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 HCC>3 ~ 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 14CC>2 in the overlying gas phase. The assay simulates the exposure of blood passing through the gills, and measured CO2 excretion rates are representa-tive of those occurring in vivo. Rates are linear over the 3min 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 ' explain-ing observed increases in blood PCO2 in vivo after exhaustive exercise and catecholamine infusions in fish