Wesson D E, Elliott S J
Department of Pediatrics, Baylor College of Medicine, Houston, Texas.
J Pharmacol Exp Ther. 1994 Sep;270(3):1197-207.
The effect of xanthine oxidase (XO)-mediated oxidant stress on endothelial cell signal transduction was determined in bradykinin-stimulated cells loaded with the Ca+(+)-sensitive probe fura-2. Calf pulmonary artery endothelial cells were incubated with a reaction mixture containing XO (50 mU/ml) and its substrate, hypoxanthine (HX) (0.5 mM), for periods of 0.5 to 2.0 hr. HX/XO time dependently increased basal cytosolic free Ca++ ([Ca++]i) and decreased the response of [Ca++]i to bradykinin, so that incubation of cells with HX/XO for 1.5 hr or longer eliminated responsiveness to agonist. In presence of XO, HX dose dependently increased basal [Ca++]i (EC50 approximately 3 x 10(-5) M) and decreased the response of [Ca++]i to bradykinin. Sequential application of bradykinin and Ca++ to cells suspended in Ca+(+)-free/EGTA buffer was performed to characterize the effects of HX/XO on receptor-activated Ca++ entry and release of Ca++ from internal stores. HX/XO attenuated internal store Ca++ release and inhibited the bradykinin-stimulated Ca++ influx pathway in a time-dependent manner. When the HX dose was decreased by an order of magnitude, HX/XO selectively inhibited the agonist-stimulated influx pathway with little effect on internal store Ca++ release. Coincubation with superoxide dismutase tended to potentiate the effects of HX/XO, whereas catalase provided almost complete protection. Similar results to HX/XO-induced alterations in Ca++ signaling were observed when glucose-glucose oxidase (G/GO) was used as the oxidant-generating system. Inhibition of Ca++ signaling by HX/XO and G/GO occurred in the absence of decreased cell viability. Together, these results suggest that HX/XO-induced inhibition of signal transduction in endothelial cells is a function of H2O2-mediated oxidant stress and represents an early dysfunction in the process of oxidant injury.
在加载了Ca+(+)-敏感探针fura-2的缓激肽刺激细胞中，测定了黄嘌呤氧化酶（XO）介导的氧化应激对内皮细胞信号转导的影响。将小牛肺动脉内皮细胞与含有XO（50 mU/ml）及其底物次黄嘌呤（HX）（0.5 mM）的反应混合物孵育0.5至2.0小时。HX/XO以时间依赖性方式增加基础胞质游离Ca++（[Ca++]i），并降低[Ca++]i对缓激肽的反应，因此将细胞与HX/XO孵育1.5小时或更长时间可消除对激动剂的反应性。在XO存在下，HX剂量依赖性地增加基础[Ca++]i（EC50约为3×10(-5) M），并降低[Ca++]i对缓激肽的反应。对悬浮在无Ca+(+)/EGTA缓冲液中的细胞依次施加缓激肽和Ca++，以表征HX/XO对受体激活的Ca++内流和从内部储存释放Ca++的影响。HX/XO以时间依赖性方式减弱内部储存Ca++释放，并抑制缓激肽刺激的Ca++内流途径。当HX剂量降低一个数量级时，HX/XO选择性抑制激动剂刺激的内流途径，对内部储存Ca++释放影响很小。与超氧化物歧化酶共同孵育倾向于增强HX/XO的作用，而过氧化氢酶提供几乎完全的保护。当使用葡萄糖-葡萄糖氧化酶（G/GO）作为氧化剂生成系统时，观察到与HX/XO诱导的Ca++信号改变相似的结果。在细胞活力未降低的情况下，HX/XO和G/GO对Ca++信号的抑制作用发生。总之，这些结果表明，HX/XO诱导的内皮细胞信号转导抑制是H2O2介导的氧化应激的作用，代表氧化损伤过程中的早期功能障碍。
