Vinculin phosphorylation and barrier failure of coronary endothelial monolayers under energy depletion.
We studied the hypothesis that, in energy-depleted endothelial cells, Ca(2+)-dependent activation of protein kinase C (PKC) causes phosphorylation of vinculin and that this effect is involved in the early loss of endothelial barrier function. Vinculin localization and phosphorylation, PKC activity, and albumin permeability were studied in cultured coronary endothelial monolayers from rats. Ten minutes after the onset of metabolic inhibition by 5 mM potassium cyanide and 5 mM 2-deoxy-D-glucose, immunofluorescence of vinculin at cell-to-cell and cell-to-matrix contacts faded, whereas total cellular vinculin content remained unchanged. During the same time period, vinculin phosphorylation at tyrosine and serine sites increased by 3.9- and 3.5-fold, respectively. Vinculin phosphorylation was related to activation of PKC and an unidentified tyrosine kinase and was elicited by a rise in cytosolic Ca2+ within energy-depleted endothelial cells. Conditions inhibiting vinculin phosphorylation also reduced monolayer permeability induced by energy depletion. These data indicate that vinculin phosphorylation is involved in the progression of hyperpermeability during energy depletion in coronary endothelial monolayers.
Muhs
A
A
Physiologisches Institut, Justus-Liebig-Universität, Giessen, Germany.
Noll
T
T
Piper
H M
HM
D000818:Animals; D002199:Capillary Permeability; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004734:Energy Metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D007211:Indoles / Q000494:pharmacology; D008297:Male; D008301:Maleimides / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012709:Serum Albumin / Q000493:pharmacokinetics; D016596:Vinculin / Q000378:metabolism*
D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D012709:Serum Albumin; D016596:Vinculin; D011493:Protein Kinase C; C088060:bisindolylmaleimide