CO(2) produced within skeletal muscle has to leave the body finally via ventilation by the lung. To get there, CO(2) diffuses from the intracellular space into the convective transport medium blood with the two compartments, plasma and erythrocytes. Within the body, CO(2) is transported in three different forms: physically dissolved, as HCO(3)(-), or as carbamate. The relative contribution of these three forms to overall transport is changing along this elimination pathway. Thus the kinetics of the interchange have to be considered. Carbonic anhydrase accelerates the hydration/dehydration reaction between CO(2), HCO(3)(-), and H(+). In skeletal muscle, various isozymes of carbonic anhydrase are localized within erythrocytes but are also bound to the capillary wall, thus accessible to plasma; bound to the sarcolemma, thus producing catalytic activity within the interstitial space; and associated with the sarcoplasmic reticulum. In some fiber types, carbonic anhydrase is also present in the sarcoplasm. In exercising skeletal muscle, lactic acid contributes huge amounts of H(+) and by these affects the relative contribution of the three forms of CO(2). With a theoretical model, the complex interdependence of reactions and transport processes involved in CO(2) exchange was analyzed.

We report here the application of a previously described method to directly determine the CO2 permeability (P(CO2)) of the cell membranes of normal human red blood cells (RBCs) vs. those deficient in aquaporin 1 (AQP1), as well as AQP1-expressing Xenopus laevis oocytes. This method measures the exchange of (18)O between CO2, HCO3(-), and H2O in cell suspensions. In addition, we measure the alkaline surface pH (pH(S)) transients caused by the dominant effect of entry of CO2 vs. HCO3(-) into oocytes exposed to step increases in [CO2]. We report that 1) AQP1 constitutes the major pathway for molecular CO2 in human RBCs; lack of AQP1 reduces P(CO2) from the normal value of 0.15 +/- 0.08 (SD; n=85) cm/s by 60% to 0.06 cm/s. Expression of AQP1 in oocytes increases P(CO2) 2-fold and doubles the alkaline pH(S) gradient. 2) pCMBS, an inhibitor of the AQP1 water channel, reduces P(CO2) of RBCs solely by action on AQP1 as it has no effect in AQP1-deficient RBCs. 3) P(CO2) determinations of RBCs and pH(S) measurements of oocytes indicate that DIDS inhibits the CO2 pathway of AQP1 by half. 4) RBCs have at least one other DIDS-sensitive pathway for CO2. We conclude that AQP1 is responsible for 60% of the high P(CO2) of red cells and that another, so far unidentified, CO2 pathway is present in this membrane that may account for at least 30% of total P(CO2).

We have determined CO2 permeabilities, P(CO2), of red cells of normal human blood and of blood deficient in various blood group proteins by a previously described mass spectrometric technique. While P(CO2) of normal red cells is approximately 0.15 cm/s, we find in red blood cells (RBCs) lacking the Rh protein complex (Rh(null)) a significantly reduced P(CO2) of 0.07 cm/s +/-0.02 cm/s (P<0.02). This value is similar to the value we have reported previously for RBCs lacking aquaporin-1 protein (AQP-1(null)), suggesting that each of the Rh and AQP-1 proteins is responsible for approximately 1/2 of the normal CO2 permeability of the RBC membrane. Four other blood group deficiencies tested lack diverse membrane proteins but exhibit normal CO2 permeability. The CO2 pathway constituted by Rh proteins was inhibitable at pH(e)= 7.4 by NH4Cl with an I50 of approximately 10 mM corresponding to an I50 for NH3 of approximately 0.3 mM. The pathway independent of Rh proteins, presumably that constituted by AQP-1, was not inhibitable by NH4Cl/NH3. However, both pathways were strongly inhibited by DIDS, which accounts for the marked inhibitory effect of DIDS on normal P(CO2), while in contrast another AE1 inhibitor, DiBAC, does not inhibit P(CO2), although it markedly reduces P(HCO3-). We conclude that Rh protein, presumably the Rh-associated glycoprotein RhAG, possesses a gas channel that allows passage of CO2 in addition to NH3.