Flounder Erythrocytes Research Articles

Expression, purification, and characterization of thermolabile hemolysin (TLH) from Vibrio alginolyticus

Hemolysin is a putative pathogenicity factor in many bacterial pathogens. In this study, a DNA fragment containing the open reading frame (1254 bp) of the thermolabile hemolysin gene (tlh) from Vibrio alginolyticus V05 was amplified and cloned into the expression plasmid pET-24d(+). The deduced amino acid sequence of the thermolabile hemolysin (TLH) shared 94 and 83% identity with the lecithin-dependent hemolysin (LDH)/TLH of V. parahaemolyticus and V. harveyi thermolabile hemolysin (VHH), respectively. The sequence analysis also indicated that it contained a GDSL lipase domain like VHH. The recombinant protein with a predicted molecular mass of 47.2 kDa was expressed in the Escherichia coli strain BL21 (DE3) as a His-tag fused protein. TLH purified by the nickel-nitrilotriacetic acid (Ni-NTA) His-Bind Resin method showed phospholipase activity on an egg yolk emulsion plate and hemolytic activity against flounder erythrocytes with a specific activity of 18 hemolytic units microg(-1). The addition of divalent cations at different concentrations decreased hemolytic activity of the purified TLH, but monavalent cations did not affect hemolytic activity. The hemolytic activity of TLH was also markedly inhibited by protein modification reagents, i.e. beta-mercaptoethanol, phenylmethylsulfonyl fluoride, and 5,5'-dithio-bis(2-nitrobenzoic acid). Moreover, TLH was toxic to zebrafish when injected intraperitoneally, with a median lethal dose (LD50) of 0.8 microg protein g(-1) fish. This work shows that TLH could potentially be developed as a vaccine and used as a diagnostic tool for vibriosis.

Effects of a fermented vegetable product on hemolysis and lipid peroxidation of Japanese flounder erythrocytes

: We investigated the protective effects of a fermented vegetable product (FVP) against the hemolysis and lipid peroxidation of Japanese flounder (Paralichthys olivaceus) erythrocytes in vitro. 2,2′-Azobis (2-aminopropane) dihydrochloride (AAPH) and tert-butyl hydroperoxide were used as model compounds that cause oxidative stress through the generation of free radicals and induction of lipid peroxidation, respectively, in erythrocytes. The hemolysis and lipid peroxidation of erythrocytes induced with 125 mM AAPH and 25 mM tert-butyl hydroperoxide, respectively, were suppressed by the administration of 1–2 mg/mL FVP (P < 0.01). These results suggested that the FVP reduces oxidative stress in fish and may be useful for the rearing of cultured fish.

Na+-dependent transport of pyruvate in erythrocytes of the Pacific hagfish (Eptatretus stouti)

We investigated the mechanisms of pyruvate transport in the erythrocytes of an ancient marine agnathan, the Pacific hagfish (Eptatretus stouti), and a sedentary euryhaline teleost, the starry flounder (Platichthys stellatus). Uptake of [14C]pyruvate (50 µM, 10°C) by flounder erythrocytes was slow (t1/2(half-life) ~ 30 min), nonconcentrative, and mediated by the band 3 Cl-/HCO-3exchanger in combination with a process similar to the H+/monocarboxylate symporter present in freshwater teleosts and mammalian erythrocytes. In contrast, pyruvate uptake by hagfish erythrocytes (50 µM, 10°C) was rapid (t1/2~ 1.5 min) and, in 10 min, reached an intracellular concentration more than 20-fold higher than that present in the extracellular medium. Pyruvate accounted for almost 90% of the accumulated intracellular radioactivity, the remaining label being incorporated into tricarboxylic acid cycle intermediates and glutamate. Influx of pyruvate was saturable (apparent Km= 12 mM) and inhibited by p-chloromercuriphenylsulphonate (PCMBS) (Ki= 71 µM) and 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) (Ki= 0.49 mM). Transport was inhibited poorly by α-cyano-4-hydroxycinnamate (CIN) (Ki&gt; 4 mM) and was not coupled to the movement of protons. Instead, the influx of pyruvate was Na+dependent. A sigmoidal relationship between pyruvate transport and extracellular Na+concentration was observed, suggesting a Na+:pyruvate coupling ratio greater than 1:1. In contrast with previously described Na+-dependent monocarboxylate transport activities in mammalian renal and intestinal epithelia, the hagfish erythrocyte system did not transport lactate.

Hemagglutinating Activity of Extracellular Alkaline Metalloendopeptidases fromVibriosp. NUF-BPP1

Alkaline metalloendopeptidase (metalloprotease) AP1 (48 kDa) from Vibrio sp. isolated from the intestine of a five-barred goatfish (Parupeneus trifasciatus) was reported in our previous paper to produce AP2 (36 kDa) by releasing a peptide fragment (molecular mass of about 12 kDa) from the C-terminal end of AP1 by autodigestion. AP1 strongly agglutinated fish (flounder, Paralichthys olivaceus) and rabbit erythrocytes, and weakly chicken erythrocytes. In contrast, AP2 had no significant hemagglutinating activity toward any erythrocytes tested, except for weak activity on flounder erythrocytes, suggesting that the C-terminal region of AP1 may be required for the strong hemagglutinating activity. The optimum temperature for the hemagglutinating activity of AP1 was found to be lower than that for the proteolytic activity. At acidic pHs (below pH 7.5), the hemagglutinating activity of AP1 decreased, and its pH profile resembled that of the proteolytic activity. The hemagglutinating activity of AP1 was not observed in the presence of o-phenanthroline or synthetic and proteinous substrates, but different kinds of saccharides and lipids had no effect. While the proteolytic activity of AP1 was not affected by CaCl2, the hemagglutinating activity of AP1 decreased with increases in CaCl2 concentrations. These results suggested that the hemagglutinating activity of these proteases (AP1 and AP2) was most likely caused by their proteolytic action on erythrocyte cell surfaces.

The osmolality-sensitive taurine channel in flounder erythrocytes is strongly stimulated by noradrenaline under hypo-osmotic conditions

Stimulation of flounder erythrocytes by noradrenaline under isosmotic conditions (330 mosmol kg-1) and physiological Na+ concentration (113 mmol l-1) caused swelling of the cells. The EC50 of this cell swelling was 0.65 &micro;mol l-1 noradrenaline. The effect of the noradrenaline-induced cell swelling on the taurine channel under isosmotic conditions was negligible. However, when the cells were stimulated by noradrenaline (1.0 &micro;mol l-1) before, simultaneously with or after reduction of osmolality (255 mosmol kg-1), the volume regulatory efflux of taurine mediated by the taurine channel was transiently accelerated. The rate coefficient for taurine efflux was more than four times higher than in osmolality-stimulated cells not exposed to noradrenaline. The present paper deals with the accelerating effect of noradrenaline on the taurine channel under hypo-osmotic conditions and the lack of effect of noradrenaline-induced cell swelling on the channel under iso-osmotic conditions. Noradrenaline initiated the cell swelling by interacting with &szlig;-receptors which appeared to be more related to the mammalian &szlig;1-receptors than to the &szlig;2-receptors. The receptor interaction activated the adenylate cyclase system and, in the presence of 1.0 &micro;mol l-1 noradrenaline, the cellular cyclic AMP concentration increased about 23 times. Noradrenaline also stimulated the Na+/H+ and Cl-/HCO3- antiporters and this affected the extracellular pH as well as the cell volume. Depending on the extracellular Na+ concentration, the incubation medium was acidified (113 mmol l-1 Na+) or alkalized (2.7 mmol l-1 Na+). Under these two conditions, the accelerating effects of noradrenaline on the taurine efflux were of similar magnitude. Similar effects on the cell volume, the extracellular pH and the volume regulatory taurine efflux were obtained in the presence of the cyclic AMP analogue 8-bromo-cyclic AMP. Under hypo-osmotic conditions in the absence of noradrenaline, the cellular level of cyclic AMP was not elevated. There was no significant positive correlation between the water content of the cells (cell volume) under different conditions in the presence or absence of noradrenaline and the state of activation of the osmolality-sensitive taurine channel. We conclude that the mechanism(s) which activate(s) the osmolality-sensitive taurine channel in flounder erythrocytes is transiently and strongly accelerated by noradrenaline, but not triggered by the noradrenaline-induced events. The acceleration does not appear to be due to increased activity of the antiporters, but to increased cellular levels of cyclic AMP.

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.

The Na(+)-independent taurine influx in flounder erythrocytes and its association with the volume regulatory taurine efflux.

95% of the Na(+)-independent influx of taurine in flounder erythrocytes at normal osmolality (330 mosmol kg-1) and 0.30 mmol l-1 taurine was mediated by a saturable system (Vmax = 0.689 nmol g-1 dry mass min-1; Km = 0.47 mmol l-1). The influx was inhibited by taurine analogues, but was not significantly affected by reduced osmolality. This saturable influx of taurine was probably mediated by the so-called Na(+)-dependent influx system for taurine operating in the 0 Na+: 1 taurine mode. The remaining 5% of the Na(+)-independent influx was mediated by a diffusional pathway (Kd = 0.050 microliter g-1 dry mass min-1), since it did not show saturation kinetics, was not inhibited by taurine analogues and did not mediate counter-exchange. This non-saturable influx system for taurine was strongly, but transiently, stimulated by reduction of osmolality. The time course for this stimulatory effect was the same as that for the system that mediates the volume regulatory efflux of taurine. The relative inhibitory effect of bumetanide, furosemide, DIDS and quinine on the fluxes mediated by these two transport systems were also the same. We suggest that these unidirectional fluxes of taurine were mediated by only one transport system: a taurine channel. The effect of reduction of osmolality on the rate coefficient for efflux of beta-alanine was equal to the effect on the efflux of taurine, but greater than the effect on the efflux of choline. This difference probably reflects structural and/or electrical restrictions on the substrates to be transported by the taurine channel. The volume regulatory efflux of taurine was inhibited in the presence of the anti-calmodulin drug trifluoperazine and, in a Ca(2+)-free medium, added EGTA. The 5-lipoxygenase inhibitor nordihydroguaiaretic acid completely blocked the volume regulatory efflux of taurine. We suggest that both Ca2+/calmodulin and leukotrienes contribute to the control of the transport mediated by the taurine channel.

Characterization of the Na+-dependent taurine influx in flounder erythrocytes

About 92% of the taurine influx in flounder erythrocytes at physiological conditions in vitro (330 mosmol·l-1, 145 mmol·l-1 Na+, 0.30 mmol·l-1 taurine) is Na+-dependent. This influx is highly specific for taurine. The β-amino compounds hypotaurine and β-alanine were the only compounds which mimicked the inhibitory effect of taurine on influx of [14C]taurine, the former more than the latter. Counterexchange of taurine was also mediated by the taurine transporters. Reduction of osmolality per se did not affect the activity of these transporters. Non-linear regression analysis of the influx values revealed the presence of two different influx systems: a system with high affinity and low capacity and another with low affinity and high capacity. However, we cannot exclude the possibility that this influx of taurine was mediated by only one transporter which operated in different modes depending on the extracellular Na+ concentration. On the assumption that the Na+-dependent influx was mediated by two separate systems, the maximal velocity of the low capacity system was 2.55 nmol·g dry weight-1·min-1 at 145 mmol·ll-1 extracellular Na+. This capacity was about 50% lower than that of the high capacity system. The Michaelis constants were 0.013 and 1.34 mmol·l-1, respectively. Reduction of the extracellular Na+ concentration reduced maximal velocity and the affinity to taurine of both transport systems. At 10 mmol·l-1 Na+ or lower concentrations the high capacity system did not seem to operate. The activation method suggested that each taurine molecule transported by the high capacity system was accompanied by two Na+. The stoichiometry of the low capacity system was 1 taurine: 1 Na+. The Hill-coefficient for both transport systems was 1.00.

Transport of organic substrates via a volume-activated channel.

We have investigated the volume-activated transport of organic solutes in flounder erythrocytes. Osmotic swelling of cells suspended in a Na(+)-free medium led to increased membrane transport of taurine, glucose, and uridine. For each compound there was a significant lag period (1-2 min at 10 degrees C) between cell swelling and activation of the flux. The volume-activated fluxes of each of the substrates increased in parallel with increasing cell volume, and those of taurine and uridine increased linearly with concentration (up to 19 mM). The volume-activated fluxes of each of the three compounds showed similar sensitivities to a number of anion-selective channel blockers (5-nitro-2-(3-phenylpropylamino)benzoic acid > 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid approximately MK-196 > niflumic acid > furosemide); the IC50 for the inhibition of the volume-activated fluxes by NPPB was around 12 microM. The results are consistent with the hypothesis that the volume-activated transport of organic osmolytes is via a pathway with the characteristics of a volume-activated "chloride channel." This raises the question of whether the transport of organic substrates might represent a physiological role for such channels in other cell types.

Taurine transport associated with cell volume regulation in flounder erythrocytes under anisosmotic conditions.

The taurine transport of flounder erythrocytes is associated with a cell volume regulation in anisosmotic media. An osmolality reduction leads to a cell volume increase, which is followed by a volume readajustment towards the original level. A 75 mosM reduction is accompanied by a 33 mumol g dry wt.-1 reduction in the cellular taurine content. The reduction in osmolality activates the taurine release mechanism by transiently increasing the rate coefficient for taurine efflux. The rate coefficient for taurine influx is similarly stimulated. This influx is mediated by a Na+-independent transport system. The concomitant activation of influx and efflux suggests a coupling between these two systems. Higher taurine efflux and influx rate coefficients which decayed more slowly with time were measured in cells suspended in Na+-free (choline replacement) media than in the presence of Na+. This suggests that Na+ may play a role in the taurine release mechanism. Noradrenaline induced a cellular swelling at normal osmolality (330 mosM), but had only a minor effect on the taurine efflux and influx and the cellular taurine content. Urea-induced cellular swelling at normal osmolality initiated a volume regulatory process and activated the taurine release mechanism, similarly to an osmolality reduction. These results show that osmolality reduction and cellular swelling are no prerequisites for the activation of the taurine release mechanism and the cell volume readajustment. It is suggested that the dimension of an intracellular solute compartment determines the activation level of this mechanism.

Some properties of the Na + + K +-linked Mg 2+-dependent adenosine triphosphatase from the erythrocyte plasma membrane of the flounder ( Platichthys Flesus L.)

1. Cell membranes of flounder erythrocytes contain both a Na +, K + and Mg 2+ dependent ATPase which is inhibited by ouabain and Ca 2+, and also ouabain insensitive ATPase activity in the presence of either Ca 2+ or Mg 2+. 2. The effect of the concentrations of ATP, Na +, K + and Mg 2+ on activity are described. 3. The enzyme appears to be specific for Na + whereas K + can be replaced by Li + or NH 4 +. There is a high specificity for ATP but a significant activity with ADP. 4. Arrhenius plots of activity show a break at 12°C with apparent activation energies of 11 kcal/mol above this temperature and 28 kcal/mol below it.

The effect of Na+ and osmolality on the influx and steady state distribution of taurine and gamma-aminobutyric acid in flounder (Platichthys flesus) erythrocytes

1.1. The effect of Na+ and osmolality on influx of taurine and GABA, and steady state distribution of Na+, K+, Cl−, taurine and GABA, has been measured in flounder (Platichthys flesus) erythrocytes.2.2. A reduction in osmolality only, lead to a net release of taurine, GABA, K+ and Cl− during the restoration of normal volume.3.3. Taurine and GABA influx was Na-dependent, a minor fraction was Na-independent.4.4. A reduced osmolality stimulated taurine influx.5.5. Present work supports the hypothesis that flounder erythrocyte taurine and GABA concentrations are controlled by factors correlated to plasma osmolality.

Lysosomes in erythrocytes from the flounder ( Platichthys flesus)

1. 1. Activities of the lysosomal enzymes β-N- acetylglucosaminidase , cathepsin D and β-glucuronidase, were found in hemolyzates of purified erythrocytes from the flounder Platichthys flesus. 2. 2. Latent enzyme activities were found for cathepsin D and β-glucuronidase in homogenates of the flounder erythrocytes, but no consistent latent activity was found for β-N- acetylglucosaminidase . 3. 3. Neutral red was shown to be taken up by the erythrocytes from the flounder. 4. 4. The possibility that lysosomes are present in flounder erythrocytes is discussed.

Volume regulation by flounder red blood cells in anisotonic media.

The nucleated high K, low Na red blood cells of the winter flounder demonstrated a volume regulatory response subsequent to osmotic swelling or shrinkage. During volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation after osmotic swelling is referred to as regulatory volume decrease (RVD) and was characterized by net K and water loss. Since the electrochemical gradient for K is directed out of the cell there is no need to invoke active processes to explain RVD. When osmotically shrunken, the flounder erythrocyte demonstrated a regulatory volume increase (RVI) back toward control cell volume. The water movements characteristic of RVI were a consequence of net cellular NaCl and KCl uptake with Na accounting for 75 percent of the increase in intracellular cation content. Since the Na electrochemical gradient is directed into the cell, net Na uptake was the result of Na flux via dissipative pathways. The addition of 10(-4)M ouabain to suspensions of flounder erythrocytes was without effect upon net water movements during volume regulation. The presence of ouabain did however lead to a decreased ration of intracellular K:Na. Analysis of net Na and K fluxes in the presence and absence of ouabain led to the conclusion that Na and K fluxes via both conservative and dissipative pathways are increased in response to osmotic swelling or shrinkage. In addition, the Na and K flux rate through both pump and leak pathways decreased in a parallel fashion as cell volume was regulated. Taken as a whole, the Na and K movements through the flounder erythrocyte membrane demonstrated a functional dependence during volume regulation.

Carbonic anhydrase in flounder erythrocytes

1. 1. The blood carbonic anhydrase activity of the starry flounder was found to be approximately 40 enzyme units per milliliter of blood. These findings are in contrast with previously published reports on the absence of carbonic anhydrase activity in flounder blood. 2. 2. Carbonic anhydrase in the blood of Rainbow trout is not found exclusively in the cytosol, as in mammalian erythrocytes. It is suggested that the major portion of erythrocytic carbonic anhydrase is associated with the plasma membrane. 3. 3. The applicability of previous assay methods are discussed.

The distribution of taurine, gamma-aminobutyric acid and inorganic ions between plasma and erythrocytes in flounder ( Platichthys flesus) at different plasma osmolalities

1. 1. In vivo variation in erythrocyte and plasma concentrations of Na +, K +, Cl − and free amino acids were studied in relation to plasma osmolality in flounder ( Platichthys flesus L.) adapted to sea water and fresh water, respectively. 2. 2. A positive linear correlation was found between intracellular K +, Cl −, taurine, GABA, respectively, and plasma osmolality. 3. 3. A negative linear correlation was found between the electrochemical potential difference of Na − in relation to the chemical potential difference of taurine and GABA, respectively. 4. 4. The results support the theory that the energy contained in the Na + gradient supplies energy for the cellular accumulation of taurine and GABA. However, the same results support too that the osmolality might be a determining factor in the regulation of taurine and GABA concentrations in flounder erythrocytes.

Synthesis of gamma-aminobutyric acid in fish erythrocytes.

WE have found evidence of high activity of the enzyme glutamate decarboxylase (GAD) outside the nervous system. This enzyme catalyses the formation of γ-amino-butyric acid (GABA), which is believed to be an inhibitory neurotransmitter1. Recently, however, a high concentration of GABA (11 mmoles/kg wet weight) was found in flounder (Pleuronectes flesus L.) erythrocytes, where GABA may participate in the maintenance of a steady cell volume during changes in plasma osmolality2,3.

Regulation of cell volume in flounder ( Pleuronectes flesus) erythrocytes accompanying a decrease in plasma osmolarity

1. 1. Cell volume regulation was studied in isolated blood cells from flounder where plasma osmolarity was reduced (10 and 20 per cent) proportional to the in vivo reduction found to occur in the plasma of flounder adapted to fresh water. 2. 2. After an initial osmotic swelling caused by the blood dilution, the cell volume regained the undiluted volume in teh course of 4 hr. 3. 3. Concomitant with the cell volume regulation the cellular content of free ninhydrin-positive substances (mean 45 mmoles/1. cells, measured in protein-free aqueous extracts) decreased about 16 per cent after 20 per cen dilution and about 8 per cent after 10 per cent dilution. The plasma content of the same substances increased proportional to the decrease in cellular content. 4. 4. Oxygenating the venous blood cells decreased the cell volume about 10 per cent. During this volume reduction no derease in cellular content or increas in plasma content of ninhydrin-positive substances could be detected. 5. 5. It is concluded that the flouder erythrocytes are capable of regulating their volume to counteract an osmotic swelling, and that some of the intracellular osmotically active solutes consist of free ninhydrin-positive substances which to some degree leave the cell during the volume regulation.