Stimulation of Cl- uptake and morphological changes in gill mitochondria-rich cells in freshwater tilapia (Oreochromis mossambicus)

The purpose of the present article is to examine the relationships between ion uptakes and morphologies of gill mitochondria-rich (MR) cells in freshwater tilapia. Tilapia were acclimated to three different artificial freshwaters (high Na [10 mM], high Cl [7.5 mM]; high Na, low Cl [0.02-0.07 mM], and low Na [0.5 mM], low Cl) for 1 wk, and then morphological measurements of gill MR cells were made and ion influxes were determined. The number and the apical size of wavy-convex MR cells positively associated with the level of Cl(-) influx. Conversely, Na(+) influx showed no positive correlation with the morphologies of MR cells. The dominant MR cell type in tilapia gills changed from deep-hole to wavy-convex within 6 h after acute transfer from a high-Cl(-) to a low-Cl(-) environment. Deep-hole MR cells became dominant 24-96 h after acute transfer from a low-Cl(-) to a high-Cl(-) environment. We conclude that wavy-convex MR cells associate with Cl(-) uptake but not Na(+) uptake, and the rapid formation of wavy-convex MR cells reflects the timely stimulation of Cl(-) uptake to recover the homeostasis of internal Cl(-) levels on acute challenge with low environmental Cl(-).     

Ion-deficient environment induces the expression of basolateral chloride channel, ClC-3-like protein, in gill mitochondrion-rich cells for chloride uptake of the tilapia Oreochromis mossambicus

Gill mitochondrion-rich (MR) cells contain different molecules to carry out functionally distinct mechanisms. To date, the putative mechanism of Cl(-) uptake through the basolateral chloride channel, however, is less understood. To clarify the Cl(-)-absorbing mechanism, this study explored the molecular and morphological alterations in branchial MR cells of tilapia acclimated to seawater (SW), freshwater (FW), and deionized water (DW). Scanning electron microscopic observations revealed that three subtypes of MR cells were exhibited in gill filament epithelia of tilapia. Furthermore, in DW-acclimated tilapia, the subtype I (ion-absorbing subtype) of MR cells predominantly occurred in gill filament as well as lamellar epithelia. Whole-mount double immunofluorescent staining revealed that branchial ClC-3-like protein and Na(+)/K(+)-ATPase (NKA), the basolateral marker of MR cells, were colocalized in tilapia. In SW-acclimated tilapia, all MR cells of gill filament epithelia exhibited faint fluorescence of ClC-3-like protein. In contrast, only some MR cells in gill filament epithelia of FW and DW tilapia expressed basolateral ClC-3-like protein; however, the fluorescence was more intense in FW and DW tilapia than in SW fish. In hyposmotic groups, the number of MR cells immunopositive for ClC-3-like protein was significantly higher in DW-exposed tilapia. Meanwhile, in gill lamellar epithelia of DW tilapia, all MR cells (subtype I) were ClC-3-like protein immunopositive. Double immunostaining of ClC-3-like protein and Na(+)/Cl(-) cotransporter (NCC) revealed that basolateral ClC-3-like protein and apical NCC were colocalized in some MR cells in FW and DW tilapia. Moreover, both mRNA and protein amounts of branchial ClC-3-like protein were significantly higher in DW-acclimated tilapia. To identify whether the expression of branchial ClC-3-like protein responded to changes in environmental [Cl(-)], tilapia were acclimated to artificial waters with normal [Na(+)]/[Cl(-)] (control), lower [Na(+)] (low Na), or lower [Cl(-)] (low Cl). Immunoblotting of crude membrane fractions for gill ClC-3-like protein showed that the protein abundance was evidently enhanced in tilapia acclimated to the low-Cl environment compared with the other groups. Our findings integrated morphological and functional classifications of ion-absorbing MR cells and indicated that ion-deficient water elevated the numbers of subtype I MR cells in both filament and lamellar epithelia of gills with positive ClC-3-like protein immunostaining and increased the expression levels of ClC-3-like protein. This study is the first to illustrate the exhibition of a basolateral chloride channel potentially responsible for Cl(-) absorption in the ion-absorbing subtype of gill MR cells of tilapia.     

Cl- uptake mechanism in freshwater-adapted tilapia (Oreochromis mossambicus)

In this study, the correlation between Cl(-) influx in freshwater tilapia and various transporters or enzymes, the Cl(-)/HCO(3)(-) exchanger, Na(+),K(+)-ATPase, V-type H(+)-ATPase, and carbonic anhydrase were examined. The inhibitors 2x10(-4) M ouabain (a Na(+),K(+)-ATPase inhibitor), 10(-5) M NEM (a V-type H(+)-ATPase inhibitor), 10(-2) M ACTZ (acetazolamide, a carbonic anhydrase inhibitor), and 6x10(-4) M DIDS (a Cl(-)/HCO(3)(-) exchanger inhibitor) caused 40%, 60%-80%, 40%-60%, and 40%-60% reduction in Cl(-) influx of freshwater tilapia, respectively. The inhibitor 2x10(-4) M ouabain also caused 50%-65% inhibition in gill Na(+),K(+)-ATPase activity. Western blot results showed that protein levels of gill Na(+),K(+)-ATPase, V-type H(+)-ATPase, and carbonic anhydrase in tilapia acclimated in low-Cl(-) freshwater were significantly higher than those acclimated to high-Cl(-) freshwater. Based on these data, we conclude that Na(+),K(+)-ATPase, V-H(+)-ATPase, the Cl(-)/HCO(3)(-) exchanger, and carbonic anhydrase may be involved in the active Cl(-) uptake mechanism in gills of freshwater-adapted tilapia.     

Morphology and function of gill mitochondria-rich cells in fish acclimated to different environments

The objective of this study is to test the hypothesis that morphologically different mitochondria-rich (MR) cells may be responsible for the uptake of different ions in freshwater-adapted fish. Tilapia (Oreochromis mossambicus) were acclimated to high-Ca, mid-Ca, low-Ca, and low-NaCl artificial freshwater, respectively, for 2 wk. Cell densities of wavy-convex, shallow-basin, and deep-hole types of gill MR cells as well as whole-body Ca(2+), Na(+), and Cl(-) influxes were measured. Low-Ca fish developed more shallow-basin MR cells in the gills and a higher Ca(2+) influx than those acclimated to other media. However, fish acclimated to low-NaCl artificial freshwater predominantly developed wavy-convex cells, and this was accompanied by the highest Na(+) and Cl(-) influxes. Relative abundance of shallow-basin and wavy-convex MR cells appear to be associated with changes in Ca(2+) and Na(+)/Cl(-) influxes, suggesting that shallow-basin and wavy-convex MR cells are mainly responsible for the uptake of Ca(2+) and Na(+)/Cl(-), respectively.     

Differential design for hopping in two species of wallabies

We explored molecular and morphological alteration in gill mitochondria-rich (MR) cells of Mozambique tilapia, Oreochromis mossambicus, acclimated to deionized freshwater (DFW), freshwater (FW), 1/3-diluted seawater (1/3 SW) and seawater (SW). Scanning electron microscopic observations revealed that the apical membrane of MR cells appeared as a flat or slightly projecting disk in DFW and FW, being larger in DFW than in FW. In contrast, the apical membrane typically formed a pit structure in 1/3 SW and SW. The mRNA expression levels of Na(+)/H(+) exchanger-3 (NHE3) and Na(+)/Cl(-) cotransporter (NCC) in the gills were increased with decreasing environmental salinity, whereas Na(+)/K(+)/2Cl(-) cotransporter-1a (NKCC1a) expression was upregulated by increasing salinity. Immunofluorescence staining showed that the MR cell population of DFW- and FW-acclimated tilapia consisted mostly of MR cells with apical NHE3 and those with apical-NCC; MR cells with basolateral NKCC1a dominated in SW-acclimated tilapia. These results indicated that apical-NHE3 and apical-NCC MR cells were ion-absorbing cells, and that basolateral-NKCC1a MR cells were ion-secreting cells. In fish acclimated to 1/3 SW, both ion-absorbing and secreting cells existed in the gills, suggesting that fish in near-isotonic water were equipped with mechanisms of both hyper- and hypoosmoregulation to prepare for environmental salinity changes.     

Responses of gill mitochondria-rich cells in Mozambique tilapia exposed to acidic environments (pH 4.0) in combination with different salinities

On exposure to hyposmotic acidic water, teleost fish suffer from decreases in blood osmolality and pH, and consequently activate osmoregulatory and acid-base regulatory mechanisms to restore disturbed ion and acid-base balances. In Mozambique tilapia Oreochromis mossambicus exposed to acidic (pH 4.0) or neutral (pH 7.4-7.7) freshwater in combination with 0mM or 50mM NaCl, we examined functional and morphological changes in gill mitochondria-rich (MR) cells. We assessed gene expression of Na(+)/H(+) exchanger-3 (NHE3), Na(+)/Cl(-) cotransporter (NCC), vacuolar-type H(+)-ATPase (V-ATPase) and Na(+)/HCO(3)(-) cotransporter-1 (NBC1) in the gills. The mRNA expression of NHE3 and NCC in tilapia gills were higher in acidic freshwater than in that supplemented with 50mM NaCl, while there was no significant difference in mRNA levels of V-ATPase and NBC1. In addition, immunocytochemical observations showed that apical-NHE3 MR cells were enlarged, and frequently formed multicellular complexes with developed deep apical openings in acidic freshwater with 0mM and 50mM NaCl. These findings suggest that gill MR cells respond to external salinity and pH treatments, by parallel manipulation of osmoregulatory and acid-base regulatory mechanisms.     

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.     

Proton pump-driven cutaneous chloride uptake in anuran amphibia

Krogh introduced the concept of active ion uptake across surface epithelia of freshwater animals, and proved independent transports of Na(+) and Cl(-) in anuran skin and fish gill. He suggested that the fluxes of Na(+) and Cl(-) involve exchanges with ions of similar charge. In the so-called Krogh model, Cl(-)/HCO(3)(-) and Na(+)/H(+) antiporters are located in the apical membrane of the osmoregulatory epithelium. More recent studies have shown that H(+) excretion in anuran skin is due to a V-ATPase in mitochondria-rich (MR) cells. The pump has been localized by immunostaining and H(+) fluxes estimated by pH-stat titration and mathematical modelling of pH-profiles in the unstirred layer on the external side of the epithelium. H(+) secretion is voltage-dependent, sensitive to carbonic-anhydrase inhibitors, and rheogenic with a charge/ion-flux ratio of unity. Cl(-) uptake from freshwater is saturating, voltage independent, and sensitive to DIDS and carbonic-anhydrase inhibitors. Depending on anuran species and probably on acid/base balance of the animal, apical exit of protons is coupled to an exchange of Cl(-) with base (HCO(3)(-)) either in the apical membrane (gamma-type of MR cell) or in the basolateral membrane (alpha-type MR cell). The gamma-cell model accounts for the rheogenic active uptake of Cl(-) observed in several anuran species. There is indirect evidence also for non-rheogenic active uptake accomplished by a beta-type MR cell with apical base secretion and basolateral proton pumping. Several studies have indicated that the transport modes of MR cells are regulated via ion- and acid/base balance of the animal, but the signalling mechanisms have not been investigated. Estimates of energy consumption by the H(+)-ATPase and the Na(+)/K(+)-ATPase indicate that the gamma-cell accomplishes uptake of NaCl in normal and diluted freshwater. Under common freshwater conditions with serosa-positive or zero V(t), the K(+) conductance of the basolateral membrane would have to maintain the inward driving force for Na(+) uptake across the apical membrane. With the K(+) equilibrium potential across the basolateral membrane estimated to -105 mV, this would apply to external Na(+) concentrations down to 40-120 micromol/l. NaCl uptake from concentrations down to 10 micromol/l, as observed by Krogh, presupposes that the H(+) pump hyperpolarizes the apical membrane, which would then have to be associated with serosa-negative V(t). In diluted freshwater, exchange of cellular HCO(3)(-) with external Cl(-) seems to be possible only if the proton pump has the additional function of keeping the external concentration of HCO(3)(-) low. Quantitative considerations also lead to the conclusion that with the above extreme demand, at physiological intracellular pH of 7.2, the influx of Cl(-) via the apical antiporter and the passive exit of Cl(-) via basolateral channels would be possible within a common range of intracellular Cl(-) concentrations.     

Ambient salinity modulates the expression of sodium pumps in branchial mitochondria-rich cells of Mozambique tilapia,Oreochromis mossambicus

Na,K-ATPase (sodium pumps) provide the primitive driving force for ion transport in branchial epithelial cells. Immunoblots of epithelial homogenates of both seawater (SW)- and freshwater (FW)-adapted tilapia gills as well as rat brain homogenate, a positive control, revealed one major band with a molecular weight of about 100 kDa. SW-adapted tilapia gills possessed larger (about 2-fold) amounts of sodium pumps compared with FW-adapted tilapia gills. (3)H-ouabain binding representing functional binding sites of Na,K-ATPase was also higher (about 3.5-fold) in gills of SW-adapted tilapia compared to that of FW-adapted fish. Moreover, specific activities of SW fish were higher (about 2-fold) than those of FW fish. Double labeling of Na,K-ATPase and Con-A, a fluorescent marker of MR cells, in tilapia gills followed by analysis with confocal microscopy showed that sodium pumps were localized mainly in MR cells, including the SW type and different FW types. Although more-active expression of Na,K-ATPase was demonstrated in gills of SW-adapted tilapia, no significant differences in densities of apical openings of MR cells were found between SW- and FW-adapted fish. These results indicate that, during salinity challenge, tilapia develop more "functional" Na,K-ATPase in SW-type MR cells to meet physiological demands.     

Influence of temperature and membrane lipid composition on the osmotic water permeability of teleost gills.

Osmotic water uptake was measured gravimetrically in isolated, ligated gill arches from trout (acclimated to and incubated at 5 degrees and 20 degrees C) and tilapia (21.5 degrees and 33 degrees C). For both species, incubation of arches at the higher temperature led to 1.5- to 3-fold greater measures of water weight gain. However, gills from warmer-acclimated trout and tilapia had 1- to >3-fold lower the initial rate and 1.5- to >2.5-fold lower the extent of water uptake seen in colder-acclimated conspecifics. Both the incubation temperature sensitivity and the acclimation effects are consistent with transmembrane water permeation. Calcium-free incubations (permitting paracellular water movement) also indicated that interfacial cell membranes contribute to gill permeability characteristics; without calcium, trout gill osmotic water uptake values increased 1.5- to 2-fold, and the temperature dependence of water uptake decreased (initial rate) or was eliminated (extent). The specific contribution of cholesterol to restricting barrier membrane water permeability was indicated by concentration-dependent increases in water uptake in the presence of either nystatin (a cholesterol-complexing, pore-forming agent) or methyl-beta-cyclodextrin (which selectively depletes membrane cholesterol). In addition, a cholesterol-specific cytochemical probe (filipin) intensely labeled the apical surface membranes of trout and tilapia gill epithelium. In summary, these studies implicate membrane cholesterol in determining water permeability in fish gills.     

Biochemical characterization of isolated branchial mitochondria-rich cells of Oreochromis mossambicus acclimated to fresh water or hyperhaline sea water

Mitochondria-rich cells have been separated from other epithelial cells of tilapia (Oreochromis mossambicus) gills by density gradient centrifugation on Percoll. During centrifugation two main bands of cells formed. The viability of the cells in both bands was high (>90%). In one band, 45–47% of the total cell number was mitochondria-rich cells. The other band contained at least 80% pavement cells, representing the majority of other gill epithelial cell types. A comparison of the activities of four enzymes involved in major metabolic and ion regulatory functions was made between these two different fractions of cells. Furthermore, the separation of gill epithelial cells and determination of enzymatic activity was carried out in tilapia after the fish were acclimated to fresh water or hyperhaline sea water (60 mg·ml^-1 S) to gain an indication of the relative contribution of mitochondria-rich cells and pavement cells to both NaCl excretion and absorption. Regardless of acclimation salinity, the activities of Na⁺/K⁺-ATPase, glutamate dehydrogenase and glucose-6-phosphate dehydrogenase were significantly higher in mitochondria-rich cells than in pavement cells. However, tilapia acclimated to hyperhaline sea water possessed significantly lower carbonic anhydrase activity in mitochondria-rich cells than in pavement cells. In contrast, no significant difference of carbonic anhydrase activity was observed between the two cell fractions in tilapia acclimated to fresh water.Abbreviations ATPase adenosine triphosphatase - CA carbonic anhydrase - DASPMI dimethylaminostyrylmethylpyridinium iodine - FW fresh-water - GIDH glutamate dehydrogenase - G6PDH glucose-6-phosphate dehydrogenase - HSW hyperhaline sea water (60 mg·ml^-1) - MR cells, mitochondria-rich cells - S salinity     

Comparisons of calcium regulation in fish larvae

The purpose of the present study was to compare the ability of larvae of different species, goldfish (Carassius auratus), zebrafish (Danio rerio), and ayu (Plecoglossus altivelis), to regulate their calcium balance. Whole-body Ca(2+) content and Ca(2+) influx in the larvae of the three species, which were incubated in low- (0.02 mM), mid- (0.2 mM), and high- (2.0 mM) Ca(2+) artificial fresh water from embryonic stages, were compared. The Ca(2+) uptake kinetics were determined in zebrafish and goldfish incubated in high- or low-Ca(2+) artificial fresh water. Ca(2+) content of both zebrafish and ayu acclimated to low-Ca(2+) media were significantly lower than those acclimated to mid- or high-Ca(2+) media. However, Ca(2+) contents of goldfish in low-, mid-, and high-Ca(2+) groups showed no significant differences. In goldfish, Ca(2+) influx in the low-Ca(2+) group was significantly higher than those of the mid- and high-Ca(2+) groups. In contrast, the Ca(2+) influx rate in the low-Ca(2+) group was significantly lower than those in the mid- and high-Ca(2+) groups in zebrafish and ayu. Compared to the high-Ca(2+) group, the low-Ca(2+) group of goldfish showed a 13% increase in the maximal velocity (J(max)) and an 84% decrease in the Michaelis constant (K(m)) for Ca(2+) influx. Smaller changes, i.e., an 8% increase in J(max) and a 67% decrease in K(m), were found in zebrafish larvae. Goldfish possess a more effective Ca(2+) regulatory capacity than do zebrafish and ayu. Differences in the strategies for Ca(2+) balance may be associated with different development patterns and environments in which these fish naturally occur.     

Proton pump activity is required for active uptake of chloride in isolated amphibian skin exposed to freshwater

Net proton secretion and unidirectional chloride fluxes were measured in isolated skin of toads ( Bufo bufo) and frogs ( Rana esculenta) mounted in an Ussing chamber and exposed to a Ringer's solution on the serosal side and a freshwater-like solution (1-3 mM Cl(-)) on the external side. Active proton secretion was 34.2+/-2.0 pmol.cm(-2).s(-1) ( n=18) in frog skin, and 16.7+/-1.7 pmol.cm(-2).s(-1) ( n=10) in toad skin. Proton secretion by toad skin was dependent on the transepithelial potential ( V(T)), and an amiloride-insensitive short-circuit current was stimulated by exogenous CO(2)/HCO(3)(-), indicating the presence of a rheogenic proton pump. Cl(-) influx was 37.4+/-7.5 pmol.cm(-2).s(-1) ( n=14) in frog skin and 19.5+/-3.5 pmol.cm(-2).s(-1) ( n=11) in toad skin. In toad skin, the mean Cl(-) flux ratio was larger than expected for simple electro-diffusion. In 8 of 11 sets of paired skins, influx was greater than the efflux indicating active uptake of Cl(-). Cl(-) influx in toad skin was unaffected by large perturbations (100-150 mV) of V(T), which was accomplished by adding amiloride to the outer bath under open circuit conditions. A component of the Cl(-) efflux seemed to be dependent on V(T). 4,4'-Diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS; 0.3 mM or 1.3 mM) inhibited Cl(-) influx and, surprisingly, increased Cl(-) efflux in toad skin. Influx and efflux of Cl(-) in toad skin were highly dependent on the external [Cl(-)] in the freshwater range (0.1-4 mM). (36)Cl(-) influx decreased whereas the total Cl(-) efflux increased as a function of external [Cl(-)]. These data indicate the presence of a DIDS-sensitive, electroneutral carrier mechanism with an external binding site for Cl(-). Ethoxzolamide (100 micro M), an inhibitor of carbonic anhydrase, reduced proton secretion and Cl(-) influx in frog skin. Concanamycin A (0.1-10 micro M), a specific vacuolar-type proton pump (V-ATPase) inhibitor, significantly reduced proton secretion in frog skin. In addition, concanamycin A (1 micro M) significantly reduced Cl(-) influx in frog skin. We suggest that the active proton secretion and Cl(-) influx are coupled. We hypothesise that an apical V-ATPase is capable of energising active Cl(-) uptake in fresh water by creating a favourable gradient for an apical HCO(3)(-) exit in exchange for external Cl(-). The data also suggest that a carbonic anhydrase activity provides H(+) and HCO(3)(-) for apically co-expressed proton pumps and Cl(-)/HCO(3)(-) exchangers.     

Sodium and chloride transport in soft water and hard water acclimated zebrafish (Danio rerio)

While the zebrafish is commonly used for studies of developmental biology and toxicology, very little is known about their osmoregulatory physiology. The present investigation of Na(+) and Cl(-) transport revealed that the zebrafish is able to tolerate extremely low ambient ion concentrations and that this is achieved at least in part by a greatly enhanced apparent uptake capacity and affinity for both ions. Zebrafish maintain plasma and whole body electrolyte concentrations similar to most other freshwater teleosts even in deionized water containing only 35 microM NaCl, i.e soft water. We recorded an extremely low transport affinity constant (K(m)) of 8+/-1 microM for the active uptake of Cl(-) in soft water acclimated fish, while other transport kinetic parameters were in agreement with reports for other freshwater organisms. While both Na(+) and Cl(-) uptake in soft water clearly depends on apical proton pump activity, changes in abundance and possibly localization of this protein did not appear to contribute to soft water acclimation. Active Cl(-) uptake was strongly dependent on branchial carbonic anhydrase (CA) activity regardless of water type, while the response of Na(+) transport to a CA inhibitor was more variable. Differential response of Na(+) uptake to amiloride depending on acclimation medium suggests that different Na(+) transport mechanisms are employed by zebrafish acclimated to soft and hard water.     

Cellular and epithelial adjustments to altered salinity in the gill and opercular epithelium of a cichlid fish (Oreochromis mossambicus)

Morphological features of the gill and opercular epithelia of tilapia (Oreochromis mossambicus) have been compared in fish acclimated to either fresh water (FW) or hypersaline water (60 S) by scanning electron and fluorescence microscopy. In hyperosmoregulating, i.e., FW-acclimated, tilapia only those mitochondria-rich (MR) cells present on the filament epithelium of the gill were exposed to the external medium. After acclimation of fish to hypersaline water these cells become more numerous, hypertrophy extensively, and form apical crypts not only in the gill filament but also in the opercular epithelium. Regardless of salinity, MR cells were never found to be exposed to the external medium on the secondary lamellae. In addition, two types of pavement cells were identified having distinct morphologies, which were unaffected by salinity. The gill filaments and the inner operculum were generally found to be covered by pavement cells with microridges, whereas the secondary lamellae were covered exclusively by smooth pavement cells.     

Phenotypic changes in mitochondrion-rich cells and responses of Na+/K+-ATPase in gills of tilapia exposed to deionized water

The aim of this study was to illustrate the phenotypic modification of mitochondrion-rich (MR) cells and Na(+)/K(+)-ATPase (NKA) responses, including relative protein abundance, specific activity, and immunolocalization in gills of euryhaline tilapia exposed to deionized water (DW) for one week. The plasma osmolality was not significantly different between tilapia of the local fresh water (LFW) group and DW group. Remodeling of MR cells occurred in DW-exposed fish. After transfer to DW for one week, the relative percentage of subtype-I (wavy-convex) MR cells with apical size ranging from 3 to 9 microm increased and eventually became the dominant MR cell subtype. In DW tilapia gills, relative percentages of lamellar NKA immunoreactive (NKIR) cells among total NKIR cells increased to 29% and led to significant increases in the number of NKIR cells. In addition, the relative protein abundance and specific activity of NKA were significantly higher in gills of the DW-exposed fish. Our study concluded that tilapia require the development of subtype-I MR cells, the presence of lamellar NKIR cells, and enhancement of NKA protein abundance and activity in gills to deal with the challenge of an ion-deficient environment.     

Functional plasticity of mitochondrion-rich cells in the skin of euryhaline medaka larvae (Oryzias latipes) subjected to salinity changes

A noninvasive technique, the scanning ion-selective electrode technique (SIET) was applied to measure Na(+) and Cl(-) transport by the yolk-sac skin and individual mitochondrion-rich cells (MRCs) in intact medaka larvae (Oryzias latipes). In seawater (SW)-acclimated larvae, significant outward Na(+) and Cl(-) gradients were measured at the yolk-sac surface, indicating secretions of Na(+) and Cl(-) from the yolk-sac skin. With Na(+) pump immunostaining and microscopic observation, two groups of MRCs were identified on the yolk-sac skin of SW-larvae. These were single MRCs (s-MRCs), which do not have an accompanying accessory cell (AC), and multicellular complex MRCs (mc-MRCs), which usually consist of an MRC and an accompanying AC. The percentage of mc-MRC was ～60% in 30 parts per thousand of SW, and it decreased with the decrease of external salinity. By serial SIET probing over the surface of the MRCs and adjacent keratinocytes (KCs), significant outward fluxes of Na(+) and Cl(-) were detected at the apical opening (membrane) of mc-MRCs, whereas only outward Cl(-) flux, but not Na(+) flux, was detected at s-MRCs. Treatment with 100 μM ouabain or bumetanide effectively blocked the Na(+) and Cl(-) secretion. Following freshwater (FW) to SW transfer, Na(+) and Cl(-) secretions by the yolk-sac skin were fully developed in 5 h and 2 h, respectively. In contrast, both Na(+) and Cl(-) secretions downregulated rapidly after SW to FW transfer. Sequential probing at individual MRCs found that Na(+) and Cl(-) secretions declined dramatically after SW to FW transfer and Na(+)/Cl(-) uptake was detected at the same s-MRCs and mc-MRCs after 5 h. This study provides evidence demonstrating that ACs are required for Na(+) excretion and MRCs possess a functional plasticity in changing from a Na(+)/Cl(-)-secreting cell to a Na(+)/Cl(-)-absorbing cell.     

Presence of Na-K-ATPase in mitochondria-rich cells in the yolk-sac epithelium of larvae of the teleost Oreochromis mossambicus

The purpose of this study is to provide biochemical evidence for the functions of the mitochondria-rich cell (MR cell) in the yolk-sac epithelium of the developing larvae of tilapia Oreochromis mossambicus. Western blotting with the antibody (6F) raised against avian Na-K-ATPase alpha1 subunit demonstrated the presence of Na-K-ATPase in yolk-sac epithelium of tilapia larvae and about 1. 46-fold more of the enzyme in seawater larvae than in freshwater ones. The yolk-sac MR cells were immunoreacted to the antibody (alpha5) against the alpha subunit of avian Na-K-ATPase and were double-labeled with anthroylouabain and dimethylaminostyrylethyl-pyridiniumiodine, suggesting the existence and activity of Na-K-ATPase in these cells. Binding of 3H-ouabain in the yolk sac of seawater larvae was much higher than in that of freshwater larvae (4.183+/-0.143 pmol/mg protein versus 1.610+/-0. 060 pmol/mg protein or 0.0508+/-0.0053 pmol/yolk sac versus 0. 0188+/-0.0073 pmol/yolk sac). These biochemical results are further evidence that yolk-sac MR cells are responsible for a major role in the osmoregulatory mechanism of early developmental stages before the function of gills is fully developed.     

Immunolocalization of Vacuolar-Type H(+)-ATPase in the Yolk-Sac Membrane of Tilapia (Oreochromis mossambicus) Larvae

To investigate the involvement of the yolk-sac membrane in ion absorption, developmental changes in whole-body cation contents, cellular localization of vacuolar-type H(+)-ATPase (V-ATPase), and size and density of pavement and chloride cells in the yolk-sac membrane were examined in tilapia (Oreochromis mossambicus) larvae in fresh water (FW) and those transferred to seawater (SW) at 2 days before hatching (day-2). The whole-body content of Na(+) in embryos and larvae adapted to both FW and SW increased constantly from day-2 to day 10, although they were not fed through the experiment. The yolk-sac membrane of FW larvae at days 0 and 2 showed V-ATPase immunoreactivity in pavement cells, but not in chloride cells. No positive immunoreactivity was detected in SW larvae. Whole-mount immunocytochemistry showed that some pavement cells were intensively immunoreactive, whereas others were less or not immunoreactive. Electron-microscopic immunocytochemistry revealed that V-ATPase immunoreactivity was present in the apical regions of pavement cells in FW larvae, especially in their ridges. The pavement cells in FW larvae were significantly smaller in size but higher in density than those in SW. These results suggest that pavement cells are the site of active Na(+) uptake in exchange for H(+) secretion through V-ATPase in FW-adapted tilapia during early life stages.     

The influence of zinc on apical uptake of cadmium in the gills and cadmium influx to the circulatory system in zebrafish (Danio rerio)

Zn (0-16 microM) effects on apical Cd uptake from the water into the branchial epithelium and influx of Cd from the water to the circulatory system in zebrafish (Danio rerio) were studied in three experiments. Apical Cd uptake was decreased by Zn in all three experiments. In fish exposed to 1-600 nM Cd (experiment 1), apical Cd uptake showed saturation kinetics at 2 and 4 microM Zn, and a competitive interaction was indicated. At 16 microMZn, Cd uptake increased linearly. Cadmium influx did not show saturation kinetics, but was inhibited by 16 microM Zn at low Cd exposure concentrations. In fish exposed to 0.1-600 nM Cd (experiment 2), Cd uptake was inhibited by 16 microM Zn, whereas at 30 nM Cd uptake was inhibited by 2 microM Zn. Similarly, 2 microM Zn did not influence Cd uptake in fish exposed to 0.1-2 nM Cd (experiment 3), whereas 2 microM Zn inhibited uptake at 8-30 nM Cd. Zinc also inhibited Cd influx at higher Cd concentrations. However, at lower Cd exposures, a Zn-induced increased influx was indicated. Zinc influences the Cd uptake and influx processes at several sites in the branchial epithelial cells, indicating that influx of Zn2+ and Cd2+ occurs through common pathways.