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 CO2 excretion 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 14CO2 in the overlying gas phase. The assay simulates the exposure of blood passing through the gills, and measured CO2 excretion 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 PCO2. Large variations in plasma [HCO3-] have only a small effect on CO2 excretion 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 CO2 excretion. When the blood is acidified, to duplicate typical post-exercise metabolic acidosis, adrenaline causes a marked inhibition of RBC CO2 excretion. 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 ‘CO2 retention theory’ explaining observed increases in blood PCO2 in vivo after exhaustive exercise and catecholamine infusions in fish.