Trout Red Blood Cells Research Articles

Piscine birnavirus triggers antiviral immune response in trout red blood cells, despite not being infective.

Background: Some fish viruses, such as piscine orthoreovirus and infectious salmon anemia virus, target red blood cells (RBCs), highly replicate inside them and induce an immune response. However, the implications of RBCs in the context of birnavirus infection (i.e, infectious pancreatic necrosis virus (IPNV)) have not yet been studied. Methods:Ex vivo trout RBCs were obtained from peripheral blood, ficoll purified and exposed to IPNV in order to analyze infectivity and induced immune response using RT-qPCR, immune fluorescence imaging, flow cytometry and western-blotting techniques. Results: IPNV could not infect RBCs; however, IPNV-exposed RBCs increased the expression of the INF1-related genes ifn-1, pkr and mx genes. Moreover, conditioned media from IPNV-exposed RBCs conferred protection against IPNV infection in CHSE-214 fish cell line. Conclusions: Trout RBCs could trigger an antiviral immune response against IPNV infection despite not being infected. Fish RBCs could be considered mediators of the antiviral response and therefore targets of novel DNA vaccines and new strategies against fish viral infections. Further research is ongoing to completely understand the molecular mechanism that triggers this immune response in trout RBCs.

Identification of diverse defense mechanisms in trout red blood cells in response to VHSV halted viral replication

Background:It has been described that fish nucleated red blood cells (RBCs) generate a wide variety of immune-related gene transcripts when viruses highly replicate inside them and are their main target cell. The immune response and mechanisms of fish RBCs against viruses targeting other cells or tissues has not yet been explored and is the objective of our study.Methods:Trout RBCs were obtained from peripheral blood, ficoll purified and exposed toViral Haemorrhagic Septicaemia virus(VHSV). Immune response was evaluated by means of RT-qPCR, flow cytometry, immunofluorescence and isobaric tag for relative and absolute quantification (iTRAQ) protein profilingResults:VHSV N gene transcripts incremented early postexposure and were drastically decreased after 6 hours postexposure (hpe). The expression of the type I interferon (ifn1) gene was significantly downregulated at early postexposure (3 hpe), together with a gradual downregulation of interferon-induciblemxandpkrgenes until 72 hpe. Type I IFN protein was downregulated and interferon-inducible Mx protein was maintained at basal levels. Co-culture assays of RBCs with TSS (stromal cell line from spleen) revealed the IFN crosstalk between both cell types. On the other hand, anti-microbial peptide β-defensin 1 and neutrophil chemotactic factor interleukin 8 were slightly upregulated in VHSV-exposed RBCs Isobaric tag for relative and absolute quantification (iTRAQ) revealed that VHSV exposure can induce a global protein downregulation in trout RBCs, mainly related to RNA stability and proteasome pathways. The antioxidant/antiviral response is also suggested to be involved in the response of trout RBCs to VHSV.Conclusions:A variety of mechanisms are proposed to be implicated in the antiviral response of trout RBCs against VHSV halted infection. Ongoing research is focused on understanding the mechanisms in detail. To our knowledge, this is the first report that implicates fish RBCs in the antiviral response against viruses not targeting RBCs.

Expression of Hemoglobin Related Proteins in Nucleated and Enucleated Red Blood Cells

In vertebrates a total of eight different globins are known which are expressed in a highly species‐ and tissue‐specific manner. Globins are small molecules with a heme prosthetic group, by which they reversibly bind O2 and other gaseous ligands. They are all involved in cellular processes related to O2 transport (e.g. hemoglobin) and storage (e.g. myoglobin), detoxification of reactive oxygen and nitrogen species, sensing and signaling or may have yet unknown functions. The functions of hemoglobin and myoglobin are well understood and known since several years, but data on the other globins are fragmentary. In particular, the factors controlling globin mRNA stability, gene transcription and translation of these globins as well as their physiological functions are currently unknown. Here we examined the expression of globin genes in nucleated and enucleated erythrocytes of different vertebrate model species by quantitative real‐time reverse transcription PCR (qRT‐PCR). Furthermore we studied the transcription rate and mRNA levels of globin genes and other genes (e.g. hypoxia inducible factor 1α, sodium/proton exchanger) under hypoxia, temperature stress and adrenergic stimulation by the nuclear run‐off assay in rainbow trout red blood cells. We surprisingly detected transcripts of the whole gnathostome globin superfamily in mature circulating red blood cells of all vertebrate species but the mRNA expression levels varied among species. The transcription rate of the genes depended on the treatment. Our results add new and unexpected aspects to the current knowledge on the expression specificity of globins in vertebrate tissues and organs.

Linking physiological and cellular responses to thermal stress: β-adrenergic blockade reduces the heat shock response in fish.

When faced with stress, animals use physiological and cellular strategies to preserve homeostasis. We were interested in how these high-level stress responses are integrated at the level of the whole animal. Here, we investigated the capacity of the physiological stress response, and specifically the β-adrenergic response, to affect the induction of the cellular heat shock proteins, HSPs, following a thermal stress in vivo. We predicted that blocking β-adrenergic stimulation during an acute heat stress in the whole animal would result in reduced levels of HSPs in red blood cells (RBCs) of rainbow trout compared to animals where adrenergic signaling remained intact. We first determined that a 1 h heat shock at 25 °C in trout acclimated to 13 °C resulted in RBC adrenergic stimulation as determined by a significant increase in cell swelling, a hallmark of the β-adrenergic response. A whole animal injection with the β2-adrenergic antagonist, ICI-118,551, successfully reduced this heat-induced RBC swelling. The acute heat shock caused a significant induction of HSP70 in RBCs of 13 °C-acclimated trout as well as a significant increase in plasma catecholamines. When heat-shocked fish were treated with ICI-118,551, we observed a significant attenuation of the HSP70 response. We conclude that circulating catecholamines influence the cellular heat shock response in rainbow trout RBCs, demonstrating physiological/hormonal control of the cellular stress response.

Comparison of clinical staging of benign and malignant ovarian tissues with DNA flow cytometry

The prevention, diagnosis and treatment of ovarian cancer are major issues. The outcome of patients with advanced ovarian cancer is poor despite aggressive therapy including surgery, combination drug chemotherapy and radiation treatments. From the literature, the direct correlation between DNA ploidy and survival is greatly enhanced using high resolution DNA measurements, which results in a coefficient of variation (CV) range between 1%-2% (1.42 ± 0.19, n=66) for trout red blood cells (TRBC) and 2%-3% (2.18 ± 0.46 SD, n=22) for tonsil nuclei derived from formalin-fixed, paraffin-embedded tissues (deparaffinated). DNA nuclear determinations from 50 ovarian cancer and 21 benign patients is presented. This was accomplished by measuring the DNA content of nuclei simultaneously isolated from deparaffinated tissues, stained with the fluorescent DNA specific dye, 4’, 6-diamidino-1-phenylindole (DAPI) and analyzed on a high resolution flow cytometer. A high percentage of aneuploidy (92.0%) was determined from the ovarian cancer patients, especially of the aneuploid DNA histogram types, such as hypodiploid, multiploid and hypertetraploid (64.0%), which have shown poor prognosis in a variety of cancers including ovarian. Furthermore, aneuploidy was detected in 23.8% of the benign patients. DNA flow cytometry may complement pathological assessment of ovarian cancer to better determine malignancy, thus justifying a closer follow-up with more specialized approaches to treatment in clinical trials that involve new therapies including the use of chemically defined, natural products, which may help offset the overall poor prognosis seen in ovarian cancer.

Changes in the Gut Microbiome of the Sea Lamprey during Metamorphosis

Vertebrate metamorphosis is often marked by dramatic morphological and physiological changes of the alimentary tract, along with major shifts in diet following development from larva to adult. Little is known about how these developmental changes impact the gut microbiome of the host organism. The metamorphosis of the sea lamprey (Petromyzon marinus) from a sedentary filter-feeding larva to a free-swimming sanguivorous parasite is characterized by major physiological and morphological changes to all organ systems. The transformation of the alimentary canal includes closure of the larval esophagus and the physical isolation of the pharynx from the remainder of the gut, which results in a nonfeeding period that can last up to 8 months. To determine how the gut microbiome is affected by metamorphosis, the microbial communities of feeding and nonfeeding larval and parasitic sea lamprey were surveyed using both culture-dependent and -independent methods. Our results show that the gut of the filter-feeding larva contains a greater diversity of bacteria than that of the blood-feeding parasite, with the parasite gut being dominated by Aeromonas and, to a lesser extent, Citrobacter and Shewanella. Phylogenetic analysis of the culturable Aeromonas from both the larval and parasitic gut revealed that at least five distinct species were represented. Phenotypic characterization of these isolates revealed that over half were capable of sheep red blood cell hemolysis, but all were capable of trout red blood cell hemolysis. This suggests that the enrichment of Aeromonas that accompanies metamorphosis is likely related to the sanguivorous lifestyle of the parasitic sea lamprey.

Fusion of images in multistep analysis of neovascularisation

Purpose: The aim of our study was to develop a method for the fusion of images received after repeated staining of the same sample taking into account spatial differences between the images.Material and methods: A method of objective fusion performance was investigated on the images receiving during multistep staining of the xenograft tumour cross-sections.Results: It was shown that several images receiving from different steps of staining procedures may be successfully fused by fluorescent marking of slide position with Trout red blood cells before analysis.Conclusions: Proposed technique provides an accurate rigid fusion of light and fluorescent images receiving during multistep image analysis under microscope and may be applied for study of neovascularisation.

β‐Adrenergic Stimulation Enhances the Heat‐Shock Response in Fish

We have taken advantage of the unique properties of nucleated rainbow trout (Oncorhynchus mykiss) red blood cells (rbcs) to demonstrate that beta-adrenergic stimulation with the agonist, isoproterenol, significantly enhanced the heat-induced induction of heat-shock proteins (Hsps) in trout rbcs without affecting hsp expression on its own. Furthermore, this beta-adrenergic potentiation of hsp expression occurred only at physiologically relevant concentrations of adrenergic stimulation. In further experiments, we found that adrenaline increased 100-fold and noradrenaline increased 50-fold in trout after a 1-h heat shock at 25 degrees C, approximately 12 degrees C above acclimation temperature. This is the first time the adrenergic heat-shock response has been described for a temperate fish species. We conclude that beta-adrenergic stimulation enhances hsp expression in trout rbcs following heat stress, indicating physiological regulation of the cellular stress response in fish.

Oxygen delivery to the fish eye: Root effect as crucial factor for elevated retinalPO2

Although the retina has one of the highest metabolic rates among tissues, certain teleost fishes lack any vascular supply to this organ which, in combination with the overall thickness of the organ, results in extremely long diffusion distances. As the only way to compensate for these obstacles, oxygen partial pressure (PO2) in the eyes of such fish is elevated far above atmospheric values. Although not supported by any direct evidence, the enhancement of PO2 is considered to be related to the Root effect, the release upon acidification of Hb-bound O2 into physical dissolution, possibly supported by counter-current multiplication similar to the loop of Henle. The present study evaluates the magnitude of intraocular PO2 enhancement under tightly controlled physiological conditions, to directly confirm the involvement of the Root effect on intraocular PO2 in the retina of rainbow trout Oncorhynchus mykiss. Intraocular PO2 was determined with special polarographic microelectrodes inserted into the eye. PO2 profiles established in vivo by driving electrodes through the entire retina yielded average PO2 values between 10 mmHg (1.3 kPa) at the inner retinal surface and 382 mmHg (50.9 kPa) close to the outer retinal limit (Bruch's membrane). According to estimates on the basis of the diffusion distances determined from sections of the retina (approximately 436 microm at the site of PO2 measurement) and literature data on specific oxygen consumption, the in vivo determined values would be sufficient to cover the oxygen demand of the retina with some safety margin. For a clear and direct in-tissue-test as to the involvement of the Root effect, an isolated in vitro eye preparation was established in order to avoid the problem of indirect blood supply to the eye from the dorsal aorta only via the pseudobranch, a hemibranch thought to modulate blood composition before entry of the eye. Any humoral effects (e.g. catecholamines) were eliminated by perfusing isolated eyes successively with standardized red blood cell (RBC) suspensions in Ringer, using trout (with Root) and human (lacking any Root effect) RBC suspension. To optimize perfusate conditions for maximal Root effect, the Root effect of trout RBCs was determined in vitro via graded acidification of individual samples equilibrated with standardized gas mixtures. During perfusion with trout RBC, PO2 at the outer retinal limit was 99 mmHg (13.2 kPa), but fell by a factor of 3.3 upon perfusion with human RBC in spite of higher total oxygen content (TO2 2.8 for trout vs 3.9 mmol l-1 for human RBC). Upon reperfusion with trout RBC, PO2 was restored immediately to the original value. This regularly observed pattern indicated a highly significant difference (P=0.003) between perfusion with trout (with Root effect; high retinal PO2) and perfusion with human (no Root effect; low retinal PO2) RBC suspension, thus clearly demonstrating that the Root effect is directly involved and a crucial prerequisite for the enhancement of PO2 in the retina of the teleost eye.

DNA index, genome size, and electronic nuclear volume of vertebrates from the Miami Metro Zoo

Flow cytometry is a rapid and reliable method for measuring nuclear DNA content and genome size. Fluorochrome binding characteristics, sample preparation and differences in DNA condensation, and availability of binding sites can cause variations in results obtained. Blood samples from 82 vertebrate species were collected in 10% dimethyl sulfoxide and stained with propidium iodide/hypotonic citrate or 4,6-diamidino-2-phenylindole dihydrochloride for analysis of DNA content and electronic nuclear volume (ENV). Trout red blood cells (TRBCs), human peripheral blood lymphocytes, and human buccal cavity cells were used as internal standards. Mean fluorescence channel (MFC) values of TRBC and buccal cavity cells used as internal standards were stable at 15 to 120 min of propidium iodide staining. TRBCs mixed with other cells especially human peripheral blood cells showed an increase in MFC. ENV and MCF values were less variable in different species of birds than in reptiles or mammals. Genome size based on use of buccal cavity cells as the internal standard showed a high degree of correlation with previous reports. Proper selection and use of internal standards and sample preparation are essential for reliable determination of DNA content and genome size in vertebrates by flow cytometry.

Analysis of Stress-Induced Gene Expression in Trout Red Blood cells following Tributyltinchloride Exposure

Submicromolar concentrations of tributyltinchloride (TBTC), an organotin compound, is able to impair mitochondrial activity in trout erythrocytes, as detected by ultrastructural transmission electron microscopy investigations and exhibits pro apoptotic activity in a variety of cells including nucleated red blood cells. Here we show that TBTC is able to induce the expression of hsp70 in rainbow trout (Onchorynchus mykiss) erythrocytes in a time- and dose-dependent manner. This induction was demonstrated by quantification of the relative abundance of mRNA, and the synthesis of hsp70 was detected by means of an immunological test. This study could provide a practical assay of environmental stress at sublethal toxicant concentrations.

Activity of the unique β-adrenergic Na+/H+exchanger in trout erythrocytes is controlled by a novel β3-AR subtype

beta-Adrenoceptors (beta-ARs) are seven-transmembrane domain, G protein-coupled receptors that transduce the cellular effects of epinephrine and norepinephrine and play a pivotal role in the vertebrate stress response. This study reports the cloning and characterization of two previously unreported beta-ARs from the rainbow trout (Oncorhynchus mykiss). Phylogenetic analysis of amino acid sequences indicates that both beta-ARs are homologs of the mammalian beta3-AR. Analysis of tissue expression patterns indicates that one of these trout beta3-adrenoceptors (beta3a-AR) is highly expressed in gill and heart, whereas the second (beta3b-AR) is highly expressed by red blood cells (RBC). Expression of the beta3b-AR in the RBC coupled with the finding of a single category of beta-AR binding sites on RBC membranes provides strong evidence for the control of the trout RBC beta-AR Na+/H+ exchanger (beta-NHE) activity by signaling through this beta3b-subtype and not through a beta1-subtype as previously proposed. The RBC-specific trout beta3b-AR exhibits binding characteristics that distinguish this receptor from each of the three pharmacologically defined categories of mammalian beta-ARs (beta1-, beta2-, and beta3-AR). This study is the first to report the presence of a beta3-AR subtype in a fish species, and the proposal that the beta3b-AR controls RBC beta-NHE activity represents a novel role for the beta3-AR subtype in vertebrates.

Effect of tributyltin on trout blood cells: changes in mitochondrial morphology and functionality

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 μM 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 μM. 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 physiological effects of heat stress and the role of heat shock proteins in rainbow trout (Oncorhynchus mykiss) red blood cells

The effects of in vitro thermal stress and the potential role ofheat shock proteins in thermoprotection were examined in the nucleated red blood cells (rbcs) of rainbow trout (Oncorhynchusmykiss). Oxygen consumption, a general indicator of cellular metabolism, was maintained in trout rbcs until 30 °C, but was markedly reduce by 35 °C. Subsequent experiments were then conducted which involved exposing rbcs to 30 °C to determine which physiological variables might be compromised by an extendedheat stress. Although this temperature challenge caused an induction of Hsp 70 mRNA, and a significant reduction in the amount of oxygen bound to hemoglobin, carbonic anhydrase (CA) activity was unaffected by an 8 h, 30 °C exposure.heat stress also caused a rise in methemoglobin formation, but the increase in rbc methemoglobin concentration did not appear to be a stimulus for Hsp 70 induction. Rbc oxygen consumption, CA activity and hemoglobin/oxygen binding ability were unaltered when hsp synthesis was inhibited by the translational inhibitor, cycloheximide. These results indicate that hsps do not have a role in protecting rbc metabolism or the function of important rbc proteins, such as hemoglobin and CA. Extended 30 °Cheat stress did cause a significant leakage of potassium from the rbcs, which appeared to be intensified in the presence of cycloheximide. These latter results suggest that hsps may play a limited role in preserving rbc membrane integrity and/or ionoregulatory processes.

Influence of hyperosmotic shrinkage and β-adrenergic stimulation on red blood cell volume regulation and oxygen binding properties in rainbow trout and carp

Whole blood from rainbow trout and carp was subjected to hyperosmotic shock and subsequent beta-adrenergic stimulation (isoprenaline) at different oxygen tension ( PO(2)) and carbon dioxide tension ( PCO(2)) levels with the aim to evaluate changes in red blood cell (RBC) volume, pH and ion concentrations and their ultimate effect on blood O(2) transport characteristics. Hyperosmolality (addition of NaCl) induced RBC shrinkage, which was followed by a regulatory volume increase (RVI) that was larger at low than at high PO(2)and more complete in carp than in trout. Carp RBC showed practically full volume recovery within 140 min at low PO(2)and partial recovery at high PO(2), whereas RVI was partial under all PO(2)and PCO(2)conditions in trout. The RVI increased intracellular [Na(+)], water content, and, in carp, also pH (pHi), suggesting activation of Na(+)/H(+) exchange. In trout RBCs, activation of RVI was rapid but succeeded by deactivation. In carp RBCs, activation of Na(+) influx was slower but it continued, allowing full volume recovery. Shrinkage of the RBCs was associated with only minor decreases in blood oxygen saturation and oxygen affinity in both species. Thus, the oxygen affinity decrease expected on the basis of the increased concentration of intracellular haemoglobin and organic phosphates was small, and it appeared to some extent countered during RVI by an oxygen affinity increase via increased pHi. Addition of isoprenaline increased RBC volume and pHi and increased Hb oxygen saturation. The beta-adrenergic response was stronger at low compared to high PO(2) and at high compared to low PCO(2). The PO(2) dependency was largest in carp, whereas the PCO(2) (pH) dependency was more expressed in trout. The adrenergic response of trout RBCs was similar under isoosmotic and hyperosmotic conditions. In carp RBCs, the response was absent at high PO(2) under isoosmotic conditions, but interestingly it could be induced under hyperosmotic conditions. The data suggest that the RBC shrinkage occurring in fish moving from freshwater to seawater has minimal impact on blood O(2) binding properties.

In vitro Regulation of β-adrenoceptor Signaling in the Rainbow Trout, Oncorhynchus mykiss

We review recent advances in the characterization of the β-adrenoceptor (AR) system of the trout and present new data on the mechanisms of β-AR desensitization. Phosphorylation of specific amino acids located in the third intracellular loop and cytoplasmic tail regions triggers β-AR desensitization. Analysis of trout β-AR sequences reveals a difference in putative phosphorylation profiles between β-AR subtypes, this suggests that each subtype (β2-, β3a- and β3b-AR) possesses different sensitivity to desensitization. β-Adrenoceptor kinase (βARK) and β-arrestin play a pivotal role in β-AR desensitization. The tissue distribution of β-arrestin and βARK mRNA in trout is reported for the first time and interestingly, both genes appear to be highly expressed in the trout red blood cell. Previous studies reported loss of β-AR binding sites in trout red blood cells following prolonged receptor activation. Reduction of β-AR mRNA levels is reported to contribute to down-regulation of β-ARs in some species. However, we did not detect a significant change in β-AR mRNA levels in trout subjected to environmental hypoxia, clenbuterol (a β-agonist) administration, or cortisol administration; therefore, changes in mRNA levels may not contribute to β-AR desensitization in trout.

Inhibition of hemolytic activity of Aeromonas salmonicida GCAT in rainbow trout red blood cells by a monoclonal antibody.

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.

Adrenergic receptors, Na+/H+ exchange and volume regulation in lungfish erythrocytes.

Aestivation in African and South American lungfish (Protopterus and Lepidosiren, respectively) is associated with elevations of extracellular osmolarity. Osmotic shrinkage of Protopterus red blood cells (RBCs) caused a small but significant stimulation of the Na influx that was amiloride-sensitive. suggesting involvement of the Na+/H+ exchanger (NHE). The associated in vitro regulatory volume increase was insignificant within a time frame of 120 min, but the shrinkage-activated Na+ influx may be sufficient for slow regulatory volume increase during aestivation in vivo. Osmotic swelling of the RBCs induced an incomplete regulatory volume decrease that was statistically significant after 180 min. The RBCs of Protopterus were very large (mean cellular volume of 6939 +/- 294 microm3) and possessed 23,066 +/- 7,326 beta-adrenoceptors cell(-1) with a Kd value of 6.1 +/- 3.2 nM. The number of receptors per unit surface area of lungfish RBCs was calculated to be twice that of trout RBCs and 70% that of cod RBCs. There was, however, no adrenergic stimulation of the NHE in either Protopterus or Lepidosiren. Acidification of the extracellular medium also failed to activate the NHE.

Effects of sulfide on K + flux pathways in red blood cells of crucian carp and rainbow trout

The effect of sulfide on K + influx pathways was measured in red blood cells (RBCs) of sulfide-sensitive rainbow trout (Oncorhynchus mykiss) and sulfide-tolerant crucian carp (Carassius carassius). In trout RBCs, maximal inhibition of Na + ,K + -ATPase was attained at 10 µmol l −1 sulfide and amounted to 32% without being influenced by pH between 6.7 and 8.3. Ouabain-resistant K + influx in the absence and presence of sulfide was insignificant at pH values between 6.7 and 7.7. At higher pH values ouabain-resistant K + influx increased, but was inhibited to about 15% by 30 µmol l −1 sulfide. In RBCs of crucian carp neither Na + ,K + -ATPase nor ouabain-resistant K + influx were affected by sulfide concentrations up to 850 µmol l −1 . Differences in sulfide-sensitivity of K + influx between both species can be based upon different properties of the membrane transporter themselves. The reduced Na + ,K + -ATPase activity in trout RBCs may also result from a slightly reduced (by 9%) ATP level after sulfide exposure. In addition, intracellular sulfide concentrations were higher in trout RBCs as compared to crucian carp. In trout, intracellular sulfide concentrations reached extracellular levels within 5 min of incubation whereas sulfide concentrations in crucian carp RBCs remained about 2-fold lower than extracellular concentrations. Although the physiological basis of sulfide-insensitive K + influx in crucian carp RBCs is currently unknown it may contribute to the extremely high sulfide-tolerance of this species. Abbreviations: DIOA – R(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid; DMSO – dimethylsulfoxide; HEPES – N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; HCT – haematocrit; PCA – perchloric acid; RBC – red blood cells.

The role of anion and cation channels in volume regulatory responses in trout red blood cells

(1) An outwardly rectifying chloride channel (ORCC) of large conductance has been detected under isotonic conditions (320 mosM l −1) in the plasma membrane of trout red blood cells (RBCs) using the excised inside-out configuration. The channel, with a permeability ratio P Cl/ P cation of 12, was inhibited by the Cl − channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) (50 μM), and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) (100 μM) in the bathing solution. (2) In hypotonic conditions (215 mosM l −1), 44% of cell-attached patches showed spontaneous single channel activity identified as nonselective cationic (NSC) channels. A second group, corresponding to 7% of cell-attached patches, showed spontaneous activity corresponding to a channel type presenting outward rectification and anionic selectivity. Finally, 49% of patches displayed a complex spontaneous signal corresponding to the superimposition of inward and outward currents probably due to activation of different channel types. (3) Giga-seals obtained without suction in intact cells under isotonic conditions possessed NSC channels that were quiescent but which could be activated either by mechanical deformation of cell membrane or by hypotonic cell swelling. (4) Hypotonically swollen RBCs exhibited regulatory volume decrease (RVD) over 3 h, which was linked to a fivefold to sixfold increase in unidirectional fluxes of K +, a net loss of intracellular K + and net gain of extracellular Na +. RVD and the hypotonically activated, unidirectional K + influx continued after replacement of Cl − by methylsulfonate (MeSF) albeit more slowly. (5) The NSC channel inhibitor, barium, and the Cl − channel inhibitor, NPPB, both inhibited the RVD response by ∼50% in Cl − containing saline. When Cl − was replaced by MeSF, the inhibition was >90% suggesting that NSC channels and ORCC play key roles in the chloride-independent component of RVD.