Raina H, Zacharia J, Li M, Wier W G
Department of Physiology, School of Medicine, University of Maryland, Baltimore, MD 21201, USA.
J Physiol. 2009 Jun 1;587(Pt 11):2599-612. doi: 10.1113/jphysiol.2008.165258. Epub 2009 Apr 29.
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 Ca(2+)/calmodulin (Ca(2+)/CaM) induces an increase in MLCK activity and a change in FRET. After exposure to high external [K(+)], intracellular [Ca(2+)] (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 [Ca(2+)] and MLCK activation. Inhibition of PKCalpha and PKCbeta with Gö6976 had no effect. KN-93, an inhibitor of CaMK II, markedly reduced force, MLCK FRET and [Ca(2+)]. Applied during tonic force, forskolin caused a rapid decrease in MLCK FRET ratio and force, but no change in Ca(2+), suggesting a cAMP mediated decrease in affinity of MLCK for Ca(2+)/CaM. However, receptor (beta-adrenergic) activated increases in cAMP during KCl were ineffective in causing relaxation, changes in [Ca(2+)], or MLCK FRET. At the same tonic force, MLCK FRET ratio activated by alpha(1)-adrenoceptors was approximately 60% of that activated by KCl. In conclusion, MLCK activity of arterial smooth muscle during KCl-induced contraction is determined primarily by Ca(2+)/CaM. Rho-kinase is activated, by unknown mechanisms, and increases 'Ca(2+) sensitivity' significantly. Forskolin mediated increases in cAMP, but not receptor mediated increases in cAMP cause a rapid decrease in the affinity of MLCK for Ca(2+)/CaM.
在表达光学荧光共振能量转移（FRET）肌球蛋白轻链激酶（MLCK）活性生物传感器的转基因小鼠动脉中，研究了MLCK和其他激酶的激活情况。Ca(2+)/钙调蛋白（Ca(2+)/CaM）的结合会诱导MLCK活性增加以及FRET发生变化。暴露于高细胞外[K(+)]后，细胞内[Ca(2+)]（fura-2比率或fluo-4荧光）和MLCK活性均迅速增加至初始峰值，然后下降，起初下降迅速，随后非常缓慢。在初始峰值（“相性”）力之后，力保持恒定或缓慢增加（称为“紧张性”力）。抑制Rho激酶（Y-27632）对紧张性力的降低作用大于相性力，但对[Ca(2+)]和MLCK激活影响很小。用Gö6976抑制PKCα和PKCβ没有效果。CaMK II抑制剂KN-93显著降低了力、MLCK FRET和[Ca(2+)]。在紧张性力期间施加福斯高林会导致MLCK FRET比率和力迅速下降，但Ca(2+)没有变化，这表明cAMP介导MLCK对Ca(2+)/CaM的亲和力降低。然而，在氯化钾刺激期间，受体（β-肾上腺素能）激活导致的cAMP增加在引起舒张、[Ca(2+)]变化或MLCK FRET方面无效。在相同的紧张性力下，α(1)-肾上腺素能受体激活的MLCK FRET比率约为氯化钾激活的60%。总之，氯化钾诱导收缩期间动脉平滑肌的MLCK活性主要由Ca(2+)/CaM决定。Rho激酶通过未知机制被激活，并显著增加“Ca(2+)敏感性”。福斯高林介导的cAMP增加，但不是受体介导的cAMP增加，会导致MLCK对Ca(2+)/CaM的亲和力迅速降低。
