This proposal focuses on the mechanism by which passive sodium (Na) and potassium (K) permeability is increased upon deoxygenation of sickle red blood cells (SSRBC). These deoxy cation fluxes act in concert with the Na pump to effect cation depletion in vitro of SSRBC. Thus deoxy cation fluxes may contribute to SSRBC destruction in vivo, since dehydration increases the cells' tendency to sickle and directly results in abnormal rheologic behavior. Preliminary results indicate that the inhibitor of anion transport 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) blocks deoxy cation fluxes. Therefore, one of the working hypotheses on which this proposal is based is that deoxy cation fluxes are mediated by the anion exchange protein. This hypothesis will be tested by examining the effects of known anion transport inhibitors on deoxy cation fluxes in SSRBC. The inhibitory potency of several aryl sulfonates against anion transport (35SO4) influx in intact SSRBC) will be compared to their potency against deoxy cation fluxes (net Na and K movements). Chemical modifications and protease treatments with well described effects on Band 3 and anion exchange will be tested for effects on deoxy cation fluxes. In addition, several functions of the anion exchanger will be examined for modification by deoxygenation of SSRBC. The increase in Ca influx in deoxy SSRBC will be tested for DIDS sensitivity. A model system employing inside out vesicles (IOVs) from normal RBC will be developed for the measurement of cation fluxes (86Rb influx) and their stimulation by deoxy HbS. Chemical modification of both HbS and IOVs will allow testing of the hypothesis that deoxy HbS binds to the cytoplasmic domain of Band 3 to activate deoxy cation fluxes. In addition, the physiologic characteristics of the deoxy cation transport pathway (time of onset, deoxy HbS concentration dependence, effects of other deoxy hemoglobins) will be determined in IOVs. Finally, the discovery of an irreversible inhibitor (DIDS) of deoxy cation fluxes in SSRBC permits testing of the hypothesis that deoxy cation fluxes contribute in vivo to early destruction of SSRBC. The effect of in vitro DIDS treatment of SSRBC on red cell life span in sickle patients will be examined by 51Cr survival studies. The proposed studies promise not only to enhance the understanding of the cellular pathophysiology of sickle cell disease, but also to lay the ground work for new pharmacologic therapies.
