A New in Vitro Assay for Carbon Dioxide Excretion by Trout Red Blood Cells: Effects of Catecholamines

A new in vitro assay was developed and critically characterized to measure the rate of CO₂excretion by trout red blood cells (RBCs) from HCO₃^- in their natural plasma under normal in vivo conditions of acid-base status. The assay is based on the addition of [¹⁴C]bicarbonate to the whole blood and collection of the resultant ¹⁴CO₂in the overlying gas phase. The assay simulates the exposure of blood passing through the gills, and measured CO₂excretion rates are representative of those occurring in vivo. Rates are linear over the 3 min time course of the assay, related to haematocrit in a non-linear fashion, elevated by the addition of carbonic anhydrase, reduced by blockade with acetazolamide, and sensitive to variations of equilibration ⁠. Large variations in plasma [HCO₃^-] have only a small effect on CO₂excretion rates when the blood is chronically equilibrated at these levels. Acute elevations in [HCO₃^-], however, create a non-equilibrium situation, resulting in large increases in CO₂excretion. When the blood is acidified, to duplicate typical post-exercise metabolic acidosis, adrenaline causes a marked inhibition of RBC CO₂excretion. The response is transient, reaching a peak 5–8 min after addition of adrenaline and disappearing by 30–60 min. The magnitude of the adrenergic inhibition is correlated with the magnitude of the RBC pHi regulatory response, expressed as the RBC transmembrane pH difference (pHe—pHi). These results support the ‘CO₂retention theory’ explaining observed increases in blood vivo after exhaustive exercise and catecholamine infusions in fish.