Dehydration of sickle red blood cells (SS RBC) contributes to pathology because sickling is highly dependent on Hb S concentration. Accordingly, inhibiting SS dehydration represents a potential therapeutic strategy. Three transport pathways mediate the cation loss which leads to dehydration: >1. Deoxygenation-induced (DI) fluxes of Na+, K+, and Ca++, 2. A Ca++-dependent K+ channel activated by DI Ca++ influx; 3. The KCI co- transporter (KCC). This project focuses on determining the therapeutic potential of several pharmacological inhibitors the DI pathway, identifying the membrane constituent which mediates the DI flux, and determining the basis for abnormal KCC activity in S RBC.
In Specific Aim 1, the potency of dipyridamole and Ca channel antagonists as inhibitors of the DI pathway 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. To identify the membrane constituent associated with inhibition of DI fluxes, Specific Aim 2 employs covalent binding to SS RBC of another inhibitor of the DA pathway, 3H2-DIDS (4,4'-diisothiocyano- 2,2'-dihydrostilbene disulfonate).
In Specific Aim 3, the hypothesis that increased KCC activity of SS RBC results either from a direct perturbation by Hb S or as a consequence of erythropoietic stress will be tested by comparing KCC in AA and SS reticulocytes with tightly defined age ranges. The rate and volume set point of KCC activated by a variety of stimuli will be measured by density shift assays using stractan gradients, and three subsets of reticulocytes identified by thiazole orange fluorescence using flow cytometry. RBC progenitors (CFUe) will be cultured from peripheral blood BFUe in vitro under defined conditions, and KCC activity of cultured AA and SS erythroblasts and reticulocytes compared. The direct effects of hematopoietic stimulation by growth factors on KCC expression will be examined in this culture system. The effects of hematopoietic stimulation by growth factors on KCC expression will be examined in this culture system. The effect of hydroxyurea will also be tested and the results integrated with measurements of KCC activity and hydration state of reticulocytes of patients taking HU (Project 4). The appearance of KCC activity during erythroid differentiation will be determined by density shift assay in SS and AA RBC precursors in bone marrow. These studies will elucidate the molecular mechanisms os SS RBC dehydration and will lay the groundwork for clinical trials aimed at preventing cellular dehydration of SS RBC in vivo by pharmacological inhibition of these cation transport pathways.
