NO/PKG介导Ca^(2+)/Calcineurin信号通路调控血管平滑肌细胞增殖

Proliferation of vascular smooth muscle cells regulated by NO/PKG mediated via Ca^(2+)/calcineurin signaling pathway

目的研究NO/PKG介导Ca2+/钙调神经磷酸酶(CaN)信号通路对血管平滑肌细胞(VSMCs)增殖的调控。方法植块法原代培养W istar大鼠VSMCs。W estern blot测定CaN表达,定磷法测定CaN活性。MTT法测定VSMCs的增殖,丫啶橙/溴化乙锭荧光染色法测定VSMCs的活性。结果SNAP和Sp-8-pCPT-cGMPS使苯肾上腺素(PE)诱发的VSMCs吸光度分别降低,而Rp-8-pCPT-cGMPS则可使吸光度增加。维拉帕米预处理VSMCs后,PE诱发的VSMCs吸光度降低,并且上述吸光度可以被SNAP或Sp-8-pCPT-cGMPS进一步抑制,但可被Rp-8-pCPT-cGMPS轻微升高。环胞素A预处理VSMCs后,PE诱发的VSMC吸光度降低,但是这一数值不能被SNAP、Sp-8-pCPT-cGMPS或Rp-8-pCPT-cGMPS进一步抑制或升高。SNAP和Sp-8-pCPT-cGMPS抑制PE诱发的CaN表达与活性。结论NO/PKG抑制血管SMCs增殖,但并不影响SMCs存活率;NO/PKG这一作用是介导SMCs内游离钙/CaN信号通路实现的。

Objective To study the regulation of VSMC proliferation by NO/PKG via modulating intracellular Ca^2＋ calcineurin （CAN） signaling pathway. Methods Primary VSMCs from rat aorta were used as the experimental model. CaN protein and its activity were assayed using immunoblotting and free inorganic phosphate content analy- sis respectively. Growth and viability of cells were determined by MTT assay and acridine orange and ethidium bromide staining. Results The addition of SNAP and Sp-8-pCPT-cGMPS decrease absorbance of cells stimulated by phenylephrine （PE）, whereas the addition of Rp-8-pCPT-cGMPS increases it. In SMCs p retreated with Vet, absorbance of cells stimulated by PE decreased and was further inhibited by the additional treatment of SNAP and Sp- 8-pCPT-cGMPS. In SMCs pretreated with CsA, absorbance of cells stimulated by PE decrease, but it can not be further altered by the additional treatment of SNAP, Sp-8-pCPT-cGMPS and Rp-8-pCPT-cGMPS. Moreover, expression and activities of CaN induced by PE is inhibited by SNAP and Sp-8-pCPT-cGMPS. Conclusion NO/PKG inhibits the proliferation of vascular SMCs without decreasing cell survival rate, which is mediated via intracellular Ca^2＋/CaN signaling pathway.