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.
Clinic of Neonatology, Department of Pediatrics, University of Heidelberg, Heidelberg, Germany. peter.ruef@tonline.de
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Ruef
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Ehrhard
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Frommhold
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Koch
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Fritzsching
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Poeschl
D000328:Adult; D000577:Amides / Q000494:pharmacology*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D020013:Calcium Signaling / Q000187:drug effects*; D003864:Depression, Chemical; D004347:Drug Interactions; D004907:Erythrocyte Deformability / Q000187:drug effects*; D004926:Escherichia coli / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D008050:Lipid A / Q000302:isolation & purification / Q000494:pharmacology*; D008297:Male; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology*; D033081:Shear Strength; D019311:Staurosporine / Q000494:pharmacology*; D014700:Verapamil / Q000494:pharmacology*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors*
D000577:Amides; D002121:Calcium Channel Blockers; D008050:Lipid A; D047428:Protein Kinase Inhibitors; D011725:Pyridines; C108830:Y 27632; D014700:Verapamil; D054460:rho-Associated Kinases; D019311:Staurosporine