CYTOSKELETAL MODULATION OF THE RESPONSE TO MECHANICAL STIMULATION IN HUMAN VASCULAR ENDOTHELIAL-CELLS

Possible interactions of cytoskeletal elements with mechanically induc ed membrane currents and Ca2+ signals were studied in human endothelia l cells by using a combined patch-clamp and Fura II technique. for mec hanical stimulation, cells were exposed to hypotonic solution (HTS). T he concomitant cell swelling activates as Cl- current releases Ca2+ fr om intracellular stores and activates Ca2+ influx. To interfere with t he cytoskeleton, cells were loaded either with the F-actin-stabilizing agent phalloidin (10 mu mol/l), or the F-actin-depolymerizing substan ce cytochalasin B (50 mu mol/l). These were administered either in the bath or the pipette solutions. The tubulin structure of the endotheli al cells was modulated by taxol (50 mu mol/l), which supports polymeri zation of tubulin, or by the depolymerizing agent colcemid (10 mu mol/ l) both applied to the bath. Immunofluorescence experiments show that under the chosen experimental conditions the cytoskeletal modifiers em ployed disintegrate the F-actin and microtubuli cytoskeleton. Neither of these cytoskeletal modifiers influenced the HTS-induced Cl- current . Ca2+ release was not affected by cytochalasin B, taxol or colcemid, but was suppressed if the cells were loaded with phalloidin. Depletion of intracellular Ca2+ stores by thapsigargin renders intracellular [C a2+] sensitive to the extracellular [Ca2+], which is indicative of a C a2+ entry pathway activated by store depletion. Neither cytochalasin B nor phalloidin affected this Ca2+ entry. We conclude that F-actin tur nover or depolymerization is necessary for Ca2+ release by mechanical activation. The tubulin network is not involved. The Ca2+ release-acti vated Ca2+ entry is not modulated by the F-actin cytoskeleton.