PKC-α、δ亚型在AVP改善缺氧处理VSMC反应性中的作用及其与MLC20磷酸化调节的关系

Role of PKC-α and δ isoforms in AVP improved contractile response of VSMC after hypoxia and its relations to MLC_(20) phosphorylation

目的:探讨蛋白激酶C(PKC)-α、δ亚型在精氨酸血管加压素(AVP)改善缺氧处理血管平滑肌细胞(VSMC)反应性中的作用及其与肌球蛋白轻链(MLC20)磷酸化、肌球蛋白轻链激酶(MLCK)和肌球蛋白轻链磷酸酶(MLCP)的关系。方法:贴块法取大鼠肠系膜上动脉(SMA)血管平滑肌细胞原代培养并传代至第3-5代后用于实验,观察缺氧1.5 h后PKC-α、δ亚型抑制剂处理对AVP调节VSMC收缩反应性的影响,以荧光素标记的牛血清白蛋白在Transwell小室中的渗透率变化反映VSMC的收缩反应性;用酶反应法测定缺氧VSMC MLCK/MLCP活性。同时取失血性休克大鼠(30 mmHg,2 h)SMA,观察PKC-α、δ亚型在AVP调节休克血管MLC20磷酸化水平(Western blotting)中的作用。结果:PKC-α抑制剂G 6976预处理可明显抑制AVP引起的缺氧VSMC收缩反应的升高,而PKC-δ抑制剂rottlerin也部分抑制AVP的作用。缺氧VSMC的MLCP活性明显升高、MLCK活性降低,同时休克血管平滑肌的MLC20磷酸化水平降低;AVP预处理可使缺氧VSMC MLCP活性降低、MLC20磷酸化水平升高,而G 6976可明显拮抗AVP的作用,rottlerin仅有轻度的抑制作用。AVP和PKC-α、δ抑制剂对MLCK活性的变化无明显影响。结论:AVP通过调节血管平滑肌细胞MLCP活性和MLC20磷酸化水平来恢复休克后血管反应性和钙敏感性,PKC-α是其中重要的调节分子。

AIM： The present study was to investigate the roles of protein kinase Cα and δ isoforms （PKC-α,δ） in arginine vasopressin （AVP） improved contractile response of vascular smooth muscle ceils （VSMC） to norepinephrine （NE） after hypoxia and its relations to myosin light chain （MLC20） phosphorylation, myosin light chain phosphatase （MLCP） and myosin light chain kinase （MLCK） activity. METHODS： Primary cultures of VSMC were obtained from the superior mesenteric artery （SMA） of rats by explanting technique and the cells in third to fifth passage were used in the study. The effects of PKC -α and δ antagonists on AVP induced contractile response of VSMC to NE after 1.5 h hypoxia were observed by measuring the ratio of accumulative infiltration of fluorescent isothiocyanate - conjugated bovine serum albumin with transwell, and their effect on the activity of MLCP/MLCK in VSMC was assayed by enzymatic catalysis. At the same time, with the SMA from hemorrhagic shock rats （30 mmHg for 2 h）, the effects of PKC α and δ isoforms in the regulation of AVP on MLC20 phosphorylation of SMA after shock were observed by Western blotting. RESULTS： Go 6976 （5 × 10^-6 mol/L, PKC -α isoform inhibitor） significantly antagonized AVP （5 × 10^-10 mol/L） -induced increase in the contractile response of VSMC to NE after hypoxia, and rottlerin （ 10^-5 mol/L, PKC -δ isoform inhibitor） also partly inhibited this effect. Hypoxia resulted in a significant increase in MLCP activity, with a decrease in MLCK activity of VSMC, and at the same time, the MLC20 phosphorylation of SMA following hemorrhagic shock was significantly decreased. AVP inhibited the activity of MLCP and increased the phosphorylation of MLC20, which was inhibited by Go 6976, while rottlerin treatment only showed a slightly inhibitory effect. AVP and PKC -α,δ inhibitor had no significant influence on MLCK activity. CONCLUSION ： AVP up - regulates vascular reactivity and calcium sensitivity of VSMC possibly through inhibiting the activity of MLCP and increasing the phosphorylation of MLC20 by PKC -α isoform.