Reiko Takahashi

An essential role of myosin light-chain kinase in the regulation of agonist- and fluid flow-stimulated Ca2+ influx in endothelial cells

Cytosolic Ca2+ ([Ca2+];) 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 pM), 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.—Watanabe, H., Takahashi, R., Zhang, X.‐X., Goto, Y., Hayashi, H., Ando, J., Isshiki, M., Seto, M., Hidaka, H., Niki, I., Ohno, R. An essential role of myosin light‐chain kinase in the regulation of agonist‐ and fluid flow‐stimulated Ca2+ influx in endothelial cells. FASEB J. 12, 341–348 (1998)

Involvement of myosin light-chain kinase in chloride-sensitive Ca2+ influx in porcine aortic endothelial cells

OBJECTIVES This study was designed to investigate the involvement of myosin light-chain kinase (MLCK) in bradykinin- and thapsigargin-induced changes in intracellular Cl- and Ca2+ concentrations ([Cl-]i; [Ca2+]i) in porcine aortic endothelial cells. METHODS Using the fluorescent probes N-ethoxycarbonylmethyl-6-methoxyquinolinium bromide (MQAE) and fura-2/AM, the effects of different MLCK inhibitors on bradykinin- and thapsigargin-induced changes in [Cl-]i and [Ca2+]i were assessed. RESULTS Bradykinin and thapsigargin significantly decreased the MQAE fluorescence intensity, which indicates increased [Cl-]i; these changes were reversed by removal of extracellular chloride (Cl-o) and were significantly inhibited by Cl(-)-channel inhibitor N-phenylanthranilic acid but not by Na(+)-K(+)-Cl- cotransport inhibitor furosemide. Pretreatment with ML-9 and wortmannin, two different selective inhibitors of MLCK, significantly reduced these changes in a dose-dependent manner. The inhibitory effects of ML-9 and wortmannin on the Cl- responses were not significantly different and were not additive. Bradykinin and thapsigargin provoked large increases in [Ca2+]i, which were significantly diminished by removal of Cl-o and by pretreatment with the Cl(-)-channel inhibitor N-phenylanthranilic acid. CONCLUSIONS The study shows that an increase in [Cl-]i may be involved in the Ca2+ influx in response to bradykinin and thapsigargin and that MLCK might be involved in the Cl- response. We suggest that MLCK might be involved in the Cl(-)-sensitive endothelial Ca2+ responses to bradykinin and thapsigargin.

Inhibitory effect of insulin on bradykinin-induced venodilation.

Background Excessive insulin is one of the risk factors of hypertension and arteriosclerosis despite its vasodilative properties shown in recent studies. Although many vasoactive substances contribute and interact with each other in the development of hypertension, the interactions between insulin and other vasoactive substances have yet to be elucidated. Objective To assess the effect of insulin on the action of bradykinin. Methods The vasodilating effect of bradykinin was evaluated, with or without coadministration of insulin, in human dorsal hand veins of healthy volunteers. In cultured porcine aortic endothelial cells, the bradykinin-induced increase of intracellular calcium was also investigated before and after insulin administration. Results Insulin significantly attenuated bradykinin-induced increase in intracellular calcium and venodilation in cultured endothelial cells and human dorsal hand veins, respectively. Conclusions These findings suggest that insulin attenuates the bradykinin-induced calcium elevation in endothelial cells and may decrease the production of vasodilative substances from endothelial cells, resulting in the reduction of vasodilation. This effect may contribute to the development of hypertension in patients with hyperinsulinaemia.