Fukami M, Tani E, Takai A, Yamaura I, Minami N
Department of Neurosurgery, Hyogo College of Medicine, Japan.
Stroke. 1995 Dec;26(12):2321-7. doi: 10.1161/01.str.26.12.2321.
Subarachnoid hemorrhage frequently leads to a long-term cerebral artery narrowing called vasospasm. Recently, the involvement of myosin light chain kinase has been found in experimental vasospasm in our laboratory. We therefore measured the activity of serine/threonine protein phosphatases 1 and 2A in the rabbit basilar artery in vasospasm and in vasocontraction to study their role, particularly in regard to vasospasm compared with vasocontraction.
Vasospasm was produced in the rabbit basilar artery by a two-hemorrhage method. Vasocontraction was induced by local application of KCl or serotonin to the rabbit basilar artery after a transclival exposure. The control animals were treated with saline instead of fresh blood. Serine/threonine protein phosphatase activity in the basilar artery was assayed with the use of [32P]phosphorylase-a as a substrate; protein phosphatase 1 activity was evaluated as protein phosphatase activity in the presence of 1 nmol/L okadaic acid, whereas protein phosphatase 2A activity was assessed as protein phosphatase activity inhibited by 1 nmol/L okadaic acid.
Values of mean activity of protein phosphatase 1 in myofibrillar extract were 3.58 +/- 0.26 nmol/min per milligram in the control group, 3.22 +/- 0.12 nmol/min per milligram in the spastic group on day 2, and 3.01 +/- 0.16 nmol/min per milligram in the spastic group on day 4 (a significant decrease in protein phosphatase 1 activity in the spastic group on days 2 and 4). In contrast, these values did not show any significant changes in the KCl and serotonin groups. Values of mean activity of protein phosphatase 2A in cytosolic extract were 0.90 +/- 0.07 nmol/min per milligram in the control group, 0.75 +/- 0.10 nmol/min per milligram in the spastic group on day 2, and 0.62 +/- 0.17 nmol/min per milligram in the spastic group on day 4 (a significant reduction in protein phosphatase 2A in the spastic group on days 2 and 4). There was no evidence of significant changes of protein phosphatase 2A in cytosolic extract in the KCl and serotonin groups.
Protein phosphatase 1 in myofibrillar extract is reported to catalyze the dephosphorylation of myosin light chain and calponin, whereas protein phosphatase 2A in cytosolic extract catalyzes the dephosphorylation of calponin and caldesmon. In addition, the phosphorylation of calponin and caldesmon results in the loss of their ability to inhibit smooth muscle contraction. Therefore, the significant decrease in activity of protein phosphatases 1 and 2A in vasospasm may result in uninterrupted vascular smooth muscle contraction by the preservation of phosphorylation of not only myosin light chain but also calponin and caldesmon.
蛛网膜下腔出血常导致一种称为血管痉挛的长期脑动脉狭窄。最近，我们实验室在实验性血管痉挛中发现肌球蛋白轻链激酶与之有关。因此，我们测量了兔基底动脉在血管痉挛和血管收缩状态下丝氨酸/苏氨酸蛋白磷酸酶1和2A的活性，以研究它们的作用，特别是与血管收缩相比在血管痉挛中的作用。
采用两次出血法在兔基底动脉诱发血管痉挛。经斜坡暴露后，通过向兔基底动脉局部应用氯化钾或5-羟色胺诱导血管收缩。对照组动物用生理盐水而非新鲜血液处理。以[32P]磷酸化酶-a为底物测定基底动脉中的丝氨酸/苏氨酸蛋白磷酸酶活性；蛋白磷酸酶1活性通过在1 nmol/L冈田酸存在下的蛋白磷酸酶活性来评估，而蛋白磷酸酶2A活性通过被1 nmol/L冈田酸抑制的蛋白磷酸酶活性来评估。
肌原纤维提取物中蛋白磷酸酶1的平均活性值在对照组为3.58±0.26 nmol/（min·mg），在第2天痉挛组为3.22±0.12 nmol/（min·mg），在第4天痉挛组为3.01±0.16 nmol/（min·mg）（第2天和第4天痉挛组蛋白磷酸酶1活性显著降低）。相比之下，氯化钾组和5-羟色胺组的这些值没有任何显著变化。胞质提取物中蛋白磷酸酶2A的平均活性值在对照组为0.90±0.07 nmol/（min·mg），在第2天痉挛组为0.75±0.10 nmol/（min·mg），在第4天痉挛组为0.62±0.17 nmol/（min·mg）（第2天和第4天痉挛组蛋白磷酸酶2A显著降低）。氯化钾组和5-羟色胺组的胞质提取物中没有蛋白磷酸酶2A显著变化的证据。
据报道，肌原纤维提取物中的蛋白磷酸酶1催化肌球蛋白轻链和钙调蛋白的去磷酸化，而胞质提取物中的蛋白磷酸酶2A催化钙调蛋白和钙结合蛋白的去磷酸化。此外，钙调蛋白和钙结合蛋白的磷酸化导致它们抑制平滑肌收缩能力的丧失。因此，血管痉挛中蛋白磷酸酶1和2A活性的显著降低可能通过不仅保留肌球蛋白轻链而且保留钙调蛋白和钙结合蛋白的磷酸化而导致血管平滑肌持续收缩。
