The role of fish erythrocytes in transport and excretion of carbon dioxide

Involvement of the red blood cells of fish in CDs exchange was investigated by examination of the CO₂ transport properties of fish blood, of ion movements (HCO₃₋, CI⁻, H⁺) across the red cell membrane, and of erythrocyte carbonic anhydrase activity. Adrenergic modulation of erythrocyte function in vitro and the effects of catecholamines on branchial CO₂ exchange in vivo also were studied. Approximately 92% of the CO₂ content of venous blood of rainbow trout (Salma gairdneri) was HCO₃₋, which at an haematocrit of 25% was distributed between the plasma water space and the red cell water space in a ratio of about 9:1. Plasma HCO₃₋ accounted for some 82% of the CO₂ excreted during branchial blood transit, while erythrocyte HCO₃₋ accounted for only 9%. The remainder of CO₂ excreted at the gills was derived from, in descending order of importance, carbamino compounds (R—NH CO₂₋, molecular CO₂, and CO₃²⁻. The erythrocyte of rainbow trout was freely permeable to HCO₃₋, CI⁻ and H⁺, all of which were distributed passively across the red cell membrane. HCO₃₋ traversed the erythrocyte membrane in an one-f or—one exchange with CI⁻ via a SITS-sensitive mechanism analogous to the Band 3 anion exchange pathway of mammalian red cells. The transmembrane equilibrium distributions of HCO₃₋ , Cl⁻ and H⁺, however, were complicated by the presence of a cell nucleus. The nuclear compartment of trout erythrocytes appeared to be more acidic and to contain less HCO₃₋ and CI⁻than the cytosol. The kinetics of the uncatalysed HCO₃₋ : CO₂ conversion were found to be at. least one order of magnitude too slow to account for the observed branchial CO₂ movements. Fish erythrocytes however, contained sufficient carbonic anhydrase activity to catalyse the i nterconversi on of HCO₃₋ and CO₂, increasing the rate of reaction by several orders of magnitude. Fish plasma contained inhibitors of carbonic anhydrase which were active against the enzyme activity of both erythrocyte and gill homogenates. These inhibitors lacked access to intracellular carbonic anhydrase and had no direct effect on membrane transport of anions. It is suggested that these inhibitors probably function to immobilize carbonic anhydrase released into the plasma during the normal destruction of erythrocytes or during injury, but have no effect, on intact red cells. These data, together with evidence that the basolateral membrane of the gill is largely impermeable to HCO₃₋ (Perry et al- 1982), clearly indicate that the principal pathway for COK excretion in fish is via the movement of plasma HCO₃₋ into the red cell by way of a 'chloride shift'. This bicarbonate then is rapidly dehydrated to form CO₂ in the presence of erythrocyte carbonic anhydrase. The resultant diffuses down its concentration gradient out of the red cell and across the gill epithelium. CO₂ loading of fish blood during tissue capillary transit involves a simple reversal of these transport and chemical mechanisms. The present information conclusively refutes the gill model of CO₂ excretion (Haswell et al . 1980) which asserts that fish erythrocytes have no functional role in branchial CO₂ exchange. Evidence was found which indicated that the original studies of Haswell and coworkers suffered from technique-related artifacts. Catecholamines had profound effects on both erythrocyte function and branchial CO₂ exchange. Beta-adrenergic agonists appeared to stimulate coupled Na⁺/H⁺ and Cl⁻/ HCO₃₋ exchangers on the cell membrane of trout erythrocytes, similar to the ion exchangers involved in volume regulatory ion movements in amphibian red cells (Cala 1980). The adrenergic responses of rainbow trout erythrocytes included a net cellular gain of Na⁺, Cl⁻ and H₂O, a net cellular loss of H⁺ and HCO₃₋, a pronounced cell swelling, and a functional reduction in net HCO₃₋ flux through the red cell. In vivo, these adrenergic responses were accompanied by a transient reduction in CO₂ excretion, an increase in body CO₂ stores, and a disruption of the HCO₃₋ : CO₂ chemical equilibrium in arterial blood immediately downstream of the gill. Oxygen uptake was unaffected by adrenaline. The adrenergic responses of fish red cells probably are important in regulating erythrocyte pH during periods of stress, and hence serve to maintain O₂ transport to the tissues under such conditions. At the same time, these responses slow net HCO₃₋ flux through the red cells during branchial blood transit, and thus serve to maintain an extracellular pool of HCO₃₋ in stress which then may be used to enhance the intracellular buffering capacities of other tissues.Science, Faculty ofZoology, Department ofGraduat