Mechanisms of phorbol ester-induced endothelial cells (EC) barrier dysfunction

Receptor-mediated agonists such as thrombin increase EC permeability via a sequence involving myosin light chain kinase (MLCK) activation, myosin light chain (MLC) phosphorylation, actin-myosin complex formation, contraction and intercellular gap formation. Although phorbol myristate acetate (PMA), a direct protein kinase C (PKC) activator, also induces gap formation and compromises barrier integrity, our recent evidence utilizing urea gel electrophoresis suggested that PMA-induced EC barrier dysfunction does not involve MLCK-mediated MLC phosphorylation. To investigate mechanisms regulating PMA-induced EC barrier dysfunction, we studied the effect of PMA on the distribution of key EC contractile proteins in the detergent (1% NP-40)-soluble (cytoplasmic) and -insoluble (cytoskeletal) fractions. PMA (100 nM) caused a time-dependent shift in the redistribution of actin, caldesmon (CaD, acalmodulin-, actin-, and myosin-binding protein), myosin heavy chains (HMM), MLC, but not MLCK from the cytoplasmic to the cytoskeletal fractions as demonstrated by immunoblotting. This redistribution coincided wiüi PMA-induced increase in CaD phosphorylation (immunoprecipitation with specific antiCaD antibodies) in ³²P-labeled EC monolayers. Fractionation of ³²P-labeled EC followed by immunoblotting with specific antibodies showed that PMA-stimulated CaD phosphorylation occured predominantly in the detergent-insoluble fraction. Western immunoblotting of EC proteins with specific anti-MLC antibodies showed that SDS homogenates included 2 unmunoreactive MLC proteins with an apparent difference in molecular weights of ∼ 1 kD, an MLC_A (MLC₂₁) and MLC_B (MLC10). In contrast, the detergent-soluble fraction included only MLC_A and the detergent-insoluble fraction was enriched in MLC_B. Immunoprecipitation of MLC under non-denaturing conditions or urea buffer treatment of EC TCA precipitates leads to extraction only MLC_A, but not MLC_B. Thrombin, but not PMA, altered MLC_A phosphorylation in this fraction, whereas MLC immunoprecipitation under denaturing conditions (in the presense of SDS) from ³²P-labeled EC leads to significant MLC_B phosphorylation in both PMA- and thrombin-stimulated EC. These data indicate that EC activation by PMA may increase EC permeability by inducing contractile protein reorganization which unlike thrombin, does not require MLC_A phosphorylation, but may depend upon CaD and MLC_B phosphorylation.