Rainbow Trout Red Cells Research Articles

Inhibition of adrenergic proton extrusion in rainbow trout red cells by nitrite-induced methaemoglobinaemia.

The effects of nitrite-induced methaemoglobinaemia on adrenergic proton extrusion from rainbow trout red blood cells were studied using the pH-stat method. In control conditions adrenergic proton extrusion was completely inhibited by amiloride and was greater in deoxygenated than in oxygenated erythrocytes. Nitrite-induced methaemoglobinaemia was associated with a pronounced reduction in the catecholamine-stimulated proton efflux from both deoxygenated and oxygenated erythrocytes. In deoxygenated erythrocytes the initial proton efflux upon catecholamine stimulation decreased by 60-70%, while the percentage of methaemoglobin in the red cells increased from the control level of 1-3% to 20%. In oxygenated erythrocytes the decrease was 30% at the same methaemoglobin percentage range. It is suggested that the pronounced influence of nitrite-induced methaemoglobinaemia on adrenergic proton efflux results from an inhibition of the red cell sodium/proton exchanger by the R-like haemoglobin conformations.

Effect of noradrenaline on the methaemoglobin concentration of rainbow trout red cells

We studied the effect of noradrenaline on the methaemoglobin (metHb) concentration in rainbow trout red cells. The erythrocytes were incubated in physiological medium with or without noradrenaline and the percentage of metHb of total Hb content was measured. Noradrenaline lowered the metHb content significantly as compared to controls. To study if the effect of noradrenaline was caused by adrenergic intracellular alkalinization, cells were treated with noradrenaline + carbonic anhydrase or noradrenaline + acetazolamide. Carbonic anhydrase inhibits the adrenergic increase in intracellular pH, but did not reduce the effect of noradrenaline on the metHb concentration. Acetazolamide accentuates the increase in intracellular pH. However, there was no difference in the methaemoglobin content of noradrenaline-incubated and noradrenaline + acetazolamide-incubated cells. These results show that the effect of noradrenaline on the methaemoglobin content is independent from the adrenergic increase in intracellular pH. However, amiloride treatment inhibited the effect of noradrenaline on the methaemoglobin content, suggesting that the protein mediating sodium/proton exchange may also be involved in controlling cellular methaemoglobin levels.

Adrenergic Control of Red Cell pH in Salmonid Fish: Roles of the Sodium/Proton Exchange, Jacobs-Stewart Cycle and Membrane Potential

ABSTRACT We investigated the mechanisms by which adrenergic activation of sodium/proton exchange reduces the pH gradient across the membrane of rainbow trout red cells. In untreated cells, adrenergic stimulation caused a significant increase in the proton distribution ratio ([H+]e/[H+]i) across the red cell membrane. The increase in the proton distribution ratio caused by adrenergic stimulation was inhibited by the protonophore 2,4-dinitrophenol (2,4-DNP). Thus, sodium/proton exchange displaces protons from electrochemical equilibrium. Active regulation of intracellular pH by sodium/proton exchange is possible, because the extracellular dehydration of carbonic acid to carbon dioxide is uncatalyzed. The increase in proton distribution ratio caused by adrenergic stimulation was inhibited in red cell suspensions to which extracellular carbonic anhydrase had been added before stimulation. In contrast, inhibition of intracellular carbonic anhydrase markedly increased the pH changes induced by adrenergic stimulation, suggesting that the net direction of the intracellular hydration/dehydration reaction may markedly affect the intracellular pH changes. Membrane potential changes are not a necessary component of the adrenergic response. The increases in red cell volume and sodium and chloride concentrations induced by adrenergic stimulation were unaffected in cells ‘voltage-clamped’ by valinomycin.

Energy metabolism in trout red cells: consequences of adrenergic stimulation in vivo and in vitro.

beta-Adrenergic stimulation of salmonid red cells results in a rapid decrease (within 5 min) in the nucleotide triphosphate:haemoglobin ratio (NTP:Hb), which is thereafter maintained at a constant level, presumably through increased ATP turnover via matched aerobic metabolism and energy-consuming processes. Addition of the beta-adrenergic agonist isoproterenol to rainbow trout red cells in vitro leads to a rise in intracellular pH (pHi), a corresponding decrease in extracellular pH (pHe) and an increase in red cell oxygen consumption (MO2). Moreover, the extent to which red cell pHi is maintained constant in the face of an acute extracellular acidosis in vitro or in vivo is proportional to the adrenergically stimulated increase in red cell MO2. In the absence of oxygen, these red cells remain capable of pH regulation, but cannot maintain NTP:Hb constant. As a result, membrane and metabolic functions become uncoupled in the stimulated deoxygenated cells.

Effects of plasma proteins on the dehydroabietic acid-induced red cell breakdown

Since red cell hemolysis may play a role in the development of jaundice in fishes exposed to resin acids, we studied the effects of dehydroabietic acid (DHAA) on rainbow trout red cells in vitro. In incubations with physiological saline DHAA caused red cell breakdown at concentrations above 5 mg/liter within 24 hr. In plasma incubations red cell breakdown was observed only after 48 hr at concentration of 240 mg/liter, and DHAA at concentrations of 60 and 120 mg/liter dissolved lipids from red cell membranes within 24–48 hr. In addition, no changes were seen in 6 hr in red cell function when cells were treated in plasma with 210 mg DHAA/liter. The addition of albumin to saline incubations markedly reduced hemolysis and showed that the protection against DHAA in plasma results from binding of DHAA to plasma proteins.

Blood oxygen equilibria and theoretical models. I. effect of protons in trout ( Salmo gairdneri) and human red cells, in absence of organic phosphates

1. 1. Oxygen equilibrium curves were measured on red cells that had been depleted of organic phosphates, for rainbow trout red cells between pH 7–9, at 15 and 20°C, and for human red cells between pH 6.8–8.0, at 37°C. 2. 2. The data were fitted to the models of Adair and of Monod et al. (MWC model). Parameters were estimated by the non-linear least-squares method, from which the number of Bohr protons released during oxygenation was calculated. 3. 3. For trout as for human red cells, the pH affects the first step of oxygenation and the overall oxygenation in nearly equal proportion. For human red cells, the association constant for binding of the last oxygen, expressed by k 4 or k R , is not affected by pH, indicating that the R structure is free of constraints. For trout red cells, there is a pronounced pH dependence of this constant at low pH, which is about 10-fold increased between pH 7–9. This corresponds to the Root effect that impairs the T → R transition, and confirms previously published data for normal trout and human red cells. 4. 4. For trout red cells, small functional heterogeneity is evidenced, despite Hb multiplicity, and effect seems linked to the level of temperature. 5. 5. The specific effect of organic phosphates, evident for human red cells, contrasts with a lack of effect for trout red cells, when taking into account the intracellular pH values.

Adrenergic regulation of haemoglobin oxygen affinity in rainbow trout red cells

Adrenaline (initial concentration 5×10−6 M) caused an increase in haemoglobin oxygen affinity of rainbow trout red cells incubated in vitro in physiological saline. The increase was more pronounced at low than at high extracellular pH values. Adrenaline also induced an increase in intracellular pH, swelling of red cells and a metabolic decrease in cellular ATP concentration. The most important reason for increased oxygen affinity was the increase in intracellular pH resulting, to a great extent, from decreased concentration of impermeable polyanions, ATP and haemoglobin, in the cell. In addition, the decrease in cellular ATP concentration as such increases oxygen affinity of haemoglobin.

Oxygen uptake by rainbow trout blood, Salmo gairdneri

Oxygen uptake rate by rainbow trout red cells was measured in the temperature range 5° to 40° C. Between 5° and 20° C there was a steady increase in rate and at 20° C a maximum rate of 2.5 μl O2/min/ml red cells occurred, followed by a decline in the rate as the temperature increased. A morphological investigation of the red cell was conducted and possible functions of the nucleus in fish red blood cells are discussed.