Cytoskeletal modulation of the response to mechanical stimulation in human vascular endothelial cells

Masahiro Oike, Gero Schwarz, Jan Sehrer, Matthias Jost, Volker Gerke, Klaus Weber, Guy Droogmans, Bernd Nilius

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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 μmol/l), or the F-actin-depolymerizing substance cytochalasin B (50 μmol/l). These were administered either in the bath or the pipette solutions. The tubulin structure of the endothelial cells was modulated by taxol (50 μmol/l), which supports polymerization of tubulin, or by the depolymerizing agent colcemid (10 μmol/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.

Original languageEnglish
Pages (from-to)569-576
Number of pages8
JournalPflügers Archiv European Journal of Physiology
Volume428
Issue number5-6
DOIs
Publication statusPublished - Oct 1 1994
Externally publishedYes

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All Science Journal Classification (ASJC) codes

  • Physiology
  • Clinical Biochemistry
  • Physiology (medical)

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