Garcia J G, Schaphorst K L, Shi S, Verin A D, Hart C M, Callahan K S, Patterson C E
Department of Medicine, Physiology, and Biophysics, Indiana University School of Medicine, Indianapolis, USA.
Am J Physiol. 1997 Jul;273(1 Pt 1):L172-84. doi: 10.1152/ajplung.1997.273.1.L172.
Myosin light chain (MLC) phosphorylation catalyzed by the Ca(2+)- calmodulin-dependent MLC kinase (MLCK) is critical to thrombin-mediated endothelial cell gap formation and barrier dysfunction. We have tested the hypothesis that the Ca2+ ionophore ionomycin stimulates MLCK-dependent endothelial cell contraction and permeability. Ionomycin significantly increased albumin clearance and decreased electrical resistance across confluent bovine pulmonary microvascular and macrovascular endothelial cell monolayers in a concentration-dependent manner that was temporally similar to that produced by thrombin. In contrast, however, ionomycin produced a significant Ca(2+)-dependent reduction in the levels of phosphorylated MLC with evidence of serine/threonine phosphatase activation. Potential MLCK-independent mechanisms of endothelial cell permeability were examined with little evidence to support a role for stimulated nitric oxide synthase or phospholipase A2 activities. Importantly, ionomycin produced 1) reductions in the activities of the barrier protective adenylate cyclase and the adenosine 3',5'-cyclic monophosphate-dependent protein kinase A, 2) dramatic dose- and time-dependent inhibition of endothelial cell tyrosine kinase activities, and 3) marked decreases in the phosphotyrosine content of the p125 focal adhesion kinase. These data indicate that ionomycin produces endothelial cell barrier dysfunction by mechanisms that are independent of MLCK activation and may involve reductions in endothelial cell tethering forces via inhibition of protein kinase A and tyrosine kinase activities, especially the p125 focal adhesion kinase.
由钙调蛋白依赖性肌球蛋白轻链激酶（MLCK）催化的肌球蛋白轻链（MLC）磷酸化对于凝血酶介导的内皮细胞间隙形成和屏障功能障碍至关重要。我们已经验证了以下假设：钙离子载体离子霉素可刺激依赖MLCK的内皮细胞收缩和通透性。离子霉素以浓度依赖的方式显著增加白蛋白清除率，并降低汇合的牛肺微血管和大血管内皮细胞单层的跨膜电阻，其时间效应与凝血酶产生的效应相似。然而，相比之下，离子霉素使磷酸化MLC水平出现显著的钙依赖性降低，并有丝氨酸/苏氨酸磷酸酶激活的证据。我们研究了内皮细胞通透性的潜在非MLCK依赖性机制，但几乎没有证据支持一氧化氮合酶或磷脂酶A2活性受刺激发挥作用。重要的是，离子霉素导致：1）屏障保护性腺苷酸环化酶和3',5'-环磷酸腺苷依赖性蛋白激酶A的活性降低；2）对内皮细胞酪氨酸激酶活性产生显著的剂量和时间依赖性抑制；3）p125粘着斑激酶的磷酸酪氨酸含量显著降低。这些数据表明，离子霉素通过独立于MLCK激活的机制导致内皮细胞屏障功能障碍，可能涉及通过抑制蛋白激酶A和酪氨酸激酶活性，尤其是p125粘着斑激酶，来降低内皮细胞的连接力。
