Disintegration of cytoskeletal structure of actin filaments in energy-depleted endothelial cells

Abstract

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.

Cadherin function probed by laser tweezer and single molecule fluorescence in vascular endothelial cells

“…These observations are in line with studies reporting disturbance of junctional integrity of endothelial and epithelial monolayers by both, cytochalasin D (Hirano et al, 1987;Stevensen and Begg, 1994;Nybom and Magnusson, 1986;Drenckhahn and Ness, 1997) and A23187 (Suttorp et al, 1989;Kuhne et al, 1993;Schnittler et al, 1997;Michel and Curry, 1999). A23187-induced barrier breakdown was suggested to be caused by Ca 2+ -calmodulinmediated myosin-based contractility (Schnittler et al, 1990;Goeckler and Wysolmerski, 1995) and by Ca 2+ -gelsolininduced actin depolymerisation (Kuhne et al, 1993). Whereas these studies could not discriminate between an essential facilitative role of cytoskeletal activity on overall monolayer structure [cytochalasin-treated cells undergo shape changes and display contraction of stress fibres and of actin-myosin-based gels (Kolega et al, 1991)] versus a direct modulatory action of the actin-based cytoskeleton on adhesive strength, the laser tweezer experiments performed in the present study allow to conclude that drug-induced depolymerisation of actin exerts a direct inhibitory action on cadherin-mediated adhesion.…”

“…Stabilisation of F-actin by pretreatment of cells with jasplakinolide prevented drug-induced weakening of bead adhesion demonstrating that both, cytochalasin D and A23187 mediate anti-adhesive activity via destabilisation of the actin filament system. These observations are in line with studies reporting disturbance of junctional integrity of endothelial and epithelial monolayers by both, cytochalasin D (Hirano et al, 1987;Stevensen and Begg, 1994;Nybom and Magnusson, 1986;Drenckhahn and Ness, 1997) and A23187 (Suttorp et al, 1989;Kuhne et al, 1993;Schnittler et al, 1997;Michel and Curry, 1999). A23187-induced barrier breakdown was suggested to be caused by Ca 2+ -calmodulinmediated myosin-based contractility (Schnittler et al, 1990;Goeckler and Wysolmerski, 1995) and by Ca 2+ -gelsolininduced actin depolymerisation (Kuhne et al, 1993).…”

“…In the present study we provide direct evidence that two permeability-increasing compounds known to cause gradual opening of endothelial junctions, that is, the inhibitor of actin polymerisation, cytochalasin D, and the Ca 2+ -ionophore A23187 (Suttorp et al, 1989;Schnittler et al, 1990;Kuhne et al, 1993;Drenckhahn and Ness, 1997;Dejana, 1997;Hordijk et al, 1999;Michel and Curry, 1999), have a strong negative impact on the adhesion of VE-cadherin-coated microbeads to the surface of cultured endothelial cells. Drug-induced reduction of adhesion was not caused by changes of the affinity for trans-interaction but depended on the decay of F-actin, and correlated with the increase of the lateral mobility of cadherins and dispersal of cadherin-enriched plasmalemmal microdomains.…”

“…Treatment of monolayers and microperfused capillaries with the Ca 2+ -ionophore A23187 (1-10 µM) resulted in similar changes (gap formation, increase in permeability), which was explained by Ca 2+ -stimulated contraction of the cellular actomyosin system, which was assumed to be strong enough to pull the cells apart by overcoming cadherin-mediated adhesion (Suttorp et al, 1989;Schnittler et al, 1990;Goeckeler and Wysolmerski, 1995). However, another model for explaining A23187-induced junctional dissociation implies Ca 2+ -gelsolininduced severing of F-actin as a major cause for junctional barrier breakdown (Kuhne et al, 1993). To obtain direct insight in the effect of cytochalasin D and A23187 on cadherin-mediated adhesion, we determined binding of VE-cadherin-coated beads to MyEnd-cells preincubated for 30 minutes with 10 µM cytochalasin D and 10 µM A23187, respectively.…”

Protein phosphatase inhibitors and heat preconditioning prevent Hsp27 dephosphorylation, F‐actin disruption and deterioration of morphology in ATP‐depleted endothelial cells

“…In vivo, depletion of cellular ATP can take place under acute ischemia and in case of surgical operations connected with bypass perfusion or transplantation of organs. The Factin collapse within ATP-deprived EC leads to dysfunction of the endothelial barrier that aggravates ischemic lesions [23,24]. Evidently, EC tolerance to lack of ATP should improve outcome of an ischemic onset as well as recovery after 'surgical stress'.…”