Inhibition of contractile activation reduces reoxygenation-induced endothelial gap formation.
OBJECTIVE
Barrier function of coronary endothelium becomes disturbed by ischemia-reperfusion. We investigated the mechanism of reperfusion-induced endothelial gap formation in monolayers of cultured endothelial cells (CEC) of the rat, exposed to simulated ischemia (40 min anoxia, pH(o) 6.4) and reperfusion (30 min reoxygenation, pH(o) 7.4).


METHODS
Cytosolic Ca(2+) (fura-2) and intercellular gap formation (planimetrical analysis) were determined. Reoxygenation conditions were varied: (a) continuing perfusion at pH(o) 6.4, (b) with or without glucose (2.5 mM), (c) in presence of NaCN (2 mM), (d) with Ca(2+) (10 mM) or BAPTA/AM (25 microM), (e) in the presence of myosin light chain kinase inhibitors ML-7 (5 microM) or wortmannin (1 microM).


RESULTS
During anoxia, CEC developed cytosolic Ca(2+) overload which was not reversed during 30 min reoxygenation. Intercellular gap formation started during anoxia, but was increased during reoxygenation. Reoxygenation-related gap formation was largest in presence of glucose, lower when glucose was withdrawn or NaCN was added. Presence of ML-7 or wortmannin also reduced gap formation during reoxygenation.


CONCLUSIONS
Reoxygenation induces gap formation. This is dependent on (i) Ca(2+) overload during reoxygenation, (ii) energy production and (iii) activation of myosin light chain kinase. Together these results indicate that activation of the endothelial contractile machinery is the underlying cause.
Schäfer
C
C
Institute of Physiology, Justus-Liebig-University Giessen, Germany.
Walther
S
S
Schäfer
M
M
Dieterich
L
L
Kasseckert
S
S
Abdallah
Y
Y
Piper
H M
HM
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001381:Azepines / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003331:Coronary Vessels; D003600:Cytosol / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D008297:Male; D015428:Myocardial Reperfusion Injury / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors; D009281:Naphthalenes / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D012966:Sodium Cyanide / Q000494:pharmacology; D000077191:Wortmannin
D000730:Androstadienes; D001381:Azepines; D004791:Enzyme Inhibitors; D009281:Naphthalenes; C070571:ML 7; D009219:Myosin-Light-Chain Kinase; D005947:Glucose; D012966:Sodium Cyanide; D002118:Calcium; D000077191:Wortmannin