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Phorbol esters increase MLC phosphorylation and actin remodeling in bovine lung endothelium without increased contraction

Division of Pulmonary and Critical Care Medicine, Center for Translational Regulatory Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224

Submitted 21 September 2001 ; accepted in final form 16 April 2003

Direct protein kinase C (PKC) activation with phorbol myristate acetate (PMA) results in the loss of endothelial monolayer integrity in bovine lung endothelial cells (EC) but produces barrier enhancement in human lung endothelium. To extend these findings, we studied EC contractile events and observed a 40% increase in myosin light chain (MLC) phosphorylation in bovine endothelium following PMA challenge. The increase in PMA-mediated MLC phosphorylation occurred at sites distinct from Ser¹⁹/Thr¹⁸, sites catalyzed by MLC kinase (MLCK), and immunoblotting with antibodies specific to phosphorylated Ser¹⁹/Thr¹⁸ demonstrated profound time-dependent Ser¹⁹/Thr¹⁸ dephosphorylation. These events occurred in conjunction with rearrangement of stress fibers into a grid-like network, but without an increase in cellular contraction as measured by silicone membrane wrinkling assay. The PMA-induced MLC dephosphorylation was not due to kinase inhibition but, rather, correlated with rapid increases in myosin-associated phosphatase 1 (PPase 1) activity. These data suggest that PMA-mediated EC barrier regulation may involve dual mechanisms that alter MLC phosphorylation. The increase in bovine MLC phosphorylation likely occurs via direct PKC-dependent MLC phosphorylation in conjunction with decreases in Ser¹⁹/Thr¹⁸ phosphorylation catalyzed by MLCK due to PMA-induced increases in PPase 1 activity. Together, these events result in stress fiber destabilization and profound actin rearrangement in bovine endothelium, which may result in the physiological alterations observed in these models.

phorbol myristate acetate; myosin light chain kinase; myosin phosphatase; protein kinase C

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