Possible role of protein kinase-dependent smooth muscle contractile mechanism in the occurrence of delayed spasm after subarachnoid hemorrhage

我们已经报道了以下实验证据，表明蛋白激酶C(PKC)的激活参与了蛛网膜下腔出血后慢性血管痉挛的发生。[1]犬基底动脉(BA)有一个PKC系统，即使在无钙介质中也能充分发挥作用，在体内或体外条件下，应用PKC抑制剂如H-7和星形孢子素均可逆转暴露于“二次出血”的BA张力变窄或增大。1，2-二酰甘油是一种内源性PKC激活剂，通过刺激痉挛的BA内的磷脂酰胆碱和磷脂酰乙醇胺的周转而显著增加。3)在痉挛的4天和7天，细胞内的PKC活性比非痉挛的对照动脉降低了40-45%，而膜上的PKC活性没有明显变化。20 kDa肌球蛋白轻链磷酸化…的程度在第14天，随着血管痉挛的缓解，动脉胞浆PKC活性恢复到正常对照水平。蛋白质印迹分析显示，痉挛动脉中PKCα和PKCε表达下调，而PKCζ表达无下调，提示在慢性血管痉挛过程中，PKCα和PKCε被激活。4]可能由PKC激活引起的主动收缩增强在慢性血管痉挛的发生中起作用，而慢性血管痉挛的发生比动脉壁被动物理性质的改变(动脉壁的可分性降低)更重要。此外，还发现氧化二酰甘油可引起正常和皮肤BA的持续收缩。在PCA=5.5时，棕榈酰-花生四烯基二酰甘油-氢过氧化氢(5x10；-5&gt；M)孵育皮肤基底动脉可显著下调PKCa和e的表达。已知该蛋白的下调是其先行激活的直接证据，故认为氧化二酰甘油引起的收缩是由PKCa或ε激活所介导的。6]在实验性蛛网膜下腔出血后的脑组织中发现PAG=OH和PLG-OH作为氧化的DAG。上述研究表明，pkc的激活，尤其是在非钙依赖的平滑肌收缩机制中发挥核心作用的e型，在蛛网膜下腔出血后慢性血管痉挛的发生中所起的作用较小。

We have already reported the following experimental evidence suggesting that PKC (protein kinase C) activation participates in the occurrence of chronic vasospasm following subarachnoid hemorrhage. 1] The canine basilar artery (BA) possesses a PKC system which fully functions even in the Ca^<2+>-free medium, and that the narrowing or the augmented tonus of the BA exposed to 'two-hemorrhage' can be reversed by application of PKC inhibitors such as H-7 and staurosporine either in vivo or in vitro condition. 2] The content of 1,2-diacylglycerol, an intrinsic PKC activator, is significantly increased through the stimulated turnover of phosphatidylcholine and phosphatidylethanolamine in the spastic BA in comparison with that in the nonspastic BA.3] In spastic arteries at Days 4 and 7, the cytosolic PKC activity showed a decrease of 40-45% with no significant changes in membrane PKC activity as compared with nonspastic control arteries. The extent of 20 kDa myosin light chain phosphorylation … More was unchanged between the spastic and nonspastic BA.On Day 14, the cytosolic PKC activity of arteries returned toward the normal control level with the remission of vasospasm. Western blot analysis of the PKC isoforms revealed that the amounts of PKCα and PKCε but not PKCζ were down-regulated in spastic arteries, suggesting the activation of PKCα and PKCε in the course of chronic vasospasm. 4] The augmentation of active smooth muscle contraction, possibly induced by PKC activation, plays a role in the occurence of chronic vasospasm which is more important than the altered passive physical properties of the arterial wall (decreased dispensability of arterial wall). 5] In addition, it is revealed that oxidized diacylglycerol induces sustained contraction of normal and skinned BA.Under the pCa=5.5, an incubation of skinned basilar artery with palmitoyl-arachidonyl diacylglycerol-hydroperoxide (5xl0^<-5>M) shows a remarkable down-regulation of PKC a and e. As it is known that the down-regulation of the protein is the direct evidence to show its preceding activation, the oxidized-diacylglycerol induced contraction is thought to be mediated by an activation of PKC a orε. 6] Finally, PAG=OH and PLG-OH as an oxidized DAG were found in the brain tissue subjected to experimental subarachnoid hemorrhage. The above line of investigations render it likely that PKC activation, especially type e which is though to play a central role in the calciun-independent mechanism of smooth muscle contraction, participates in the occurrence of chronic vasospasm following subarachnoid hemorrhage Less