Role of the cardiovascular system in ammonia excretion in early life stages of zebrafish (Danio rerio).

The purpose of this study was to investigate if the cardiovascular system is important for ammonia excretion in the early life stages of zebrafish. Morpholino knockdowns of cardiac troponin T (TNNT2) or vascular endothelial growth factor A (VEGFA) provided morphants with nonfunctional circulation. At the embryonic stage [30-36 h postfertilization (hpf)], ammonia excretion was not constrained by a lack of cardiovascular function. At 2 days postfertilization (dpf) and 4 dpf, morpholino knockdowns of TNNT2 or VEGFA significantly reduced ammonia excretion in all morphants. Expression of rhag, rhbg, and rhcgb showed no significant changes but the mRNA levels of the urea transporter (ut) were upregulated in the 4 dpf morphants. Taken together, rhag, rhbg, rhcgb, and ut gene expression and an unchanged tissue ammonia concentration but an increased tissue urea concentration, suggest that impaired ammonia excretion led to increased urea synthesis. However, in larvae anesthetized with tricaine or clove oil, ammonia excretion was not reduced in the 4 dpf morphants compared with controls. Furthermore, oxygen consumption was reduced in morphants regardless of anesthesia. These results suggest that cardiovascular function is not directly involved in ammonia excretion, but rather reduced activity and external convection may explain reduced ammonia excretion and compensatory urea accumulation in morphants with reduced cardiovascular function.

Tissue Accumulation and the Effects of Long-Term Dietary Copper Contamination on Osmoregulation in the Mudflat Fiddler Crab Minuca rapax (Crustacea, Ocypodidae).

We examined copper accumulation in the hemolymph, gills and hepatopancreas, and hemolymph osmolality, Na+ and Cl- concentrations, together with gill Na+/K+-ATPase and carbonic anhydrase activities, after dietary copper delivery (0, 100 or 500 Cu µg g-1) for 12 days in a fiddler crab, Minuca rapax. In contaminated crabs, copper concentration decreased in the hemolymph and hepatopancreas, but increased in the gills. Hemolymph osmolality and gill Na+/K+-ATPase activity increased while hemolymph [Na+] and [Cl-] and gill carbonic anhydrase activity decreased. Excretion likely accounts for the decreased hemolymph and hepatopancreas copper titers. Dietary copper clearly affected osmoregulatory ability and hemolymph Na+ and Cl- regulation in M. rapax. Gill copper accumulation decreased carbonic anhydrase activity, suggesting that dietary copper affects acid-base balance. Elevated gill Na+/K+-ATPase activity appears to compensate for the ion-regulatory disturbance. These effects of dietary copper illustrate likely impacts on semi-terrestrial species that feed on metal-contaminated sediments.

The effects of Deepwater Horizon crude oil on ammonia and urea handling in mahi-mahi (Coryphaena hippurus) early life stages.

Many ecologically important fishes, including mahi-mahi (Coryphaena hippurus), and their offspring were directly exposed to crude oil following the Deepwater Horizon (DWH) oil spill. Early life stage fish are especially vulnerable to the toxicity of crude oil-derived polycyclic aromatic hydrocarbons (PAHs). In teleosts, yolk sac proteins are the main energy source during development and are usually catabolized into ammonia or urea among other byproducts. Although excretion of these waste products is sensitive to oil exposure, we know little about the underlying mechanisms of this process. In this study, we examined the effects of crude oil on ammonia and urea handling in the early life stages of mahi. Mahi embryos exposed to 30-32 µg L-1 ∑PAH exhibited increased urea excretion rates and greater accumulation of urea in the tissues before hatch suggesting that ammonia, which is highly toxic, was converted into less-toxic urea. Oil-exposed embryos (6.3-32 µg L-1 ∑PAH) displayed significantly increased tissue ammonia levels at 42 hpf and upregulated mRNA levels of ammonia transporters (Rhag, Rhbg and Rhcg1) from 30 to 54 hpf. However, despite increased accumulation and higher expression of ammonia transporters, the larvae exposed to higher ∑PAH (30 µg L-1 ∑PAH) showed reduced ammonia excretion rates after hatch. Together, the increased production of nitrogenous waste reinforces previous work that increased energy demand in oil-exposed embryos is fueled, at least in part, by protein metabolism and that urea synthesis plays a role in ammonia detoxification in oil-exposed mahi embryos. To our knowledge, this study is the first to combine physiological and molecular approaches to assess the impact of crude-oil on both nitrogenous waste excretion and accumulation in the early life stages of any teleosts.

Regulation of apical H⁺-ATPase activity and intestinal HCO3⁻ secretion in marine fish osmoregulation.

The absorption of Cl(-) and water from ingested seawater in the marine fish intestine is accomplished partly through Cl(-)/HCO(3)(-) exchange. Recently, a H(+) pump (vacuolar-type H(+)-ATPase) was found to secrete acid into the intestinal lumen, and it may serve to titrate luminal HCO(3)(-) and facilitate further Cl(-)/HCO(3)(-) exchange, especially in the posterior intestine, where adverse concentration gradients could limit Cl(-)/HCO(3)(-) exchange. The H(+) pump is expressed in all intestinal segments and in gill tissue of gulf toadfish (Opsanus beta) maintained in natural seawater. After acute transfer of toadfish to 60 ppt salinity, H(+) pump expression increased 20-fold in the posterior intestine. In agreement with these observations was a fourfold-increased H(+)-ATPase activity in the posterior intestine of animals acclimated to 60 ppt salinity. Interestingly, Na(+)-K(+)-ATPase activity was elevated in the anterior intestine and gill, but not in the posterior intestine. Apical acid secretion by isolated intestinal tissue mounted in Ussing chambers fitted with pH-stat titration systems increased after acclimation to hypersalinity in the anterior and posterior intestine, titrating >20% of secreted bicarbonate. In addition, net base secretion increased in hypersalinity-acclimated fish and was ∼70% dependent on serosal HCO(3)(-). Protein localization by immunohistochemistry confirmed the presence of the vacuolar-type H(+)-ATPase in the apical region of intestinal enterocytes. These results show that the H(+) pump, especially in the posterior intestine, plays an important role in hypersaline osmoregulation and that it likely has significant effects on HCO(3)(-) accumulation in the intestinal lumen and, therefore, the continued absorption of Cl(-) and water.

Intestinal anion exchange in marine teleosts is involved in osmoregulation and contributes to the oceanic inorganic carbon cycle.

Marine teleost fish osmoregulation involves seawater ingestion and intestinal fluid absorption. Solute coupled fluid absorption by the marine teleost fish intestine has long been believed to be the product of Na(+) and Cl(-) absorption via the Na(+) :K(+) :2Cl(-) co-transporter (NKCC2). However, the past decade has revealed that intestinal anion exchange contributes significantly to Cl(-) absorption, in exchange for HCO(3) (-) secretion, and that this process is important for intestinal water absorption. In addition to contributing to solute coupled water absorption intestinal anion exchange results in luminal precipitation of CaCO(3) which acts to reduce luminal osmotic pressure and thus assist water absorption. Most recently, activity of apical H(+) -pumps, especially in distal segments of the intestine have been suggested to not only promote anion exchange, but also to reduce luminal osmotic pressure by preventing excess HCO(3)(-) concentrations from accumulating in intestinal fluids, thereby aiding water absorption. The present review summarizes and synthesizes the most recent advances in our view of marine teleosts osmoregulation, including our emerging understanding of epithelial transport of acid-base equivalents in the intestine, the consequences for whole organism acid-base balance and finally the impact of piscine CaCO(3) formation on the global oceanic carbon cycle.

Basolateral NBCe1 plays a rate-limiting role in transepithelial intestinal HCO3- secretion, contributing to marine fish osmoregulation.

Although endogenous CO2 hydration and serosal HCO3- are both known to contribute to the high rates of intestinal HCO3- secretion important to marine fish osmoregulation, the basolateral step by which transepithelial HCO3- secretion is accomplished has received little attention. Isolated intestine HCO3- secretion rates, transepithelial potential (TEP) and conductance were found to be dependent on serosal HCO3- concentration and sensitive to serosal DIDS. Elevated mucosal Cl- concentration had the unexpected effect of reducing HCO3- secretion rates, but did not affect electrophysiology. These characteristics indicate basolateral limitation of intestinal HCO3- secretion in seawater gulf toadfish, Opsanus beta. The isolated intestine has a high affinity for serosal HCO3- in the physiological range (Km=10.2 mmol l(-1)), indicating a potential to efficiently fine-tune systemic acid-base balance. We have confirmed high levels of intestinal tract expression of a basolateral Na+/HCO3- cotransporter of the electrogenic NBCe1 isoform in toadfish (tfNBCe1), which shows elevated expression following salinity challenge, indicating its importance in marine fish osmoregulation. When expressed in Xenopus oocytes, isolated tfNBCe1 has transport characteristics similar to those in the isolated tissue, including a similar affinity for HCO3- (Km=8.5 mmol l(-1)). Reported affinity constants of NBC1 for Na+ are generally much lower than physiological Na+ concentrations, suggesting that cotransporter activity is more likely to be modulated by HCO3- rather than Na+ availability in vivo. These similar functional characteristics of isolated tfNBCe1 and the intact tissue suggest a role of this cotransporter in the high HCO3- secretion rates of the marine fish intestine.

The involvement of SLC26 anion transporters in chloride uptake in zebrafish (Danio rerio) larvae.

After demonstrating phylogenetic relatedness to orthologous mammalian genes, tools were developed to investigate the roles of three members (A3, A4 and A6c) of the SLC26 anion exchange gene family in Cl- uptake and HCO3 excretion in embryos and larvae of zebrafish (Danio rerio). Whole-mount in situ hybridization revealed the presence of SLC26 mRNA in gill primordia, mesonephros and heart (slc26a3 and a4 only) at 5-9 days postfertilization (d.p.f.). SLC26A3 protein was highly expressed in lateral line neuromasts and within the gill, was localized to a sub-population of epithelial cells, which often (but not always) coexpressed Na+/K+-ATPase. SLC26 mRNA levels increased with developmental age, peaking at 5-10 d.p.f.; the largest increases in rates of Cl- uptake (JinCl-) preceded the mRNA spike, occurring at 2-5 d.p.f. Raising zebrafish in water with a low [Cl-] caused marked increases in JinCl- at 3-10 d.p.f. and was associated with increased levels of SLC26 mRNA. Raising fish in water of high [Cl-] was without effect on JinCl- or SLC26 transcript abundance. Selective gene knockdown using morpholino antisense oligonucleotides demonstrated a significant role for SLC26A3 in Cl- uptake in larval fish raised in control water and roles for A3, A4 and A6c in fish raised in water with low [Cl-]. Prolonged (7 days) or acute (24 h) exposure of fish to elevated (2 or 5 mmol l(-1)) ambient [HCO3-] caused marked increases in Cl- uptake when determined in water of normal [HCO3-] that were accompanied by elevated levels of SLC26 mRNA. The increases in JinCl- associated with high ambient [HCO3-] were not observed in the SLC26 morphants (significant only at 5 mmol l(-1) HCO3- for A4 and 2 mmol l(-1) HCO3- for A6c). Net base excretion was markedly inhibited in the slc26a3 and a6c morphants thereby implicating these genes in Cl-/HCO3- exchange. The results suggest that under normal conditions, Cl- uptake in zebrafish larvae is mediated by SLC26A3 Cl-/HCO3- exchangers but under conditions necessitating higher rates of high affinity Cl- uptake, SlC26A4 and SLC26A6c may assume a greater role.

The involvement of H+-ATPase and carbonic anhydrase in intestinal HCO3- secretion in seawater-acclimated rainbow trout.

Pyloric caeca and anterior intestine epithelia from seawater-acclimated rainbow trout exhibit different electrophysiological parameters with lower transepithelial potential and higher epithelial conductance in the pyloric caeca than the anterior intestine. Both pyloric caeca and the anterior intestine secrete HCO(3)(-) at high rates in the absence of serosal HCO(3)(-)/CO(2), demonstrating that endogenous CO(2) is the principal source of HCO(3)(-) under resting control conditions. Apical, bafilomycin-sensitive, H(+) extrusion occurs in the anterior intestine and probably acts to control luminal osmotic pressure while enhancing apical anion exchange; both processes with implications for water absorption. Cytosolic carbonic anhydrase (CAc) activity facilitates CO(2) hydration to fuel apical anion exchange while membrane-associated, luminal CA activity probably facilitates the conversion of HCO(3)(-) to CO(2). The significance of membrane-bound, luminal CA may be in part to reduce HCO(3)(-) gradients across the apical membrane to further enhance anion exchange and thus Cl(-) absorption and to facilitate the substantial CaCO(3) precipitation occurring in the lumen of marine teleosts. In this way, membrane-bound, luminal CA thus promotes the absorption of osmolytes and reduction on luminal osmotic pressure, both of which will serve to enhance osmotic gradients to promote intestinal water absorption.

Effects of copper, cadmium, and zinc on the hatching success of brine shrimp (Artemia franciscana).

Previous studies indicate that the hatching success of brine shrimp (Artemia franciscana) cysts is surprisingly sensitive to ambient metal concentrations. These studies estimated median effective concentrations (EC50s) of 7, 5, and 28 microg l-1 for Cd, Cu, and Zn, suggesting that the hatching end point for A. franciscana is the most sensitive tested to date for Cd and Zn in saline environments and comparable in sensitivity with the most sensitive tested to date for Cu. Furthermore, these data suggest that brine shrimp are at significant risk from Cu and Zn in Great Salt Lake (GSL), UT, where ambient concentrations as high as 10 and 14 microg l-1, respectively, have been measured. Given that brine shrimp appear to be successfully reproducing in GSL, we hypothesized that these toxicity values were either biased low as a result of an artifact of the test method used or that site-specific water-quality conditions in the lake had decreased metal bioavailability such that brine shrimp could successfully reproduce. To test these hypotheses, we initiated a step-wise series of experiments. First we investigated the effects of pretreatment of brine shrimp cysts with antibiotics on brine shrimp sensitivity to metals because previous investigators as part of their test methods have used antibiotics. Next we considered the effect of ionic composition of the artificial test media on sensitivity. Finally, we evaluated the effects of the site-specific water quality of the GSL on metal bioavailability and toxicity. Results indicate that pretreatment of cysts with antibiotics had no effect on sensitivity. However, we were unable to repeat the previous values for Cd and Zn, obtaining EC50s of 11,859 and 289 microg l-1 for Cd and Zn, respectively. For Cu, however, we estimated an EC50 of 12 microg l-1, so we conducted further testing on the artificial media, adjusting the media composition to better reflect the Ca2+ and HCO3- concentration of normal seawater. This increased the EC50 to 28 microg l-1. Finally we evaluated the toxicity of Cu in GSL water and obtained an EC50 of 68 microg l-1, suggesting that the increased dissolved organic carbon in GSL has a significant protective effect. Overall, the results of this study suggest that brine shrimp hatching success is not particularly sensitive to Cd and Zn, but it is sensitive to Cu. However, site-specific water-quality conditions ensure that brine shrimp cyst hatching success is not significantly affected by any of these metals at the normal background concentrations that occur in GSL (<15 microg l-1).

Ouabain-sensitive bicarbonate secretion and acid absorption by the marine teleost fish intestine play a role in osmoregulation.

The gulf toadfish (Opsanus beta) intestine secretes base mainly in the form of HCO3- via apical anion exchange to serve Cl- and water absorption for osmoregulatory purposes. Luminal HCO3- secretion rates measured by pH-stat techniques in Ussing chambers rely on oxidative energy metabolism and are highly temperature sensitive. At 25 degrees C under in vivo-like conditions, secretion rates averaged 0.45 micromol x cm(-2) x h(-1), of which 0.25 micromol x cm(-2) x h(-1) can be accounted for by hydration of endogenous CO2 partly catalyzed by carbonic anhydrase. Complete polarity of secretion of HCO3- and H+ arising from the CO2 hydration reaction is evident from equal rates of luminal HCO3- secretion via anion exchange and basolateral H+ extrusion. When basolateral H+ extrusion is partly inhibited by reduction of serosal pH, luminal HCO3- secretion is reduced. Basolateral H+ secretion occurs in exchange for Na+ via an ethylisopropylamiloride-insensitive mechanism and is ultimately fueled by the activity of the basolateral Na+-K+-ATPase. Fluid absorption by the toadfish intestine to oppose diffusive water loss to the concentrated marine environment is accompanied by a substantial basolateral H+ extrusion, intimately linking osmoregulation and acid-base balance.

The effects of pH and the iron redox state on iron uptake in the intestine of a marine teleost fish, gulf toadfish (Opsanus beta).

In the marine teleost intestine the secretion of bicarbonate increases pH of the lumen (pH 8.4 -9.0) and importantly reduces Ca2+ and Mg2+ concentrations by the formation of insoluble divalent ion carbonates. The alkaline intestinal environment could potentially also cause essential metal carbonate formation reducing bioavailability. Iron accumulation was assessed in the Gulf toadfish (Opsanus beta) gut by mounting intestine segments in modified Ussing chambers fitted to a pH-stat titration system. This system titrates to maintain lumen pH constant and in the process prevents bicarbonate accumulation. The luminal saline pH was clamped to pH 5.5 or 7.0 to investigate the effect of proton concentrations on iron uptake. In addition, redox state was altered (gassing with N2, addition of dithiothreitol (DTT) and ascorbate) to evaluate Fe3+ versus Fe2+ uptake, enabling us to compare a marine teleost intestine model for iron uptake to the mammalian system for non-haem bound iron uptake that occurs via a ferrous/proton (Fe2+/H+) symporter called Divalent Metal Transporter 1 (DMT1). None of the redox altering strategies affected iron (Fe3+ or Fe2+) binding to mucus, but the addition of ascorbate resulted in a 4.6-fold increase in epithelium iron accumulation. This indicates that mucus iron binding is irrespective of valency and suggests that ferrous iron is preferentially transported across the apical surface. Altering luminal saline pH from 7.0 to 5.5 did not affect ferric or ferrous iron uptake, suggesting that if iron is entering via DMT1 in marine fish intestine this transporter works efficiently under circumneutral conditions.

Effects of water hardness on the physiological responses to chronic waterborne silver exposure in early life stages of rainbow trout (Oncorhynchus mykiss).

Early life stages of rainbow trout were exposed to 0, 0.1 and 1 microg/L Ag (as AgNO(3)) in very soft water (2mg/L CaCO(3)), moderately hard water (150 mg/L CaCO(3)) and hard water (400mg/L CaCO(3)) of low dissolved organic carbon concentration (0.5mg C/L) from fertilization to swim-up (64 days) under flow-through conditions, and monitored for whole embryo/larval silver accumulation, Na(+) and Cl(-) concentrations, Na(+) uptake and Na(+)K(+)-ATPase activity. The objective of the study was to investigate potential protective effects of water hardness on the physiological responses to chronic silver exposure. In the absence of silver, there was little effect of hardness on the ionoregulatory parameters studied, though higher hardness did improve survival post-hatch. At all three water hardness levels, whole embryo/larval Na(+) uptake was low and relatively constant prior to 50% hatch, but dramatically increased following 50% hatch, whereas Na(+)K(+)-ATPase activity steadily increased over development. Whole embryo/larval Na(+) and Cl(-) concentrations were low and constant prior to 50% hatch, but following 50% hatch Na(+) concentration increased, while Cl(-) concentration decreased. Following 50% hatch, exposure to 0.1 and 1 microg/L Ag resulted in a decrease in whole embryo/larval Na(+) concentration, Cl(-) concentration, Na(+) uptake and Na(+)K(+)-ATPase activity, indicating that the mechanism of chronic silver toxicity involves an ionoregulatory disturbance, and is similar to the mechanism of acute silver toxicity. An increase in water hardness reduced or eliminated the effect of silver on these parameters while enhancing survival, suggesting that the nature of the protective effect of hardness involves effects on the ionoregulatory disturbance associated with silver exposure. An increase in water hardness did not fully protect against the accumulation of silver associated with silver exposure. These results suggest that it may be possible to model chronic silver toxicity using a biotic ligand type model, and that a physiologically based model may be more appropriate because Na(+)K(+)-ATPase activity or Na(+) uptake is an endpoint for prediction rather than whole embryo or larval silver accumulation.

Effects of prolonged copper exposure in the marine gulf toadfish (Opsanus beta) II: copper accumulation, drinking rate and Na+/K+ -ATPase activity in osmoregulatory tissues.

Gulf toadfish were exposed to sublethal levels of copper (12.8 or 55.2 microM) for 30 days. Drinking in control fish averaged 1 ml kg(-1)h(-1) but exposure to 55.2 microM copper resulted in a complex biophasic pattern with initial (3 h and 1 day) inhibition of drinking rate, followed by an elevation of drinking rate from day 3 onwards. Drinking led to copper accumulation in the intestinal fluids at levels three to five times higher than the ambient copper concentrations, which in turn resulted in intestinal copper accumulation. The gill exhibited more rapid accumulation of copper than the intestine and contributed to early copper uptake leading to accumulation in internal organs. Muscle, spleen and plasma exhibited little if any disturbance of copper homeostasis while renal copper accumulation was evident at both ambient copper concentrations. The liver exhibited the highest copper concentrations and the greatest copper accumulation of all examined internal organs during exposure to 55.2 microM. Elevated biliary copper excretion was evident from measurements of gall bladder bile copper concentrations and appeared to protect partially against internal accumulation in fish exposed to 12.8 microM copper. No inhibition of Na+/K+ -ATPase activity in either gills or intestine was seen despite copper accumulation in these organs. Calculations of inorganic copper speciation suggest that Cu(CO3)(2)2- complexes which dominate in seawater and intestinal fluids are of limited availability for uptake while the low levels of ionic Cu2+, CuOH+ and CuCO3 may be the forms taken up by the gill and the intestinal epithelium.

Plasma clearance of cadmium and zinc in non-acclimated and metal-acclimated trout.

Adult rainbow trout were pre-exposed to a sublethal concentration of waterborne cadmium (Cd, 26.7 nmol/l) or waterborne zinc (Zn, 2294 nmol/l) for 30 days to induce acclimation. A single dose of radiolabeled Cd (64.4 nmol/kg) or Zn (183.8 nmol/kg) was injected into the vascular system of non-acclimated and Cd- or Zn-acclimated trout through indwelling arterial catheters. Subsequently, repetitive blood samples over 10 h and terminal tissue samples (liver, heart, bile, stomach, intestine, kidney, gills, muscle, and spleen) were taken to characterize the effect of metal acclimation on clearance kinetics in vivo. Plasma clearance of Cd in Cd-acclimated fish (0.726+/-0.015 and 0.477+/-0.012 ml/min per kg for total and newly accumulated Cd, respectively), was faster than that in non-acclimated trout (0.493+/-0.013 and 0.394+/-0.009 ml/min per kg). Unlike plasma Cd, the levels of Cd in red blood cells (RBCs) were 1.2-2.2 times higher in Cd-acclimated fish than in non-acclimated fish. At 10 h post-injection, the liver accumulated the highest proportion ( approximately 22%) of the injected Cd dose in both non-acclimated and Cd-acclimated fish but did not account for the difference in plasma levels of Cd between two groups. Plasma clearance of Zn ( approximately 0.23 ml/min per kg for new Zn) was substantially lower than Cd clearance. Pre-acclimation to waterborne Zn reduced the new Zn levels in RBCs, but did not affect the clearance of Zn from blood plasma or tissue burdens of Zn in fish. Bile concentrations of both Cd and Zn were elevated in acclimated fish, but total bile burden accounted for <1% of the injected metal dose. The results suggest that the detoxification process of injected plasma Cd is stimulated by pre-acclimation to waterborne Cd, and that Zn levels are homeostatically controlled in both non-acclimated and acclimated trout.

Branchial versus intestinal silver toxicity and uptake in the marine teleost Parophrys vetulus.

Exposure to elevated waterborne silver as AgNO3 (4.07 microM=448 microg l(-1)) in seawater resulted in osmoregulatory disturbance in the lemon sole (Parophrys vetulus). The main effects were increased plasma Na+ and Cl- concentrations which translated into increased plasma osmolality. Plasma Mg2+ levels were also slightly increased after 96 h exposure. Using radioisotopic flux measurements, a 50% reduction in branchial unidirectional Na+ extrusion was observed after 48 h silver exposure. By applying an intestinal perfusion approach, we were able to separate and thus quantify the intestinal contribution to the observed silver-induced physiological disturbance and internal silver accumulation. This analysis revealed that the intestinal contribution to silver-induced ionoregulatory toxicity was as high as 50-60%. In marked contrast, internal silver accumulation (in liver and kidney) was found to be derived exclusively from uptake across the gills. Drinking of silver-contaminated seawater resulted in substantial silver accumulation in the intestinal tissue (but apparently not silver uptake across the intestine), which probably explains the intestinal contribution to silver-induced physiological disturbance.

Intestinal iron uptake in the European flounder (Platichthys flesus).

Iron is an essential element because it is a key constituent of the metalloproteins involved in cellular respiration and oxygen transport. There is no known regulated excretory mechanism for iron, and homeostasis is tightly controlled via its uptake from the diet. This study assessed in vivo intestinal iron uptake and in vitro iron absorption in a marine teleost, the European flounder Platichthys flesus. Ferric iron, in the form (59)FeCl(3), was reduced to Fe(2+) by ascorbate, and the bioavailability of Fe(3+) and Fe(2+) were compared. In vivo Fe(2+) uptake was significantly greater than Fe(3+) uptake and was reduced by the iron chelator desferrioxamine. Fe(2+) was also more bioavailable than Fe(3+) in in vitro studies that assessed the temporal pattern and concentration-dependency of iron absorption. The posterior region, when compared with the anterior and mid regions of the intestine, was the preferential site for Fe(2+) uptake in vivo. In vitro iron absorption was upregulated in the posterior intestine in response to prior haemoglobin depletion of the fish, and the transport showed a Q(10) value of 1.94. Iron absorption in the other segments of the intestine did not correlate with haematocrit, and Q(10) values were lower. Manipulation of the luminal pH had no effect on in vitro iron absorption. The present study demonstrates that a marine teleost absorbs Fe(2+) preferentially in the posterior intestine. This occurs in spite of extremely high luminal bicarbonate concentrations recorded in vivo, which may be expected to reduce the bioavailability of divalent cations as a result of the precipitation as carbonates (e.g. FeCO(3)).

Sensitivity of the spiny dogfish (Squalus acanthias) to waterborne silver exposure.

The physiological effects of waterborne silver exposure (added as AgNO(3)) on spiny dogfish, Squalus acanthias, were evaluated at 30, 200 and 685 microg silver per l in 30 per thousand seawater. These concentrations cover the toxic range observed for freshwater teleosts, where silver is extremely toxic, to seawater teleosts which tolerate higher silver concentrations. However, these levels are considerably higher than those that occur in the normal environment. At 685 microg l(-1), dogfish died within 24 h. Causes of death were respiratory as well as osmoregulatory failure. Arterial P(a)O(2) rapidly declined below 20 Torr, and blood acidosis (both respiratory and metabolic) occurred. Urea excretion increased dramatically and plasma urea dropped from 340 to 225 mM. There were pronounced increases in plasma Na(+), Cl(-), and Mg(2+), indicative of ionoregulatory failure due to increased diffusive permeability as well as inhibited NaCl excretion. At 200 microg l(-1), fish died between 24 and 72 h of silver exposure. The same physiological events occurred with a small time delay. At 30 microg l(-1), effects were much less severe, although slight mortality (12.5%) still occurred. Respiratory alkalosis occurred, together with moderate elevations in plasma Na(+) and Cl(-) levels. Silver accumulated to the highest concentrations on gills, with only low levels in the intestine, in accord with the virtual absence of drinking. Na(+)/K(+)-ATP-ase activities of gill and rectal gland tissue were impaired at the highest silver concentration. Normal gill function was impaired due to swelling and fusion of lamellae, lamellar aneurism and lifting of the lamellar epithelium. Our results clearly indicate that this elasmobranch is much more sensitive (about 10-fold) to silver than marine teleosts, with silver's toxic action exerted on the gill rather than on the intestine, in contrast to the latter.

Plasma copper clearance and biliary copper excretion are stimulated in copper-acclimated trout.

Nonacclimated and Cu-acclimated rainbow trout (Oncorhynchus mykiss) exhibited equally rapid clearance of a single bolus of injected (64)Cu (3,780 nmol/kg) from the plasma (32-40 min to half- concentration). Eight hours after Cu injection, approximately 80% of the injected Cu was found in the liver. However, when Cu labeled with (64)Cu was presented intravascularly via continuous infusion at a rate of 158 nmol x kg(-1) x h(-1) for 72 h, Cu-acclimated fish cleared plasma Cu more effectively than nonacclimated fish. The use of chronically implanted cystic bile duct cannulas revealed a fourfold increase in hepatobiliary Cu excretion in Cu-acclimated fish during infusion, demonstrating the important homeostatic role of the liver in Cu metabolism. Extrahepatobiliary Cu excretion, likely through the gills and apparently exceeding biliary Cu excretion, was evident from appearance of (64)Cu in the ambient water but was not altered by Cu acclimation. Cu accumulation in white muscle also played an important a role in copper homeostasis.

Hepatic versus gallbladder bile composition: in vivo transport physiology of the gallbladder in rainbow trout.

Ion and water transport across the teleost Oncorhynchus mykiss gallbladder were studied in vivo by comparing flow and composition of hepatic bile, collected by chronic catheter, to volume and composition of terminally collected gallbladder bile. Differences in composition were comparable with those of other vertebrates, whereas bile flow (75 microl. kg(-1). h(-1)) was below values reported for endothermic vertebrates. The gallbladder concentrates bile acids five- to sevenfold and exhibits higher net Cl(-) than Na(+) transport in vivo, in contrast to the 1:1 transport ratio from gallbladders under saline/saline conditions. Transepithelial potential (TEP) in the presence of bile, at the apical surface, was -13 mV (bile side negative) but +1.5 mV in the presence of saline. Bile acid in the apical saline reversed the TEP, presumably by a Donnan effect. We propose that ion transport across the gallbladder in vivo involves backflux of Na(+) from blood to bile resulting in higher net Cl(-) than Na(+) flux. This Na(+) backflux is driven by a bile side negative TEP and low Na(+) activity in bile due to the complexing effects of bile acids.

ATP-dependent silver transport across the basolateral membrane of rainbow trout gills.

Silver has been shown to be extremely toxic to freshwater teleosts, acting to inhibit Na(+) uptake at the gills, due to the inactivation of branchial Na(+)/K(+)-ATPase activity. However, the gills are also a route by which silver may enter the fish. Therefore, this study focuses on the mechanism of transport of this nonessential metal across the basolateral membrane of the gill cell, using basolateral membrane vesicles (BLMV) prepared from the gills of freshwater rainbow trout. Uptake of silver by BLMV was via a carrier-mediated process, which was ATP-dependent, reached equilibium over time, and followed Michaelis-Menten kinetics, with maximal transport capacity (V(max)) of 14.3 +/- 5.5 (SE) nmol mg membrane protein(-1) min(-1) and an affinity (K(m)) of 62.6 +/- 43.7 microM, and was inhibited by 100 microM sodium orthovanadate (Na(3)VO(4)). The ionophore monensin (10 microM) released transported silver from the BLMV. Acylphosphate intermediates, of a 104 kDa size, were formed from the BLMV preparations in the presence of ATP plus Ag. These results demonstrate that there is a P-type ATPase present in the basolateral membrane of the gills of rainbow trout that can actively transport silver, a process which will remove this heavy metal from its site of toxic action, the gill.