Disintegration of cytoskeletal structure of actin filaments in energy-depleted endothelial cells.
In a previous study [Watanabe, H., W. Kuhne, R. Spahr, P. Schwartz, and H. M. Piper. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H1344-H1352, 1991] metabolic inhibition (5 mM KCN + 5 mM 2-deoxy-D-glucose, for 2 h) was found to cause disintegration of F-actin filaments, cell retraction, and augmented paracellular macromolecule permeability in monolayer cultures of porcine aortic endothelial cells after a rapid depletion of ATP stores (90% in 5 min). These changes were reversible. In the present study, the nature of this cytoskeletal disintegration was investigated. 1) Disintegration of F-actin filaments within 2-h incubation under metabolic inhibition was accompanied by appearance of F-actin clumps in the cells, but total contents of F-actin remained unaltered. 2) Cytosolic Ca2+ levels rapidly rose in metabolically inhibited cells; after 2 h a 10-fold increase was observed. 3) Presence of the Ca2+ ionophore A23187 (10 microM) mimicked the reversible effect of metabolic inhibition on F-actin filaments and monolayer permeability but not the extensive depletion of ATP stores. 4) Existence of the Ca(2+)-activatable actin-severing protein gelsolin in endothelial cells was demonstrated. The results show that during the reversible phase of endothelial energy depletion disintegration of F-actin filaments is only partial, since it is based on their fragmentation and not depolymerization. Increase in cytosolic Ca2+ levels seems to be the primary cause for the fragmentation, possibly through the activation of gelsolin.
Kuhne
W
W
Physiologisches Institut I, Universität Düsseldorf, Germany.
Besselmann
M
M
Noll
T
T
Muhs
A
A
Watanabe
H
H
Piper
H M
HM
D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000648:ultrastructure*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure*; D004734:Energy Metabolism; D018260:Gelsolin; D046911:Macromolecular Substances; D008840:Microfilament Proteins / Q000378:metabolism
D000199:Actins; D002135:Calcium-Binding Proteins; D018260:Gelsolin; D046911:Macromolecular Substances; D008840:Microfilament Proteins; D000001:Calcimycin; D000255:Adenosine Triphosphate; D002118:Calcium