Tran Q K, Watanabe H, Zhang X X, Takahashi R, Ohno R
Department of Internal Medicine III, Hamamatsu University School of Medicine, Japan.
Cardiovasc Res. 1999 Dec;44(3):623-31. doi: 10.1016/s0008-6363(99)00238-2.
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.
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.
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.
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.
本研究旨在探讨肌球蛋白轻链激酶（MLCK）在缓激肽和毒胡萝卜素诱导的猪主动脉内皮细胞内氯离子（[Cl-]i）和钙离子（[Ca2+]i）浓度变化中的作用。
使用荧光探针N-乙氧羰基甲基-6-甲氧基喹啉溴化物（MQAE）和fura-2/AM，评估不同MLCK抑制剂对缓激肽和毒胡萝卜素诱导的[Cl-]i和[Ca2+]i变化的影响。
缓激肽和毒胡萝卜素显著降低了MQAE荧光强度，这表明[Cl-]i升高；去除细胞外氯离子（Cl-o）可逆转这些变化，且Cl(-)通道抑制剂N-苯基邻氨基苯甲酸可显著抑制这些变化，而钠钾氯共转运抑制剂呋塞米则无此作用。用两种不同的MLCK选择性抑制剂ML-9和渥曼青霉素预处理，可显著降低这些变化，且呈剂量依赖性。ML-9和渥曼青霉素对氯离子反应的抑制作用无显著差异，且无相加作用。缓激肽和毒胡萝卜素引起[Ca2+]i大幅升高，去除Cl-o和用Cl(-)通道抑制剂N-苯基邻氨基苯甲酸预处理可显著减弱这种升高。
该研究表明，[Cl-]i升高可能参与了缓激肽和毒胡萝卜素诱导的钙离子内流，且MLCK可能参与了氯离子反应。我们认为MLCK可能参与了缓激肽和毒胡萝卜素引起的对氯离子敏感的内皮细胞钙离子反应。
