SPIN LABEL STUDY OF ERYTHROCYTE DEFORMABILITY Ca2"-Induced Loss of Deformability and the Effects of Stomatocytogenic Reagents on the Deformability Loss in Human Erythrocytes in Shear Flow SUMIHARE Noji, *$ SHIGEHIKO TANIGUCHI t AND HIDEO KON * *Laboratory of Chemical Physics, National Institute of Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892; and $Department of Biochemistry, Okayama University Dental School, Shikatacho, Okayama 700, Japan ABSTRACT The Ca2"-induced loss of deformability in human erythrocytes and the recovery of the lost deformability by stomatocytogenic reagents were investigated by means of a new flow electron paramagnetic resonance (EPR) spin label method, which provides information on deformation and orientation characteristics of spin labeled erythrocytes in shear flow. The Ca2"-induced loss of deformability is attributed mainly to the increase in intracellular viscosity resulting from efflux of intracellular potassium ions and water (Gardos effect). Partial recovery of the lost deformability is demonstrated in the presence of stomatocytogenic reagents, such as chlorpromazine, trifluoperazine, W-7, and calmidazolium (R2457 1). The recovery can not be explained solely by suppression of the Gardos effect due to the reagents. Incorporation of an optimal amount of the reagents into the membrane appears to compensate for the membrane modification due to Ca2+ ions to restore a part of the lost deformability. INTRODUCTION Human erythrocytes maintain a low concentration of intracellular Ca 2+ ions at -0. 1-1 uM by the calcium pump (Ca2`-Mg2`)-ATPase. When the intracellular Ca2" con- centration is increased by a treatment with the calcium ionophore A23187, a series of complex biochemical reac- tions occurs including the activation of (Ca2+-Mg2+)- ATPase by Ca2+-binding calmodulin (21, 28), the modifi- cation of the metabolism and composition of membrane lipids (1-3, 13), and the efflux of K+ ions and water (28, 30) (Gardos effect [16]). It is also known that with an increase in the intracellular Ca 2+ ion concentration, the cells suffer from a partial loss of the whole cell deformabil- ity (11, 20, 22, 26, 40, 43). The erythrocyte deformability is assumed to be generally determined by the following factors: (a) physical properties of the membrane and cytoskeleton (e.g., elasticity); (b) the state of the cytoplasm (e.g., viscosity, hemoglobin concentration and aggrega- tion); and (c) the geometry of the cell (e.g., shape, the surplus of the membrane surface area of the cell relative to its given volume) (14, 19, 23, 32, 35). Of the three factors, the second has been proposed in the past to be the principal mechanism of the Ca 2+-induced loss of deformability as the result of the cell dehydration and the consequent high internal viscosity (increase in hemoglobin concentration), Correspondence should be addressed to Dr. H. Kon, Room B1-14, Building 2, National Institutes of Health, Bethesda, MD 20892. BIOPHYS. J. © Biophysical Society * 0006-3495/87/08/221/07 Volume 52 August 1987 221-227 because no loss of deformability was observed in a high potassium medium in which dehydration and K+ effilux are prevented (11, 20). Since it was reported recently that some stomatocytog- enic calmodulin inhibitors are able to suppress the Ca2+- activated K+ efflux in both intact cells and inside-out vesicles (27, 38, 39), it is expected that these reagents may indeed suppress the Ca 2k-induced deformability loss with concomitant inhibition of the Gardos effect. In the present work, we used the stomatocytogenic calmodulin inhibitors chlorpromazine (CPZ), trifluopera- zine (TFP), W-7 (N-[6-aminohexyl]-5-chloro-1-naphthal- ene sulfonamide), and calmidazolium (CMZ) (R24571, 1- [bis(p-chlorophenyl)methyl]-3- [2,4-dichloro-f-(2,4- dichlorobenzyloxyl)phenethyl]imidazolinium chloride) in an attempt to suppress the Ca 2k-induced K+ efflux, and examine the effects of such reagents on deformability loss induced by Ca 2+ loading. To measure the deformability of red blood cells in laminar shear flow, we have utilized the electron paramagnetic resonance (EPR) spin label tech- nique, combined with a computer-regulated variable flow device (23-25, 34, 35). It has been demonstrated experi- mentally and theoretically that the change in the EPR spectrum of a spin-labeled cell suspension observed in shear flow as compared with that at rest is closely related to the whole cell deformation and orientation (7-9, 23, 34, 35, 37). The method provides a means to assess the degree of deformation and orientation as a function of volume flow rate in intact as well as variously treated cells. $2.00 221