Nakamura Kensei, Koga Yasuhiko, Sakai Hiroyasu, Homma Kazuaki, Ikebe Mitsuo
University of Massachusetts Medical School, Department of Physiology, 55 Lake Avenue North, Worcester, MA 01655, USA.
Circ Res. 2007 Sep 28;101(7):712-22. doi: 10.1161/CIRCRESAHA.107.153981. Epub 2007 Aug 2.
Nitric oxide/cGMP pathway induces vasodilatation, yet the underlying mechanism is obscure. In the present study, we studied the mechanism of cGMP-induced relaxation of the smooth muscle contractile apparatus using permeabilized rabbit femoral arterial smooth muscle. 8-Br-cGMP-induced relaxation was accompanied with a decrease in myosin light chain (MLC) phosphorylation. MLC phosphatase (MLCP) activity, once decreased by agonist-stimulation, recovered to the resting level on addition of 8-Br-cGMP. Because MLCP activity is regulated by the phosphorylation of a MLCP-specific inhibitor, CPI17 at Thr38 and MBS (myosin binding subunit of MLCP) at Thr696, we examined the effect of 8-Br-cGMP on the phosphorylation of these MLCP modulators. Whereas CPI17 phosphorylation was unchanged after addition of 8-Br-cGMP, MBS phosphorylation at Thr696 was significantly decreased by 8-Br-cGMP. We found that 8-Br-cGMP markedly increased MBS phosphorylation at Ser695 in the fiber pretreated with phenylephrine. MBS phosphorylation of Thr696 phosphorylated MBS at Ser695 partially resumed MLCP activity inhibited by Thr696 phosphorylation. Whereas Ser695 phosphorylation was markedly increased, the extent of diphosphorylated MBS at Ser695 and Thr696 in fibers was unchanged after cGMP-stimulation. We found that MBS phosphatase activity in arteries for both diphosphorylated MBS and monophosphorylated MBS at Thr696 significantly increased by 8-Br-cGMP, whereas MBS kinase activity was unchanged. These results suggest that the phosphorylation at Ser640 induced by cGMP shifted the equilibrium of the Thr641 phosphorylation toward dephosphorylation, thus increasing MLCP activity. This results in the decrease in MLC phosphorylation and smooth muscle relaxation.
一氧化氮/cGMP途径可诱导血管舒张，但其潜在机制尚不清楚。在本研究中，我们使用通透化的兔股动脉平滑肌研究了cGMP诱导平滑肌收缩装置舒张的机制。8-溴-cGMP诱导的舒张伴随着肌球蛋白轻链（MLC）磷酸化的减少。MLC磷酸酶（MLCP）活性一旦因激动剂刺激而降低，在加入8-溴-cGMP后会恢复到静息水平。由于MLCP活性受MLCP特异性抑制剂CPI17在苏氨酸38位点和MLCP的肌球蛋白结合亚基（MBS）在苏氨酸696位点的磷酸化调节，我们研究了8-溴-cGMP对这些MLCP调节剂磷酸化的影响。加入8-溴-cGMP后，CPI17的磷酸化没有变化，而苏氨酸696位点的MBS磷酸化则被8-溴-cGMP显著降低。我们发现，8-溴-cGMP使预先用去氧肾上腺素处理的纤维中丝氨酸695位点的MBS磷酸化显著增加。苏氨酸696位点磷酸化的MBS在丝氨酸695位点的磷酸化部分恢复了受苏氨酸696磷酸化抑制的MLCP活性。虽然丝氨酸695位点的磷酸化显著增加，但cGMP刺激后纤维中丝氨酸695和苏氨酸696双磷酸化的MBS程度没有变化。我们发现，8-溴-cGMP使双磷酸化的MBS和苏氨酸696单磷酸化的MBS在动脉中的MBS磷酸酶活性显著增加，而MBS激酶活性没有变化。这些结果表明，cGMP诱导的丝氨酸640位点磷酸化使苏氨酸641位点磷酸化的平衡向去磷酸化方向移动，从而增加了MLCP活性。这导致MLC磷酸化减少和平滑肌舒张。
