An Evaluation of Factors Limiting Carbon Dioxide Excretion by Trout Red Blood Cells in Vitro

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 pretreatment (30min) of blood with the carbonic anhydrase inhibitor acetazolamide (final nominal concentration 1024 mol l21) or the Cl/HCO3 exchange inhibitor SITS (4acetamido-49-isothiocyanatostilbene-2,29-disulphonic acid; 1024 mol l21). 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 3mgml21. 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 (3mgml21) 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 3min 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