Cytoskeletal modulation of the response to mechanical stimulation in human vascular endothelial cells.
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 mumol/l), or the F-actin-depolymerizing substance cytochalasin B (50 mumol/l). These were administered either in the bath or the pipette solutions. The tubulin structure of the endothelial cells was modulated by taxol (50 mumol/l), which supports polymerization of tubulin, or by the depolymerizing agent colcemid (10 mumol/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.
KU Leuven, Laboratorium voor Fysiologie, Belgium.
M
M
Oike
G
G
Schwarz
J
J
Sehrer
M
M
Jost
V
V
Gerke
K
K
Weber
G
G
Droogmans
B
B
Nilius
D000199:Actins; D002118:Calcium / Q000502:physiology; D002712:Chlorides / Q000502:physiology; D003571:Cytochalasin B / Q000494:pharmacology; D003599:Cytoskeleton / Q000502:physiology*; D004730:Endothelium, Vascular / Q000502:physiology*; D006801:Humans; D007038:Hypotonic Solutions / Q000494:pharmacology; D010590:Phalloidine / Q000494:pharmacology
D000199:Actins; D002712:Chlorides; D007038:Hypotonic Solutions; D010590:Phalloidine; D003571:Cytochalasin B; D002118:Calcium