An evaluation of several potential factors limiting carbon dioxide excretion by rainbow trout (Oncorhynchus mykiss) red blood cells was performed in vitro using a recently developed radioisotopic assay. Red blood cell (RBC) CO2 excretion was reduced by pre-treatment (30 min) of blood with the carbonic anhydrase inhibitor acetazolamide (final nominal concentration 10–4 mol l-1) or the Cl-/HCO3- exchange inhibitor SITS (4-acetamido-4′-isothiocyanatostilbene-2,2′-disulphonic acid; 10-4 mol l-1). The addition of bovine carbonic anhydrase to plasma stimulated CO2 excretion in a dose-dependent manner, with maximal levels of CO2 excretion achieved at a concentration of 3 mg ml-1. These results confirmed that carbonic anhydrase activity and/or Cl-/HCO3- exchange velocity are potential limiting factors in CO2 excretion. Increasing the haematocrit elevated the rate of RBC CO2 excretion, although the effect was apparent only between 0 and 15 % haematocrit; the rate of CO2 excretion was unaffected by further increases in haematocrit between 15 and 35 %. Acute elevation of plasma HCO3- levels increased the rate of CO2 excretion in blood but not in plasma (with or without added carbonic anhydrase). These data suggest that HCO3- availability may limit CO2 excretion at higher haematocrits when the Cl-/HCO3- exchange sites are most plentiful. Lysis of RBCs and the accompanying release of intracellular carbonic anhydrase into the plasma significantly increased CO2 excretion at all haematocrit and HCO3- levels, indicating that the velocity of Cl-/HCO3- exchange does indeed limit trout RBC CO2 excretion. The addition of carbonic anhydrase (3 mg ml-1) to lysed blood caused a further increase in the rate of CO2 excretion but only at the low haematocrit of 5 %. This result suggests that the activity of RBC carbonic anhydrase does not normally limit CO2 excretion except at unusually low haematocrits, such as might occur during severe anaemia. The rapid oxygenation of partially deoxygenated blood during the 3 min assay caused a marked stimulation of CO2 excretion that was concurrent with a significant decrease of RBC intracellular pH (pHi). These data indicate that the supply of Bohr protons during the oxygenation of the blood is a key factor limiting CO2 excretion. Oxygenation of the blood prior to performing the assay also lowered RBC pHi, although CO2 excretion was actually reduced, indicating a possible specific effect of pHi on Cl-/HCO3- exchange activity or HCO3- dehydration. The results are discussed with reference to the control of carbon dioxide excretion in fish.
THE CATALYSIS OF THE HYDRATION OF CARBON DIOXIDE AND DEHYDRATION OF CARBONIC ACID BY AN ENZYME ISOLATED FROM RED BLOOD CELLS