Oxygen dependency of the adrenergic Na/H exchange in rainbow trout erythrocytes is diminished by a hydroxyl radical scavenger.

AIM: Potassium transport via the potassium chloride cotransporter in rainbow trout erythrocytes is increased by high oxygen tension. It appears that the effect of oxygen is mediated by reactive oxygen species, especially hydroxyl radicals. In contrast, the activity of adrenergically stimulated sodium proton exchange decreases with increasing oxygen tension. As available data suggest that the two transporters are regulated reciprocally, the present study was undertaken to evaluate, if hydroxyl radicals may inhibit sodium transport via the adrenergically stimulated sodium proton exchanger. METHODS: The effects of the hydroxyl radical scavenger, 2 mm mercaptopropionyl glycine (MPG), on the activity of the adrenergically activated sodium proton exchange in rainbow trout erythrocytes were examined by measuring unidirectional sodium flux, using radioactive isotope, and cellular water content. RESULTS: The activity of the sodium proton exchange increased with decreasing oxygen tension after adrenergic stimulation. When MPG was present during incubation, there was no statistically significant effect of oxygen tension on the adrenergically stimulated sodium proton exchange, whereby the activity of the transporter at atmospheric oxygen tension was markedly higher in the presence than in the absence of MPG. In the absence of adrenergic stimulation, MPG did not influence the transporter activity significantly at any oxygen tension. CONCLUSION: The data suggest that hydroxyl radicals are involved in the inhibition of the adrenergically stimulated sodium proton exchange at elevated oxygen tensions.

Intestinal iron uptake in the European flounder (Platichthys flesus).

Iron is an essential element because it is a key constituent of the metalloproteins involved in cellular respiration and oxygen transport. There is no known regulated excretory mechanism for iron, and homeostasis is tightly controlled via its uptake from the diet. This study assessed in vivo intestinal iron uptake and in vitro iron absorption in a marine teleost, the European flounder Platichthys flesus. Ferric iron, in the form (59)FeCl(3), was reduced to Fe(2+) by ascorbate, and the bioavailability of Fe(3+) and Fe(2+) were compared. In vivo Fe(2+) uptake was significantly greater than Fe(3+) uptake and was reduced by the iron chelator desferrioxamine. Fe(2+) was also more bioavailable than Fe(3+) in in vitro studies that assessed the temporal pattern and concentration-dependency of iron absorption. The posterior region, when compared with the anterior and mid regions of the intestine, was the preferential site for Fe(2+) uptake in vivo. In vitro iron absorption was upregulated in the posterior intestine in response to prior haemoglobin depletion of the fish, and the transport showed a Q(10) value of 1.94. Iron absorption in the other segments of the intestine did not correlate with haematocrit, and Q(10) values were lower. Manipulation of the luminal pH had no effect on in vitro iron absorption. The present study demonstrates that a marine teleost absorbs Fe(2+) preferentially in the posterior intestine. This occurs in spite of extremely high luminal bicarbonate concentrations recorded in vivo, which may be expected to reduce the bioavailability of divalent cations as a result of the precipitation as carbonates (e.g. FeCO(3)).

The adrenergic volume changes of immature and mature rainbow trout (Oncorhynchus mykiss) erythrocytes.

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.

Seasonal and temperature effects on the adrenergic responses of Arctic charr (Salvelinus alpinus) erythrocytes.

In the present study, we have examined the adrenergic responses of Arctic charr (Salvelinus alpinus) erythrocytes acclimated to different temperatures (2, 8 and 14 degrees C) during different seasons. We measured the changes in cellular water and ion contents after noradrenaline stimulation using different noradrenaline concentrations and external pH values. Furthermore, the effects of acute temperature changes on the magnitude of the adrenergic response were studied. The adrenergic response of Arctic charr erythrocytes showed pronounced seasonal variation. The [Na(+)]/[Cl(-)] accumulation ratio after adrenergic stimulation was greatest in May, indicating an enhanced activity of the Na(+)/H(+) exchanger. The noradrenaline-induced change in [Na(+)](i) was greatest in spring. In addition to a seasonal effect, the exchanger seemed to be most active in erythrocytes from charr acclimated to low temperature (2 degrees C) early in May: the EC(50) value was lower and the calculated maximal increase in [Na(+)](i) was greater in the 2 degrees C-acclimated group than in the other acclimation groups. In contrast, acclimation to different temperatures did not affect these responses (measured at a constant temperature) in February. An acute temperature change has a smaller effect on the adrenergic response of Arctic charr erythrocytes than on rainbow trout (Oncorhynchus mykiss) erythrocytes.

Effect of temperature on the resistance of individual red blood cells to flow through capillary-sized apertures.

Low temperature can be expected to increase the resistance to deformation of red blood cells, but the effect of such changes on microcirculatory perfusion are unknown. We therefore analysed resistance to flow through capillary-sized apertures for individual human red blood cells, by micropipette aspiration (approximately 3 microm aperture) and pore transit analysis (approximately 5 microm), as well as average resistance to flow of red cell suspension through multipore filters (5-microm pores). Over a range decreasing from 37 to 0 degrees C, rates of flow of single cells through the 3- and 5-microm apertures decreased monotonically by 2.5- to 3-fold. The changes were similar in magnitude to that expected for the viscosity of aqueous fluid (2.5-fold increase). Average flow resistance measured by bulk filtration also increased in line with viscosity of water, while tendency to block pores was not increased. Micropipette aspiration of small membrane tongues showed that membrane rigidity increased as temperature was lowered, but by a factor rather less than the viscosity. Cell volume also responded rapidly to change in temperature, with lower temperature being associated with swelling, although this effect was much reduced in plasma compared with saline buffer. We conclude that, although increased resistance to deformation of red cells may impair microcirculation at low temperature, there is no structural change likely to induce more dramatic occlusion of flow. Moreover, the effect is comparable in magnitude to the increase predicted for changes in plasma and blood viscosity.

Do fish acclimated to low temperature improve microcirculatory perfusion by adapting red cell rheology?

We have measured the rheological properties of individual red blood cells from fishes inhabiting different thermal environments and have also investigated the effects on red cells of acute in vitro temperature changes. The membrane shear elastic modulus (rigidity) increased markedly with decreasing temperature, and the dependence was similar if temperature was varied acutely in vitro or if cells were measured at normal body temperatures. Red cells from trout and Notothenia coriiceps had almost equal membrane rigidity at comparable temperatures and showed similar temperature-sensitivity in acute experiments. Entry times of trout cells into narrow (approximately 4 microm diameter) micropipettes also increased during in vitro reduction of temperature, and this could be explained largely by the temperature-dependence expected of aqueous solutions. Perhaps surprisingly, entry times did not vary when trout living at different temperatures were tested at these temperatures. Transit times of individual cells through somewhat larger pores (5 microm) in oligopore filters again increased with decreasing temperature in vitro (partly due to increasing fluid viscosity), but such transit times did show a similar temperature-dependence for cells from trout living at different temperatures. Thus, the temperature-dependence of cellular flow resistance appears to arise from variations in membrane rigidity and in the viscosity of fluid components, along with unquantified variations in components such as microtubular structures (which we found did not influence membrane rigidity but did affect pore entry time) and the cell nucleus. Thermal acclimation did not involve adaptation to compensate for increased membrane rigidity or a large pore transit time, with, at most, minor compensation in entry times into smaller pores. We conclude that impaired cellular rheology is not a major factor influencing circulation in fish at low temperature.