R. G. Boutilier

Acute exposure to graded levels of hypoxia in rainbow trout (Salmo gairdneri): metabolic and respiratory adaptations

We have studied the mechanisms of acute hypoxia tolerance in rainbow trout (Salmo gairdneri). Fish held at 9 degrees C were exposed to various levels of hypoxia for 24 h. At an environmental PO2 of 30 Torr, the fish showed an initial plasma acidosis probably of metabolic origin which was subsequently offset such that blood pH returned to normal within about 4 h. Over this time period, red cell pH was maintained constant. Comparing the effects of different levels of hypoxia following 24 h exposure, oxygen consumption of the animal remained unchanged over a broad range of inspired oxygen tensions but declined by over 30% of normoxic values at inspired water PO2 levels of 80 Torr. This appeared to be a true metabolic depression because signs of increased anaerobic metabolism did not occur until there was a further reduction in water oxygen levels. Rainbow trout appear to be able to maintain a relatively high energy status in their white muscle during 24 h exposure to severe hypoxia (water PO2 = 30 Torr). As the level of hypoxia was intensified, there was a reduction in the oxygen gradient across the gills, probably facilitated in part by the release of catecholamines into the blood. The erythrocytic ATP: Hb4 molar ratios declined with increasing hypoxic stress as did the pH gradient between the erythrocyte and plasma. The overall effect was no change in Hb O2-affinity after 24 h exposure to severe hypoxia.

Determination of intracellular pH and PCO2 after metabolic inhibition by fluoride and nitrilotriacetic acid.

Mean intracellular pH (pHi) and PCO2 (PiCO2) have been analysed based on pH and total CO2 measurements in tissue homogenates. Tissues were sampled from undisturbed worms (Sipunculus nudus), squid (Illex illecebrosus), trout (Salmo gairdneri), toads (Bufo marinus), and rats. Homogenate metabolism was inhibited by the addition of potassium fluoride and nitrilotriacetic acid (NTA). Model calculations revealed that the influence of dilution, medium buffers, and contamination by extracellular fluids was negligible. In white muscle tissue the resulting pHi values were virtually the same as found in studies using DMO (dimethyloxazolidinedione). If large fractions of mitochondria were present (e.g. in heart muscle), DMO derived pHi values were considerably higher, probably representing overestimates. Homogenate derived pHi values are concluded to represent the effective mean pHi by taking into account pH gradients, and the volumes and buffering of cellular compartments. High time resolution and small variability make this method especially useful to assess rapid changes in pHi, e.g. in exercising animals.

Metabolic energy production during adrenergic pH regulation in red cells of the Atlantic salmon, Salmo salar

Whole blood from Atlantic salmon was incubated anaerobically at 10 degrees C so as to measure the metabolic activity of the nucleated erythrocytes. An acute extracellular acidosis was produced by adding either an acid solution (sham) or an acid solution with adrenaline (final concentration, 5 x 10(-4) M). The extracellular acidosis produced by the sham solution was transferred to the erythrocytes, whereas with adrenaline, intracellular pH actually increased in the face of a plasma acidosis. Indeed, the extracellular acidosis in the adrenaline-treated blood was significantly higher than that of the sham as a result of net H+ excretion from the erythrocyte. This pH response of the erythrocyte was accompanied by a proportional increase in the O2 consumption of the blood, with no change in lactate production. In comparison to sham-treated cells, the content of erythrocytic nucleotide triphosphates initially decreased upon addition of adrenaline but was thereafter maintained at a constant NTP/Hb ratio presumably due to an increased ATP turnover. In conclusion, it appears that the aerobic rather than anaerobic metabolism of erythrocytes is accelerated upon addition of adrenaline to blood, and that this increased metabolism is involved in fueling the membrane transport processes involved in adrenergic pH regulation of salmonid red cells.

CO2 transport in agnathan blood: evidence of erythrocyte Cl−/HCO3− exchange limitations

CO2 transport properties of blood were examined in the lamprey Petromyzon marinus and the hagfish Myxine glutinosa. In order to evaluate possible chloride/bicarbonate exchange limitations, experiments were conducted under control conditions and in the presence of an ionophore to permit equilibrium distribution of chloride, bicarbonate, and protons across the erythrocyte membrane. The ionophore, tri-n-propyl tin chloride, markedly altered the CO2 transport properties and apparent nonbicarbonate buffering characteristics of the blood of Petromyzon marinus. In addition, the distributions of protons, bicarbonate and chloride ions across the erythrocyte membrane were very different from each other under control conditions, but became very similar in the presence of the anionic ionophore. The CO2 transport properties of the blood of Myxine glutinosa were not significantly different in the presence of the ionophore. Small but significant changes were observed, however, in erythrocyte pH, chloride concentration and water content in the presence of tri-n-propyl tin chloride. These results demonstrate that chloride/bicarbonate exchange limitations and possibly active transport of protons contribute to the unique CO2 transport properties in the blood of the lamprey, Petromyzon marinus. In the hagfish, Myxine glutinosa, the importance of anion exchange limitations or active proton transport with regard to the CO2 carrying properties of the blood are clearly much less than in the lamprey under the in vitro conditions of this study.