The effect of 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate on CO ₂ permeability of the red blood cell membrane

Abstract

It has long been assumed that the red cell membrane is highly permeable to gases because the molecules of gases are small, uncharged, and soluble in lipids, such as those of a bilayer. as expected, but also appeared to reduce intracellular A, although separate experiments showed it has no effect on CA activity in homogenous solution. A decrease in P m,CO 2 would explain this finding. With a more advanced computational model, which solves for CA activity and membrane permeabilities to both CO 2 and HCO 3 ؊ , we found that DIDS inhibited both P m,HCO ؊ 3 and P m,CO 2 , whereas intracellular CA activity remained unchanged. The mechanism by which DIDS reduces CO 2 permeability may not be through an action on the lipid bilayer itself, but rather on a membrane transport protein, implying that this is a normal route for at least part of red cell CO 2 exchange. Ϫ and H 2 16 O in a red cell suspension at chemical equilibrium can be used in principle to measure the proportional intracellular acceleration (A) of CO 2 hydration produced inside intact red blood cells by carbonic anhydrase (CA) compared with the uncatalyzed rate (1) and the self-exchange permeability of the membrane to HCO 3 (P m,HCO Ϫ 3 ). As predicted, A was found to be the same in intact erythrocytes as in hemolysate, both under normal conditions and when exposed to varying concentrations of a membrane-permeable CA sulfonamide inhibitor (ethoxzolamide), whereas P m,HCO Ϫ 3 was not affected (2). Validation of the ability of the technique to differentiate between CA activity and HCO 3 Ϫ permeability in intact red cells was extended by exposing them to phlorizin (3), an established Band III inhibitor (1); this drug decreased P m,HCORecently we exposed red cells to a more specific inhibitor of Band III protein, 4,4Ј-diisothiocyanato-stilbene-2,2Ј-disulfonate (DIDS), to decrease P m,HCO Ϫ 3 without inhibiting CA. However, we found that whereas DIDS lowered HCO 3 Ϫ transport, it also lowered the membrane permeability to CO 2 . METHODSTwo groups of experiments were carried out, one in Philadelphia and one in Hannover, Germany. Blood from healthy adults, freshly drawn into heparin, was washed three times with 145 mM NaCl at 4°C; the red cells were diluted to approximately 40% hematocrit and used the same day. Hemolysates were prepared by freezing and thawing the cell suspension.The fractional water volume, v, of the red cells in the reaction suspension was calculated as the hematocrit of the stock cell suspension ϫ water content of red cells [0.61 (1) for the Philadelphia experiments and 0.65 (4) for the Hannover experiments] ϫ its dilution in the reaction mixture. In later experiments, v was calculated as 55.5 ϫ [cyanmethemoglobin] in mM in the reactant mixture. NaHCO 3 was prepared by incubating 2% and 5% 18O labeled HOH with unlabeled NaHCO 3 at 150°C in a pressure bomb for several days, after which the water was removed by lyophylization. DIDS was obtained from Research Organics and phlorizin (phloridzin) was obtained from Sigma.The technique of the ...

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