cell contact area. In transmission electron microscopic images two pairs of cytoplasmatic electron-dense structures confining the zonula occludens apically and basally toward the zonula adhaerens were found. Addition of cortisol increased the distance between those paired structures, hence led to deeper tight junctions. The cortisol-induced increase in barrier properties, therefore, involves a structural fortification of the tight junctions which was not generally modified by a short 24-h apical freshwater stress. These results identify cortisol as a regulator of tight junction morphology between pavement cells of euryhaline fish such as the trout.

Claudin 28b and F-actin are involved in rainbow trout gill pavement cell tight junction remodeling under osmotic stress.

Permeability of rainbow trout gill pavement cells cultured on permeable supports (single seeded inserts) changes upon exposure to freshwater or treatment with cortisol. The molecular components of this change are largely unknown, but tight junctions that regulate the paracellular pathway are prime candidates in this adaptational process. Using differential display polymerase chain reaction we found a set of 17 differentially regulated genes in trout pavement cells that had been exposed to freshwater apically for 24 h. Five genes were related to the cell-cell contact. One of these genes was isolated and identified as encoding claudin 28b, an integral component of the tight junction. Immunohistochemical reactivity to claudin 28b protein was concentrated in a circumferential ring colocalized to the cortical F-actin ring. To study the contribution of this isoform to changes in transepithelial resistance and Phenol Red diffusion under apical hypo-or hyperosmotic exposure we quantified the fluorescence signal of this claudin isoform in immunohistochemical stainings together with the fluorescence of phalloidin-probed F-actin. Upon hypo-osmotic stress claudin 28b fluorescence and epithelial tightness remained stable. Under hyperosmotic stress, the presence of claudin 28b at the junction significantly decreased, and epithelial tightness was severely reduced. Cortical F-actin fluorescence increased upon hypo-osmotic stress, whereas hyperosmotic stress led to a separation of cortical F-actin rings and the number of apical crypt-like pores increased. Addition of cortisol to the basolateral medium attenuated cortical F-actin separation and pore formation during hyperosmotic stress and reduced claudin 28b in junctions except after recovery of cells from exposure to freshwater. Our results showed that short-term salinity stress response in cultured trout gill cells was dependent on a dynamic remodeling of tight junctions, which involves claudin 28b and the supporting F-actin ring.

Chronic reduction in cardiac output induces hypoxic signaling in larval zebrafish even at a time when convective oxygen transport is not required.

In the present study, the zebrafish breakdance mutant (bre) was used to assess the role of blood flow in development because it has been previously shown that bre larvae have a chronically reduced cardiac output as a result of ventricular contraction following only every second atrial contraction in addition to an atrial bradycardia. We confirmed a 50% reduction compared with control fish and further showed that blood flow in the caudal part of the dorsal aorta decreased by 80%. Associated with these reductions in blood flow were indications of developmental retardation in bre mutants, specifically delayed hatching, reduced cell proliferation, and a transiently decreased growth rate. Surprisingly, an increased red blood cell concentration and an earlier appearance of trunk vessels in bre larvae indicated some compensation to convective oxygen transport, although in previous studies it has been shown that zebrafish larvae at this stage obtain oxygen by bulk diffusion. In bre animals immunohistochemical analyses showed a significant increase in hypoxia inducible factor 1 (HIF)-α protein expression, comparable with wild-type larvae that were raised under hypoxic conditions. Accordingly, the expression of some hif downstream genes was affected. Furthermore, Affymetrix microarray analyses revealed a large number of genes that were differently expressed comparing control and bre larvae, and the number even increased with proceeding development. The results showed that a chronic reduction in blood flow generated hypoxic molecular signals despite partial compensation by increased oxygen carrying capacity and transiently slowed the overall development of zebrafish bre larvae.

Thiolated chitosan nanoparticles: transfection study in the Caco-2 differentiated cell culture.

The aim of this study was to monitor the expression of secreted protein in differentiated Caco-2 cells after transfection with nanoparticles, in order to improve gene delivery. Based on unmodified chitosan and thiolated chitosan conjugates, nanoparticles with the gene reporter pSEAP (recombinant Secreted Alkaline Phosphatase) were generated at pH 4.0. Transfection studies of thiolated chitosan in Caco-2 cells during the exponential growth phase and differentiation growth phase of the cells led to a 5.0-fold and 2.0-fold increase in protein expression when compared to unmodified chitosan nanoparticles. The mean particle size for both unmodified chitosan and cross-linked thiolated chitosan nanoparticles is 212.2 ± 86 and 113.6 ± 40 nm, respectively. The zeta potential of nanoparticles was determined to be 7.9 ± 0.38 mV for unmodified chitosan nanoparticles and 4.3 ± 0.74 mV for cross-linked thiolated chitosan nanoparticles. Red blood cell lysis evaluation was used to evaluate the membrane damaging properties of unmodified and thiolated chitosan nanoparticles and led to 4.61 ± 0.36% and 2.29 ± 0.25% lysis, respectively. Additionally, cross-linked thiolated chitosan nanoparticles were found to exhibit higher stability toward degradation in gastric juices. Furthermore the reversible effect of thiolated chitosan on barrier properties was monitored by measuring the transepithelial electrical resistance (TEER) and is supported by immunohistochemical staining for the tight junction protein claudin. According to these results cross-linked thiolated chitosan nanoparticles have the potential to be used as a non-viral vector system for gene therapy.

Acid-base regulation in isolated gill cells of the goldfish (Carassius auratus).

Mechanisms of acid release and intracellular pH (pH(i)) homeostasis were analysed in goldfish (Carassius auratus) gill cells in primary culture. The rate of acid secretion was measured using a cytosensor microphysiometer, and pH(i) was determined using the fluorescent probe 2',7'-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF). Amiloride, a Na(+) channel and Na(+)/H(+) exchanger (NHE) inhibitor, had no effect on pH(i), but acid secretion of the gill cells was significantly impaired. In the presence of amiloride, the intracellular acidification (achieved using the NH(4)Cl pulse technique) was more severe than in the absence of amiloride, and recovery from the acidosis was slowed down. Accordingly, acid secretion of gill cells was severely reduced in the absence of extracellular Na(+). Under steady-state conditions, 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid (DIDS), a HCO(3)(-)-transport inhibitor, caused a slow acidification of pH(i), and acid secretion was significantly reduced. No recovery from intracellular acidification was observed in the presence of DIDS. Bafilomycin A(1), an inhibitor of V-ATPase, had no effect on steady-state pH(i) and recovery from an intracellular acidification, whereas the rate of acid secretion under steady-state conditions was slightly reduced. Immunohistochemistry clearly revealed the presence of the V-ATPase B-subunit in goldfish gill lamellae. Taken together, these results suggest that a Na(+)-dependent HCO(3)(-) transport is the dominant mechanism besides an NHE and V-ATPase to control pH(i) in goldfish gill cells.