Deoxygenation of red blood cells containing Hemoglobin S(SS RBC) increases membrane permeability to Na, K, and Ca, which perturbs a number of ion transport mechanisms and leads to cellular dehydration. This potentiates sicking because of its high dependence on Hb S concentration, so that inhibiting SS RBC dehydration is a potential therapeutic strategy i sickle cell disease. DIDS (4,4'-diisothiocyano-2,2'-disulfostibene), dipyridamole, and Ca channel blockers inhibit deoxygenation-induced (DI) cation fluxes associated with sickling. The goal of this project is to develop the therapeutic potential of these drugs for inhibiting cellular dehydration in sickle cell patients and to exploit their inhibitory properties to elucidate the molecular and biophysical nature of the DK pathway and its role in the perturbation of cell volume regulation during erythroid differentiation.
In Specific Aim 1, the inhibitory potency of dipyridamole and Ca channel blockers will be determined, measuring DI fluxes of Na, K and 45Ca and inhibition of density changes in SS RBC incubated in vitro during oxy/deoxy cycles.
Specific Aim 2 employs covalent 3H2-DIDS binding to SS RBC to identify the membrane constituent associated with inhibition of DI fluxes.
Specific Aim 2 will test the dual hypotheses that the DI pathway is localized to membrane spicules formed during sickling and represents the activity of a nonselective cation channel. Tracer cation fluxes will be measured in isolated membrane spicules (IMS) and IMS incorporated into artificial lipid bilayers will be analyzed electrophysiologically using single channel techniques. Vesicle fluxes and single channel activities will be tested for physiological and pharmacological properties which characterize DI fluxes in intact SS RBC.
In Specific Aim 4, normal and SS RBC progenitors (BFU-e/CFU-e) will be cultured in vitro under defined conditions, and density distribution of erythroblasts and reticulocytes determined on stractan gradients. These experiments will test the hypothesis that the presence of Hb S +/-sickling perturbs volume regulation prior to the reticulocyte stage and alters the expression of volume regulatory transporters. This system will permit testing of transport inhibitors for effects on the hydration state of erythroblasts and early reticulocytes and will shed light on which transport systems are involved in reticulocyte dehydration. These studies will lay the groundwork for clinical trials aimed at preventing cellular dehydration of SS RBC in vivo by pharmacological inhibition of the deoxygenation-induced cation transport pathway.
