Characterization of a branchial epithelial calcium channel (ECaC) in freshwater rainbow trout (Oncorhynchus mykiss).

The entry of calcium (Ca2+) through an apical membrane epithelial calcium channel (ECaC) is thought to a key step in piscine branchial Ca2+ uptake. In mammals, ECaC is a member of the transient receptor potential (TRP) gene family of which two sub-families have been identified, TRPV5 and TPRV6. In the present study we have identified a single rainbow trout (Oncorhynchus mykiss) ECaC (rtECaC) that is similar to the mammalian TRPV5 and TRPV6. Phylogenetic analysis of the protein sequence suggests that an ancestral form of the mammalian genes diverged from those in the lower vertebrates prior to the gene duplication event that gave rise to TRPV5 and TRPV6. The putative model for Ca2+ uptake in fish proposes that the mitochondria-rich cell (also termed ionocyte or chloride cell) is the predominant or exclusive site of transcellular Ca2+ movements owing to preferential localisation of ECaC to the apical membrane of these cells. However, the results of real-time PCR performed on enriched gill cell populations as well as immunocytochemistry and in situ hybridisation analysis of enriched cells, cell cultures and whole gill sections strongly suggest that ECaC is not exclusive to mitochondria-rich cells but that it is also found in pavement cells. Not only was ECaC protein localized to areas of the gill normally having few mitochondria-rich cells, but there was also no consistent co-localization of ECaC- and Na+/K+-ATPase-positive (a marker of mitochondria rich cells) cells. Taken together, the results of the present study suggest that although ECaC (mRNA and protein) does exist in trout gill, its cellular distribution is more extensive than previously thought, thus suggesting that Ca2+ uptake may not be restricted to mitochondria-rich cells as was proposed in previous models.

Modulation of calcium balance in tilapia larvae (Oreochromis mossambicus) acclimated to low-calcium environments.

This study examined how developing fish larvae regulate their Ca2+ balance for acclimation to low ambient Ca2+. Calcium balance in newly hatched larvae was examined individually. Developing larvae not only increased Ca2+ influx but also decreased Ca2+ efflux when they were acclimated to low-Ca2+ environments. After acclimation for 8 days, the influx and efflux of the low-Ca2+ (0.02 mM) group were about 106% and 43%, respectively, compared to those of the high-Ca2+ (1.0 mM) group. Sensitivity and response to low-Ca2+ environments are age-dependent. Upon acute exposure to low Ca2+. newly hatched (H0) larvae increased both Ca2+ influx (from 24% to 67% of high-Ca2+) and net uptake (from 5% to 69%) within 64 h, while 3-day-posthatching (H3) larvae managed to reach the levels of the control within 38 h. Declining Ca2+ efflux in H3 larvae occurred 14 h after exposure, much faster than those in H0 larvae (38 h). It is suggested that modulation of Ca2+-balance mechanisms in developing larvae is dependent upon the levels of Ca2+ in the larval body.

Modification of morphology and function of integument mitochondria-rich cells in tilapia larvae (Oreochromis mossambicus) acclimated to ambient chloride levels.

Similar to those of the gills of adults, three types of mitochondria-rich (MR) cells with different morphologies of apical surfaces (wavy convex, shallow basin, and deep hole) were identified on the integument of freshwater-acclimated tilapia larvae (Oreochromis mossambicus). The object of this study is to test the hypothesis that these subtype cells may represent MR cells equipped with variable efficiencies in Cl(-) uptake. Larvae acclimated to low-Cl(-) =0.001-0.007 mM) water developed higher densities of MR cells than those acclimated to high-Cl(-) =7.3-7.9 mM) water. The percentage of wavy-convex-type cells in total MR cells was higher in low-Cl(-)-acclimated larvae than in high-Cl(-)-acclimated larvae, which displayed only deep-hole type. In addition, Cl(-) influx rates of whole larva measured with (36)Cl(-) showed a coincident correlation with MR cell densities, that is, low-Cl(-) larvae displayed higher Cl(-) influx rates than did high-Cl(-) larva, suggesting that tilapia larvae develop a higher density of MR cells with larger apical surfaces (wavy-convex type) to boost Cl(-) uptake in Cl(-)-deficient water. The distinct types of apical surfaces may represent different phases of MR cells that possess different efficiencies of Cl(-) uptake. Increased apical membrane surface areas of MR cells may provide larvae with rapid regulation of Cl(-) before new MR cells differentiate.

Regulation of drinking rate in euryhaline tilapia larvae (Oreochromis mossambicus) during salinity challenges.

Euryhaline tilapia larvae are capable of adapting to environmental salinity changes even when transferred from freshwater (FW) to seawater (SW) or vice versa. In this study, the water balance of developing tilapia larvae (Oreochromis mossambicus) adapted to FW or SW was compared, and the short-term regulation of drinking rate of the larvae during salinity adaptation was also examined. Following development, wet weight and water content of both SW- and FW-adapted larvae increased gradually, while the dry weight of both group larvae showed a slow but significant decline. On the other hand, the drinking rate of SW-adapted larvae was four- to ninefold higher than that of FW-adapted larvae from day 2 to day 5 after hatching. During acute salinity challenges, tilapia larvae reacted profoundly in drinking rate, that is, increased or decreased drinking rate within several hours while facing hypertonic or hypotonic challenges, to maintain their constancy of body fluid. This rapid regulation in water balance upon salinity challenges may be critical for the development and survival of developing larvae.

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.

Metallothionein induction in early larval stages of tilapia (Oreochromis mossambicus).

Amounts of whole-body metallothionein (MT) in tilapia (Oreochromis mossambicus) larvae increased to a peak (1,500 ng mg(-1) protein) 1 d after hatching (H1), decreased rapidly thereafter, and was maintained at a constant level (700 ng mg(-1)) 3 d after hatching (H3). Waterborne Cd(2+) could stimulate MT expression in newly hatched (H0) larvae in dose-dependent and time-dependent patterns. H0 larvae, which were treated with 35 microg L(-1) Cd(2+) for 24 h, showed a 1.7-fold increase in the MT amount (174.0+/-64.7) and a 6. 5-fold increase in accumulated Cd(2+) but no significant change in Ca(2+) content, compared with the H0 control (MT, 102.6+/-48.1). H3 larvae with the same treatment revealed about a 10-fold increase in accumulated Cd(2+), a 10% decrease in Ca(2+) content, but no change in MT (261.2+/-120.0), compared with the H3 control (MT, 330+/-74.0). H0 larvae could synthesize more MT to bind Cd(2+) for detoxification in 35 microg L(-1) Cd(2+), a dose that would not affect normal physiology or survival of H0 larvae. On the other hand, 35 microg L(-1) Cd(2+) caused H3 larvae to experience hypocalcemia, an abnormal physiological condition, in which H3 larvae could not synthesize sufficient MT, thus causing greater than 25% mortality. These results indicate for the first time that the inducibility of MT by waterborne Cd(2+) is development dependent, being correlated with inconsistent sensitivities to Cd(2+) during larval development.

Complete genomic organization and promoter analysis of the round-spotted pufferfish JAK1, JAK2, JAK3, and TYK2 genes.

We have previously reported the isolation of the JAK1 gene from the round-spotted pufferfish. In the present study, we cloned and characterized genomic sequences encoding pufferfish JAK2, JAK3, and TYK2, which are other members of JAK family. To our knowledge, this is the first report to demonstrate the existence of four JAK genes in fish. All pufferfish JAK genes except JAK1 are composed of 24 exons; JAK1 has an additional exon. A comparison of the exon-intron organization of these genes revealed that the splice sites of JAK genes are nearly identical. In addition, all pufferfish JAK genes have one intron in the 5' untranslated region. Taken together, these data suggest that the pufferfish JAK genes may have evolved from a common ancestor. By 5' rapid amplification of cDNA ends and sequence analysis, we deduced the promoter regions for all JAK genes and found they do not contain typical TATA or CCAAT boxes but rather numerous other potential binding sites for transcription factors. Interestingly, the TYK2 gene is linked to CDC37 in a head-to-tail manner with a small intergenic region of 292 bp. Within this region, there are two potential binding sites for transcriptional factors such as c-Myb and NF-IL6. The putative promoter regions of all JAK genes were tested either in a carp CF cell line or in zebrafish embryos using CAT or lacZ as reporter genes. Both assays confirmed the transcriptional activities of these promoters in vitro and in vivo.

Effects of cortisol and salinity challenge on water balance in developing larvae of tilapia (Oreochromis mossambicus).

Effects of exogenous cortisol on drinking rate and water content in developing larvae of tilapia (Oreochromis mossambicus) were examined. Both freshwater- and seawater-adapted larvae showed increases in drinking rates with development. Drinking rates of seawater-adapted larvae were about four- to ninefold higher than those of freshwater-adapted larvae from day 2 to day 5 after hatching. Seawater-adapted larvae showed declines in drinking rate and water content at 4 and 14 h, respectively, after immersion in 10 mg L(-1) cortisol. In the case of freshwater-adapted larvae, the drinking rate decreased after 8 h of cortisol immersion, while the water content did not show a significant change even after 32 h of cortisol immersion. In a subsequent experiment of transfer from freshwater to 20 ppt (parts per thousand, salinity) seawater, immersion in 10 mg L(-1) cortisol for 8-24 h enhanced the drinking rate in larvae at 4 h after transfer, but no significant difference was found in water contents between cortisol-treated and control groups following transfer. These results suggest that cortisol is involved in the regulation of drinking activity in developing tilapia larvae.

Effects of cortisol on ion regulation in developing tilapia (Oreochromis mossambicus) larvae on seawater adaptation.

The yolk diameter of cortisol-treated tilapia (Oreochromis mossambicus) larvae, immersed in freshwater (FW) containing 10 mg L-1 cortisol from 48 h postfertilization to 12 d posthatching, was significantly larger than that of control larvae after 8 d of treatment, suggesting that inhibition on larval growth occurred only after a long-term treatment with cortisol. Tilapia embryos or larvae treated with 1-10 mg L-1 cortisol for 1-2 d and then transferred to 20-30 g L-1 seawater (SW) showed reduced cumulative larval mortality in SW compared with controls. Moreover, 4-5 d of cortisol treatments significantly diminished the degree of increase in larval body Na content after the transfer to SW. Significant effect of cortisol on body Na content of larvae occurred as early as 4-8 h after the transfer to SW, while no significant difference was found in the ouabain binding of yolk-sac epithelia between control and cortisol-treated larvae even 12 h after the transfer. Cortisol may be involved in the early phase of SW adaptation in developing larvae, and this mechanism may be achieved by other means than increasing the Na-K-ATPase of yolk-sac epithelia.

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.

Isoform expression of Na+-K+-ATPase alpha-subunit in gills of the teleost Oreochromis mossambicus.

Three isoform-specific antibodies, 6F against the alpha1-isoform of the avian sodium pump, HERED against the rat alpha2-isoform, and Ax2 against the rat alpha3-isoform, were used to detect the expression of Na+-K+-ATPase alpha-subunits in gills of a teleost, the tilapia (Oreochromis mossambicus). Tilapia gill tissue showed positive reactions to antibodies specific for alpha1- and alpha3-isoforms. The results of immunoblots were converted to numerical values (relative intensities) by image analysis for comparisons. Relative amounts of alpha1-like isoform alone and consequently the ratio of alpha1-like to alpha3-like isoforms were higher in gills of seawater-adapted tilapia than in those of freshwater-adapted ones, indicating that the two isoforms respond differently to environmental salinities. In the subsequent immunocytochemical experiments, gill mitochondria-rich cells were demonstrated to immunoreact with antibodies specific for alpha1- and alpha3-isoforms. alpha1-like and alpha3-like isoforms of gill Na+-K+-ATPase are suggested to be involved in the ion- and osmoregulation mechanisms in tilapia. Moreover, differential expressions of two isoforms may be associated with different functions, secretion and uptake of ions and acid-base regulation, in gills of seawater- and freshwater-adapted tilapia.

Effects of wheat germ agglutinin and colchicine on microtubules of the mitochondria-rich cells and Ca2+ uptake in tilapia (Oreochromis mossambicus) larvae.

The organization of the microtubules and actin filaments in the gills of tilapia (Oreochromis mossambicus) larvae was revealed by confocal microscopy. The fluorescence intensity of the microtubules in the gills was increased by adding wheat germ agglutinin (WGA, 40 ng ml-1) to the ambient water for 30 min, but the staining pattern of the actin filaments was not changed. The fluorescence intensity of the microtubules in the gills was decreased by treatment with the microtubule-disrupting reagent colchicine at 0.2 mmol l-1 for 4 h. WGA treatment concurrently raised Ca2+ influx rates, and the increase was particularly large when the larvae were kept in water with extremely low Ca2+ levels ([Ca2+]=0.002 mmol l-1). Colchicine treatment, in contrast, reduced the Ca2+ influx rate. These results indicate that the microtubule network in tilapia gills, particularly in mitochondria-rich cells, could play a critical role in the uptake of Ca2+ in tilapia larvae.

Ca2+ uptake and Cd2+ accumulation in larval tilapia (Oreochromis mossambicus) acclimated to waterborne Cd2+.

The present study compares the rates of Ca2+ uptake and Cd2+ accumulation in tilapia (Oreochromis mossambicus) between larvae preexposed to Cd2+ and naive larvae. Preexposure to Cd2+ induces some form of adaptation that attenuates the effects of Cd2+ later on. Exposure to Cd2+ decreased the uptake of Ca2+ but did not suppress the accumulation rate of Cd2+. A 12-fold increase in 96-h half-maximal lethal concentration was found in tilapia larvae preexposed to 0.45 microM Cd2+ from hatching for 3 days in comparison with naive 3-day-old larvae. The effects of Cd2+ on Ca2+ influx kinetics in larvae preexposed to 0.18 microM Cd2+ for 3 days were examined. The Michaelis constant for Ca2+ in the 0.18 microM Cd2+ preexposed larvae did not change significantly in the presence of Cd2+, whereas maximal velocity increased by approximately 23%. An enhanced Ca2+ uptake efficiency ( approximately 18%) was found in these Cd2+-acclimated larvae. The criterion that determines the survival of tilapia larvae encountering Cd2+ challenge is the degree of interference with Ca2+ homeostasis instead of the absolute amount of Cd2+ accumulated.

Immune localization of prolactin receptor in the mitochondria-rich cells of the euryhaline teleost (Oreochromis mossambicus) gill.

The present work demonstrates, by Western blotting and immunofluorescent staining, the presence and localization of prolactin (PRL) receptor in tilapia (ti) Oreochromis mossambicus gills. Gill epithelial cells that reacted with PRL receptor antibody were found to be labelled concomitantly with Con A, a marker of the apical crypts in mitochondria-rich (MR) cells. No positive staining was observed in pavement cells or mucus cells with PRL receptor antibody. This indicates that PRL receptors are located specifically in the gill MR cells. Further, the tiPRL receptors were found only in the MR cells of seawater-adapted tilapia gills. The effects of salinity and ions on the expression of tiPRL receptors are discussed.

Effect of environmental calcium levels on calcium uptake in tilapia larvae Oreochromis mossambicus.

Effects of environmental calcium concentrations on the survival, growth, body calcium content and calcium uptake kinetics in developing tilapia (Oreochromis mossambicus) larvae were studied. Fertilized eggs were incubated in high- and low-calcium artificial freshwater (0.88-0.96 mmol l(-1) vs. 0.02-0.03 mmol l(-1) CaCl2 or CaSO4) until 3 days after hatching. Tilapia larvae showed similar hatching rates and wet weights in either high- or low-calcium medium, indicating neither the development nor the growth in tilapia larvae was affected by the environmental calcium levels. The body calcium content in low-calcium groups was about 90-95% that of high-calcium groups, No matter what calcium source was used (CaCl2 or CaSO4), acclimation to low calcium medium caused a stimulation of calcium uptake (measured in 0.2 mmol l(-1) calcium),i.e., 1.2-1.3 fold higher than that of high calcium groups. This enhanced calcium uptake capacity was characterized by a 50% decrease in Km and a 25% increase in Jmax. Effect of different calcium salts on calcium influx was significant only in low calcium level,i.e., calcium influx in low-CaCl2 group higher than that in low-CaSO4 group. These results suggest that tilapia larvae are able to modulate their calcium uptake mechanism to maintain normal body calcium content and growth in environments with different levels of calcium.

Mitochondria-rich cells in the branchial epithelium of the teleost,Oreochromis mossambicus, acclimated to various hypotonic environments.

Branchial mitochondria-rich (MR) cells were examined on the afferent side of gill filaments in tilapia (Oreochromis mossambicus) acclimated to different hypotonic environments, local fresh water (LFW), hard fresh water (HFW) and 5‰ salt water (SW). Scanning electron micrographs (SEM) identified three types of apical surfaces of the MR cells, wavy convex, shallow basin and deep hole. In spite of the different types of apical surfaces, light microscopic (LM) and transmission electron microscopic (TEM) studies suggested that these cells were MR cells. The relative abundance of these 3 types of branchial MR cells varied with external hypotonic milieus. Wavy-convexed MR cells were dominant in the gills of fish adapted to HFW, whereas shallow-basined MR cells were evident in LFW-adapted fish. In SW-adapted fish, most of the MR cells were deep holes. Experiments on adaptation to various hypotonic milieus revealed that the changes of the branchial MR cells were reversible and occurred within 24 hours following transfer. The morphological alterations of the MR cells correlated with ionic changes in different milieus, indicating that these distinct types of MR cells may play key roles for osmoregulation in hypotonic media.