Merker M P, Olson L E, Bongard R D, Patel M K, Linehan J H, Dawson C A
Department of Anesthesiology, Medical College of Wisconsin, Milwaukee 53226, USA.
Am J Physiol. 1998 May;274(5):L685-93. doi: 10.1152/ajplung.1998.274.5.L685.
Pulmonary endothelial cells are capable of reducing certain electron acceptors at the luminal plasma membrane surface. Motivation for studying this phenomenon comes in part from the expectation that it may be important both as an endothelial antioxidant defense mechanism and in redox cycling of toxic free radicals. Pulmonary arterial endothelial cells in culture reduce the oxidized forms of thiazine compounds that have been used as electron acceptor probes for studying the mechanisms of transplasma membrane electron transport. However, they reduce another commonly studied electron acceptor, ferricyanide, only very slowly by comparison. In the present study, we examined the influence of ascorbate [ascorbic acid (AA)] and dehydroascorbate [dehydroascorbic acid (DHAA)] on the ferricyanide and thiazine reductase activities of the bovine pulmonary arterial endothelial cell surface. The endothelial cells were grown on microcarrier beads so that the reduction of ferricyanide and methylene blue could be studied colorimetrically in spectrophotometer cuvettes and in flow-through cell columns. The ferricyanide reductase activity could be increased 80-fold by adding DHAA to the medium, with virtually no effect on methylene blue reduction. The DHAA effect persisted after the DHAA was removed from the medium. AA also stimulated the ferricyanide reductase activity but was less potent, and the relative potencies of AA and DHAA correlated with their relative rates of uptake by the cells. The results are consistent with the hypothesis that AA is an intracellular electron donor for an endothelial plasma membrane ferricyanide reductase and that the stimulatory effect of DHAA is the result of increasing intracellular AA. Adding sufficient DHAA to markedly increase extracellular ferricyanide reduction had little effect on the plasma membrane methylene blue reductase activity, suggesting that pulmonary arterial endothelial cells have at least two separate transplasma membrane electron transport systems.
肺内皮细胞能够在管腔质膜表面还原某些电子受体。研究这一现象的动机部分源于这样的期望，即它可能作为一种内皮抗氧化防御机制以及在有毒自由基的氧化还原循环中都很重要。培养的肺动脉内皮细胞可还原噻嗪化合物的氧化形式，这些化合物已被用作电子受体探针来研究跨质膜电子转运机制。然而，相比之下，它们还原另一种常用的电子受体铁氰化物的速度非常慢。在本研究中，我们研究了抗坏血酸盐[抗坏血酸（AA）]和脱氢抗坏血酸盐[脱氢抗坏血酸（DHAA）]对牛肺动脉内皮细胞表面铁氰化物和噻嗪还原酶活性的影响。内皮细胞在微载体珠上生长，以便在分光光度计比色皿和流通式细胞柱中通过比色法研究铁氰化物和亚甲蓝的还原。向培养基中添加DHAA可使铁氰化物还原酶活性提高80倍，而对亚甲蓝还原几乎没有影响。从培养基中去除DHAA后，DHAA的作用仍然存在。AA也刺激铁氰化物还原酶活性，但效力较小，并且AA和DHAA的相对效力与其被细胞摄取的相对速率相关。这些结果与以下假设一致：AA是内皮质膜铁氰化物还原酶的细胞内电子供体，并且DHAA的刺激作用是细胞内AA增加的结果。添加足够的DHAA以显著增加细胞外铁氰化物还原对质膜亚甲蓝还原酶活性几乎没有影响，这表明肺动脉内皮细胞至少有两个独立的跨质膜电子转运系统。
