Water chloride provides partial protection during chronic exposure to waterborne silver in rainbow trout (Oncorhynchus mykiss) embryos and larvae

Rainbow trout embryos and larvae were continuously exposed (at 12.5 degrees C) to waterborne silver in a flow-through setup, from fertilization to swim-up, at nominal silver concentrations of 0, 0.1, or 1.0 microg/L total silver (as AgNO(3)) at three different water Cl(-) levels (30, 300, and 3,000 microM, added as KCl). Exposures were conducted in synthetic soft water (hardness 20 mg CaCO(3)/L generated from reconstituted reverse osmosis freshwater). Continuous exposure to 1.0 microg/L total silver for 58 d at 30 microM water Cl(-) resulted in a pronounced ionoregulatory disturbance (as indicated by a reduction in whole body Na(+),K(+)-ATPase activity, unidirectional Na(+) uptake [Jin Na(+)], and whole body Na(+) and Cl(-) levels) and a reduction in extractable protein and wet weight. Thus, the mechanism of chronic silver toxicity appears to be similar to that observed during acute silver exposure in juvenile and adult fish, specifically an ionoregulatory disturbance. Higher water Cl(-) levels (300 and 3,000 microM Cl(-)) offered some degree of protection from the ionoregulatory disturbance, with only minor protective effects in terms of mortality. The protective effects of water Cl(-) on the toxicity of silver (as AgNO(3)) appear to be far less during chronic than during acute exposure. Mortality and larval Na(+) concentration, Jin Na(+), and Na(+),K(+)-ATPase activity all appear to be correlated with silver body burden and calculated water Ag(+) during chronic silver exposure. Thus, there appears to be potential to model chronic toxicity but not simply by recalibration of an acute model. A chronic model must be based on real chronic data because the protective effects of various ligands appear to be quantitatively very different from those in the acute situation.     

Ionoregulatory development and the effect of chronic silver exposure on growth, survival, and sublethal indicators of toxicity in early life stages of rainbow trout (Oncorhynchus mykiss)

Rainbow trout embryos and larvae were continuously exposed, in a flow-through system, to 0, 0.1 microg/l (measured=0.098 +/- 0.002 microg/l) or 1.0 microg/l (measured=0.853+/-0.022 microg/l) total silver (as AgNO3) in moderately hard water (120 mg CaCO3/l, 0.70 mM Cl, 1.3 mg/l dissolved organic matter and 13.7 +/- 0.1 degrees C) from fertilization to I week post-hatch. The objectives of the study were to investigate the effects of chronic silver exposure on mortality, time to hatch and growth, and on sublethal physiological indicators of toxicity. Exposure to 1.0 microg/l total silver resulted in a small, but statistically significant, increase in mortality (16%) relative to controls (12%) but interestingly, resulted in an increased rate of growth (as indicated by larval weight, length and extractable protein) and ionoregulatory development over the duration of this study. Whole body unidirectional Na uptake (J(in)Na+) increased with silver exposure concentration (both 0.1 microg/l and 1.0 microg/l total silver) just prior to and following hatch, with up to a three-fold elevation in J(in)Na+ in the 1.0 microg/l treatment relative to controls. Qualitatively similar changes in whole body Na+,K-ATPase activity (per mg protein or per whole embryo or larvae) also occurred over this period. By 1 week post-hatch, there were no differences in J(in)Na among treatments and Na+,K+-ATPase activity levels in silver exposed groups were significantly reduced relative to controls. Within 2 days following hatch, there was an elevation in whole larval ammonia levels, while cortisol levels were elevated at 1 week post-hatch in the 1.0 microg/l treatment relative to controls. Ionoregulatory disturbance and elevations in both cortisol and ammonia have also been observed during acute silver exposure in adult rainbow trout, indicating that chronic and acute mechanisms of toxicity may be similar.     

Physiological effects of chronic silver exposure in Daphnia magna

Daphnia magna were exposed to a total concentration of 5.0+/-0.04 microg Ag/l, added as AgNO(3) (dissolved concentration, as defined by 0.45 microm filtration = 2.0+/-0.01 microg Ag/l) in moderately hard synthetic water under static conditions (total organic carbon = 4.80+/-1.32 mg/l) with daily feeding and water renewal, for 21 days. There was no mortality in control daphnids and 20% mortality in silver-exposed animals. Silver exposure caused a small but significant reduction of reproductive performance manifested as a 13.7% decrease in the number of neonates produced per adult per reproduction day over the 21-day exposure. However, silver exposed daphnids also exhibited a much more marked ionoregulatory disturbance, which was characterized by a 65% decrease in whole body Na(+) concentration, and an 81% inhibition of unidirectional whole body Na(+) uptake. Previous work on the acute toxicity of Ag(+) to daphnids has shown that Na(+) uptake inhibition is directly related to inhibition of Na(+),K(+)-ATPase activity. Therefore, we suggest that the Na(+) uptake inhibition seen in response to chronic silver exposure was explained by an inhibition of the Na(+) channels at the apical 'gill' membrane, since a 60% increase in whole body Na(+),K(+)-ATPase activity was observed in the chronically silver-exposed daphnids. Our findings demonstrate that, in broad view, the key mechanism involved in chronic silver toxicity in D. magna, the most acutely sensitive freshwater organism tested up to now, resembles that described for acute toxicity-i.e. ionoregulatory disturbance associated with inhibition of active Na(+) uptake, though the fine details may differ. Our results provide encouragement for future extension of the current acute version of the Biotic Ligand Model (BLM) to one that predicts chronic silver toxicity for environmental regulation and risk assessment. The results strongly suggest that Na(+) uptake inhibition is the best endpoint to determine sensitivity to both acute and chronic toxicity in the scope of future versions of the BLM for silver.     

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.     

Physiological interactions of silver and humic substances in Daphnia magna: effects on reproduction and silver accumulation following an acute silver challenge

Silver (Ag) in aquatic environments mediates its toxic actions by inhibiting sodium influx. Humic substances protect against silver toxicity by complexing the toxic, ionic form of the metal, but may also directly stimulate sodium influx in aquatic organisms. This study investigated the effects of silver and humic substances on the water flea Daphnia magna. Acute silver challenge (24 h; 1 microg L(-1)) and the chronic exposure to humic substances (Aldrich humic acid; 7 mg C L(-1)) had considerable influence on daphnid physiology and reproduction. In particular silver exposure in the absence of humic substances stimulated reproduction, resulted in enhanced adult mass, and altered both the response of the animal to subsequent silver exposure and a physiological surrogate measure of silver toxicity (whole body sodium concentration). The presence of humic substances countered the effects on adult mass and reproduction, returning these parameters to control levels. Humic substances also lowered silver body burden, but with significantly improved whole body sodium status than previously silver-exposed animals. These changes may distort the correlation between silver body burden and indicators of toxic action, an important tenet of site-specific risk assessment tools such as the biotic ligand model.     

Influence of Ca, humic acid and pH on lead accumulation and toxicity in the fathead minnow during prolonged water-borne lead exposure

The present study examines the influence of Ca2+ as (CaSO4), dissolved organic carbon (DOC) and pH on chronic water-borne lead (Pb) toxicity to the larval fathead minnow (Pimephales promelas) under flow-through conditions. The 30 day LC50 for low hardness basic test water (19 mg CaCO3 L(-1)) was 39 (range: 27-51) microg dissolved Pb L(-1) and was greatly increased by increasing concentrations of CaSO4 and DOC to as much as 1903 (range: 1812-1992) mug dissolved Pb L(-1). Both reduced and increased pH (6.7 and 8.1, respectively) compared to control pH of 7.4 appeared to increase Pb toxicity substantially. Whole body Pb accumulation did not reflect water chemistry and thus exhibited no correlation with Pb induced mortality. One possible explanation for this lack of correlation is that mortality occurred predominantly during the first 4-6 days of exposure, whereas Pb accumulation was determined in surviving fish at the end of 30 days of exposure. Chronic Pb exposure resulted in a general iono-regulatory disturbance affecting K+, Na+ and Ca2+ homeostasis. However, recovery of Na+ and K+ levels and reversal of effects on Ca2+ homeostasis during continued exposure strongly suggest fathead minnow can acclimate to Pb. The gills accumulate the highest Pb concentrations during chronic exposure but the skeleton contains the largest mass of Pb by contributing up to approximately 80% of whole body Pb. In conclusion, water chemistry characteristics like Ca2+ and DOC should be considered for chronic water quality criteria.     

Time course analysis of the mechanism by which silver inhibits active Na+ and Cl- uptake in gills of rainbow trout

A time course analysis using (110m)Ag, (24)Na(+), and (36)Cl(-) examined gill silver accumulation and the mechanism by which waterborne silver (4.0 x 10(-8) M; 4.3 microg/l) inhibits Na(+) and Cl(-) uptake in gills of freshwater rainbow trout. Analyses of gill and body fluxes allowed calculation of apical uptake and basolateral export rates for silver, Na(+), and Cl(-). To avoid changes in silver bioavailability, flow-through conditions were used to limit the buildup of organic matter in the exposure water. For both Na(+) and Cl(-) uptake, apical entry, rather than basolateral export, was the rate-limiting step; Na(+) and Cl(-) uptake declined simultaneously and equally initially, with both uptakes reduced by approximately 500 nmol.g(-1).h(-1) over the 1st h of silver exposure. There was a further progressive decline in Na(+) uptake until 24 h. Carbonic anhydrase activity was inhibited by 1 h, whereas Na(+)-K(+)-ATPase activity was not significantly inhibited until 24 h of exposure. These results indicate that carbonic anhydrase inhibition can explain the early decline in Na(+) and Cl(-) uptake, whereas the later decline is probably related to Na(+)-K(+)-ATPase blockade. Contrary to previous reports, gill silver accumulation increased steadily to a plateau. Despite the rapid inhibition of apical Na(+) and Cl(-) uptake, apical silver uptake (and basolateral export) increased until 10 h, before decreasing thereafter. Thus silver did not inhibit its own apical uptake in the short term. These results suggest that reduced silver bioavailability is the mechanism behind the pattern of peak and decline in gill silver accumulation previously reported for static exposures to silver.     

The mechanisms and costs of physiological and toxicological acclimation to waterborne silver in juvenile rainbow trout (Oncorhynchus mykiss)

Juvenile rainbow trout were exposed to 0, 0.1, 1, 3, and 5 micro g/l silver (Ag, as AgNO3) for 23 days. Specific growth rate, cumulative food consumption, food-conversion efficiency, and critical swimming speed (U(crit)) were significantly reduced during 5 micro g/l Ag exposure, demonstrating a physiological cost of silver acclimation. Only the 5 microg/l Ag treatment had significant cumulative mortality (5.2%). Fish were most susceptible to silver on days 5 and 15. Exposure to 5 microg/l Ag significantly lowered plasma Na+ and Cl- on days 5 and 10, but plasma ion concentration recovered thereafter. Unidirectional Na+ uptake and gill Na/K-ATPase activity were significantly inhibited by 3 and 5 microg/l Ag exposure. Na+ uptake was inhibited by 3 micro g/l Ag at day 5 alone, whereas the effects at the highest Ag exposure persisted until day 15. Gill Na/K-ATPase was inhibited on day 5 in both the 3 and 5 microg/l Ag treatments but increased to approx. 1.5 times of control levels by day 23. Only the 3 and 5 microg/l Ag treatments produced toxicological acclimation (at least twofold elevations in 168-h LC50 values in fish subsampled on days 15 and 23). We conclude that physiological acclimation results from compensatory changes in Na+ transport at the gills, and that these changes may eventually lead to toxicological acclimation.     

Acclimation of Daphnia magna to environmentally realistic copper concentrations

It may be hypothesised that as the bioavailable background concentration of an essential metal increases (within natural limits), the natural tolerance (to the metal) of the acclimated/adapted organisms and communities will increase. In this study the influence of acclimation to different copper concentrations on the sensitivity of the freshwater cladoceran Daphnia magna Straus was investigated. D. magna was acclimated over three generations to environmentally relevant copper concentrations ranging from 0.5 to 100 microg Cu/l (copper activity: 7.18 x 10(-15) to 3700 x 10(-12) M Cu2+). A modified standard test medium was used as culture and test medium. Medium modifications were: reduced hardness (lowered to 180 mg CaCO3/l) and addition of Aldrich humic acid at a concentration of 5 mg DOC/l (instead of EDTA). The effects of acclimation on these organisms were monitored using acute mortality assays and long-term assays in which life table parameters, copper body concentrations and energy reserves were used as test endpoints. Our results showed a two-fold increase in acute copper tolerance with increasing acclimation concentration for second and third generation organisms. Copper acclimation concentrations up to 35 microg Cu/l (80 pM Cu2+) did not affect the net reproduction and the intrinsic growth rate. The energy reserves of the acclimated daphnids revealed an Optimal Concentration range (OCEE) and concentrations between 5 and 12 microg Cu/l (0.5-4.1 pM Cu2+) and 1 and 35 microg Cu/l (0.023-80 pM Cu2+) seemed to be optimal for first and third generation daphnids, respectively. Lower and higher copper concentrations resulted in deficiency and toxicity responses. It was also demonstrated that up to 35 microg Cu/l, third generation daphnids were able to regulate their total copper body concentration. These results clearly indicate that bioavailable background copper concentrations present in culture media have to be considered in the evaluation of toxicity test results, especially when the toxicity data are used for water quality guideline derivation and/or ecological risk assessment for metals.     

The role of dissolved organic carbon in moderating the bioavailability and toxicity of Cu to rainbow trout during chronic waterborne exposure

We examined the influence of dissolved organic carbon (DOC) on the bioavailability of waterborne Cu to rainbow trout (Oncorhynchus mykiss) during chronic sublethal exposure. Juvenile rainbow trout were exposed to Cu (as CuSO(4)) and DOC as humic acid (HA, as sodium salt) for one month in synthetic soft water to give treatments with varying combinations of free ionic and HA complexed Cu. The total Cu concentration was 7 microg/l for all treatments (except controls) and HA was added at levels of 0, 2.5 and 7.5 mg/l which corresponded to DOC levels of 1.2, 2.2 and 4.0 mg/l. Fish grew well in all treatments and no mortalities occurred. Cu was highly bioavailable in the treatment with no added HA; gill and liver Cu accumulation occurred as well as a disruption of Na(+) regulation. In Cu treatments with additions of both 2.5 and 7.5 mg/l HA, there was no significant tissue accumulation of Cu. The addition of HA alleviated and delayed the disruption of iono-regulatory mechanisms. A recovery of plasma Na(+) losses was observed and this was associated with an increase in gill Na(+)/K(+) ATPase activity by the end of the exposure. Following the month of chronic exposure the uptake and turnover rates of Cu at the gills and into various tissue compartments were measured through radioisotopic techniques ((64)Cu). While chronic Cu exposure did not result in acclimation (i.e. increased LC50), the uptake rate and extent of Cu uptake into the gills and liver was increased. This study demonstrates that growth and tissue accumulation of Cu are poor predictors of the chronic effects of Cu, and illustrates that HA moderates chronic Cu bioavailability. The lack of a link between Cu bioaccumulation and Cu impact and the role of organic matter in reducing the bioavailability of Cu are important considerations in the context of ecological risk assessment.     

Short-term exposure to waterborne free silver has acute effects on membrane current of Xenopus oocytes

Waterborne free silver can cause osmo- and ionoregulatory disturbances in freshwater organisms. The effects of a short-term exposure to extracellular Ag+ ions on membrane currents were investigated in voltage-clamped defolliculated Xenopus oocytes. At a holding potential of -60 mV, ionic silver (1 microM Ag+) increased inward currents (=I(Ag)) from -8+/-2 nA to -665+/-41 nA (n=74; N=27). I(Ag) activated within 2 min of silver exposure and then rose impetuously. This current was largely reversible by washout and repeatable. I(Ag) reversed around -30 mV and rectified slightly at more positive potentials. Na+-free bath conditions reduced the silver-induced current to a smaller but sustained current. The response to silver was abolished by the Cl- channel blockers DIDS and SITS, whereas niflumic acid strongly potentiated I(Ag). Intraoocyte injection of AgNO3 to about 1 mM [Ag]i strongly potentiated I(Ag). Extracellular application of either dithiothreitol (DTT), a compound known to reduce disulfide bridges, or L-cysteine abolished Ag+-activated increase of membrane current. In contrast, n-ethylmaleimide (NEM) which oxidizes SH-groups potentiated I(Ag). Hypoosmotic bath solution significantly increased I(Ag) whereas hyperosmolar conditions attenuated I(Ag). The activation of I(Ag) was largely preserved after chelation of cytosolic Ca2+ ions with BAPTA/AM. Taken together, these data suggest that Xenopus oocytes are sensitive to short-term exposure to waterborne Ag+ ions and that the elicited membrane currents result from extra- and intracellular action of Ag+ ions on peptide moieties at the oocyte membrane but may also affect conductances after internalization.     

Protection by natural blackwater against disturbances in ion fluxes caused by low pH exposure in freshwater stingrays endemic to the Rio Negro

Stenohaline freshwater stingrays (Potamotrygon spp.) are endemic to the very dilute (Na(+), Cl(-), Ca2(+) 300 micromol L(-1) in reference water (low DOC) to about 100 micromol L(-1) in blackwater (high DOC). In reference water, both JNain and JClin were inhibited >90%, both JNaout and JClout more than doubled, and J(Amm) did not change at pH 4.0. In blackwater, the inhibition of influxes was attenuated, the increases in outflux did not occur, and J(Amm) increased by 60% at pH 4.0. Addition of 100 micromol L(-1) Ca(2+) to reference water prevented the increases in JNaout and JClout and allowed J(Amm) to increase at pH 4.0, which demonstrates that the gills are sensitive to Ca(2+). However, addition of Ca(2+) to blackwater had no effect on the responses to pH 4.0. Addition of commercial humic acid to reference water did not duplicate the effects of natural Rio Negro blackwater at the same DOC level; instead, it greatly exacerbated the increases in JNaout and JClout at low pH and prevented any protective influence of added Ca(2+). Thus, blackwater DOC appears to be very different from commercial humic acid. Biogeochemical modeling indicated that blackwater DOC prevents Ca(2+) binding, but not H(+) binding, to the gills and that the protective effects of blackwater cannot be attributed to its higher buffer capacity or its elevated Al or Fe levels. Natural DOC may act directly at the gills at low pH to exert a protective effect and, when doing so, may override any protective action of Ca(2+) that might otherwise occur.     

草甸湿地土壤溶解有机碳淋溶动态及其影响因素

采用土柱淋溶试验研究了草甸湿地有机土层（2～13 cm）DOC的淋溶动态,并探讨了土壤呼吸、NH₄⁺产生速率和淋溶液pH与DOC生成速率的关系.试验第一周,小叶章湿草甸（Ⅺ）和小叶章沼泽化草甸（Ⅻ）土壤DOC的释放速率经历了一个快速下降的过程,而后达到平稳水平,其DOC的释放动态可用一次指数衰减方程进行描述（R²>0.96,P<0.05）.整个试验期间（35 d）,两种草甸湿地土壤DOC的累积释放量分别为2109（Ⅺ）和506.58 μgC·g^-1（Ⅻ）,CO₂的累积释放量为679.64（Ⅺ）和455.54 μgC·g^-1（Ⅻ）,表明Ⅺ的低DOC释放可能与高CO₂释放所造成的微生物碳源受限有关.DOC的释放速率与NH₄⁺的生成速率呈显著正相关（r_Ⅺ=0.886,P<0.05;r_Ⅻ=0.972,P<0.01）,而与淋溶液pH无相关性.多元回归分析表明,草甸湿地DOC的生成主要受土壤氮矿化潜势制约（P<0.05）.     

HCO3-secretion by bullfrog duodenum: dependence on nutrient Na+ during secretory stimulation

HCO3(-) secretion across in vitro duodenal mucosa of Rana catesbeiana was investigated under baseline conditions and during secretory stimulation. Baseline secretion was abolished by removal of CO2-HCO3(-)and reduced approximately 60% by removal of nutrient Na+, but was not sensitive to changes in Cl- or K+. Baseline secretion was not directly altered by exposure to 10(-3) M amiloride or 10(-3) M H2DIDS (dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) in the nutrient solution and only mildly reduced by acetazolamide. Following removal and restoration of Na+, recovery of secretion was impaired by exposure to acetazolamide (5 x 10(-4) M) or H2DIDS (5 x 10(-4) M) in the nutrient solution. Secretion stimulated by glucagon (10(-6) M) or 16,16-dimethyl prostaglandin E2 (10 microg.mL(-1)) was markedly attenuated by removal of Na+ or by exposure to H2DIDS, but secretion was not altered by acetazolamide (5 x 10(-4) M) or nutrient amiloride (1 mM). Thus, the HCO3(-) that is secreted under nonstimulated conditions derives partly from basolateral Na(+)-dependent uptake and partly from cellular CO2 hydration. Secretagogue-stimulated secretion by duodenal surface epithelium depends on stilbene-sensitive Na+(HCO3(-))n uptake across the basolateral membrane.     

Toxicity of dissolved Cu, Zn, Ni and Cd to developing embryos of the blue mussel (Mytilus trossolus) and the protective effect of dissolved organic carbon

Marine water quality criteria for metals are largely driven by the extremely sensitive embryo-larval toxicity of Mytilus sp. Here we assess the toxicity of four dissolved metals (Cu, Zn, Ni, Cd) in the mussel Mytilus trossolus, at various salinity levels while also examining the modifying effects of dissolved organic carbon (DOC) on metal toxicity. In 48 h embryo development tests in natural seawater, measured EC50 values were 6.9-9.6 microg L(-1) (95% C.I.=5.5-10.8 microg L(-1)) for Cu, 99 microg L(-1) (86-101) for Zn, 150 microg L(-1) (73-156) for Ni, and 502 microg L(-1) (364-847) for Cd. A salinity threshold of >20 ppt (approximately 60% full strength seawater) was required for normal control development. Salinity in the 60-100% range did not alter Cu toxicity. Experimental addition of dissolved organic carbon (DOC) from three sources reduced Cu toxicity; for example the EC50 of embryos developing in seawater with 20 mg C L(-1) was 39 microg Cu L(-1) (35.2-47.2) a 4-fold increase in Cu EC50. The protective effects of DOC were influenced by their distinct physicochemical properties. Protection appears to be related to higher fulvic acid and lower humic acid content as operationally defined by fluorescence spectroscopy. The fact that DOC from freshwater sources provides protection against Cu toxicity in seawater suggests that extrapolation from freshwater toxicity testing may be possible for saltwater criteria development, including development of a saltwater Biotic Ligand Model for prediction of Cu toxicity.     

Acid-base balance during social interactions in rainbow trout (Oncorhynchus mykiss)

Socially subordinate rainbow trout (Oncorhynchus mykiss) experience chronic stress that impacts upon a variety of physiological functions, including Na(+) regulation. Owing to the tight coupling between Na(+) and Cl(-) uptake and, respectively, H(+) and HCO(3)(-) loss at the gill, ionoregulatory changes associated with social status may affect acid-base regulation. The present study assessed the responses of dominant, subordinate and control trout to hypercapnia (1% CO(2)) to test this hypothesis. Social status appeared to impact net acid excretion (J(net)H(+)) as subordinate individuals failed to increase net acid flux in response to hypercapnia. However, blood acid-base status was found to be unaffected by social status before or during hypercapnic exposure, indicating that subordinate fish were as effective as dominant or control trout in achieving compensation for the acid-base disturbance induced by hypercapnic exposure. Compensation in all groups involved decreasing Cl(-) uptake in response to hypercapnia. The branchial activities of both Na(+),K(+)-ATPase (NKA) and V-type H(+)-ATPase were affected by social interactions and/or exposure to hypercapnia. Branchial NKA activity was higher but V-ATPase activity was lower in control fish than in dominant or subordinate trout. In addition, control and subordinate but not dominant trout exposed to 24h of hypercapnia exhibited significantly higher branchial V-ATPase activity than fish maintained in normocapnia. Collectively, the data suggest that subordinate trout are able to regulate blood pH during a respiratory acidosis.     

Evidence of UCP1-independent regulation of norepinephrine-induced thermogenesis in brown fat

To study the thermal response of interscapular brown fat (IBF) to norepinephrine (NE), urethan-anesthetized rats (1.2 g/kg ip) maintained at 28-30 degrees C received a constant venous infusion of NE (0-2 x 10(4) pmol/min) over a period of 60 min. IBF temperatures (T(IBF)) were recorded with a small thermistor fixed under the IBF pad. Data were plotted against time and expressed as maximal variation (Deltat degrees C). Saline-injected rats showed a decrease in T(IBF) of approximately 0.6 degrees C. NE infusion increased T(IBF) by a maximum of approximately 3.0 degrees C at a dose of 10(4) pmol x min(-1) x 100 g body wt(-1). Surgically thyroidectomized (Tx) rats kept on 0.05% methimazole showed a flat response to NE. Treatment with thyroxine (T(4), 0.8 microg x 100 g(-1) x day(-1)) for 2-15 days normalized mitochondrial UCP1 (Western blotting) and IBF thermal response to NE, whereas iopanoic acid (5 mg x 100 g body wt(-1) x day(-1)) blocked the effects of T(4). Treatment with 3,5, 3'-triiodothyronine (T(3), 0.6 microg x 100 g body wt(-1) x day(-1)) for up to 15 days did not normalize UCP1 levels. However, these animals showed a normal IBF thermal response to NE. Cold exposure for 5 days or feeding a cafeteria diet for 20 days increased UCP1 levels by approximately 3.5-fold. Nevertheless, the IBF thermal response was only greater than that of controls when maximal doses of NE (2 x 10(4) pmol/min and higher) were used. Conclusions: 1) hypothyroidism is associated with a blunted IBF thermal response to NE; 2) two- to fourfold changes in mitochondrial UCP1 concentration are not necessarily translated into heat production during NE infusion.     

Determination of carotenoid and vitamin A concentrations in everted salmonid intestine following exposure to solutions of carotenoid in vitro

Carotenoid (astaxanthin and canthaxanthin) concentrations in everted intestine from rainbow trout (Oncorhynchus mykiss, Walbaum) and Atlantic salmon (Salmo salar, L.) exposed to micelle solubilised carotenoid, have been determined. Following exposure (1 h) to astaxanthin solution (5 mg l(-1)), trout pyloric caeca and mid intestine had higher (P<0.05) mean tissue astaxanthin concentrations (0.50+/-0.08 microg g(-1) and 0.54+/-0.09 microg g(-1), respectively) compared to hind intestine (0.04+/-0.01 microg g(-1); n=11+/-S.E.). Furthermore, the astaxanthin concentration in pyloric caeca (0.50+/-0.08 microg g(-1)) was greater (P<0.05) than that of canthaxanthin (0.11+/-0.01 microg g(-1); n=11, +/-S.E.) when exposed to solutions of similar carotenoid concentration (5.11+/-0.16 mg l(-1) and 5.35+/-0.16 mg l(-1), respectively; n=3+/-S.E.). However, no differences (P>0.05) were recorded between trout and salmon intestinal tissue in terms of astaxanthin concentration following exposure. Trout caeca exposed to astaxanthin solution had significantly (P<0.05) more vitamin A (514.1+/-36.4 microg g(-1)) compared to control tissues (316.5+/-61.7 microg g(-1); n=8+/-S.E.). Vitamin A(1) concentrations in caeca (287.7+/-11.0 microg g(-1)) exposed to astaxanthin solution were significantly higher (P<0.05) compared to controls (174.9+/-26.9 microg g(-1)). However, vitamin A(2) concentrations were not significantly (P>0.05) different (226.3+/-28.2 microg g(-1) and 141.6+/-35.2 microg g(-1), respectively).     

Urinary selenium excretion in selenite-loaded sheep and subsequent Se dynamics in blood constituents

Renal selenium excretion in sheep was measured during intravenous infusion of sodium selenite, and the post-infusion dynamics of Se levels in whole blood, plasma and red blood cells (RBC) were investigated for the next 5 days. The plasma Se level increased almost twenty fold with the infusion of Na2SeO3 (from 0.39 +/- 0.02 to 7.83 +/- 0.33 micromol x L(-1), P < 0.001) compared with the baseline value. The selenium concentration in urine (0.07 +/- 0.02 vs. 18.53 +/- 2.56 micromol x L(-1), P < 0.001), the amount of Se excreted (0.14 +/- 0.07 vs. 21.40 +/- 2.31 nmol x min(-1), P < 0.001) and the renal clearance of Se (0.1 9 +/- 0.03 vs. 3.01 +/- 0.34 mL x min(-1), P < 0.001) were found to be highly significantly elevated during selenite loading. The clearance measurements showed no changes in the urinary flow rate or in the glomerular filtration rate. During and at the end of infusion the highest Se level was attained in plasma, followed by whole blood and RBC. The plasma Se level fell rapidly within 10 min after the end of infusion, but the concentration of Se in RBC was stable up to the fourth hour, when it started to decrease too. On day 5 the Se concentrations in plasma, RBC and whole blood were found to be only slightly but still significantly higher than before the selenite infusion. The large disproportion between the infusion rate of Se (8.76 microg x min(-1)) and its renal excretion rate (1.69 microg x min(-1)) found in clearance measurements suggests low glomerular filtration of infused selenium, which might primarily be caused by the binding of selenite metabolites to blood constituents. The presented results confirm the low bioavailability to ruminants of Se from sodium selenite.     

In vivo regulation of Na/Ca exchanger expression by adrenergic effectors

The Na/Ca exchanger encoded by the NCX1 gene plays an important role in calcium homeostasis in cardiac muscle. We previously identified three in vitro signaling pathways that are of major importance in the regulation of Na/Ca exchanger gene expression in neonatal cardiac myocytes, the protein kinase A (PKA) and protein kinase C (PKC) pathways, and intracellular Ca(2+). To determine whether these pathways are important in vivo, we stimulated the PKA and PKC pathways and examined functional expression of the Na/Ca exchanger in adult rat heart. After a 3- and 7-day treatment, norepinephrine (200 microg x kg(-1) x h(-1)), isoproterenol (150 microg x kg(-1) x h(-1)), and phenylephrine (200 microg x kg(-1) x h(-1)) each stimulated a significant increase in NCX1 mRNA levels (35-85%, P < 0.05). Norepinephrine also stimulated a 35% increase in protein abundance (P < 0.05), a 20% decrease in relaxation duration (P < 0.05), and a 25% reduction in the fluorescence decay constant (P < 0.05) after a 7-day treatment. We conclude that a 7-day treatment of alpha- and beta-adrenergic agonists increases the expression of functional Na/Ca exchangers in adult rat heart.