The as yet undefined membrane defect leading to sickle RBC K+ loss and dehydration is arguably the most important defect of the sickle RBC membrane. The proposed studies will examine a specific hypothesis regarding the etiology of K+ leak in sickle RBC cells and they will provide an accurate biophysical description of the K+ leak pathway(s) in these cells. Specifically, I hypothesize that autoxidative membrane damage induces a defect which makes the sickle RBC membrane abnormally susceptible to the potentially adverse effects of cellular deformation accompanying deoxygenation. This could explain the apparently reversible nature of sickling-induced abnormalities of cation homeostasis. Four approaches will be used. (1) RBC from different sickle patients and from density-subpopulations of single patients will be examined to see if the dehydration abnormality (as defined by size of the abnormally dense subpopulation and by monovalent cation content) correlates with various """"""""footprints"""""""" of sickle RBC oxidation (radical generation, membrane-bound heme, thiol oxidation, lipid peroxidation). (2) It will be determined whether in vitro oxidative perturbation accurately models the K+ leak of unmanipulated sickle RBC. Oxidative insults will include intracellular generation of superoxide, thiol oxidation without lipid peroxidation and lipid peroxidation without thiol oxidation. (3) It will be determined whether minimal oxidative insult (which itself does not cause K+ leak in the absence of deformation) predisposes RBC to K+ leak during minimal deformation (which itself does not cause K+ leak in the absence of oxidation). In these studies deformation will be induced by quantitative application of shear stress in a concentric cylinder viscometer. (4) Finally, the K+ leak pathway of normal, sickle and minimally-oxidized normal RBC will be compared under oxygenated and deoxygenated conditions, and during and after deformation. Under these conditions, K+ leak will be described in terms of actual pore size and pore number per cell (using differential seiving of radio-probes), the possible role of calcium, the ion selectivity sequence of the K+ leak pore, and its flux kinetics and activation energy.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL037528-02
Application #
3353266
Study Section
(SRC)
Project Start
1986-09-30
Project End
1989-12-31
Budget Start
1988-01-01
Budget End
1988-12-31
Support Year
2
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Type
Schools of Medicine
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Shalev, O; Hileti, D; Nortey, P et al. (1999) Transport of 14C-deferiprone in normal, thalassaemic and sickle red blood cells. Br J Haematol 105:1081-3
Browne, P; Shalev, O; Hebbel, R P (1998) The molecular pathobiology of cell membrane iron: the sickle red cell as a model. Free Radic Biol Med 24:1040-8
Sheng, K; Shariff, M; Hebbel, R P (1998) Comparative oxidation of hemoglobins A and S. Blood 91:3467-70
Cragg, L; Hebbel, R P; Miller, W et al. (1998) The iron chelator L1 potentiates oxidative DNA damage in iron-loaded liver cells. Blood 92:632-8
Shalev, O; Hebbel, R P (1996) Extremely high avidity association of Fe(III) with the sickle red cell membrane. Blood 88:349-52
Shalev, O; Hebbel, R P (1996) Catalysis of soluble hemoglobin oxidation by free iron on sickle red cell membranes. Blood 87:3948-52
Enright, H; Miller, W J; Hays, R et al. (1996) Preferential targeting of oxidative base damage to internucleosomal DNA. Carcinogenesis 17:1175-7
Browne, P V; Mosher, D F; Steinberg, M H et al. (1996) Disturbance of plasma and platelet thrombospondin levels in sickle cell disease. Am J Hematol 51:296-301
Shalev, O; Repka, T; Goldfarb, A et al. (1995) Deferiprone (L1) chelates pathologic iron deposits from membranes of intact thalassemic and sickle red blood cells both in vitro and in vivo. Blood 86:2008-13
Sugihara, T; Yawata, Y; Hebbel, R P (1994) Deformation of swollen erythrocytes provides a model of sickling-induced leak pathways, including a novel bromide-sensitive component. Blood 83:2684-91

Showing the most recent 10 out of 22 publications