Influence of calcium permeabilization and membrane‐attached hemoglobin on erythrocyte deformability

Abstract

The present study was designed to evaluate the influence of intracellular calcium [Ca]i regulated membrane attached hemoglobin (Hbm) on the deformability of human RBC and ghosts. [Ca]i of RBC was elevated via the ionophore A23187 (10 microM); the deformability of RBC and resealed ghosts was determined via measuring RBC and ghost transit times through 5 microns diameter pores with the Cell Transit Analyzer (CTA). Salient results included: (1) significantly increased RBC levels of Hbm following ionophore treatment; (2) elevated Hbm with increasing lysing medium calcium concentration (0-5 mM); (3) decreased deformability of both intact RBC and ghosts with increasing Hbm and significant (P less than 0.02 or better) linear relationships between Hbm and RBC or ghost transit times; and (4) an increased sensitivity to ionophore treatment/membrane attached hemoglobin for the higher percentiles of the CTA transit time distribution (i.e., for more rigid subpopulations). Our results thus indicate that calcium-induced interaction of hemoglobin with the RBC membrane produces cellular rheological changes; in addition, they demonstrate the usefulness of the CTA system in measuring both average RBC rheologic behavior and the distribution of cellular rheologic properties within an erythrocyte population.

Ryanodine receptor 1 mediates Ca2+ transport and influences the biomechanical properties in RBCs

“…It is accepted now that RyR channels are activated by Ca 2 + at nanomolar level and inhibited by Ca 2 + at micromolar level (Lai et al, 1988;Bull and Marengo, 1993;Wayne Chen et al, 1997;Sonnleitner et al, 1998;Friederichs et al, 2006). Several groups reported that the activity of RyR1 could be almost entirely inhibited as [Ca 2 + ] ex concentration rises to 1 mM (Bull and Marengo, 1993;Yin et al, 2005).…”

“…The lipid peroxidation, proteins cross-linking and the attachment of hemoglobins increase the membrane viscoelasticity. Evidence shows that [Ca 2 + ] in increase also causes the perturbations of membrane skeletal protein network (Friederichs et al, 2006), which would give rise to membrane instability and lead to the loss of cell surface area and then the deformability, and even cell lysis (Zail, 1986;Kalfa et al, 2006). It was also reported that [Ca 2 + ] in could affect the elastic modulus and RBC deformability through changing the transmembrane potential (Trayko and Rakesh, 1986).…”

“…The elevation of [Ca 2 + ] in causes the increase of cytoplasmic viscosity and irreversible alterations in the membrane including peroxidation of membrane, cross-linking and degradation of membrane proteins and leads to the attachment of hemoglobins onto the membrane (Smith et al, 1981;Friederichs et al, 2006;Friederichs and Meiselman, 1994). All biphasic variations of RBC's mechanic properties in responding for the changes of [Ca 2 + ] in with [Ca 2 + ] ex increase, in our study, may be ascribed to these biochemical and biophysical changes in the membrane.…”

“…Of the three RyRs, RyR1 is the most well characterized one (Wayne Chen et al, 1997;Yin et al, 2008 the membrane. Previous in vitro studies indicated that the calcium ionophore, such as A23187, could facilitate Ca 2 + transport across the membrane in RBCs under in vitro condition (Tanabe and Doi, 1989;Mark et al, 2000;Wang et al, 2001;Friederichs et al, 2006). In these experiments, the researchers incubated RBCs with A23187/Ca 2 + containing buffer, and found that [Ca 2 + ] in levels increased transiently.…”

Isolation of Protein Subpopulations Undergoing Protein-Protein Interactions

“…Although hemoglobin binding to other hemoglobin subunits depends on Fe 2ϩ and O 2 , it is not known to bind calcium; however, hemoglobin can bind to reticulocyte membranes in the presence of calcium (32), suggesting that it may be a partner for some other calcium binding membrane-bound protein. Similarly, it is possible that citrate synthase and carbonic anhydrase can associate with some other as yet uncharacterized protein in the presence of calcium.…”