Neppl Ronald L, Lubomirov Lubomir T, Momotani Ko, Pfitzer Gabriele, Eto Masumi, Somlyo Avril V
Department of Molecular Physiology and Biological Physics, Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, Virginia 22908, USA.
J Biol Chem. 2009 Mar 6;284(10):6348-60. doi: 10.1074/jbc.M807040200. Epub 2008 Dec 17.
Myosin light chain phosphatase plays a critical role in modulating smooth muscle contraction in response to a variety of physiologic stimuli. A downstream target of the RhoA/Rho-kinase and nitric oxide (NO)/cGMP/cyclic GMP-dependent kinase (cGKI) pathways, myosin light chain phosphatase activity reflects the sum of both calcium sensitization and desensitization pathways through phosphorylation and dephosphorylation of the myosin phosphatase targeting subunit (MYPT1). As cerebral blood flow is highly spatio-temporally modulated under normal physiologic conditions, severe perturbations in normal cerebral blood flow, such as in cerebral vasospasm, can induce neurological deficits. In nonpermeabilized cerebral vessels stimulated with U-46619, a stable mimetic of endogenous thromboxane A2 implicated in the etiology of cerebral vasospasm, we observed significant increases in contractile force, RhoA activation, regulatory light chain phosphorylation, as well as phosphorylation of MYPT1 at Thr-696, Thr-853, and surprisingly Ser-695. Inhibition of nitric oxide signaling completely abrogated basal MYPT1 Ser-695 phosphorylation and significantly increased and potentiated U-46619-induced MYPT1 Thr-853 phosphorylation and contractile force, indicating that NO/cGMP/cGKI signaling maintains basal vascular tone through active inhibition of calcium sensitization. Surprisingly, a fall in Ser-695 phosphorylation did not result in an increase in phosphorylation of the Thr-696 site. Although activation of cGKI with exogenous cyclic nucleotides inhibited thromboxane A2-induced MYPT1 membrane association, RhoA activation, contractile force, and regulatory light chain phosphorylation, the anticipated decreases in MYPT1 phosphorylation at Thr-696/Thr-853 were not observed, indicating that the vasorelaxant effects of cGKI are not through dephosphorylation of MYPT1. Thus, thromboxane A2 signaling within the intact cerebral vasculature induces "buffered" vasoconstrictions, in which both the RhoA/Rho-kinase calcium-sensitizing and the NO/cGMP/cGKI calcium-desensitizing pathways are activated.
肌球蛋白轻链磷酸酶在响应多种生理刺激调节平滑肌收缩中起关键作用。作为RhoA/Rho激酶和一氧化氮（NO）/环磷酸鸟苷（cGMP）/环磷酸鸟苷依赖性激酶（cGKI）途径的下游靶点，肌球蛋白轻链磷酸酶活性通过肌球蛋白磷酸酶靶向亚基（MYPT1）的磷酸化和去磷酸化反映钙敏化和脱敏途径的总和。由于脑血流在正常生理条件下受到高度的时空调节，正常脑血流的严重扰动，如脑血管痉挛，可导致神经功能缺损。在用U-46619刺激的非通透化脑血管中，U-46619是一种与脑血管痉挛病因有关的内源性血栓素A2的稳定模拟物，我们观察到收缩力、RhoA激活、调节性轻链磷酸化以及MYPT1在Thr-696、Thr-853以及令人惊讶的Ser-695位点的磷酸化显著增加。一氧化氮信号传导的抑制完全消除了基础MYPT1 Ser-695磷酸化，并显著增加和增强了U-46619诱导的MYPT1 Thr-853磷酸化和收缩力，表明NO/cGMP/cGKI信号传导通过主动抑制钙敏化维持基础血管张力。令人惊讶的是，Ser-695磷酸化的下降并未导致Thr-696位点磷酸化的增加。尽管用外源性环核苷酸激活cGKI可抑制血栓素A2诱导的MYPT1膜结合、RhoA激活、收缩力和调节性轻链磷酸化，但未观察到预期的MYPT1在Thr-696/Thr-853位点磷酸化降低，表明cGKI的血管舒张作用不是通过MYPT1的去磷酸化实现的。因此，完整脑血管内的血栓素A2信号传导诱导“缓冲”血管收缩，其中RhoA/Rho激酶钙敏化和NO/cGMP/cGKI钙脱敏途径均被激活。
