In this competing renewal application, we propose to extend our previous work on the uncoupling of the lipid bilayer from the underlying skeleton during sickling, with large areas of the lipid bilayer being directly laminated by polymerized hemoglobin S. We now propose to test a hypothesis that such membrane regions represent critical sites of sickle cell membrane injury. Such injury may lead to localized alterations in (a) lipid bilayer asymmetry and lipid loss, (b) aggregation of band 3 protein, the mobility of which is no longer restrained by the skeleton, (c) aggregation of glycophorin followed by surface charge clustering, and (d) increased membrane cation permeability. To examine this hypothesis, we propose the following studies: (a) Are changes in phospholipid asymmetry confined to the regions of lipid-skeleton uncoupling? This will be studied by visualizing regions of altered lipid organizations with merocyanine fluorescence (which principally measures lipid packing) as well as autoradiography of 14C lysophosphatidylcholine. (b) Are surface charges redistributed in the areas of lipid bilayer uncoupling? Surface charge will be visualized by cationized ferritin electron microscopy. (c) Is band 3 preferentially clustered in the skeleton free areas: Band 3 clustering will be studied by freeze fraction electron microscopy, as well as ultrastructural and chemical studies of band 3 aggregates in the membranes and membrane skeletons, including a chemical crosslinking of the band 3 in the membrane. (d) Are the sickling induced alterations in cation permeability confined to skeleton free areas? Passive fluxes of 45Ca will be studied by autoradiography and by use of fluorescent probes. In addition to studies in sickle cells, we will employ a model system in which attachment of normal red cells to nucleopore filters under a negative pressure produces deformation with long tongue-like protrusions at the tip of which the lipid bilayer separates from the underlying skeleton. In this system, changes in the monovalent cation permeability and lipid bilayer asymmetry in the deformed membrane regions can be readily compared to the parts of the cell membrane which are morphologically intact. If indeed the membrane protrusions of sickled cells represent the major site of membrane damage, it is likely that conditions which minimize the sickle cell deformation will diminish, or event prevent, the above outlined membrane abnormalities. To test the role of the degree of morphological sickling, we propose the following studies: (a) Studies of hemoglobin-red cell ghost hybrids employing ghosts of cells with Southeast Asian ovalocytosis, which have very rigid membranes. (b) Pretreatment of sickle cells or normal membranes containing encapsulated hemoglobin S with agents which increase membrane rigidity by stabilizing the skeleton (mild crosslinking agents, antiglycophorin antibodies) or agents altering lipid bilayer asymmetry. (c) Conditions which inhibit binding of hemoglobin S to the membrane. (d) Lastly, the degree of morphological sickling will be modified by varying the rate of deoxygenation or the osmolarity to the suspending medium.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Comprehensive Center (P60)
Project #
5P60HL015157-26
Application #
6241521
Study Section
Project Start
1997-04-01
Project End
1998-03-31
Budget Start
1996-10-01
Budget End
1997-09-30
Support Year
26
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Boston Medical Center
Department
Type
DUNS #
005492160
City
Boston
State
MA
Country
United States
Zip Code
02118
Steinberg, Martin H; Sebastiani, Paola (2012) Genetic modifiers of sickle cell disease. Am J Hematol 87:795-803
Loscalzo, Joseph (2012) Irreproducible experimental results: causes, (mis)interpretations, and consequences. Circulation 125:1211-4
Vandorpe, David H; Xu, Chang; Shmukler, Boris E et al. (2010) Hypoxia activates a Ca2+-permeable cation conductance sensitive to carbon monoxide and to GsMTx-4 in human and mouse sickle erythrocytes. PLoS One 5:e8732
Jahangir, Eiman; Vita, Joseph A; Handy, Diane et al. (2009) The effect of L-arginine and creatine on vascular function and homocysteine metabolism. Vasc Med 14:239-48
Casula, Sabina; Zolotarev, Alexander S; Stuart-Tilley, Alan K et al. (2009) Chemical crosslinking studies with the mouse Kcc1 K-Cl cotransporter. Blood Cells Mol Dis 42:233-40
Steinberg, Martin H; Voskaridou, Ersi; Kutlar, Abdullah et al. (2003) Concordant fetal hemoglobin response to hydroxyurea in siblings with sickle cell disease. Am J Hematol 72:121-6
Ware, Russell E; Eggleston, Barry; Redding-Lallinger, Rupa et al. (2002) Predictors of fetal hemoglobin response in children with sickle cell anemia receiving hydroxyurea therapy. Blood 99:10-4
Mirchev, R; Golan, D E (2001) Single-particle tracking and laser optical tweezers studies of the dynamics of individual protein molecules in membranes of intact human and mouse red cells. Blood Cells Mol Dis 27:143-7
Jiang, L; Jha, V; Dhanabal, M et al. (2001) Intracellular Ca(2+) signaling in endothelial cells by the angiogenesis inhibitors endostatin and angiostatin. Am J Physiol Cell Physiol 280:C1140-50
Shmukler, B E; Brugnara, C; Alper, S L (2000) Structure and genetic polymorphism of the mouse KCC1 gene. Biochim Biophys Acta 1492:353-61

Showing the most recent 10 out of 39 publications