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 MLCA (MLC21) and MLCB (MLC10). In contrast, the detergent-soluble fraction included only MLCA and the detergent-insoluble fraction was enriched in MLCB. Immunoprecipitation of MLC under non-denaturing conditions or urea buffer treatment of EC TCA precipitates leads to extraction only MLCA, but not MLCB. Thrombin, but not PMA, altered MLCA phosphorylation in this fraction, whereas MLC immunoprecipitation under denaturing conditions (in the presense of SDS) from 32P-labeled EC leads to significant MLCB 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 MLCA phosphorylation, but may depend upon CaD and MLCB phosphorylation.
|Original language||English (US)|
|Journal||Journal of Investigative Medicine|
|State||Published - Jan 1 1996|
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)