Abstract

In this study, we characterized the effects of the protein phosphatases type 1 (PP 1) and type 2A (PP 2A) inhibitor cantharidin in endothelial cells. We identified catalytic subunits of PP 1α, PP 2Aα, and PP 2Aβ immunologically in bovine aortic endothelial cells. Moreover, we detected mRNAs coding for catalytic subunits of PP 1α, PP 1β, and PP 2Aα by hybridization with specific DNA probes in total RNA from these cells. Okadaic acid and cantharidin inhibited the activities of catalytic subunits of PP 1 (okadaic acid, 0.01–1 μM; cantharidin, 1–100 μM) and PP 2A (okadaic acid, 0.1 nM to 1 μM; cantharidin, 0.1–100 μM) separated by column chromatography in a concentration-dependent manner. Moreover, cantharidin (1 μM to 1 mM) increased the phosphorylation state of endothelial proteins including the regulatory light chains of myosin without affecting cytosolic calcium concentrations. Cantharidin (5–100 μM) increased the permeability of cultured endothelial cells in a time- and concentration-dependent manner. We suggest that inhibition of PP 1 and PP 2A activities by cantharidin increases endothelial permeability by enhancing the phosphorylation state of endothelial regulatory proteins. Thus, cantharidin might be a useful tool to study the function of protein phosphatases in endothelial barrier function.