Whorton A R, Montgomery M E, Kent R S
J Clin Invest. 1985 Jul;76(1):295-302. doi: 10.1172/JCI111960.
Oxidative damage to the vascular endothelium may play an important role in the pathogenesis of atherosclerosis and aging, and may account in part for reduced vascular prostacyclin (PGI2) synthesis associated with both conditions. Using H2O2 to induce injury, we investigated the effects of oxidative damage on PGI2 synthesis in cultured endothelial cells (EC). Preincubation of EC with H2O2 produced a dose-dependent inhibition (inhibitory concentration [IC50] = 35 microM) of PGI2 formation from arachidonate. The maximum dose-related effect occurred within 1 min after exposure although appreciable H2O2 remained after 30 min (30% of original). In addition, H2O2 produced both a time- and dose-dependent injury leading to cell disruption, lactate dehydrogenase release, and 51Cr release from prelabeled cells. However, in dramatic contrast to H2O2 effects on PGI2 synthesis, loss of cellular integrity required doses in excess of 0.5 mM and incubation times in excess of 1 h. The superoxide-generating system, xanthine plus xanthine oxidase, produced a similar inhibition of PGI2 formation. Such inhibition was dependent on the generation of H2O2 but not superoxide in that catalase was completely protective whereas superoxide dismutase was not. H2O2 (50 microM) also effectively inhibited basal and ionophore A23187 (0.5 microM)-stimulated PGI2 formation. However, H2O2 had no effect on phospholipase A2 activity, because ionophore A23187-induced arachidonate release was unimpaired. To determine the effects on cyclooxygenase and PGI2 synthase, prostaglandin products from cells prelabeled with [3H]arachidonate and stimulated with ionophore A23187, or products formed from exogenous arachidonate were examined. Inhibition of cyclooxygenase but not PGI2 synthase was observed. Incubation of H2O2-treated cells with prostaglandin cyclic endoperoxide indicated no inhibition of PGI2 synthase. Thus, in EC low doses of H2O2 potently inhibit cyclooxygenase after brief exposure whereas larger doses and prolonged exposure are required for classical cytolytic effects. Surprisingly, PGI2 synthase, which is known to be extremely sensitive to a variety of lipid peroxides, is not inhibited by H2O2. Lipid solubility, enzyme location within the EC membrane, or the local availability of reducing factors may explain these results, and may be important determinants of the response of EC to oxidative stress.
血管内皮的氧化损伤可能在动脉粥样硬化和衰老的发病机制中起重要作用，并且可能部分解释了与这两种情况相关的血管前列环素（PGI2）合成减少的原因。我们使用过氧化氢（H2O2）诱导损伤，研究了氧化损伤对培养的内皮细胞（EC）中PGI2合成的影响。用H2O2对EC进行预孵育会产生剂量依赖性抑制（抑制浓度[IC50]=35微摩尔），抑制花生四烯酸生成PGI2。尽管在30分钟后仍有相当数量的H2O2残留（为初始量的30%），但最大剂量相关效应在暴露后1分钟内就出现了。此外，H2O2产生了时间和剂量依赖性损伤，导致细胞破裂、乳酸脱氢酶释放以及预先标记细胞中51Cr的释放。然而，与H2O2对PGI2合成的影响形成鲜明对比的是，细胞完整性丧失需要超过0.5毫摩尔的剂量和超过一小时的孵育时间。超氧化物生成系统，即黄嘌呤加黄嘌呤氧化酶，对PGI2形成产生了类似的抑制作用。这种抑制作用依赖于H2O2的生成而不是超氧化物，因为过氧化氢酶具有完全的保护作用，而超氧化物歧化酶则没有。H2O2（50微摩尔）也有效抑制了基础状态下以及离子载体A23187（0.5微摩尔）刺激下的PGI2形成。然而，H2O2对磷脂酶A2活性没有影响，因为离子载体A23187诱导的花生四烯酸释放未受损害。为了确定对环氧化酶和PGI2合酶的影响，检测了用[3H]花生四烯酸预先标记并经离子载体A23187刺激的细胞产生的前列腺素产物，或由外源性花生四烯酸形成的产物。观察到对环氧化酶有抑制作用，但对PGI2合酶没有抑制作用。用过氧化前列腺素环内过氧化物孵育经H2O2处理的细胞，未显示对PGI2合酶有抑制作用。因此，在EC中，低剂量的H2O2在短暂暴露后能有效抑制环氧化酶，而经典的细胞溶解效应则需要更大剂量和更长时间的暴露。令人惊讶的是，已知对多种脂质过氧化物极其敏感的PGI2合酶并未被H2O2抑制。脂质溶解性、酶在EC膜内的位置或还原因子的局部可用性可能解释了这些结果，并且可能是EC对氧化应激反应的重要决定因素。
