title: An essential role of myosin light-chain kinase in the regulation of agonist- and fluid flow-stimulated Ca2+ influx in endothelial cells.
abstract: Cytosolic Ca2+ ([Ca2+]i) plays an important role in endothelial cell signaling. Although it has been suggested that the influx of Ca2+ can be triggered by depletion of intracellular Ca2+ stores, the mechanism (or mechanisms) underlying this phenomenon needs further elaboration. In the present study, involvement of myosin light-chain kinase (MLCK) in the regulation of Ca2+ signaling was investigated in agonist- and fluid flow-stimulated endothelial cells loaded with Ca2+-sensitive dyes. Bradykinin (BK) and thapsigargin caused an increase in [Ca2+]i followed by a sustained rise due to Ca2+ influx from extracellular space and shifted total myosin light-chain (MLC) from the unphosphorylated to the diphosphorylated form. ML-9 (100 microM), an inhibitor of MLCK, abolished Ca2+ influx and prevented MLC diphosphorylation in BK- and thapsigargin-treated cells, but did not affect Ca2+ mobilization from internal stores. Fluid flow stimulation (shear stress=5 dynes/cm2) increased [Ca2+]i and enhanced MLC phosphorylation. ML-9 also inhibited Ca2+ response and MLC phosphorylation in fluid flow-stimulated cells. The Ca2+ influx in response to BK was linearly correlated with the diphosphorylation of MLC in ML-9 treated cells. Effects of ML-5 and ML-7, analogs of ML-9, to inhibit Ca2+ influx paralleled their potencies to inhibit MLCK activity. These findings demonstrate that MLCK plays an essential role in regulating the plasmalemmal Ca2+ influx in agonist- and fluid flow-stimulated endothelial cells. This study is the first to report the close relationship between Ca2+ influx and MLC diphosphorylation.
authors.affiliation: Internal Medicine III, Hamamatsu University School of Medicine, Japan. hwat@hama-med.ac.jp
authors.forename: H
authors.initials: H
authors.lastname: Watanabe
authors.forename: R
authors.initials: R
authors.lastname: Takahashi
authors.forename: X X
authors.initials: XX
authors.lastname: Zhang
authors.forename: Y
authors.initials: Y
authors.lastname: Goto
authors.forename: H
authors.initials: H
authors.lastname: Hayashi
authors.forename: J
authors.initials: J
authors.lastname: Ando
authors.forename: M
authors.initials: M
authors.lastname: Isshiki
authors.forename: M
authors.initials: M
authors.lastname: Seto
authors.forename: H
authors.initials: H
authors.lastname: Hidaka
authors.forename: I
authors.initials: I
authors.lastname: Niki
authors.forename: R
authors.initials: R
authors.lastname: Ohno
mesh_terms: D000818:Animals; D001381:Azepines / Q000494:pharmacology; D001920:Bradykinin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002120:Calcium Channel Agonists / Q000494:pharmacology*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015394:Molecular Structure; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009281:Naphthalenes / Q000494:pharmacology; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D013552:Swine; D019284:Thapsigargin / Q000494:pharmacology*
chemical_list: D001381:Azepines; D002120:Calcium Channel Agonists; D004791:Enzyme Inhibitors; D018994:Myosin Light Chains; D009281:Naphthalenes; D047428:Protein Kinase Inhibitors; C056218:ML 9; C070571:ML 7; D019284:Thapsigargin; D009219:Myosin-Light-Chain Kinase; D001920:Bradykinin; D002118:Calcium