Lipid A decreases human erythrocytes deformability by increasing intracellular Ca2+: Effects of verapamil, staurosporine and the rho-kinase inhibitor Y-27632
Issue title: Selected Proceedings of the 16th Conference of the European Society for Clinical Hemorheology and Microcirculation (ESCHM), 18–21 June, 2011, Munich, Germany
Affiliations: Clinic of Neonatology, Department of Pediatrics, University of Heidelberg, Heidelberg, Germany
Note: [] Corresponding author: PD Dr. med. P. Ruef, Klinik für Neonatologie, Zentrum für Kinder- und Jugendmedizin, Im Neuenheimer Feld 430, 69120 Heidelberg, Germany. Tel.: +49(0)6221 561983; Fax: +49(0)6221 565071; E-mail: [email protected]
Note: [] These authors contributed equally to this work.
Note: [] These authors contributed equally to this work.
Abstract: There are several reports demonstrating an involvement of bacterial toxins in the rigidity of red blood cells (RBC). The present study investigates the influence of E. coli F-583-Rd lipid A on RBC deformability under mechanical shear stress. Verapamil (Ca2+ channel inhibitor), staurosporine (protein kinase inhibitor) and Y-27632 (rho-kinase inhibitor) were used to modify the effect of lipid A on RBC deformability. We also determined if E. coli F-583-Rd Lipid A could induce an increase of intracellular Ca2+ concentration. For the deformation measurements RBC (10 adult donors) were incubated with E. coli F-583-Rd lipid A (100μg/ml) and also co-incubated with either verapamil (10−7 mol/l), staurosporine (10−7 mol/l) or Y-27632 (10−7 mol/l). The deformation of the RBC under different shear stresses (0.6–60 Pa) was measured by a shear stress diffractometer (Rheodyne SSD). Intracellular Ca2+ was determinded by flow cytometry in RBC incubated with Lipid A and labeled with fluorescent Fluo-4/AM which binds intracellular Ca2+ with high affinity resulting in enhanced green fluorescence intensity. At increasing shear stresses Lipid A induced a significantly lower elongation. Co-incubation of the erythrocytes with verapamil or staurosporine inhibited lipid A induced decrease in elongation while Y-27632 had no effect. Verapamil, Staurosporine and Y-27632 did not influence the elongation response of the cells under control conditions. Lipid A induced a marked increase in fluorescence Fluo-4/AM indicating increased intracellular Ca2+. These results suggest that E. coli F-583-Rd lipid A is able to influence red blood cell rigidity by a rapid and significant increase of intracellular Ca2+ concentration. Verapamil and staurosporine abolished the decrease in deformability of Lipid A incubated RBC.
Keywords: Red blood cell, sepsis, lipid A, Ca$^{2+}$, deformability, staurosporine, verapamil, rho-kinase-inhibitor
