Dixon B S, Sharma R V, Dickerson T, Fortune J
Department of Medicine, University of Iowa College of Medicine, Iowa City.
Am J Physiol. 1994 May;266(5 Pt 1):C1406-20. doi: 10.1152/ajpcell.1994.266.5.C1406.
The effects of bradykinin (BK) and angiotensin II (ANG II) were compared in cultured rat mesenteric arterial smooth muscle cells. BK and ANG II activated a phosphoinositide-specific phospholipase C, leading to the rapid release of [3H]inositol phosphates, an increase in intracellular calcium, and formation of sn-1,2-diacylglycerol (DAG). DAG formation was biphasic with a transient peak at 5 s followed by a sustained increase from 60 to 600 s. The BK-mediated increases in inositol triphosphate and DAG were dose dependent with half-maximal increases at concentrations of 5 and 2 nM, respectively. Both hormones were found to activate protein kinase C (PKC) as assessed by phosphorylation of the 68- to 72-kDa intracellular PKC substrate myristoylated alanine-rich C kinase substrate. However, despite similar phosphorylation of this substrate, only ANG II produced a significant increase in membrane-bound PKC activity. The mechanism accounting for the inability of BK to increase membrane-bound PKC activity is unclear. Our studies excluded differential translocation of PKC to the nuclear membrane, production of an inhibitor of membrane-bound PKC activity, and expression of BK and ANG II receptors on different cells as the mechanism. Vascular smooth muscle cells were found to express at least four different PKC isozymes: alpha, delta, zeta, and a faint band for epsilon. All of the isozymes except zeta-PKC were translocated by treatment with the phorbol ester 4 beta-phorbol 12-myristate 13-acetate. However, neither ANG II nor BK produced significant translocation of any measured isozyme; therefore, we could not exclude the possibility that ANG II and BK activate different isozymes of PKC. Both hormones were found to have a similar small and inconsistent effect in stimulating [3H]thymidine incorporation. These observations demonstrate that BK and ANG II have similar biochemical effects on vascular smooth muscle cells and imply that, in selected vessels, the vasodilatory effects of BK mediated by the endothelium may be partially counterbalanced by a vasoconstrictor effect on the underlying vascular smooth muscle cells.
在培养的大鼠肠系膜动脉平滑肌细胞中比较了缓激肽（BK）和血管紧张素II（ANG II）的作用。BK和ANG II激活了一种磷酸肌醇特异性磷脂酶C，导致[3H]肌醇磷酸迅速释放、细胞内钙增加以及sn-1,2-二酰甘油（DAG）形成。DAG形成呈双相性，在5秒时出现短暂峰值，随后从60秒到600秒持续增加。BK介导的肌醇三磷酸和DAG增加呈剂量依赖性，半最大增加浓度分别为5 nM和2 nM。通过68至72 kDa细胞内PKC底物肉豆蔻酰化富含丙氨酸的C激酶底物的磷酸化评估发现，两种激素均能激活蛋白激酶C（PKC）。然而，尽管该底物的磷酸化相似，但只有ANG II能使膜结合的PKC活性显著增加。BK不能增加膜结合PKC活性的机制尚不清楚。我们的研究排除了PKC向核膜的差异转位、膜结合PKC活性抑制剂的产生以及BK和ANG II受体在不同细胞上的表达作为该机制。发现血管平滑肌细胞表达至少四种不同的PKC同工酶：α、δ、ζ和一条微弱的ε带。除ζ-PKC外，所有同工酶在用佛波酯4β-佛波醇12-肉豆蔻酸酯13-乙酸酯处理后均发生转位。然而，ANG II和BK均未使任何检测到的同工酶发生显著转位；因此，我们不能排除ANG II和BK激活不同PKC同工酶的可能性。发现两种激素在刺激[3H]胸苷掺入方面具有相似的微小且不一致的作用。这些观察结果表明，BK和ANG II对血管平滑肌细胞具有相似的生化作用，并暗示在特定血管中，内皮介导的BK的血管舒张作用可能会被对下层血管平滑肌细胞的血管收缩作用部分抵消。
