Activation by Ca2+/calmodulin of an exogenous myosin light chain kinase in mouse arteries
Activation by Ca2+/calmodulin of an exogenous myosin light chain kinase in mouse arteries
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DOI：
10.1113/jphysiol.2008.165258
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发表时间：
2009-06-01
影响因子：
5.5
通讯作者：
Wier, W. G.
中科院分区：
文献类型：
--
作者：
Raina, H.;Zacharia, J.;Wier, W. G.
在表达光荧光共振能量转移(FRET)MLCK活性生物传感器的转基因小鼠的动脉中，研究了肌球蛋白轻链激酶(MLCK)和其他激酶的激活。钙/钙调素结合可引起MLCK活性增加和FRET的改变。细胞外高K+暴露后，细胞内[Ca~(2+)](Fura-2比值或Fluo-4荧光强度)和MLCK活性均迅速升高，达到一个初始峰值，而后下降，先快后慢。在最初的峰值(“相”)之后，力是恒定的或缓慢增加的(称为“紧张力”)。抑制Rho-Kinase(Y-27632)对紧张力的抑制作用大于时相作用，但对[Ca~(2+)]和MLCK的激活影响不大。Go6976对PKCα和PKCβ的抑制作用不明显。CaMK II抑制剂KN-93显著降低肌张力、MLCK FRET和[Ca~(2+)]。在紧张力作用下，Forskolin引起MLCK FRET比率和肌张力的迅速下降，但对钙离子无影响，提示cAMP介导的MLCK对钙/CaM的亲和力降低。然而，受体(β-肾上腺素能)激活的cAMP在KCl时的升高并不能引起松弛、[Ca~(2+)]的变化或MLCK FRET。在相同的紧张力下，α(1)-肾上腺素能受体激活的MLCK FRET比率与KCl激活的MLCK FRET比率相近。综上所述，在KCl2诱导的收缩过程中，动脉平滑肌MLCK活性主要由钙/钙调素决定。Rho-Kinase被激活，其机制未知，并显著增加“对钙离子的敏感性”。Forsklin介导的cAMP增加，而不是受体介导的cAMP增加，导致MLCK对钙/钙调素的亲和力迅速下降。
Activation of myosin light chain kinase (MLCK) and other kinases was studied in the arteries of transgenic mice that express an optical fluorescence resonance energy transfer (FRET) MLCK activity biosensor. Binding of Ca2+/calmodulin (Ca2+/CaM) induces an increase in MLCK activity and a change in FRET. After exposure to high external [K+], intracellular [Ca2+] (fura-2 ratio or fluo-4 fluorescence) and MLCK activity both increased rapidly to an initial peak and then declined, rapidly at first and then very slowly. After an initial peak ('phasic') force was constant or increased slowly (termed 'tonic' force). Inhibition of rho-kinase (Y-27632) decreased tonic force more than phasic, but had little effect on [Ca2+] and MLCK activation. Inhibition of PKC alpha and PKC beta with Go6976 had no effect. KN-93, an inhibitor of CaMK II, markedly reduced force, MLCK FRET and [Ca2+]. Applied during tonic force, forskolin caused a rapid decrease in MLCK FRET ratio and force, but no change in Ca2+, suggesting a cAMP mediated decrease in affinity of MLCK for Ca2+/CaM. However, receptor (beta-adrenergic) activated increases in cAMP during KCl were ineffective in causing relaxation, changes in [Ca2+], or MLCK FRET. At the same tonic force, MLCK FRET ratio activated by alpha(1)-adrenoceptors was similar to 60% of that activated by KCl. In conclusion, MLCK activity of arterial smooth muscle during KCl-induced contraction is determined primarily by Ca2+/CaM. Rho-kinase is activated, by unknown mechanisms, and increases 'Ca2+ sensitivity' significantly. Forskolin mediated increases in cAMP, but not receptor mediated increases in cAMP cause a rapid decrease in the affinity of MLCK for Ca2+/CaM.