Modulation of ion transporter expression in gill mitochondrion-rich cells of eels acclimated to low-Na(+) or-Cl(-) freshwater

In this study, we aimed to establish an experimental model to study the role of the gill mitochondrion-rich cells (MRCs) of freshwater fish in Na(+) uptake and to examine the effect of adjusting external Na(+) and Cl(-) ions on selected ion transporters in gill MRCs. Japanese eels (Anguilla japonica) acclimated to deionized (DI) water for 2 weeks were transferred directly to (a) ion-supplemented artificial freshwater (AF), (b) Na(+) -deficient AF, or (c) Cl(-) -deficient AF for 2 days. The effects of the transfer on the expression levels of ion transporters in isolated gill cells were investigated. Our data demonstrated that the 2-day acclimation in ion-supplemented AF, Na(+) -deficient AF, or Cl(-) -deficient AF led to a significant increase in serum osmolarity attributed mainly to an increase in serum Na(+) and/or Cl(-) levels when compared with DI-acclimated eel. Significant inductions of V-type H(+) -ATPase (V-H(+) -ATPase) and cotransporter (NBC1) mRNA expression in gill MRCs were detected in AF-acclimated fish. In fish acclimated to Na(+) -deficient AF, mRNA expression levels of V-H(+) -ATPase, NBC1, and Na(+) /H(+) -exchanger-3 (NHE3) were significantly increased in MRCs. Fish acclimated to Cl(-) -deficient AF showed no observable change in expression levels of ion transporters in gill MRCs. In addition, expression levels of ion transporters in pavement cells were stable throughout the 2-day experiments. These data indicate that the level of Na(+) in freshwater is important for altering the mRNA expression of ion transporters in gill MRCs, which supports the notion that gill MRCs play important roles in freshwater Na(+) uptake.     

Morphochemical analysis of mucosubstances in some epithelial tissues of the eel (Anguilla japonica).

In attempts to gain insight into the nature of mucosubstances in the eel epithelial tissues, the epidermis and epithelia lining the gill and intestine of the fish have been reacted for a series of light microscopic stainings of mucosaccharide and protein histochemistry under normal and experimental conditions. The present morphochemical analyses indicate that (1) the mucosubstances of the epidermis are found within two well differentiated cell types, goblet and clavate cells which elaborate a neuraminic acid containing mucosaccharide with vicinal hydroxyl, sulfate and carboxyl groupings and a glycoprotein respectively and (2) the mucosubstances of the gill and intestinal epithelia are found within goblet cells which elaborate a mucosaccharide with histochemical properties comparable to those of mucosaccharide within the epidermal goblet cells. The histochemical characteristics of all these mucosubstances have been briefly discussed in relation to the activities of the epithelial tissues in the eel.     

Vacuolar-type H(+)-ATPase and Na+, K(+)-ATPase expression in gills of Atlantic salmon (Salmo salar) during isolated and combined exposure to hyperoxia and hypercapnia in fresh water

Changes in branchial vacuolar-type H(+)-ATPase B-subunit mRNA and Na+, K(+)-ATPase alpha- and beta-subunit mRNA and ATP hydrolytic activity were examined in smolting Atlantic salmon exposed to hyperoxic and/or hypercapnic fresh water. Pre-smolts, smolts, and post-smolts were exposed for 1 to 4 days to hyperoxia (100% O2) and/or hypercapnia (2% CO2). Exposure to hypercapnic water for 4 days consistently decreased gill vacuolar-type H(+)-ATPase B-subunit mRNA levels. Salmon exposed to hyperoxia had either decreased or unchanged levels of gill B-subunit mRNA. Combined hyperoxia + hypercapnia decreased B-subunit mRNA levels, although not to the same degree as hypercapnic treatment alone. Hyperoxia generally increased Na+, K(+)-ATPase alpha- and beta-subunit mRNA levels, whereas hypercapnia reduced mRNA levels in presmolts (beta) and smolts (alpha and beta). Despite these changes in mRNA levels, whole tissue Na+, K(+)-ATPase activity was generally unaffected by the experimental treatments. We suggest that the reduced expression of branchial vacuolar-type H(+)-ATPase B-subunit mRNA observed during internal hypercapnic acidosis may lead to reduction of functional V-type H(+)-ATPase abundance as a compensatory response in order to minimise intracellular HCO3- formation in epithelial cells.     

Regulation of membrane lipid bilayer structure during salinity adaptation: a study with the gill epithelial cell membranes of Oreochromis niloticus

A significant variation in the membrane fluidity (as assessed by DPH-fluorescence polarisation) and membrane lipid bilayer composition is noticed in the subcellular membranes of the gill epithelial cells of Oreochromis niloticus due to exposure of the fish to 1% saline water for 1 month. Also, a 70% enhanced activity of Na(+)-K(+)-ATPase in plasma membranes and a 2.5-fold increase of glucose-6-phosphate dehydrogenase in microsomal membranes are recorded in the treated fish. The changed membrane structure and fluidity along with the changed enzymatic activity of Na(+)-K(+)-ATPase help the influx the Na(+) rather than the efflux of K(+) through the gill epithelial cells during salinity adaptation.     

RT-PCR detection of the Na,K-ATPase beta3-isoform in human heart.

The Na,K-ATPase is a heterodimer composed of an alpha-catalytic and a beta-glycoprotein subunit. At present, three different alpha-polypeptides (alpha1, alpha2, alpha3) and two distinct beta-isoforms (beta1 and beta2) have been detected in human heart. The aim of the present study was to determine whether or not the beta3-isoform of the Na,K-ATPase can be detected in human heart. Using the highly sensitive method of RT-PCR, we here show that human heart expresses the beta3-isoform of the Na,K-ATPase. Given the differences in pharmacological properties of the nine different Na,K-ATPase isoenzymes (containing all combinations of the subunit isoforms), the study of beta3-isoform regulation in human heart may be of interest in understanding the altered response of human myocardium to digitalis therapy during heart failure.     

Teleost chloride cell. I. Response of pupfish Cyprinodon variegatus gill Na,K-ATPase and chloride cell fine structure to various high salinity environments

Certain euryhaline teleosts can tolerate media of very high salinity, i.e. greater than that of seawater itself. The osmotic gradient across the integument of these fish is very high and the key to their survival appears to be the enhanced ability of the gill to excrete excess NaCl. These fish provide an opportunity to study morphological and biochemical aspects of transepithelial salt secretion under conditions of vastly different transport rates. Since the cellular site of gill salt excretion is believed to be the "chloride cell" of the branchial epithelium and since the enzyme Na,K-ATPase has been implicated in salt transport in this and other secretory tissues, we have focused our attention on the differences in chloride cell structure and gill ATPase activity in the variegated pupfish Cyprinodon variegatus adapted to half-strength seawater (50% SW), seawater (100% SW), or double-stregth seawater (200% SW). The Na,K-ATPase activity in gill homogenates was 1.6 times greater in 100% SW. When 50% SW gills were compared to 100% SW gills, differences in chloride cell morphology were minimal. However, chloride cells from 200% SW displayed a marked hypertrophy and a striking increase in basal-lateral cell surface area. These results suggest that there are correlations among higher levels of osmotic stress, basal-lateral extensions of the cell surface, and the activity of the enzyme Na,K-ATPase.     

Up-regulation of Na(+),K(+)-ATPase alpha 3-isoform and down-regulation of the alpha1-isoform in human colorectal cancer

We investigated expression levels of Na(+),K(+)-ATPase alpha-isoforms and their ATPase activities in human colorectal cancer tissue and the accompanying normal mucosa. A decrease in expression of the alpha1-isoform protein was observed in all sampled cancer tissues compared with the normal mucosae. The level of ouabain (5 microM)-sensitive Na(+),K(+)-ATPase activity in carcinomas was 81+/-5% that of in the normal mucosae. The mRNA expression of alpha2- and alpha 4-isoforms was decreased in almost all the carcinoma samples. Interestingly, the expression level of the alpha 3-isoform protein in the cancer tissue was higher than that of the normal mucosa. These results indicate that a decrease in the alpha1-isoform expression and an increase in the alpha 3-isoform expression may be associated with colorectal cancer.     

Exercise training differentially modifies age-associated alteration in expression of Na+-K+-ATPase subunit isoforms in rat skeletal muscles

The present study tests the hypothesis that endurance exercise training (ETr) reverses age-associated alterations in expression of Na+-K+-ATPase subunit isoforms in rat skeletal muscles. Expression of the isoforms was examined in 16-mo-old sedentary middle-aged, 29-mo-old sedentary senescent, and 29-mo-old treadmill exercise-trained senescent Fischer 344 x Brown Norway rats. Levels of the alpha1-isoform increased with age in red gastrocnemius (GR), white gastrocnemius (GW), and extensor digitorum longus (EDL) muscles, and ETr further increased its levels. Levels of the alpha2-isoform were unchanged in GR, had a strong trend for a decrease in GW, and decreased significantly in EDL. ETr increased expression of the alpha2-isoform in all three muscle groups. There was no increase in expression of the beta1-isoform in GR, GW, or EDL with age, whereas ETr markedly increased its levels in the muscles. There was a marked decrease with age in expression of the beta2-isoform in the muscle groups that was not reversed by ETr. By contrast, beta3-isoform levels increased with age in GR and GW, and ETr was able to reverse this increase. Na+-K+-ATPase enzyme activity was unchanged with age in GR and GW but increased in EDL. ETr increased enzyme activity in GR and GW and did not change in EDL. Myosin heavy chain isoforms in the muscle groups did not change significantly with age; ETr caused a general shift toward more oxidative fibers. Thus ETr differentially modifies age-associated alterations in expression of Na+-K+-ATPase subunit isoforms, and a mechanism(s) other than physical inactivity appears to play significant role in some of the age-associated changes.     

Cultured gill epithelia from freshwater tilapia (Oreochromis niloticus): effect of cortisol and homologous serum supplements from stressed and unstressed fish

Procedures for the preparation and culture of branchial epithelia from dispersed gill cells of freshwater tilapia (Oreochromis niloticus) are described. Epithelia were cultured on permeable supports (terephthalate membranes, "filters") and bathed on both the apical and basolateral side with isotonic media containing 6% fetal bovine serum (FBS). When the apical medium was replaced with freshwater (pseudo in vivo asymmetrical culture conditions), transepithelial resistance (TER) increased markedly, transepithelial potential became negative, and paracellular permeability decreased. The physiological effects of cortisol and 10% homologous (tilapia) serum were investigated. Tilapia serum (TS) was prepared from unstressed and stressed fish and therefore allowed comparison between the effects of homologous serum derived from fish in differing physiological states. Under both symmetrical and asymmetrical culture conditions, cortisol significantly elevated TER across cultured tilapia gill epithelia, indicative of a significant increase in epithelial "tightness." Cortisol reduced transepithelial Na + and Cl? movement and paracellular permeability. The glucocorticoid agonist dexamethasone elicited a similar response, which was inhibited by the glucocorticoid antagonist (receptor blocker) RU486. Cortisol did not stimulate active ion transport across epithelia under either symmetrical or asymmetrical culture conditions. In epithelia supplemented with TS from stressed fish, physiological changes in cultured preparations were consistent with those observed in FBS + cortisol-supplemented epithelia. Differences between the physiological status of epithelia supplemented with TS from unstressed and stressed fish could be abolished with RU486. Using TS as a medium supplement did not stimulate active ion transport under asymmetrical culture conditions, although Na +-K +-ATPase activity increased in TS-supplemented epithelia relative to FBS-supplemented preparations.     

Acute changes in gill Na+-K+-ATPase and creatine kinase in response to salinity changes in the euryhaline teleost, tilapia (Oreochromis mossambicus)

Some freshwater (FW) teleosts are capable of acclimating to seawater (SW) when challenged; however, the related energetic and physiological consequences are still unclear. This study was conducted to examine the changes in expression of gill Na(+)-K(+)-ATPase and creatine kinase (CK) in tilapia (Oreochromis mossambicus) as the acute responses to transfer from FW to SW. After 24 h in 25 ppt SW, gill Na(+)-K(+)-ATPase activities were higher than those of fish in FW. Fish in 35 ppt SW did not increase gill Na(+)-K(+)-ATPase activities until 1.5 h after transfer, and then the activities were not significantly different from those of fish in 25 ppt SW. Compared to FW, the gill CK activities in 35 ppt SW declined within 1.5 h and afterward dramatically elevated at 2 h, as in 25 ppt SW, but the levels in 35 ppt SW were lower than those in 25 ppt SW. The Western blot of muscle-type CK (MM form) was in high association with the salinity change, showing a pattern of changes similar to that in CK activity; however, levels in 35 ppt SW were higher than those in 25 ppt SW. The activity of Na(+)-K(+)-ATPase highly correlated with that of CK in fish gill after transfer from FW to SW, suggesting that phosphocreatine acts as an energy source to meet the osmoregulatory demand during acute transfer.     

Linkage arrangement of Na,K-ATPase genes in the tetraploid-derived genome of the rainbow trout (Oncorhynchus mykiss)

As part of our efforts to characterize Na,K-ATPase isoforms in salmonid fish, we investigated the linkage arrangement of genes coding for the alpha and beta-subunits of the enzyme complex in the tetraploid-derived genome of the rainbow trout (Oncorhynchus mykiss). Genetic markers were developed from four of five previously characterized alpha-subunit isoforms (alpha1b, alpha1c, alpha2 and alpha3) and four expressed sequence tags derived from yet undescribed beta-subunit isoforms (beta1a, beta1b, beta3a and beta3b). Sex-specific linkage analysis of polymorphic loci in a reference meiotic panel revealed that Na,K-ATPase genes are generally dispersed throughout the rainbow trout genome. A notable exception was the colocalization of two alpha-subunit genes and one beta-subunit gene on linkage group RT-12, which may thus share a conserved orthologous segment with linkage group 1 in zebrafish (Danio rerio). Consistent with previously reported homeologous relationships among the chromosomes of the rainbow trout, primers designed from the alpha3-isoform detected a pair of duplicated genes on linkage groups RT-27 and RT-31. Similarly, the evolutionary conservation of homeologous regions on linkage groups RT-12 and RT-16 was further supported by the map localization of gene duplicates for the beta1b isoform. The detection of homeologs within each gene family also raises the possibility that novel isoforms may be discovered as functional duplicates.     

Tissue-Specific Induction of Hsp90 mRNA and Plasma Cortisol Response in Chinook Salmon following Heat Shock, Seawater Challenge, and Handling Challenge

In studying the whole-body response of chinook salmon (Oncorhynchus tshawytscha) to various stressors, we found that 5-hour exposure to elevated temperature (mean 21.6°C; + 10.6°C over ambient) induced a marked increase in Hsp90 messenger RNA accumulation in heart, brain, gill, muscle, liver, kidney, and tail fin tissues. The most vital tissues (heart, brain, gill, and muscle) showed the greatest Hsp90-mRNA response, with heart tissue increasing approximately 35-fold. Heat shock induced no increase in plasma cortisol. In contrast, a standard handling challenge induced high plasma cortisol levels, but no elevation in Hsp90 mRNA in any tissue, clearly separating the physiological and cellular stress responses. We saw no increase either in tissue Hsp90 mRNA levels or in plasma cortisol concentrations after exposing the fish to seawater overnight. Received October 1, 1999; accepted January 21, 2000     

Gill Na+-K+-ATPase activity correlates with basolateral membrane lipid composition in seawater- but not freshwater-acclimated Arctic char (Salvelinus alpinus)

The successful migration of euryhaline teleost fish from freshwater to seawater requires the upregulation of gill Na+-K+-ATPase, an ion transport enzyme located in the basolateral membrane (BLM) of gill chloride cells. Following 39 days of seawater exposure, Arctic char had similar plasma sodium and chloride levels as individuals maintained in freshwater, indicating they had successfully acclimated to seawater. This acclimation was associated with an eightfold increase in gill Na+-K+-ATPase activity but only a threefold increase in gill Na+-K+-ATPase protein number, suggesting that other mechanisms may also modulate gill Na+-K+-ATPase activity. We therefore investigated the influence of membrane composition on Na+-K+-ATPase activity by examining the phospholipid, fatty acid, and cholesterol composition of the gill BLM from freshwater- and seawater-acclimated Arctic char. Mean gill BLM cholesterol content was significantly lower ( approximately 22%) in seawater-acclimated char. Gill Na+-K+-ATPase activity in individual seawater Arctic char was negatively correlated with BLM cholesterol content and positively correlated with %phosphatidylethanolamine and overall %18:2n6 (linoleic acid) content of the BLM, suggesting gill Na+-K+-ATPase activity of seawater-acclimated char may be modulated by the lipid composition of the BLM and may be especially sensitive to those parameters known to influence membrane fluidity. Na+-K+-ATPase activity of individual freshwater Arctic char was not correlated to any membrane lipid parameter measured, suggesting that different lipid-protein interactions may exist for char living in each environment.     

Water balance in the european eel (Anguilla anguilla L.). Role of the oesophagus in the utilisation of drinking water and a study of the osmotic permeability of the gills (author's transl)]

10 New experimental devices are described which allow chonic measurements of drinking rate and osmotic gill permeability in the eel. 20 The oesophagus of the seawater (SW) silver eel plays a role in osmoregulation. It decreases the concentration of Cl- and Na+ of the ingested SW without losing water in the serosal to mucosal direction. This allows for immediate water absorption in the intestine and decreases the quantity of ions actively absorbed by the intestine. In the freshwater (FW) silver eel, the oesophagus is impermeable to water, Cl- and Na+. The ionic impermeability exists only in the serosal to mucosal direction. A mucosal to serosal permeability to Cl- and Na+ exists in the FW oesophagus receiving hypertonic drinking water, this promotes seawater adaptation. 30 The osmotic gill permeability, measured in vivo in the silver eel, is very low in FW and decreases slightly in SW. Thus, the silver eel has an osmotic gill permeability preadapted to SW life. The kinetics of FW to SW adaptation are described.     

Comparative metabolism of phospholipids of osmoregulation effector organs in European eel (Anguilla anguilla)]

The phospholipid composition from various organs of the fresh water eel, such as gill, kidney, gut, liver and muscle, were determined by thin-layer chromatography. The major phosphatides found in these tissues were PC, PE and SPH and minor constituents PS, PI, DPG, AP and also LPC in the gut. A greater percentage of PS and SPH occurs in the osmoregulatory effector organs such as gill, kidney, and gut. From in vivo comparative kinetic studies of the 32P incorporation into the phospholipids, between 6 and 48 hours, certain remarkable features of phospholipid metabolism have been found in these tissues. A low uptake of inorganic 32P into the tissue lipid phosphorus was observed in the eel at 15 degrees C. The specific activity of the lipid phosphorus increased continuously in all tissues during 48 hours after 32P injection. During this experimental period, phosphatidic acid and phosphatidyl inositol fractions were labelled most rapidly in gill, kidney and gut, while the specific activity of the phosphatidyl choline fraction remained low in these organs. In liver, the rate of PC formation appears to be faster than the PI and PE biosynthesis. In gill and gut, the PE showed greater turnover than the PC as measured by 32P incorporation. In the eel, an euryhalin fish, the DPG of osmoregulatory effector organs has a high specific activity at all times. PS showed only a high specific activity in the gill. Labelling of SPH occured slowly in the various tissues only becoming evident after 24 hours. The results are compared with those published for other poikilotherm and homeotherm vertebrates. Relative differences between the turnover of various tissue phosphatides are discussed with of reference to the general scheme on phospholipid biosynthesis and to the physiological functions of the various organs.