In vitro experiments were conducted to determine the factors contributing to the unusual distribution of CO2 in the blood of the sea lamprey. When rainbow trout red blood cells (RBCs) were equilibrated with a 3 % CO2:nitrogen mixture in either normal saline or sodium-free saline, the extracellular total carbon dioxide content (CCO2ext) was highly dependent upon the fraction of RBCs in the suspension. In contrast, when lamprey RBCs were equilibrated in normal saline, the CCO2ext decreased with increasing hematocrit. In the absence of extracellular sodium, however, the CCO2ext in the lamprey RBC suspension also became positively correlated with hematocrit. These results suggest that the membrane of sea lamprey RBCs may be somewhat permeable to bicarbonate, but that transmembrane bicarbonate movements may only be detectable in vitro when Na+/H+ exchange is inactivated. Also in contrast to the results for rainbow trout, the changes in CCO2 that occurred in lamprey RBC suspensions following a step increase in PCO2 were not associated with any change in RBC chloride concentration and were not markedly affected by the chloride/bicarbonate exchange inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). When lamprey RBCs in sodium-free saline were treated with an ionophore for anions, tributyl tin chloride (TBTC), however, the distribution of anions across the RBC membrane came to resemble that of the trout. Furthermore, the relationship between CCO2ext and hematocrit in suspensions of TBTC-treated lamprey RBCs also resembled that of trout in normal saline. Thus, these results demonstrate that both the presence of Na+/H+ exchange and the absence of significant anion exchange contribute to the unique CO2 transport properties of sea lamprey blood.

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