Lipid A decreases human erythrocytes deformability by increasing intracellular Ca(2+): effects of verapamil, staurosporine and the rho-kinase inhibitor Y-27632.

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 (Ca(2+) 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 Ca(2+) concentration. For the deformation measurements RBC (10 adult donors) were incubated with E. coli F-583-Rd lipid A (100 μg/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 Ca(2+) was determinded by flow cytometry in RBC incubated with Lipid A and labeled with fluorescent Fluo-4/AM which binds intracellular Ca(2+) 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 Ca(2+). 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 Ca(2+) concentration. Verapamil and staurosporine abolished the decrease in deformability of Lipid A incubated RBC.