THE ROLE OF CADHERIN ENDOCYTOSIS IN ENDOTHELIAL BARRIER REGULATION - INVOLVEMENT OF PROTEIN-KINASE-C AND ACTIN-CADHERIN INTERACTIONS

We have previously reported that exposure of endothelial monolayers to low (0.12 mM) extracellular calcium significantly decreased the endot helial solute barrier, and that this effect was reversed by restoring 'normal' (1.2 mM) calcium (1). This effect was shown to be dependent o n cadherins, however the molecular mechanisms through which barrier wa s altered by low calcium were not characterized. Here we investigated the mechanism of increased endothelial permeability produced by low ca lcium exposure. Endothelial permeability was significantly increased b y exposure to low (0.12 mM) calcium; this effect was attenuated by pre -treatment with the protein kinase C (PKC) inhibitor, staurosporine (2 x 10(-7) M) for 30 min. Cell border retraction and gap formation prod uced by low calcium was also prevented by staurosporine. Treatment of monolayers with 0.12 mM calcium also stimulated the endocytosis of end othelial cadherins. This low calcium mediated cadherin endocytosis was also prevented by pretreatment with staurosporine. Low calcium mediat ed endocytosis was also prevented by the actin filament toxin, cytocha lasin D (1 ug/ml, 30 min). We conclude that the mechanism of low calci um mediated loss of endothelial barrier function is mediated in part b y a PKC dependent endocytosis of endothelial cadherins, which may invo lve interactions with the actin cytoskeleton. Physiological regulation of the in vivo endothelial barrier may also involve PKC dependent-act in mediated endocytosis of cadherin junctional elements.