Mitochondria-rich Research Articles

Morphological Changes and Ionic Regulations in Gills of Euryhaline Fish during Osmoregulation: A Review

Euryhaline fish can thrive in both freshwater habitat and saltwater conditions. The migration from one salinity range to another involves the use of various organs like gills, kidneys and intestine which are all osmoregulatory in function. Among them, gills are considered to be the most important osmoregulatory organ with multiple functions and various ion transporters which helps the fish to survive the adverse environmental conditions. The gills have various cells involved in osmoregulation and mainly the MR (Mitochondria Rich) cells which have many transporters for intake and excretion of ions according to the environmental salinity. This review briefs the studies of various researchers on the morphological changes and the ionic variations in the fish gills which helps in acclimating them to different salinities.

Hypothermal Effects on Energy Supply for Ionocytes in Gills of Freshwater- and Seawater-Acclimated Milkfish, Chanos chanos

The milkfish, Chanos chanos, is an important aquaculture species that can be cultured in freshwater (FW) and seawater (SW) ponds because of its high euryhalinity. In winter, cold snap leading to high mortality of this tropical species is a critical issue for the aquaculture industry in Taiwan. Under hypothermal stress, however, changes in energy supply for osmoregulation of this euryhaline species is intriguing. In this study, we used an antibody against glycogen phosphorylase (GP) to identify glycogen-rich (GR) cells distributed adjacent to the mitochondria-rich (MR) ionocytes in milkfish gills. Glucose transporter 1 (GLUT1), which plays a major role in energy supply, was also identified and localized in GR cells. Moreover, the expression of indicators of aerobic metabolism and energy production, citrate synthase (CS) and cytochrome c oxidase (COX), were analyzed in gills of FW- and SW-acclimated milkfish to reveal different strategies of energy utilization under hypothermal stress. When exposed to a low-temperature environment, SW individuals used branchial glycogen and lactate to match the energy demands of aerobic metabolism in ionocytes, and elevated aerobic capacity to support and maintain normal functions in gills. However, branchial glycogen mainly localized in the GR cells of FW milkfish was not utilized under hypothermal stress. Meanwhile, a similar level of branchial COX activity and COXIV protein abundance at low temperatures between FW and SW milkfish indicated similar energy requirements in gills. This suggested that another source, but not branchial glycogen, maintained the energy demand in FW milkfish. The present study illustrated differential energy supply strategies in gills between SW- and FW-acclimated milkfish for osmoregulation under low temperatures.

Bioconcentration and toxicological impacts of fipronil and 2,4-D commercial formulations (single and in mixture) in the tropical fish, Danio rerio.

The insecticide fipronil and the herbicide 2,4-D are the most applied pesticides in sugarcane crops leading to aquatic contamination. The whole-body bioconcentration of fipronil and 2,4-D, single and in mixture, was evaluated in Danio rerio after 96-h exposure. The activities of catalase (CAT) and glutathione S-transferase(GST) in whole body and in the gills and the acetylcholinesterase (AChE) in muscle were determined. The gill histopathology and the morphology of the pavement (PVC) and the mitochondria-rich(MRC) cells at gill surface were analyzed. Bioconcentration occurred after exposure to fipronil (2.69 L kg-1) and 2,4-D (1.73 L kg-1) single and in mixture of fipronil (3.10 L kg-1) and 2,4-D (1.27 L kg-1). Whole-body CAT activity was unchanged, and its activity decreased in the gills after exposure to fipronil and increased after exposure to 2,4-D and mixture. GST and AChE increased after single exposure to each pesticide and mixture of both. Fish exposed to mixture increased the MRC fractional area (MRCFA) which suggested possible ionic regulation disturbance and reduced the microridge of the PVC surface. Synergistic interactions occurred in the CAT activity and MRCFA after exposure to mixture of pesticides. The results indicate that the recommended application dose of fipronil and 2,4-D, single or in mixture, for sugarcane crops affects this fish species altering its homeostasis.

A Multifunction Muscle in Squid

Some striated muscles are multifunctional; they serve several different roles during locomotion and movement, including acting as motors, brakes, struts, or springs. The few multifunctional muscles that have been reported occur in the cross-striated muscles of animals with complex, jointed, skeletal support systems. In the comparatively simple muscular system of a cephalopod mollusc, we identified an obliquely striated muscle, the nuchal retractor muscle, which appears to be multifunctional. The nuchal retractor is composed of two different fiber types, mitochondria-rich (MR) and mitochondria-poor (MP) fibers; shortening of these fibers retracts the head toward the mantle. Synchronized measurements of head movement (as a proxy for nuchal retractor length) and muscle activation revealed that, while the MP nuchal retractor muscle fibers were activated only for head retractions that occurred during escape jet locomotion, the MR fibers were activated 1) as the head retracted during escape jets and a few jets used during slow swimming, 2) during brief periods of head stasis as the animal changed swimming direction, and 3) during the rapid head extensions that followed an escape jet. Our results suggest that the nuchal retractor muscle may function as a motor, a brake, and, occasionally, a strut. More broadly, our findings suggest that multifunctionality is not restricted to cross-striated fibers or to the muscles of animals with jointed skeletal support systems.

Microtubule-dependent changes in morphology and localization of chloride transport proteins in gill mitochondria-rich cells of the tilapia, Oreochromis mossambicus.

The tilapia (Oreochromis mossambicus) is a euryhaline fish exhibiting adaptive changes in cell size, phenotype, and ionoregulatory functions upon salinity challenge. Na(+) /Cl(-) cotransporter (NCC) and Na(+) /K(+) /2Cl(-) cotransporter (NKCC) are localized in the apical and basolateral membranes of mitochondria-rich (MR) cells of the gills. These cells are responsible for chloride absorption (NCC) and secretion (NKCC), respectively, thus, the switch of gill NCC and NKCC expression is a crucial regulatory mechanism for salinity adaptation in tilapia. However, little is known about the interaction of cytoskeleton and these adaptive changes. In this study, we examined the time-course of changes in the localization of NKCC/NCC in the gills of tilapia transferred from fresh water (FW) to brackish water (20‰) and from seawater (SW; 35‰) to FW. The results showed that basolateral NKCC disappeared and NCC was expressed in the apical membrane of MR cells. To further clarify the process of these adaptive changes, colchicine, a specific inhibitor of microtubule-dependent cellular regulating processes was used. SW-acclimated tilapia were transferred to SW, FW, and FW with colchicine (colchicine-FW) for 96 h. Compared with the FW-treatment group, in the MR cells of colchicine-FW-treatment group, (1) the average size was significantly larger, (2) only wavy-convex-subtype apical surfaces were found, and (3) the basolateral (cytoplasmic) NKCC signals were still exhibited. Taken together, our results suggest that changes in size, phenotype, as well as the expression of NCC and NKCC cotransporters of MR cells in the tilapia are microtubule-dependent. J. Morphol. 277:1113-1122, 2016. © 2016 Wiley Periodicals, Inc.

Effects of salinity on metabolic rate and branchial expression of genes involved in ion transport and metabolism in Mozambique tilapia (Oreochromis mossambicus)

This study investigated the effects of two rearing salinities, and acute salinity transfer, on the energetic costs of osmoregulation and the expression of metabolic and osmoregulatory genes in the gill of Mozambique tilapia. Using automated, intermittent-flow respirometry, measured standard metabolic rates (SMRs) of tilapia reared in seawater (SW, 130mgO2kg−1h−1) were greater than those reared in fresh water (FW, 103mgO2kg−1h−1), when normalized to a common mass of 0.05kg and at 25±1°C. Transfer from FW to 75% SW increased SMR within 18h, to levels similar to SW-reared fish, while transfer from SW to FW decreased SMR to levels similar to FW-reared fish. Branchial gene expression of Na+–K+–2Cl− cotransporter (NKCC), an indicator of SW-type mitochondria-rich (MR) cells, was positively correlated with SMR, while Na+–Cl− cotransporter (NCC), an indicator of FW-type MR cells, was negatively correlated. Principal Components Analysis also revealed that branchial expression of cytochrome c oxidase subunit IV (COX-IV), glycogen phosphorylase (GP), and a putative mitochondrial biogenesis regulator in fish, peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), were correlated with a higher SMR, plasma osmolality, and environmental salinity, while expression of glycogen synthase (GS), PGC-1β, and nuclear respiratory factor 1 (NRF-1) had negative correlations. These results suggest that the energetic costs of osmoregulation are higher in SW than in FW, which may be related to the salinity-dependent differences in osmoregulatory mechanisms found in the gills of Mozambique tilapia.

Feeding induces translocation of vacuolar proton ATPase and pendrin to the membrane of leopard shark (Triakis semifasciata) mitochondrion-rich gill cells

In this study we characterized mitochondrion-rich (MR) cells and regulation of acid/base (A/B) relevant ion-transporting proteins in leopard shark (Triakis semifasciata) gills. Immunohistochemistry revealed that leopard shark gills posses two separate cell populations that abundantly express either Na+/K+-ATPase (NKA) or V-H+-ATPase (VHA), but not both ATPases together. Co-immunolocalization with mitochondrial Complex IV demonstrated, for the first time in shark gills, that both NKA- and VHA-rich cells are also MR cells, and that all MR cells are either NKA- or VHA-rich cells. Additionally we localized the anion exchanger pendrin to VHA-rich cells, but not NKA-rich cells. In starved sharks, VHA was localized throughout the cell cytoplasm and pendrin was present at the apical pole (but not in the membrane). However, in a significant number of gill cells from fed leopard sharks, VHA translocated to the basolateral membrane (as previously described in dogfish), and pendrin translocated to the apical membrane. Our results highlight the importance of translocation of ion-transporting proteins to the cell membrane as a regulatory mechanism for A/B regulation.

Xenopus embryonic epidermis as a mucociliary cellular ecosystem to assess the effect of sex hormones in a non-reproductive context

BackgroundHow important are sexual hormones beyond their function in reproductive biology has yet to be understood. In this study, we analyzed the effects of sex steroids on the biology of the embryonic amphibian epidermis, which represents an easily amenable model of non-reproductive mucociliary epithelia (MCE). MCE are integrated systems formed by multiciliated (MC), mucus-secreting (MS) and mitochondrion-rich (MR) cell populations that are shaped by their microenvironment. Therefore, MCE could be considered as ecosystems at the cellular scale, found in a wide array of contexts from mussel gills to mammalian oviduct.ResultsWe showed that the natural estrogen (estradiol, E2) and androgen (testosterone, T) as well as the synthetic estrogen (ethinyl-estradiol, EE2), all induced a significant enhancement of MC cell numbers. The effect of E2, T and EE2 extended to the MS and MR cell populations, to varying degrees. They also modified the expression profile of RNA MCE markers, and induced a range of “non-typical” cellular phenotypes, with mixed identities and aberrant morphologies, as revealed by imaging analysis through biomarker confocal detection and scanning electron microscopy. Finally, these hormones also affected tadpole pigmentation, revealing an effect on the entire cellular ecosystem of the Xenopus embryonic skin.ConclusionsThis study reveals the impact in vivo, at the molecular, cellular, tissue and organism levels, of sex steroids on non-reproductive mucociliary epithelium biogenesis, and validates the use of Xenopus as a relevant model system in this field.

Expression of ion transporters in gill mitochondrion-rich cells in Japanese eel acclimated to a wide range of environmental salinity

We examined morphological changes and molecular mechanisms of ion regulation in mitochondrion-rich (MR) cells of Japanese eel acclimated to different environmental salinities. Electron microscopic observations revealed that the apical membrane of MR cells appeared as a flat or slightly projecting disk with a mesh-like structure on its surface in eel acclimated to freshwater (FW). In seawater (SW)-acclimated eel, in contrast, the apical membrane of MR cells showed a slightly concave surface without a mesh-like structure. The mRNA expression of Na+/H+ exchanger-3 (NHE3) in deionized FW and normal SW was higher than that in normal FW and 30%-diluted SW. Expression of Na+/K+/2Cl− cotransporter-1a (NKCC1a) became higher with increasing environmental salinity. Immunofluorescence staining showed that the apical NHE3 immunoreaction was stronger in deionized FW and normal SW than in the other groups. Basolateral NKCC1 immunoreaction was most intense in normal SW. These results indicate that apical NHE3 is involved in ion uptake in fish acclimated to hypotonic environments, and that basolateral NKCC1 is important for acclimation to hypertonic environments. The relatively high expression of NHE3 in SW further indicates a possible role of NHE3 in acid-base regulation in the gills in SW-acclimated fish.

Hyposmoregulatory ability and ion- and water-regulatory mechanisms during the leptocephalus stages of Japanese eel Anguilla japonica

We explored osmoregulatory ability and mechanisms of ion and water regulation in Japanese eel leptocephali. Tissue osmolality of leptocephali ranged from 360 to 540 mOsm/kg·H2O. Immunocytochemical observations revealed that Na+/K+-ATPase-immunoreactive mitochondrion-rich (MR) cells were distributed over the entire body surface of leptocephali. Using a fluorescent sodium indicator and the chloride test, we localized Na+ and Cl− secreting sites at the apical region of cutaneous MR cells. To further examine drinking behavior and water absorption in the intestine, leptocephali were exposed to seawater containing dextran labeled with Alexa Fluor. To calculate relative water absorption, fluorescent intensity was measured along the digestive tract. Whereas water was hardly absorbed in the stomach and intestine, water absorption predominantly took place in the rectum. Our findings indicate that Japanese eel exert hyposmoregulatory ability as early as during leptocephalus stages, secreting Na+ and Cl− through cutaneous MR cells and primarily absorbing water from ingested seawater in the rectum.

Environmental Regulation of Mitochondria-Rich Cells in Chalcalburnus tarichi (Pallas, 1811) During Reproductive Migration

Chalcalburnus tarichi is an anadromous cyprinid fish that has adapted to extreme conditions (salinity 22 ‰, pH 9.8 and alkalinity 153 mEq × l⁻¹) in Lake Van in eastern Turkey. Changes in immunoreactivity of Na⁺/K⁺-ATPase in gill tissue and osmolarity and ion levels in plasma were investigated in C. tarichi during reproductive migration. Physicochemical characteristics and ion levels in Lake Van were high compared freshwater. Plasma osmolality and plasma ion concentrations ([Na⁺], [K⁺] and [Cl⁻]) increased after transfer from freshwater to Lake Van. The mitochondria-rich (MR) cells of the gill were stained in both filament and lamellar epithelia of C. tarichi by immunocytochemistry with a specific antiserum for Na⁺/K⁺-ATPase in river fish samples. Density and area of MR cells were decreased in lake-adapted fishes. These results indicated that freshwater acclimation capacity is correlated with the size and distribution of MR cells in C. tarichi, in contrast to many teleost fishes.

The acute and regulatory phases of time-course changes in gill mitochondrion-rich cells of seawater-acclimated medaka (Oryzias dancena) when exposed to hypoosmotic environments

The recent model showed that seawater (SW) mitochondrion-rich (MR) cells with hole-type apical openings secrete Cl− through the transporters including the Na+, K+-ATPase (NKA), Na+, K+, 2Cl− cotransporter (NKCC), and cystic fibrosis transmembrane conductance regulator (CFTR). The present study focused on the dynamic elimination of the Cl− secretory capacity and illustrated different phases (i.e., acute and regulatory phases) of branchial MR cells in response to hypoosmotic challenge. Time-course remodeling of the cell surfaces and the altered expressions of typical ion transporters were observed in the branchial MR cells of SW-acclimated brackish medaka (Oryzias dancena) when exposed to fresh water (FW). On the 1st day post-transfer, rapid changes were shown in the acute phase: the flat-type MR cells with large apical surfaces replaced the hole-type cells, the gene expression of both Odnkcc1a and Odcftr decreased, and the apical immunostaining signals of CFTR protein disappeared. The basolateral immunostaining signals of NKCC1a protein decreased throughout the regulatory phase (>1day post-transfer). During this period, the size and number of NKA-immunoreactive MR cells were significantly reduced and elevated, respectively. Branchial NKA expression and activity were maintained at constant levels in both phases. The results revealed that when SW-acclimated brackish medaka were transferred to hypoosmotic FW for 24h, the Cl− secretory capacity of MR cells was eliminated, whereas NKCC1a protein was retained to maintain the hypoosmoregulatory endurance of the gills. The time-course acute and regulatory phases of gill MR cells showed different strategies of the euryhaline medaka when subjected to hypoosmotic environments.

Recruitment and degeneration of mitochondrion-rich cells in the gills of Mozambique tilapia Oreochromis mossambicus during adaptation to a hyperosmotic environment

Cellular recruitment and degeneration of branchial mitochondrion-rich (MR) cells were examined in Mozambique tilapia transferred from hypoosmotic to hyperosmotic water. To examine apoptosis in the gills associated with salinity change, tilapia were directly transferred from freshwater to 70% seawater. The TUNEL assay showed that apoptotic cells in the gills were significantly increased at 1day after transfer, which was supported by an electron-microscopic observation that gill MR cells underwent morphological changes characteristic of apoptosis such as an irregularly shaped electron-dense nucleus and distension of the tubular system. To further examine MR–cell recruitment, freshwater-acclimated tilapia were transferred either to freshwater or to 70% seawater after BrdU injection. Immunohistochemical detection of BrdU-labeled nuclei and Na+/K+-ATPase-rich MR cells allowed us to classify BrdU-labeled MR cells into two subtypes: a single MR cell and an MR–cell complex. Although newly generated single MR cells were observed similarly in both freshwater and 70% seawater-transferred fish, the density of MR–cell complexes was much higher in 70% seawater than in freshwater. Our findings indicated that transfer from hypoosmotic to hyperosmotic water enhanced apoptosis of freshwater-type MR cells, resulting in reduction in hyperosmoregulatory ability for freshwater adaptation, and stimulated the recruitment of MR–cell complexes to develop hypoosmoregulatory ability for seawater adaptation.

Excretion of cesium and rubidium via the branchial potassium-transporting pathway in Mozambique tilapia

Using an insolubilization technique in combination with element analysis, we examined the possible excretion of Cs+ and Rb+, both known to be biochemical analogs of K+, through the K+-transporting pathway in the gills of seawater-adapted Mozambique tilapia. To detect ion excretion from gills infused with Rb+ or Cs+, the isolated gills were treated with sodium tetraphenylborate, which reacts not only with K+ but also with Rb+ and Cs+ to form insoluble precipitates. The precipitates were formed specifically at the apical openings of mitochondrion-rich (MR) cells, which are responsible for K+ excretion in marine teleosts. Elemental analysis showed that Cs and Rb were distributed at the area of precipitates on the gill surface, and that these elements were colocalized with K. Our findings indicate that the gills eliminate unnecessary Cs+ and Rb+ in the body fluid, presumably through the same pathway as the K+ excretion in MR cells.

Morphological and functional characterization of a novel Na+/K+-ATPase-immunoreactive, follicle-like structure on the gill septum of Japanese banded houndshark, Triakis scyllium

In teleost fishes, it is well-established that the gill serves as an important ionoregulatory organ in addition to its primary function of respiratory gas exchange. In elasmobranch fish, however, the ionoregulatory function of the gills is still poorly understood. Although mitochondria-rich (MR) cells have also been found in elasmobranch fish, these cells are considered to function primarily in acid-base regulation. In this study, we found a novel aggregate structure made up of cells with basolaterally-expressed Na(+)/K(+)-ATPase (NKA), in addition to NKA-immunoreactive MR cells that have already been described in the gill filament and lamella. The cell aggregates, named follicularly-arranged NKA-rich cells (follicular NRCs), were found exclusively in the epithelial lining of the venous web in the cavernous region of the filament and the inter-filamental space of the gill septum. The follicular NRCs form a single-layered follicular structure with a large lumen leading to the external environment. The follicular NRCs were characterized by: (i) well-developed microvilli on the apical membrane, (ii) less prominent infoldings of the basolateral membrane and (iii) typical junction structures including deep tight junction between cells. In addition, large numbers of vesicles were observed in the cytoplasm and some of them were fused to the lateral membrane. The follicular NRCs expressed Na(+)/H(+) exchanger 3 and Ca(2+) transporter 1. The follicular NRCs thus have the characteristics of absorptive ionoregulatory cells and this suggests that the elasmobranch gill probably contributes more importantly to body fluid homeostasis than previously thought.

Acid secretion by mitochondrion-rich cells of medaka (Oryzias latipes) acclimated to acidic freshwater

In the present study, medaka embryos were exposed to acidified freshwater (pH 5) to investigate the mechanism of acid secretion by mitochondrion-rich (MR) cells in embryonic skin. With double or triple in situ hybridization/immunocytochemistry, the Na(+)/H(+) exchanger 3 (NHE3) and H(+)-ATPase were localized in two distinct subtypes of MR cells. NHE3 was expressed in apical membranes of a major proportion of MR cells, whereas H(+)-ATPase was expressed in basolateral membranes of a much smaller proportion of MR cells. Gill mRNA levels of NHE3 and H(+)-ATPase and the two subtypes of MR cells in yolk sac skin were increased by acid acclimation; however, the mRNA level of NHE3 was remarkably higher than that of H(+)-ATPase. A scanning ion-selective electrode technique was used to measure H(+), Na(+), and NH(4)(+) transport by individual MR cells in larval skin. Results showed that Na(+) uptake and NH(4)(+) excretion by MR cells increased after acid acclimation. These findings suggested that the NHE3/Rh glycoprotein-mediated Na(+) uptake/NH(4)(+) excretion mechanism plays a critical role in acidic equivalent (H(+)/NH(4)(+)) excretion by MR cells of the freshwater medaka.

Potassium excretion through ROMK potassium channel expressed in gill mitochondrion-rich cells of Mozambique tilapia

Despite recent progress in physiology of fish ion homeostasis, the mechanism of plasma K+ regulation has remained unclear. Using Mozambique tilapia, a euryhaline teleost, we demonstrated that gill mitochondrion-rich (MR) cells were responsible for K+ excretion, using a newly invented technique that insolubilized and visualized K+ excreted from the gills. For a better understanding of the molecular mechanism of K+ excretion in the gills, cDNA sequences of renal outer medullary K+ channel (ROMK), potassium large conductance Ca(2+)-activated channel, subfamily M (Maxi-K), K(+)-Cl(-) cotransporters (KCC1, KCC2, and KCC4) were identified in tilapia as the candidate molecules that are involved in K+ handling. Among the cloned candidate molecules, only ROMK showed marked upregulation of mRNA levels in response to high external K+ concentration. In addition, immunofluorescence microscopy revealed that ROMK was localized in the apical opening of gill MR cells, and that the immunosignals were most intense in the fish acclimated to the environment with high K+ concentration. To confirm K+ excretion via ROMK, K+ insolubilization-visualization technique was applied again in combination with K+ channel blockers. The K+ precipitation was prevented in the presence of Ba2+, indicating that ROMK has a pivotal role in K+ excretion. The present study is the first to demonstrate that the fish excrete K+ from the gill MR cells, and that ROMK expressed in the apical opening of the MR cells is a main molecular pathway responsible for K+ excretion.

HER2 status in unusual histological variants of gastric adenocarcinomas

AimTo investigate HER2 status in a cohort of 109 gastric adenocarcinomas also including unusual variants, such as 14 hepatoid (HAS) and 9 mitochondrion-rich (MRC), characterised by an opposing clinical behaviour.Methods...

Salinity-dependent expression of the branchial Na+/K+/2Cl− cotransporter and Na+/K+-ATPase in the sailfin molly correlates with hypoosmoregulatory endurance

In the branchial mitochondrion-rich (MR) cells of euryhaline teleosts, the Na(+)/K(+)/2Cl(-) cotransporter (NKCC) is an important membrane protein that maintains the internal Cl(-) concentration, and the branchial Na(+)/K(+)-ATPase (NKA) is crucial for providing the driving force for many other ion-transporting systems. Hence this study used the sailfin molly (Poecilia latipinna), an introduced aquarium fish in Taiwan, to reveal that the potential roles of NKCC and NKA in sailfin molly were correlated to fish survival rates upon salinity challenge. Higher levels of branchial NKCC were found in seawater (SW)-acclimated sailfin molly compared to freshwater (FW)-acclimated individuals. Transfer of the sailfin molly from SW to FW revealed that the expression of the NKCC and NKA proteins in the gills was retained over 7days in order to maintain hypoosmoregulatory endurance. Meanwhile, their survival rates after transfer to SW varied with the duration of FW-exposure and decreased significantly when the SW-acclimated individuals were acclimated to FW for 21days. Double immunofluorescence staining showed that in SW-acclimated sailfin molly, NKCC signals were expressed on the basolateral membrane of MR cells, whereas in FW-acclimated molly, they were expressed on the apical membrane. This study illustrated the correlation between the gradual reductions in expression of branchial NKCC and NKA (i.e., the hypoosmoregulatory endurance) and decreasing survival rates after hyperosmotic challenge in sailfin molly.

Mechanism of sodium uptake in PNA negative MR cells from rainbow trout, Oncorhynchus mykiss as revealed by silver and copper inhibition

The rate of acid-stimulated and phenamil-sensitive sodium (Na +) uptake was measured in three different cell lineages: pavement cells (PVC), total mitochondrion-rich (MR) cell populations, and peanut lectin agglutinin-negative mitochondrion-rich cells (PNA − MR) isolated from the rainbow trout gill epithelium. Despite the presence of basal levels of Na + uptake in PVC, this transport was not enhanced by acidification, nor was it inhibited by independent treatment with bafilomycin (i.e., a V-type H +-ATPase inhibitor), phenamil (i.e., a specific inhibitor of ENaC), or Ag (a specific inhibitor of active Na + transport in fish). In contrast, Na + uptake in PNA − MR cells was increased by ~ 220% above basal levels following acidification of near 0.4 pH units in the presence of 1.0 mM external Na +. Acid-stimulated Na + transport was entirely inhibited by both phenamil and bafilomycin. Silver (Ag) and copper (Cu), which are known to interfere with active Na + transport in fish, were also responsible for inhibiting acid stimulated Na + uptake in PNA − MR cells, but by themselves had no effect on basal Na + transport. Thus, we demonstrate that Ag specifically prevented acid-stimulated Na + uptake in PNA − MR cells in a dose-dependent manner. We also demonstrate rapid (< 1 min) and significant inhibition of carbonic anhydrase (CA) by Ag in PNA − MR cells, but not in PVC. These data lend further support to the idea of a PNA − MR cell type as the primary site for Na + uptake in the freshwater (FW) gill phenotype of rainbow trout. Moreover, these findings provide support for the importance of intracellular protons in regulating the movement of Na + across the apical surface of the fish gill.