VINCULIN PHOSPHORYLATION AND BARRIER FAILURE OF CORONARY ENDOTHELIAL MONOLAYERS UNDER ENERGY DEPLETION

We studied the hypothesis that, in energy-depleted endothelial cells, Ca2+-dependent activation of protein kinase C (PKC) causes phosphoryla tion of vinculin and that this effect is involved in the early loss of endothelial barrier function. Vinculin localization and phosphorylati on, PKC activity, and albumin permeability were studied in cultured co ronary endothelial monolayers from rats. Ten minutes after the onset o f metabolic inhibition by 5 mM potassium cyanide and 5 mM 2-deoxy-D-gl ucose, immunofluorescence of vinculin at cell-to-cell and cell-to-matr ix contacts faded, whereas total cellular vinculin content remained un changed. During the same time period, vinculin phosphorylation at tyro sine and serine sites increased by 3.9- and 3.5-fold, respectively. Vi nculin phosphorylation was related to activation of PKC and an unident ified tyrosine kinase and was elicited by a rise in cytosolic Ca2+ wit hin energy-depleted endothelial cells. Conditions inhibiting vinculin phosphorylation also reduced monolayer permeability induced by energy depletion. These data indicate that vinculin phosphorylation is involv ed in the progression of hyperpermeability during energy depletion in coronary endothelial monolayers.