No abstract

Specific models of vascular permeability are critically dependent on myosin light chain phosphorylation, a reaction catalyzed by a novel high molecular-weight (214 kD) Ca2+/calmodulin (CaM)-dependent myosin light chain kinase (MLCK) isoform recently cloned in human endothelium (Am. J. Respir. Cell Mol. Biol., 1997;16:489-494). To evaluate mechanisms of endothelial cell (EC) barrier dysfunction evoked by the serine protease thrombin, we studied the regulation of the 214-kD EC MLCK isoform expressed in bovine endothelium. The EC MLCK isoform bound biotinylated CaM in a Ca2+-dependent manner and co-immunoprecipitated in a functional complex with myosin, actin, and CaM. Thrombin rapidly increased MLCK activity in concert with time-dependent translocation of the enzyme to the actin cytoskeleton. To evaluate whether EC MLCK activity was regulated by direct phosphorylation, amino acid sequence analysis identified multiple potential EC MLCK sites for Ser/Thr phosphorylation, including highly conserved phosphorylation sites for cyclic adenosine monophosphate-dependent protein kinase A (PKA) adjacent to the CaM-binding region. EC MLCK activity was attenuated by either PKA-mediated MLCK phosphorylation or inhibition of Ser/Thr phosphatase activity (fluoride or calyculin), which significantly increased MLCK phosphorylation while decreasing MLCK activity (3- to 4-fold decrease). In summary, although the EC MLCK isoform exhibits multiple features intrinsic to this family of kinases, thrombin-mediated EC contraction and barrier dysfunction requires increased EC MLCK-actin interaction and MLCK translocation to the cytoskeleton. EC MLCK activity appears to be highly dependent upon the phosphorylation status of this key contractile effector.

NaF, a potent G protein activator and Ser/Thr phosphatase inhibitor, significantly increased albumin permeability and decreased transcellular electrical resistance (TER), indicating endothelial cell (EC) barrier impairment. EC barrier dysfunction induced by NaF was accompanied by the development of actin stress fibers, intercellular gap formation, and significant time-dependent increases in myosin light chain (MLC) phosphorylation. However, despite rapid, albeit transient, activation of Ca(2+)/calmodulin-dependent MLC kinase (MLCK), the specific MLCK inhibitor ML-7 failed to affect NaF-induced MLC phosphorylation, actin cytoskeletal rearrangement, and reductions in TER, suggesting a limited role of MLCK in NaF-induced EC activation. In contrast, strategies to reduce Rho (C3 exoenzyme or toxin B) or to inhibit Rho-associated kinase (Y-27632 or dominant/negative RhoK) dramatically reduced MLC phosphorylation and actin stress fiber formation and significantly attenuated NaF-induced EC barrier dysfunction. Consistent with this role for RhoK activity, NaF selectively inhibited myosin-specific phosphatase activity, whereas the total Ser/Thr phosphatase activity remained unchanged. These data strongly suggest that MLC phosphorylation, mediated primarily by RhoK, and not MLCK, participates in NaF-induced EC actin cytoskeletal changes and barrier dysfunction.