Hart C M, Andreoli S P, Patterson C E, Garcia J G
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202.
J Cell Physiol. 1993 Jul;156(1):24-34. doi: 10.1002/jcp.1041560105.
We have previously shown that supplementing cultured porcine pulmonary artery endothelial cells (PAEC) with exogenous oleic acid (18:1 omega 9) alters the fatty acid composition of the cells and reduces oxidant-mediated cytotoxicity. Because the mechanisms by which lipid alterations modulate oxidant susceptibility have not been defined, the ability of 18:1 to reduce hydrogen peroxide (H2O2)-mediated PAEC dysfunction was evaluated. PAEC monolayers on polycarbonate filters were incubated for 3 h in maintenance medium supplemented with either 0.1 mM 18.1 in ethanol vehicle (ETOH) or with an equivalent volume of vehicle alone. Twenty-four hours later monolayers were treated for 30 min with 50 or 100 microM H2O2 in Hanks' balanced salt solution (HBSS) or with HBSS alone (nonoxidant control). As a functional index of PAEC monolayer integrity, the permeability of monolayers to albumin was then measured for 3 h. Treatment with 100 microM H2O2 caused cytotoxicity and progressive increases in PAEC monolayer permeability that were attenuated by 18:1 supplementation, whereas 50 microM H2O2 caused only a transient increase in permeability without cytotoxicity. Supplementation with 18:1 also attenuated H2O2-induced reductions in PAEC adenosine triphosphate (ATP) content and disruption of PAEC microfilament architecture. The ATP content of PAEC monolayers was reversibly reduced in the absence of oxidant stress by incubation with glucose-depleted medium containing deoxyglucose and antimycin A. Metabolic inhibitor-induced ATP depletion increased monolayer permeability and altered cytoskeletal architecture, alterations that resolved during recovery of PAEC ATP content. These results demonstrate that ATP depletion plays a critical role in barrier dysfunction and suggests that the ability of 18:1 to reduce oxidant-mediated PAEC dysfunction and injury may relate directly to its ability to preserve PAEC ATP content.
我们之前已经表明，用外源性油酸（18:1 ω9）补充培养的猪肺动脉内皮细胞（PAEC）会改变细胞的脂肪酸组成，并降低氧化剂介导的细胞毒性。由于脂质改变调节氧化剂易感性的机制尚未明确，因此评估了18:1降低过氧化氢（H2O2）介导的PAEC功能障碍的能力。将聚碳酸酯滤膜上的PAEC单层在补充有0.1 mM 18.1乙醇载体（ETOH）或等体积单独载体的维持培养基中孵育3小时。24小时后，单层细胞在汉克斯平衡盐溶液（HBSS）中用50或100 μM H2O2处理30分钟，或仅用HBSS处理（非氧化剂对照）。作为PAEC单层完整性的功能指标，然后测量单层对白蛋白的通透性3小时。用100 μM H2O2处理导致细胞毒性和PAEC单层通透性逐渐增加，而18:1补充可减弱这种增加，而50 μM H2O2仅导致通透性短暂增加而无细胞毒性。补充18:1还减弱了H2O2诱导的PAEC三磷酸腺苷（ATP）含量降低和PAEC微丝结构破坏。在不存在氧化应激的情况下，通过与含有脱氧葡萄糖和抗霉素A的葡萄糖耗尽培养基孵育，PAEC单层的ATP含量可逆性降低。代谢抑制剂诱导的ATP耗竭增加了单层通透性并改变了细胞骨架结构，这些改变在PAEC ATP含量恢复过程中得到解决。这些结果表明，ATP耗竭在屏障功能障碍中起关键作用，并表明18:1降低氧化剂介导的PAEC功能障碍和损伤的能力可能与其保留PAEC ATP含量的能力直接相关。
