Aromolaran A S, Albert A P, Large W A
Department of Pharmacology and Clinical Pharmacology, St George's Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK.
J Physiol. 2000 May 1;524 Pt 3(Pt 3):853-63. doi: 10.1111/j.1469-7793.2000.00853.x.
The role of myosin light chain kinase (MLCK) in the activation of the noradrenaline-evoked non-selective cation current (Icat) was examined with the whole-cell recording technique in single rabbit portal vein smooth muscle cells. Intracellular dialysis with 5 microM MLCK(11-19)amide, a substrate-specific peptide inhibitor of MLCK, markedly reduced the amplitude and rate of activation of noradrenaline-evoked Icat. A similar result was obtained when the cells were dialysed with 10 microM AV25, which also inhibits MLCK by an action at the auto-inhibitory domain of MLCK. Inhibitors of binding of ATP to MLCK, wortmannin and synthetic naphthalenesulphonyl derivatives (ML-7 and ML-9), at micromolar concentrations, also reduced the amplitude of noradrenaline-evoked Icat. ML-7 and ML-9 (both at 5 microM) reduced the amplitude of Icat induced by both guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) and 1-oleoyl-2-acetyl-sn-glycerol (OAG). MLCK(11-19)amide, AV25 and ML-9 did not inhibit the noradrenaline-evoked Ca2+-activated potassium current at a holding potential of 0 mV. In addition, MLCK(11-19)amide and AV25 did not reduce the non-selective cation current induced by ATP in rabbit ear artery cells. Intracellular dialysis with 2 microM Ca2+ and 9 microM calmodulin activated Icat, which developed over a period of about 5 min. Intracellular dialysis with the non-hydrolysable analogue of ATP, 5'-adenylylimidodiphosphate (AMP-PNP), reduced the amplitude and rate of activation of noradrenaline-evoked Icat. The results indicate that MLCK mediates noradrenaline-activated Icat in rabbit portal vein smooth muscle cells.
采用全细胞记录技术，在单个兔门静脉平滑肌细胞中研究了肌球蛋白轻链激酶（MLCK）在去甲肾上腺素诱发的非选择性阳离子电流（Icat）激活中的作用。用5微摩尔MLCK(11 - 19)酰胺（一种MLCK的底物特异性肽抑制剂）进行细胞内透析，显著降低了去甲肾上腺素诱发的Icat的幅度和激活速率。当用10微摩尔AV25对细胞进行透析时也得到了类似结果，AV25也是通过作用于MLCK的自身抑制结构域来抑制MLCK的。微摩尔浓度的ATP与MLCK结合的抑制剂渥曼青霉素和合成萘磺酰衍生物（ML - 7和ML - 9）也降低了去甲肾上腺素诱发的Icat的幅度。ML - 7和ML - 9（均为5微摩尔）降低了由鸟苷5'-O-(3 - 硫代三磷酸)（GTPγS）和1 - 油酰 - 2 - 乙酰 - sn - 甘油（OAG）诱导的Icat的幅度。MLCK(11 - 19)酰胺、AV25和ML - 9在0 mV的钳制电位下不抑制去甲肾上腺素诱发的Ca2 + 激活的钾电流。此外，MLCK(11 - 19)酰胺和AV25不降低兔耳动脉细胞中由ATP诱导的非选择性阳离子电流。用2微摩尔Ca2 + 和9微摩尔钙调蛋白进行细胞内透析可激活Icat，其在约5分钟的时间内形成。用ATP的不可水解类似物5'-腺苷酰亚胺二磷酸（AMP - PNP）进行细胞内透析，降低了去甲肾上腺素诱发的Icat的幅度和激活速率。结果表明，MLCK介导兔门静脉平滑肌细胞中去甲肾上腺素激活的Icat。
