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Accumulating evidence is highlighting the importance of a system of enhanced hemoglobin-oxygen (Hb-O 2 ) unloading for cardiovascular O 2 transport in teleosts. Adrenergically stimulated sodium-proton exchangers (β-NHE) create H + gradients across the red blood cell (RBC) membrane that are short-circuited in the presence of plasma-accessible carbonic anhydrase (paCA) at the tissues; the result is a large arterial-venous pH shift that greatly enhances O 2 unloading from pH-sensitive Hb. However, RBC intracellular pH (pH i ) must recover during venous transit (31-90 s) to enable O 2 loading at the gills. The halftimes ( t 1/2 ) and magnitudes of RBC β-adrenergic stimulation, short-circuiting with paCA and recovery of RBC pH i , were assessed in vitro, on rainbow trout whole blood, and using changes in closed-system partial pressure of O 2 as a sensitive indicator for changes in RBC pH i . In addition, the recovery rate of RBC pH i was assessed in a continuous-flow apparatus that more closely mimics RBC transit through the circulation. Results indicate that: 1) the t 1/2 of β-NHE short-circuiting is likely within the residence time of blood in the capillaries, 2) the t 1/2 of RBC pH i recovery is 17 s and within the time of RBC venous transit, and 3) after short-circuiting, RBCs reestablish the initial H + gradient across the membrane and can potentially undergo repeated cycles of short-circuiting and recovery. Thus, teleosts have evolved a system that greatly enhances O 2 unloading from pH-sensitive Hb at the tissues, while protecting O 2 loading at the gills; the resulting increase in O 2 transport per unit of blood flow may enable the tremendous athletic ability of salmonids.

Red blood cells (RBCs) possess methemoglobin reductase activity that counters the ongoing oxidation of hemoglobin (Hb) to methemoglobin (metHb), which in circulating blood is caused by Hb autoxidation or reactions with nitrite. We describe an assay for determining metHb reductase activity in intact RBCs in physiological saline at normal Pco 2 and pH. After initial loading of oxygenated RBCs with nitrite (partly oxidizing Hb to metHb), the nitrite is removed by three washes of the RBCs in nitrite-free physiological saline to enable the detection of RBC metHb reductase activity in the absence of counteracting oxidation. This assay was used to compare metHb reduction in rainbow trout and carp RBCs under both oxygenated and deoxygenated conditions. Washing resulted in effective wash-out of nitrite to low and safe values (~2μM). The subsequent decline in [metHb] with time followed first-order kinetics, allowing characterization of metHb reductase activity through the first order rate constant k. In oxygenated RBCs at 25°C, the k values for rainbow trout and carp were slightly below or above 0.01min -1 , respectively; which is double the value reported for mammals at 37°C. We conclude the higher metHb reductase activity in fish offsets their higher Hb autoxidation and higher likelihood of encountering elevated nitrite. Deoxygenation significantly raised the rates of RBC metHb reduction, and more so in rainbow trout than in carp. The temperature sensitivity of metHb reduction in rainbow trout RBCs was high (Q 10 ~2.8) and instrumental in handling increased Hb autoxidation with temperature., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cd is known for its carcinogenic effects, however its mechanism of toxicity and in particular its ability to promote oxidative stress is debated. In fact, although it is considered a redox-inactive metal, at high concentration Cd was shown to promote indirectly oxidative stress. In this study we investigated metal accumulation in ex vivo exposed trout (Oncorhynchus mykiss) erythrocytes and Cd dose-dependent effect in terms of RBC viability, cytosolic and mitochondrial ROS levels as well as its effects on mitochondrial membrane depolarization, hemoglobin stability and precipitation. In the concentration range used, Cd did not affect cell viability. However, metal accumulation was associated with an increase in all oxidative indexes evaluated, except mitochondrial superoxide anion production that, on the contrary, was significantly decreased, probably due to a lowered respiration rate associated with interference of Cd with complex I, II and III, as suggested by the observed Cd-dependent mitochondrial membrane depolarization. On the other hand, hemoglobin destabilisation seems to be the major trigger of oxidative stress in this cell type., (Copyright © 2017. Published by Elsevier Inc.)

The induction of micronuclei (MN), nuclear buds (NB), bi-nucleated erythrocytes with nucleoplasmic bridge (BNb), vacuolated (VacNuc), blebbed (BL), 8-shaped nuclei, bi-nucleated (BN) and fragmented-apoptotic (FA) erythrocytes was analysed in the peripheral blood, cephalic kidney and liver of rainbow trout Oncorhynchus mykiss after 4-day treatment with copper (Cu) and zinc (Zn) mixture solutions and in 4-, 8- and 12-day depuration process. Fish (three treatment and one control group, N=40) were exposed to 0.0625, 0.125 and 0.25 fractions of 96-h LC50, respectively under semi-static conditions. Exposure of O. mykiss to Cu and Zn induced significant increase of MN (in blood in all test groups; in liver 0.125, 0.25 and in kidney 0.25 groups, respectively), NB and BL (in blood and kidney 0.25 group), 8-shaped (in blood 0.25; in liver 0.125, 0.25 and in kidney all test groups, respectively) and VacNuc (in liver and kidney 0.0625 and 0.125 groups). After 4-day recovery, significantly elevated levels of MN (in blood 0.0625, 0.125; in liver and kidney 0.125 group, respectively) and 8-shaped (in kidney-0.0625 group) were observed in fish. Significant recovery was observed in 0.0625 group after 12-day depuration, estimating the formation of MN in erythrocytes of blood, of 8-shaped nuclei erythrocytes in liver and kidney (after 8-, 12-day and 8-day recovery, respectively). Significant decrease of MN in blood (after 8- and 12-day recovery), in liver (after 8-day recovery), of NB in blood and kidney (after 8-day recovery) and of 8-shaped nuclei erythrocytes in blood (after 8 and 12-day recovery), kidney and liver (after 8-day recovery) was determined in 0.25 group. Changes in gross morphometric indices and biological parameters were observed. The binary metal mixture did not induce FA erythrocytes in any tissue at any test concentration., (Copyright © 2016 Elsevier B.V. All rights reserved.)

TiO2 has been widely used to promote organic compounds degradation on waste aqueous solution, however, data on TiO2 nanotoxicity to aquatic life are still limited. In this in vitro study, we compare the toxicity of two different families of TiO2 nanoparticles on erythrocytes from Oncorhynchus mykiss trout. The crystal structure of the two TiO2 nanoparticles was analyzed by XRD and the results indicated that one sample is composed of TiO2 in the anatase crystal phase, while the other sample contains a mixture of both the anatase and the rutile forms of TiO2 in a 2:8 ratio. Further characterization of the two families of TiO2 nanoparticles was determined by SEM high resolution images and BET technique. The toxicity results indicate that both TiO2 nanoparticles increase the hemolysis rate in a dose dependent way (1.6, 3.2, 4.8 μg mL(-1) ) but they do not influence superoxide anion production due to NADH addition measured by chemiluminescence. Moreover, TiO2 nanoparticles (4.8 μg mL(-1) ) induce DNA damage and the entity of the damage is independent from the type of TiO2 nanoparticles used. Modified comet assay (Endo III and Fpg) shows that TiO2 oxidizes not only purine but also pyrimidine bases. In our experimental conditions, the exposure to TiO2 nanoparticles does not affect the DNA repair system functionality. The data obtained contribute to better characterize the aqueous environmental risks linked to TiO2 nanoparticles exposure., (Copyright © 2011 Wiley Periodicals, Inc.)

Glutathione S-transferase enzyme (GST) (EC 2.5.1.18) was purified from rainbow trout erythrocytes, and some characteristics of the enzyme and effects of some metal ions on enzyme activity were investigated. For this purpose, erythrocyte glutathione S-transferase enzyme which has 16.54 EU/mg protein specific activities was purified 11,026-fold by glutathione-agarose affinity chromatography with a yield of 59%. Temperature was kept under control (+4°C) during purification. Enzyme purification was checked by performing SDS-PAGE. Optimal pH, stable pH, optimal temperature, and K(M) and Vmax values for GSH and 1-chloro-2, 4-dinitrobenzene (CDNB) were also determined for the enzyme. In addition, IC50 values, Ki constants and the type of inhibition were determined by means of Line-Weaver-Burk graphs obtained for such inhibitors as Ag(+); Cd(2+), Cr(2+) and Mg(2+).

The aim of this study was to assess the effect of the different aquatic conditions on anion transport of fish erythrocytes through the measurement of the sulphate uptake operating from band 3, the determination of reduced glutathione (GSH) and oxidized glutathione (GSSG). To this purpose, blood samples of 30 Liza aurata and 30 Salmo irideus fishes were collected via caudal vein and washed with physiological buffer. Successively, erythrocytes suspended at 3% haematocrit were used to measure the SO(4) (=) influx by atomic absorption spectrophotometry at 425 nm wavelength and the GSH concentration using an immunodiagnostic assay intended for the quantitative determination of glutathione in ethylenediaminetetraacetic acid in blood. All results were analyzed using unpaired Student's t-test and a P < 0.05 was considered statistically significant. The results of this study showed statistically significant differences about the sulphate uptake and the glutathione levels in S. irideus with respect to L. aurata (P < 0.0001). In conclusion, the different aquatic conditions play an important role on anion transport in fish erythrocytes, showing that environmental differences induce perturbations in erythrocyte membrane and should be evaluated to prevent physiological damages in fishes., (Copyright © 2012 John Wiley & Sons, Ltd.)

The retention of a nucleus in the mature state of fish red blood cells (RBCs) and the ability to easily collect and manipulate blood in nonterminal experiments make blood an ideal tissue on which to study the cellular stress response in fish. Through the use of the cGRASP 16K salmonid microarray, we investigated differences in RBC global gene transcription in fish held under control conditions (11 degrees C) and exposed to heat stress (1 h at 25 degrees C followed by recovery at 11 degrees C). Repeated blood sampling (via a dorsal aorta cannula) enables us to examine the individual stress response over time. Samples were taken preheat stress (representing individual control) and at 4 and 24 h postheat stress (representing early and late transcriptional regulation). Approximately 3,000 microarray features had signal above threshold when hybridized with RBC RNA-derived targets, and cannulation did not have a detectable effect on RBC mRNA expression at the investigated time points. Genes involved in the stress response, immune response, and apoptosis were among those showing the highest dysregulation during both early and late transcriptional regulation. Additionally, genes related to the differentiation and development of blood cells were transcriptionally upregulated at the 24 h time point. This study provides a broader understanding of the mechanisms underpinning the stress response in fish and the discovery of novel genes that are regulated in a stress specific manner. Moreover, salmonid transcripts that are consistently dysregulated in blood in response to heat stress are potential candidates of nonlethal biomarkers of exposure to this particular stressor.

Infectious salmon anaemia (ISA) virus (ISAV) is a fish orthomyxovirus that has recently been assigned to the new genus Isavirus within the family Orthomyxoviridae. It possesses the major functional characteristics of the virus family including haemagglutinating, receptor destroying enzyme (RDE), and fusion activities associated with the virion surface proteins. It is generally accepted that ISAV agglutinates erythrocytes of several fish species and that the ISAV RDE activity dissolves this haemagglutination reaction except for Atlantic salmon (Salmo salar) erythrocytes. We used electron microscopy to examine the physical interaction between ISAV and erythrocytes from Atlantic salmon and rainbow trout (Oncorhynchus mykiss) during haemagglutination. We present evidence that ISAV enters into Atlantic salmon erythrocytes. Atlantic salmon erythrocytes incubated with ISAV for 4 hours showed endocytosis of the virus particles, which is consistent with virus infection. These observations suggest that the lack of dissolution of ISAV-induced haemagglutination of Atlantic salmon erythrocytes favours virus infection of the erythrocytes. Moreover, such a haemagglutination-infection phenotype is fundamentally different from haemagglutination by avian and mammalian orthomyxoviruses, and is indicative of a different pathogenesis for the fish orthomyxovirus.

Many teleostean fish, including rainbow trout, regulate red blood cell (RBC) pH (pH(i)) in the presence of a stress-induced acidosis such as hypoxia, hypercapnia, or exhaustive exercise. This is accomplished through activation of RBC Na+/H+ exchange (beta-NHE), ultimately minimizing impairment to oxygen transport. Presence and characterization of the RBC beta-NHE in fish is best tested in blood from cannulated, resting animals; however, several studies have used blood from stressed animals drawn from the caudal vein and stored prior to use. The effects of sampling procedures and storage on the beta-NHE response is not known and is the focus of this study. Whole blood drawn from cannulated, resting rainbow trout was compared with RBCs obtained from the caudal vein rinsed and stored at 4 degrees C for 0, 6, 24, 48, 96 or 144 h. Isoproterenol (10(-5) M), a beta-adrenergic agonist, was added to hypoxia/hypercapnia incubated RBCs in vitro. In all treatments, isoproterenol induced a large beta-NHE response, and storage duration (< or =96 h) had a minimal affect, indicating that rinsing and storing is an easy and viable means by which to obtain RBCs and investigate function. Storage for 144 h still resulted in a significant RBC beta-NHE response; however, viability of RBCs may be compromised.

A method for perfusion of the isolated trout heart coronary circulation with red blood cells (RBCs) was developed. The method was used to analyse the influence of RBC perfusion on myocardial O(2) supply and O(2) consumption and to test the hypothesis that nitrite is converted to vasoactive nitric oxide in the RBC-perfused coronary circulation. Perfusion with RBCs significantly increased myocardial O(2) supply and O(2) consumption by increasing the incoming O(2) concentration and the O(2) extraction. Coronary flow did not differ between RBC perfusion and saline perfusion, but RBC perfusion established a strong linear increase in myocardial O(2) consumption with coronary flow. Nitric oxide was measured in the atrial effluent of the preparation. Perfusion with saline under hypoxic conditions was associated with NO production. The nitric oxide synthase inhibitor L-NA obliterated this NO production and significantly decreased coronary flow, showing that the NO was vasoactive and probably of endothelial origin. RBC perfusion at low P(O(2)) similarly caused an L-NA-inhibitable NO production. The change in NO production upon subsequent nitrite addition, by contrast, was not inhibited by L-NA. Nitrite entered trout erythrocytes independent of degree of oxygenation, but the O(2) saturation of RBCs showed a major decrease in the coronary circulation, and [NO(2)(-)] decreased while methaemoglobin rose, suggesting that deoxyHb-mediated reduction of nitrite to NO may have occurred. However, other possibilities (e.g. NO(2)(-)-->NO conversion in myocardial cells) cannot be excluded. The NO formation associated with nitrite had no effect on coronary flow, possibly because NO was produced after the resistance vessels.

Inhibitory effects of some drugs on glucose 6-phosphate dehydrogenase from the erythrocytes of rainbow trout (Oncorhynchus mykiss Walbaum, 1792) were investigated. The enzyme was purified 2488-fold in a yield of 76.8% using ammonium sulfate precipitation and 2',5'-ADP Sepharose 4B affinity gel at 4 degrees C. The drugs pental sodium, MgSO4, vancomycin, metamizol, marcaine, and prilocaine all exhibited inhibitory effects on the enzyme. While MgSO4 (K(i) = 12.119 mM), vancomycin (K(i) = 1.466 mM) and metamizol (K(i) = 0.392 mM) showed competitive inhibition, pental sodium (K(i) = 0.748 mM) and marcaine (K(i) = 0.0446 mM) displayed noncompetitive inhibition.

The effects of low molecular weight plasma inhibitors from rainbow trout (Oncorhynchus mykiss) (RT) were investigated on the carbonic anhydrase enzyme (CA) activities in in vitro human and in in vivo Sprague-Dawley rat erythrocytes. The RT blood was used as extracellular fluid (plasma) source and plasma inhibitors were obtained by dialysis of the plasma. For the in vitro study, human carbonic anhydrase-II (HCA-II) isozyme was obtained by Sepharose 4B-L-tyrosine-sulfanylamide affinity chromatography with an overall purification of about 646-fold. The enzyme (specific activity of 7750 EU/mg protein) was obtained with a yield of 71.1% and SDS-PAGE showed a single band. From in vitro studies, the I50 value for RT plasma inhibitors obtained was 0.37 mg/ml. From in vivo studies on rat erythrocytes, CA activity was significantly inhibited by the inhibitors from the extracellular fluid of RT for up to 3 h (p < 0.05) following intraperitoneal administration.

Erythrocytes from the rainbow trout Salmo gairdneri Richardson (Salmo g.R.) were classified into immature and mature populations, respectively, by measuring longitudinal diameters. More elongated fish erythrocytes (FE), classified as mature cells, were those interacting with Candida albicans (CA) in a higher frequency in terms of either binding to the fungus or its intracellular engulfment. At the same time, in the rosetting phenomenon more elongated mature FE surrounded macrophages (Mø) phagocytosing CA. Finally, FE activated by CA released in the supernatants cytokine-like factors able to modulate Mø functions. In particular, these active supernatants were analyzed for their capacity to inhibit Mø migration Macrophage Inhibition Factor (MIF) activity and enhance Mø phagocytosis. Both activities were detected in supernatants from CA stimulated FE but not in control supernatants. MIF activity could play a role in the accumulation of Mø in the context of functional rosettes, while the factor enhancing Mø phagocytosis could promote clearance of CA in a more efficacious way.

The aquatic environment is the largest sink for the highly toxic organotin compounds, particularly as one of the main sources is the direct release of organotins from marine antifouling paints. The aim of this study was to investigate the mitochondrial toxicity and proapoptotic activity of tributyltin chloride (TBTC) in teleost leukocytes and nucleated erythrocytes, by means of electron microscopy investigation and mitochondrial membrane potential evaluation, in order to provide an early indicator of aquatic environmental pollution. Erythrocytes and leukocytes were obtained from an inbred strain of rainbow trout (Oncorhynchus mykiss). Transmission electronic micrographs of trout red blood cells (RBC) incubated in the presence of TBTC at 1 and 5 microM for 60 min showed remarkable mitochondrial morphological changes. TBTC-mediated toxicity involved alteration of the cristae ultrastructure and mitochondrial swelling, in a dose-dependent manner. Both erythrocytes and leukocytes displayed a consistent drop in mitochondrial membrane potential following TBTC exposure at concentrations >1 microM. The proapoptotic effect of TBTC on fish blood cells, and involvement of mitochondrial pathways was also investigated by verifying the release of cytochrome c, activation of caspase-3 and the presence of "DNA laddering". Although mitochondrial activity was much more strongly affected in erythrocytes, leukocytes incubated in the presence of TBTC showed the characteristic features of apoptosis after only 1 h of incubation. Longer exposures, up to 12 h, were required to trigger an apoptotic response in erythrocytes.

The present study shows that a monoclonal antibody (MAb) directed to Aeromonas salmonicida glycerophospholipid:cholesterol acyltransferase (GCAT) is capable of protecting rainbow trout red blood cells from the cytotoxic effects of the enzyme in vitro.

Unidirectional (36)Cl(-) efflux via the red blood cell anion exchanger was measured under Cl(-) self-exchange conditions (i.e. no net flow of anions) in rainbow trout Oncorhynchus mykiss and red-eared freshwater turtle Trachemys scripta to examine the effects of acute temperature changes and acclimation temperature on this process. We also evaluated the possible adaptation of anion exchange to different temperature regimes by including our previously published data on other animals. An acute temperature increase caused a significant increase in the rate constant (k) for unidirectional Cl(-) efflux in rainbow trout and freshwater turtle. After 3 weeks of temperature acclimation, 5 degrees C-acclimated rainbow trout showed only marginally higher Cl(-) transport rates than 15 degrees C-acclimated trout when compared at the same temperature. Apparent activation energies for red blood cell Cl(-) exchange in trout and turtle were lower than values reported in endothermic animals. The Q(10) for red blood cell anion exchange was 2.0 in trout and 2.3 in turtle, values close to those for CO(2) excretion, suggesting that, in ectothermic animals, the temperature sensitivity of band-3-mediated anion exchange matches the temperature sensitivity of CO(2) transport (where red blood cell Cl(-)/HCO(3)(-) exchange is a rate-limiting step). In endotherms, such as man and chicken, Q(10) values for red blood cell anion exchange are considerably higher but are no obstacle to CO(2) transport, because body temperature is normally kept constant at values at which anion exchange rates are high. When compared at constant temperature, red blood cell Cl(-) permeability shows large differences among species (trout, carp, eel, cod, turtle, alligator, chicken and man). Cl(-) permeabilities are, however, remarkable similar when compared at preferred body temperatures, suggesting an appropriate evolutionary adaptation of red blood cell anion exchange function to the different thermal niches occupied by animals.

In this study, we examined whether the adrenergic volume response of teleost erythrocytes is related to cell maturity. Rainbow trout (Oncorhynchus mykiss) were made anaemic by reducing their haematocrit to approximately 50 % of the original value. After 3-4 weeks, small, young erythrocytes were seen in the circulation. By measuring the volume distribution of blood samples from anaemic fish before and after noradrenaline stimulation (10 min, 10(-5)mol l(-1) final concentration), we were able to show that the volume response of young, immature erythrocytes to catecholamine stimulation was greater than that of mature erythrocytes. In addition, the membrane fluidity, measured using the steady-state fluorescence polarisation method, was greater in anaemic fish after 24 days of recovery from bleeding than in control fish. Since blood from anaemic fish contained a large fraction of immature erythrocytes, this result indicates that the fluidity of the membrane of immature erythrocytes is greater than that of mature erythrocytes.

Density separated trout erythrocytes, using a discontinuous Percoll gradient, yielded three distinct subfractions (top, middle and bottom) since older cells are characterized by increasing density. Cells from each subfraction were incubated with mitochondria-specific fluorescent probe Mitotracker and JC-1 in order to assess mitochondrial mass and membrane potential by means of cytofluorimetric analysis, confocal microscopy and subsequent computer-aided image analysis allowing a detailed investigation at single cell level. Both cytofluorimetric data and image analysis revealed changes in size and redistribution of mitochondria starting from the light fraction to the bottom. In particular in young erythrocytes small mitochondria were detected localized exclusively around the nucleus in a crown-like shape, the middle fraction revealed enlarged mitochondria partially scattered throughout the cytosol, whereas the last fraction represented again mitochondria with reduced size being distinctly dispersed throughout the cytosol in the cells. Concerning membrane potential considerations, our study revealed a dramatic decrease of DeltaPsi(m) in the bottom layer cell mitochondria compared to the top and unusual membrane potential increase of a subpopulation of enlarged mitochondria. DeltapH was also investigated in the three fractions by pretreating the cells with nigericin, allowing to confirm a mitochondrial energetic impairment in older cells.

The effects of cell age on metabolism in the nucleated red blood cells of rainbow trout (Oncorhynchus mykiss) were examined. Red blood cells were separated according to age using fixed-angle centrifugation. The mean erythrocyte haemoglobin concentration in old red blood cells was found to be 120 % of that in young red blood cells. In young red blood cells, the activities of the mitochondrial enzymes citrate synthase and cytochrome oxidase were 135-200 %, respectively, of those measured in old red blood cells. The activity of the glycolytic enzyme lactate dehydrogenase in young red blood cells was 170 % of that in old red blood cells, whereas the activity of the glycolytic enzyme pyruvate kinase was not significantly affected by cell age. In addition, young red blood cells consumed over twice as much O(2) and devoted 50 % more O(2) to protein synthesis and the activity of Na(+)/K(+)-ATPase than old red blood cells. Red blood cell age did not significantly affect the rate of lactate production. This study shows that ageing in rainbow trout nucleated red blood cells is accompanied by a significant decline in aerobic energy production and the processes it supports, as well as a corresponding increase in the glycolytic contribution to metabolism.

A sudden decrease in external medium osmolality (90 mosmol/kg) causes an immediate swelling of trout erythrocytes, followed by a regulatory volume decrease (RVD) due to activation of both a KCl cotransporter and a taurine transport pathway. Here, we determined how trout red cells respond when they are exposed to a gradual and slow decrease in medium osmolality (80 mosmol/kg at a rate of 0.7 mosmol/kg per min). Erythrocytes were unable to regulate their volume efficiently when swollen gradually and it increased continuously throughout the experimental period (120 min). As long as volume was increased slowly by 15-25%, regulatory pathways remained essentially inactivated, erythrocytes losing no significant amount of intracellular osmotically active solutes. Above this swelling threshold, a response was triggered but the quantity of solutes lost via the regulatory pathways was still not sufficient to counterbalance the continuous entry of water due to the slow and gradual decrease in medium tonicity.

The physiological and biochemical signals that induce stress protein (HSP) synthesis remain conjectural. In this study, we used the nucleated red blood cells from rainbow trout, Oncorhynchus mykiss, to address the interaction between energy status and HSP gene expression. Heat shock (25 degrees C) did not significantly affect ATP levels but resulted in an increase in HSP70 mRNA. Hypoxia alone did not induce HSP transcription in these cells despite a significant depression in ATP. Inhibition of oxidative phosphorylation with azide, in the absence of thermal stress, decreased ATP by 56% and increased lactate production by 62% but did not induce HSP gene transcription. Inhibition of oxidative phosphorylation and glycolysis with azide and iodoacetic acid respectively, decreased ATP by 79% and prevented lactate production, but did not induce either HSP70 or HSP30 gene transcription in these cells. This study demonstrates that a reduction in the cellular energy status will not induce stress protein gene transcription in rainbow trout red blood cells and may, in fact, limit induction during extreme metabolic inhibition.

If swelling of a cell is induced by a decrease in external medium tonicity, the regulatory response is more complex than if swelling of similar magnitude is due to salt uptake. The present results provide an explanation. In fish erythrocytes, two distinct transport pathways were swelling activated: a channel of broad specificity and a K+-Cl- cotransporter. Each was activated by a specific signal: the channel by a decrease in intracellular ionic strength and the K+-Cl- cotransporter by cell enlargement. A decrease in ionic strength also affected K+-Cl- cotransport activity, but by acting as a negative modulator of the cotransport. Thus cells swollen by salt accumulation respond by activating exclusively the K+-Cl- cotransport, leading to a Cl--dependent K+ loss. By contrast, cells swollen by electrolyte dilution respond by activating both pathways, leading to a reduced loss of electrolytes and a large loss of taurine. Thus two swelling-sensitive pathways, differently regulated, would allow control of the ionic composition of a cell exposed to different volume perturbations.

Because previous literature reports have demonstrated that nucleated trout erythrocytes in conditions of oxidative stress are subjected to both membrane damage and a decrease in the enzymatic defense systems (glutathione peroxidase), which in turn lead to hemolysis, the present study was undertaken to determine whether DNA may be affected too, prior to the hemolytic event. Impairment of DNA in stressed trout erythrocytes was assessed using the comet assay--a rapid and sensitive, single-cell gel electrophoresis technique used to detect primary DNA damage in individual cells. In addition, indolinic and quinolinic nitroxide radicals were included in the study to determine their efficacy as antioxidants against free-radical-induced DNA damage. The parameters, tail length, tail intensity, and tail moment, used as an index of DNA damage, have shown that trout erythrocytes exposed to oxidative stress experience DNA damage prior to hemolysis and that the nitroxides significantly prevent this damage. This result provides further information about the potential use of these compounds as antioxidants in biological systems.

We studied effects of in vitro conditions on the steady-state distribution and exchange of thyroid hormones (TH) between red blood cells (RBC) and plasma of rainbow trout. At steady state at 12 degrees C the RBC contained 5-11% of L-thyroxine (T4), 14-23% of 3,5,3'-triiodo-L-thyronine (T3), and 23-24% of 3,3',5'-triiodo-L-thyronine (rT3) present in whole blood. The steady-state distribution was (i) higher in immature than in mature trout for T3, (ii) increased by incubation temperature from 0 to 22 degrees C for both T4 and T3, (iii) unaltered by blood T4 or T3 concentration (0-40 ng/ml), and (iv) increased by O2 gassing and decreased by N2 gassing for T4 and rT3, but negligibly for T3. The exchange between RBC and plasma was more rapid for T3 (50% of maximal influx or efflux at 30-40 s) than rT3 (14 min) than T4 (30 min). Efflux of T4 and T3 was greatly reduced in the absence of plasma protein. Incubation with 10% bovine serum albumin extracted > 98% of labeled T4 and T3 from RBC. We conclude that for trout (i) steady-state distribution and exchange kinetics between RBC and plasma differ greatly for T4, T3, and rT3 and vary with the in vitro conditions, (ii) almost all TH in RBC are reversibly bound to intracellular sites, (iii) efflux is strongly influenced by plasma binding sites, (iv) T3 exchange is rapid and may allow T3 access to RBC TH receptors, buffer plasma T3 levels, or aid T3 delivery to tissues, (v) T4 exchange is slow and this may prevent oxygenation state from altering T4 uptake into RBC, and (vi) rT3 uptake into RBC may contribute to low rT3 levels in trout plasma.

1. The nystatin-perforated whole-cell recording mode of the patch-clamp technique was used to investigate the membrane conductance of trout (Oncorhynchus mykiss) red blood cells in the steady state, 5 min after exposure to hyposmotic medium and 10 min after return to normal isosmotic medium. 2. Whole-cell I-V relations showed outward rectification when red blood cells were bathed in isosmotic (320 mosmol l-1) saline solution and the patch pipette was filled with 117 mM KCl. The membrane conductance was 2.58 +/- 0.59 nS (number of experiments, n = 18) between 0 and 100 mV and 1.32 +/- 0.19 nS (n = 18) between 0 and -100 mV. Removal of Cl- from the extracellular side or incubation with the Cl- channel blocker DIDS caused a reduction in whole-cell membrane conductance by more than 50%, indicating that the membrane current was generated by Cl- ions. The remaining conductance was voltage independent and probably due to non-selective cation conductance. 3. The membrane conductance increased approximately 2-fold after cell swelling induced by exposure to hyposmotic saline solution (215 mosmol l-1). This effect was abolished in Cl(-)-free hyposmotic medium or in the presence of DIDS. 4. The return to isosmotic solution produced a fall in membrane conductance to, or below, control values. 5. We conclude that trout red blood cells possess a significant Cl- conductance in the steady state which is reversibly activated during cell swelling and contributes to volume recovery.

The volume sensitivity of different K flux pathways has been determined in trout red blood cells subjected to volume perturbation. Gentle hyposmotic swelling induced a K influx in a Cl-containing saline but not in NO3- or methanesulfonate (MeSF)-containing salines, consistent with the activation of a Cl-dependent flux. Extreme hyposmotic swelling led to larger K fluxes in all salines but with reduced anion discrimination of the Cl-dependent flux. In contrast to these graded responses, isosmotic swelling using ammonium chloride or beta-adrenergic stimulation activated only Cl-dependent fluxes in an all-or-none fashion. The relationship between the hyposmotically and isosmotically induced pathways was studied by coactivation using either ammonium chloride or isoproterenol with anisosmotic treatment. Cells in ammonium chloride-containing hyposmotic salines showed no additive K flux over that induced by hyposmotic treatment alone, indicating that the isosmotically induced Cl-dependent flux was identical to the hyposmotically induced Cl-dependent flux. However, cells coactivated by hyposmotic and beta-adrenergic treatment showed a small Cl-dependent flux in addition to that induced by hyposmotic treatment alone. This small third component was unaffected by anisosmotic treatment. We conclude that the major Cl-dependent and Cl-independent K flux pathways are distinct and separate and that the former has an anion dependence that varies with cell volume and a volume sensitivity that varies with ionic strength.

The mechanisms of uptake of thyroid hormones (TH) 3,5,3'-triiodo-L-thyronine (T3) and L-thyroxine (T4) by trout red blood cells (RBC) were characterized by coincubating washed RBC with [125I]T3 or [125I]T4 and a variety of inhibitors and potentially competing amino acids and TH analogues. Nonsaturable uptake of both TH was unaffected by almost all agents tested, consistent with a diffusion process. Saturable T3 uptake was unaffected by a major (80%) cyanide- and iodoacetate-induced reduction in RBC nucleotide triphosphate content or by inhibitors of Na+ transport, but was depressed by (i) inhibitors of protein binding (bromosulfophthalein, 8-anilino-1-naphthalene sulfonic acid, phloretin, and 5,5'-diphenylhydantoin, (ii) sulfhydryl blockers (p-hydroxy-mercuribenzoate and N-ethylmaleimide), (iii) endocytotic inhibitors (chloroquine, colchicine, and monodansylcadaverine), (iv) tryptophan and phenylalanine, and (v) certain TH analogues, particularly 3,3',5'-triiodo-L-thyronine (reverse T3). In contrast, saturable T4 uptake was depressed only by inhibitors or protein binding. Cross-competition between T3 and T4 for transport occurred only at highly pharmacologic TH levels. We conclude that T3 and T4 are transported into trout RBC by two separate systems. The relatively slow T4 uptake depends on binding to certain proteins. The much more rapid T3 uptake depends on protein binding, but is Na(+)-independent, and involves a system comparable to the T-system of aromatic amino acid transport. However, participation od receptor-mediated endocytosis cannot be excluded.

Inhibition of red cell carbonic anhydrase (CA) activity resulted in the rapid development of a respiratory acidosis (0.25 pH depression within 15 min post-injection) in the blood of trout. In the lamprey, however, the onset of the respiratory acidosis was delayed and its magnitude was less (0.18 pH depression at 6 h post-injection). Erythrocyte pH of both species decreased by about 0.12 units by 1 h after CA inhibition. These data, combined with the lack of rapid anion (Cl-/HCO3-) exchange in the red cells of agnathans but not in other lower vertebrates, support the hypotheses that (1) the majority of total CO2 in lamprey is transported within the erythrocyte, and (2) the limiting step in the evolution of a functioning Jacobs-Stewart cycle, and thus the evolution of the common mechanism of systemic CO2 transport in vertebrate blood, was the incorporation of the band-3 anion exchange protein into the membrane of the red cell.