Cytoskeletal modulation of the response to mechanical stimulation in human vascular endothelial cells |
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Authors: | Masahiro Oike Gero Schwarz Jan Sehrer Matthias Jost Volker Gerke Klaus Weber Guy Droogmans Bernd Nilius |
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Institution: | (1) Laboratorium voor Fysiologie, Campus Gasthuisberg, KU Leuven, B-3000 Leuven, Belgium;(2) Department of Biochemistry, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany |
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Abstract: | Possible interactions of cytoskeletal elements with mechanically induced membrane currents and Ca2+ signals were studied in human endothelial cells by using a combined patch-clamp and Fura II technique. For mechanical stimulation, cells were exposed to hypotonic solution (HTS). The concomitant cell swelling activates a Cl– current, releases Ca2+ from intracellular stores and activates Ca2+ influx. To interfere with the cytoskeleton, cells were loaded either with the F-actin-stabilizing agent phalloidin (10 mol/l), or the F-actin-depolymerizing substance cytochalasin B (50 mol/l). These were administered either in the bath or the pipette solutions. The tubulin structure of the endothelial cells was modulated by taxol (50 mol/l), which supports polymerization of tubulin, or by the depolymerizing agent colcemid (10 mol/l) both applied to the bath. Immunofluorescence experiments show that under the chosen experimental conditions the cytoskeletal modifiers employed 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 the intracellular Ca2+] sensitive to the extracellular Ca2+], which is indicative of a Ca2+ entry pathway activated by store depletion. Neither cytochalasin B nor phalloidin affected this Ca2+ entry. We conclude that F-actin turnover or depolymerization is necessary for Ca2+ release by mechanical activation. The tubulin network is not involved. The Ca2+ release-activated Ca2+ entry is not modulated by the F-actin cytoskeleton. |
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Keywords: | Endothelium Patch clamp Intracellular Ca2+ Cytoskeleton Phalloidin Cytochalasin B Taxol Colcemid Mechanosensitivity |
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