Carp Erythrocytes Research Articles

Zinc-induced damage to carp (Cyprinus carpio L.) erythrocytes in vitro.

Fish erythrocytes were used to elucidate the effect of zinc ions on the cell antioxidant defence system. It was detected that an increase of the Zn2+ concentration (0.01-1 mM) leads to a marked decrease (p < 0.05) in the catalase and the glutathione peroxidase activities. We observed a loss of 14-39% activity of glutathione peroxidase, and 16-20% diminution for catalase. No significant changes were found in case of the superoxide dismutase. Incubation of red blood cells with zinc brought about a decrease of the erythrocyte thiol group content. Treatment of carp erythrocytes with zinc ions also resulted in enhanced hemolysis and in the induction of significant (p < 0.001) changes in the intracellular glucose level. The increase of glucose concentration in the erythrocytes was correlated with increased concentration of metal in the incubation medium. It was proposed that Zn could affect transport systems across the red blood cells and therefore increased the permeability of the membranes to small molecules (e.g. hexose), and led to hemolysis. Zinc ions could act as a potential cell toxicant, leading to disturbances in functions of the antioxidant defence system and to alterations in the erythrocyte membrane properties.

Sublethal Effects of Phenol on the Phospholipid Fatty Acid Composition of Carp Erythrocyte Plasma Membrane

The effect of different concentrations (5, 10, and 20 ppm) of phenol on the carp erythrocyte plasma membrane was examined following exposures of 48 and 96 h. The results indicated that the high concentrations of phenol pollutant led to an increase in phosphatidylcholine (PC) and eliminated phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidic acid (PA). The data also revealed that arachidonic acid (20:4n−6) was present in the greatest amounts; its quantity in both low and high doses increased throughout the experimental period. Then−3 fatty acids (eicosapentaenoic, 20:5n−3, and docosahexaenoic acids, 22:6n−3) displayed a fairly varied picture after exposure to phenol pollutant. Long-term exposure to higher phenol concentrations led to elimination of these acids and significantly decreasedn−3/n−6 ratios.

Isolation of cDNA encoding the constant region of the immuoglobulin heavy-chain from common carp (Cyprinus carpioL.)

Complementary DNA fragments encoding the constant region (CH) of immunoglobulin heavy chain were isolated from spleen of carp (Cyprinus carpioL.) by reverse transcription-polymerase chain reaction (RT-PCR) using degenerated primers corresponding to the conserved amino acid sequences in theJ-segment of the variable region, and in CH4. Sequences encoding CH4 and transmembrane domains were also obtained by a rapid amplification of cDNA end method (3′-RACE). Primary structure of the entire carp CH domain of the secreted form contained well conserved cysteine and tryptophan residues and showed 30% to 42% identity with that of other teleost species. Sequence analysis of membrane-bound CH domain revealed that the carp transmembrane domain is connected to the end of CH3, splicing out the entire CH4 exon, as in other teleosts. Interestingly, three distinct partial CH sequences which share 72–95% identity at amino acid level were obtained from a single carp. This strongly indicates the presence of duplicated CH genes, which are more divergent than those in Atlantic salmon, which carries two isotypic Ig H genes. Southern hybridisation using carp erythrocyte DNA also revealed two distinct CH genes indicating that each of the duplicated CH genes is disomicly inherited. It seems likely that the duplication in carp occurred as whole Ig H genes, in association with ancient tetraploidisation, rather than by tandem duplication, as reported for another tetraploidised teleost, Atlantic salmon.

Blood gas parameters and the responses of erythrocytes in carp exposed to deep hypoxia and subsequent recovery

The quantitative importance of the adrenergic response of carp erythrocytes during severe oxygen restriction is not clear at present. Quantitative differences between in vivo and in vitro studies suggest that the response of carp erythrocytes may be dependent on the actual hypoxic condition. To our knowledge, a clear picture of the blood gas status, erythrocytic responses and catecholamines measured simultaneously in carp exposed to deep severe hypoxia or anoxia has not yet been reported. Therefore, we studied the physiological response of carp exposed to deep hypoxia at 0.3 kPa and subsequent recovery. Carp were fitted with an indwelling cannula in the dorsal aorta for repeated blood sampling and the blood was analysed for hematocrit, hemoglobin, mean cellular hemoglobin content, intra- and extracellular pH,pO2,PCO2, total CO2 and catecholamines. Large fluctuations in arterialpO2 levels were observed in normoxic control carp, probably caused by the alternating breathing pattern of carp. Even at waterpO2 levels of 0.3 kPa, arterialpO2 levels were maintained at about 0.2–0.3 kPa. Catecholamine levels were increased during deep hypoxia with noradrenaline as the predominant catecholamine. Hematological variables showed that the number of circulating erythrocytes was increased during hypoxia. The intracellular pH of carp red cells was maintained at pre-exposure values despite a considerable decrease of pHe. In this in vivo study, a marked decrease of the proton gradient across the red cell membrane (pHe-pHi), as high as 0.35 pH units, was observed, which is quantitatively similar to that usually observed in salmonids during hypoxia. It is suggested that the regulation of the carp erythrocytic pHi is probably caused to a major extent by deoxygenation of hemoglobin (Haldane effect) while adrenergic activation of the red cells is likely to contribute significantly to the observed reduction of the proton gradient. These mechanisms result in the persistence of a capacity for aerobic metabolism in carp of about 10–20% of the energy metabolism despite environmentalpO2 values of 2–3 mm Hg.

Glucose transport in carp erythrocytes: individual variation and effects of osmotic swelling, extracellular pH and catecholamines

The characteristics of the uptake of 3-O-methyl-d-glucose (3-OMG) by carp (Cyprinus carpio) erythrocytes were studied in vitro with tracer methods. There is large individual variation in the permeability of the carp erythrocyte membrane to 3-OMG. Although transport is inhibited by cytochalasin B and phloretin, the lack of saturation kinetics for transport in a physiologically relevant concentration range suggests either that a glucose transporter does not exist or that its affinity for glucose is extremely low. The marked increase in transport after osmotic swelling and the inhibition of swelling-induced glucose transport by cytochalasin B suggest that the glucose transport pathway in carp erythrocytes (both in isotonic and hypotonic conditions) may be similar to the volume-activated channel described for flounder erythrocytes. 3-OMG transport across the carp erythrocyte membrane is increased by catecholamines by a mechanism independent of the catecholamine-induced cell swelling.

Volume- and catecholamine-dependent regulation of Na/H antiporter and unidirectional potassium fluxes in Salmo trutta red blood cells

β-Adrenergic- and volume-dependent regulation of 22Na influx and 86Rb influx and efflux in erythrocytes of brown trout (Salmo trutta m. lacustris) were studied. Norepinephrine (10-6 mol·1-1) increased the rate of 22Na influx 10-to 20-fold via the activation of a Na/H exchanger (ethyl isopropyl amiloride inhibited component of 22Na influx). Unlike carp erythrocytes the activity of the Na, K-pump (ouabain-inhibited 86Rb influx) was only slightly (25–35%) increased by norepinephrine. The norepinephrine-induced increment of Na, K-pump activity was completely abolished by ethyl isopropyl amiloride thus indicating that this effect was mediated by Na/H exchanger-induced increase of intracellular Na+ concentration. Cell shrinkage in hyperosmotic media resulted in a several-fold activation of the Na/H exchanger. Cell swelling in hypotonic media increased both the rate of K, Cl-cotransport [((dihydroindenyl)oxy)alcanaic acidsensitive components of 86Rb influxe and efflux] and passive permeability (leakage) of erythrocyte membranes for Na+ and K+. No volume-dependent regulation of Na, K, 2Cl-cotransport (bumetanide-sensitive components of 86Rb fluxes) was found. It may be concluded that the regulation of monovalent cation transport in erythrocytes of fast-moving (carnivorous) brown trout differs essentially from that in slowly moving (herbivorous) carp.

Micronuclei induced by selenium, mercury, methylmercury and their mixtures in binucleated blocked fish erythrocyte cells

The genotoxic effects of low concentrations of Se(IV), Hg 2+, and CH 3HgCl added separately or together (Se(IV)/Hg 2+ and Se(IV)/CH 3HgCl) were studied by determining the induction of micronuclei in the binucleated erythrocytes of Prussian carp. The frequencies of micronuclei were elevated in a dose-dependent manner in all treatments when compared to the relevant controls. Addition of Se(IV) reduced the frequencies of micronuclei in treatments with both forms of mercury. This novel approach to genotoxicity testing using binucleated cytokinesis-blocked erythrocytes in fish appears to be worth further study as a method for monitoring the genotoxicity of waterborne pollutants.

Catecholamine- and volume-dependent ion fluxes in carp (Cyprinus carpio) red blood cells

In carp erythrocytes, noradrenaline (10-6 mol·l-1) induces a 30- to 40-fold activation of Na+/H+ exchange (the ethylisopropylamiloride-inhibited component of the 22Na influx) and a fourfold stimulation of the Na+, K+ pump (ouabain-inhibited component of 86Rb influx). In both cases the effect of noradrenaline is blocked by propranolol but not phentolamine and is imitated by forskolin. An activator of protein kinase C (β-phorbol 12-myristate, 13-acetate) increases Na+/H+ exchange by 10 times and decreases the Na+, K+ pump activity by 20–30 percent. In the presence of ethylisopropylamiloride the increment of the Na+, K+ pump activity induced by noradrenaline is reduced by 35–45 percent, indicating the existence of a Na+/H+ exchange-independent mechanism of the Na+, K+ pump regulation by β-adrenergic catecholamines. Hypertonic shrinkage of carp erythrocytes results in a 40- to 80-fold activation of Na+/H+ exchange, whereas hypotonic swelling induces an increase in the rate of 86Rb+ efflux which is inhibited by furosemide by about 30–40 percent. The rate of pH0 recovery in response to acidification or alkalinization in rat erythrocytes is approximately 15 times as fast as in carp erythrocytes. Unlike in rat erythrocytes, valinomycin does not cause an alkalinization of incubation medium in carp erythrocytes indicating the absence of conductive pathway in the operation of anion transporter protein. A scheme is suggested which describes the interrelation of Na+/H+ exchange, Na+, K+ pump and a non-identified system providing for K+ efflux in cell swelling, regulation of cell volume and cytoplasmic pH in fish erythrocytes under conditions of deep hypoxia and high activity.

Temperature shifts induce adaptive changes in the physical state of carp (Cyprinus carpio L.) erythrocyte plasma membranes in vitro

Blood, freshly collected from warm- and cold-acclimated carp, Cyprinus carpio L., was cooled to 5°C for 4h or warmed to 25°C for 4h, respectively, and the fluorescence anisotropy of washed red blood cells was recorded using the fluorescent dye 3-(p-(6-phenyl-1,3,5-hexatrienyl) phenyl propionic acid [DPH-PA] (which is restricted to the outer leaflet of the plasma membrane) before and after the temperature shift. Despite individual variation, the plasma membrane of cold-exposed erythrocytes became more fluid while that of warm-exposed cells became more rigid following the temperature shift. This response was rapid and reversible. Cold-exposed cells from warm-acclimated fish became more fluid within 40-60 minutes and reverted to their original fluidity within the same time on warming, at a rate of 1°C/min; erythrocytes, from cold-adapted carp displayed an opposite change in fluidity over a similar time period. Cells from warm-acclimated, temperature down-shifted carp hyperfluidized their plasma membranes in the cold, whereas cells from cold-acclimated fish up-shifted in temperature showed no similar effect. These cells showed a complete adjustment of membrane physical state to the temperature. Total phospholipids obtained from warm-acclimated temperature down-shifted cells became more rigid than they were, when assayed at the acclimation temperature. In contrast, phospholipids obtained from cold-acclimated cells became more rigid when exposed to increasing temperatures. No significant changes occurred to the polar head groups, or to the fatty acid composition of the total phospholipids. It was concluded that the lipids play only a secondary role in the control of the physical state of plasma membrane in carp erythrocytes, and that some non-lipid components of these structures might be involved in these regulatory processes.

Substrate Utilization by Carp (Cyprinus Carpio) Erythrocytes

Substrate Utilization by Carp (<i>Cyprinus Carpio</i>) Erythrocytes

Adrenergic responses and the Root effect in erythrocytes of freshwater fish

The effects of adrenergic‐stimulation upon the oxygen‐binding capacity of fish erythrocytes have been investigated. The oxygen capacity of rainbow trout, Oncorhynchus mykiss (Walbaum), erythrocytes was lowered by 44% on extracellular acidification (the so‐called ‘Root effect’). Addition of isoproterenol at 20° Ccaused an acid shift of the curve relating oxygen capacity to pH0 by approximately 0.2 pH units, a value which was similar to the change in intracellular pH caused by adrenergic stimulation (Cossins &amp; Kilbey Journal of Experimental Biology, 148, 303–312, 1990). Moreover, when plotted as a function of pHi, the curves for control and adrenalinstimulated erythrocytes were superimposable suggesting that the adrenergic shift in the Root curve was a result of the change in pHi caused by activation of the adrenergic Na+/H+ exchanger.A similarly large adrenergic shift in the Root curve was observed for pike, Esox lucius L., erythrocytes, though not for erythrocytes of carp, Cyprinus carpio L., and tench, Tinea tinea (L.). The pH for the mid‐point of the Root effect in pike erythrocytes was distinctly more acid than for trout, but in both cases corresponded closely with the optimal pH for the adrenergic Na+/H+ exchange mechanism. This suggests a link between the functional characteristics of the exchanger and the oxygen‐binding properties of haemoglobin.

D-Glucose permeability in river lamprey ( lampetra fluviatilis) and carp ( cyprinus carpio) erythrocytes

1. 1. The transport of 3-O- methyl- d- glucose (3-OMG) in lamprey and carp erythrocytes was studied. 2. 2. In lamprey erythrocytes the half-time for the equilibration of 3-OMG was fast, approx. 8 min. In contrast, the erythrocytes of carp were almost impermeable to 3-OMG, with a half-time for equilibration of 14.2 hr. 3. 3. 3-OMG was taken up by lamprey erythrocytes both by facilitated diffusion and simple diffusion. The presence of carrier-mediated transport was indicated by saturation kinetics and by inhibition by phloretin. The K m and V m of the saturable component of 3-OMG transport were 1.6mmol/l and 12.4mmol/kg packed cells/hr. 4. 4. Since the 3-OMG transport in carp erythrocytes showed no sign of saturation kinetics, it appears to proceed by simple diffusion only. 5. 5. These results suggest that, as for most other teleost fish so far studied, low glucose permeability is insufficient to maintain normal energy metabolism in carp erythrocytes. In contrast, in agnathans facilitated glucose transport seems to be quite effective.

In Vitro Hemolysis and Methemoglobin Formation in Common Carp Erythrocytes Induced by Hydrogen Peroxide and Hypoxanthine-Xanthine Oxidase.

In Vitro Hemolysis and Methemoglobin Formation in Common Carp Erythrocytes Induced by Hydrogen Peroxide and Hypoxanthine-Xanthine Oxidase.

A major histocompatibility locus in fish: serological identification and segregation of transplantation antigens in the common carp (Cyprinus carpio L.).

In this study, experiments are described that were designed to investigate whether fish have an immune regulatory systems similar to the major histocompatibility complex (MHC) in higher vertebrate species. From combinations of gynogenetic carp showing either slow or fast rejection of skin transplants, the latter were chosen for alloantiserum production by hyperimmunization with peripheral blood leucocytes. The resulting alloantisera were analyzed for hemagglutinating reactivity with gynogenetic siblings and proved to be operationally monospecific in absorption experiments. The serologically determined carp erythrocyte specificities were shown to correspond to two codominantly expressed allelic products of a single locus and were designated K1 and K2, respectively. Flow cytometer analysis revealed that the same products are also present on leucocytes from peripheral blood, thymus, spleen, and pronephros. K1- and K2-homozygous second-generation gynogenetic siblings were used to study the histocompatibility nature of the K locus products. Skin transplants between K-allogeneic gynogenetic siblings were rejected significantly faster than within K-syngeneic combinations. Taken together, these data suggest that the K locus incorporates MHC class I-like characteristics.

Use of filtration methods in evaluation of the condition of fish red blood cells.

Use of filtration methods in evaluation of the condition of fish red blood cells.

Effects of temperature, oxygen and carbon dioxide on osmotic fragility of carp, Cyprinus carpio L., erythrocytes

The in vitro effects of gases and temperature on the osmotic fragility of carp erythrocytes were studied. At the three different temperatures analyzed (5, 11 and 20°C) there was no noticeable modification in erythrocyte membrane osmotic resistance. Osmotic fragility of red blood cells was altered by CO2 and air treatment, as compared to the standard procedure. This suggests the need to take into account a possible moderate hypoxia that develops in the routine procedure of nucleated erythrocyte osmotic fragility tests.

Influence of heat treatment on osmotic fragility of carp erythrocytes.

SummaryThe effects of temperature on osmotic properties of carp erythrocytes have been examined. The results show that carp erythrocytes are radically different from most mammalian cells in the relationship between temperature and osmotic fragility.

Interaction of mycoplasma mobile 163K with erythrocytes

Mycoplasma (M.) mobile 163K isolated from fish was investigated for its hemadsorbing, hemagglutinating, and hemolysing capacities and for its ability to adhere to erythrocytes. Hemadsorption to the colonies of M. mobile occurred with ovine, bovine, equine, trout, and carp erythrocytes and was inhibited by treatment of the mycoplasmas with substances acting on proteins (pronase, trypsin, glutaraldehyde), heat, UV-irradiation and homologous antiserum. Hemadsorption could be prevented also by treatment of the erythrocytes with neuraminidase. In liquid medium ovine erythrocytes were agglutinated and afterwards lysed by M. mobile. The erythrocytes which were adsorbed to the colonies of M. mobile were finally lysed also. Darkfield preparations showed the ability of M. mobile to adhere to erythrocytes and also its hemagglutinating properties.

Paraquat as an agent affecting antioxidant enzymes of common carp erythrocytes

I. The effects of paraquat on the superoxide dismutase, catalase, glutatione peroxidase activities and lipid peroxidation at different times of paraquat exposure of Cyprinus carpio morph L. erythrocytes were studied. 2. Typical characteristics were observed in the changes of the enzyme activities of the erythrocytes after exposure to paraquat. 3. The haemoglobin concentration of common carp haemolysates was decreased by exposure to paraquat.

Scanning cytophotometry of carp, Cyprinm carpio L., erythrocyte populations: the influence of short‐term hypoxic environment and the recovery period following severe bleeding

Scanning stage absorbance cytophotometry was used to examine haemoglobin contents in individual cells or carp erythrocyte populations. Changes in frequency distributional profiles in response to respiratory stress caused experimentally by hypoxia and/or bleeding were studied at intervals.Histograms were drawn of haemoglobin contents of individual red blood cells in populations obtained from the same carp on four consecutive days after the fish was temporarily exposed to hypoxic environment. The modes and means of the haemoglobin values increased during the 3 days following to the stimulus, whereas a decline was measured on the 4th day. Erythrocyte counts showed the total number of red blood cells to be approximately constant during the period of investigation. On the basis of these findings, it is concluded that the mature, nucleated red blood cells of carp are capable of resuming haemoglobin synthesis after stimuli such as reducing ambient oxygen concentration.Frequency distributional profiles covering a period of 4 weeks following severe loss of blood showed that it took 10–12 days after bleeding until masses of immature erythrocytes appeared in the peripheral blood and that there were then two distinct populations of red blood cells. In the course of about 2 weeks the precursor cells developed into mature erythrocytes.