Expression of a novel high molecular-weight myosin light chain kinase in endothelium

Myosin light chain phosphorylation results in cellular contraction and is a critical component of agonist-mediated endothelial cell (EC) junctional ga p formation and permeability. We have shown that this reaction is catalyzed by a novel high molecular-weight Ca2+/calmodulin-dependent nonmuscle myosi n light chain kinase (MLCK) isoform recently cloned in human endothelium (A m. J. Respir. Cell Mel. Biol., 1997:16:489-194). To characterize EC MLCK ex pression further in cultured and adult tissues, we employed immunoblotting techniques and reverse transcriptase-polymerase chain reaction to demonstra te that freshly isolated and cultured human macro- and microvascular EC exp ress only the EC MLCK isoform (214 kD), which is distinct From smooth-muscl e MLCK isoforms (130 to 150 kD). Immunocytochemical studies demonstrated th e presence of the high molecular-weight MLCK isoform in adult human cardiac endothelium using anti-MLCK antibodies, which preferentially recognize the high molecular-weight EC MLCK isoform. Monitoring of MLCK expression in di fferent cell types with antibodies generated against a unique human EC MLCK N-terminal sequence revealed a high level of expression of the 214-kD enzy me in endothelium, minimal level of expression in smooth muscle, and no exp ression in skeletal muscle. These data suggest that the novel 214-kD kinase , the only MLCK isoform found in endothelium, may be preferentially express ed in this nonmuscle tissue.