McDonald K K, Rouhani R, Handlogten M E, Block E R, Griffith O W, Allison R D, Kilberg M S
Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville 32610-0245, USA.
Biochim Biophys Acta. 1997 Feb 21;1324(1):133-41. doi: 10.1016/s0005-2736(96)00226-x.
A variety of N omega-monosubstituted L-arginine analogs are established inhibitors of nitric oxide synthase; in all cases, initial binding is competitive with the substrate L-arginine. The efficacy of such compounds in vivo will depend on their transport into the relevant nitric oxide synthase-containing cells; in fact, inhibition may actually be augmented if cellular uptake of L-arginine is also blocked by the analogs. Because vascular endothelial cells synthesize vasoactive nitric oxide under both physiological and pathophysiological conditions, we have performed inhibition analyses with novel arginine analogs to determine the substrate specificity of the primary L-arginine transport system. Na(+)-independent System y+, present in porcine pulmonary artery endothelial cells. As reported by others, no Na(+)-independent System bo,+ activity was detectable. For System y+. Dixon plots suggest competitive inhibition and apparent Ki values, which ranged between 0.1 and 0.8 mM, estimated for each inhibitor. Some influence of amino acid side chain structure could be detected, but in general, the data establish that this transport system accepts a broad range of arginine derivatives. Loading the cells with individual arginine analogs resulted in trans-stimulation of arginine uptake suggesting that they serve as substrates of System y+ as well as inhibitors. These results indicate that plasma membrane transport is unlikely to be a limiting factor in drug development for nitric oxide synthase inhibitors.
多种Nω-单取代L-精氨酸类似物是一氧化氮合酶的既定抑制剂；在所有情况下，初始结合与底物L-精氨酸具有竞争性。这类化合物在体内的功效将取决于它们转运到相关的含一氧化氮合酶的细胞中的能力；事实上，如果L-精氨酸的细胞摄取也被这些类似物阻断，抑制作用实际上可能会增强。由于血管内皮细胞在生理和病理生理条件下都会合成血管活性一氧化氮，我们用新型精氨酸类似物进行了抑制分析，以确定主要L-精氨酸转运系统的底物特异性。Na⁺非依赖性转运系统y⁺存在于猪肺动脉内皮细胞中。正如其他人所报道的，未检测到Na⁺非依赖性转运系统bo,+的活性。对于转运系统y⁺，狄克逊图表明存在竞争性抑制，并对每种抑制剂估计了介于0.1至0.8 mM之间的表观Ki值。可以检测到氨基酸侧链结构的一些影响，但总体而言，数据表明该转运系统接受多种精氨酸衍生物。用单个精氨酸类似物加载细胞导致精氨酸摄取的反式刺激，这表明它们既是转运系统y⁺的底物也是抑制剂。这些结果表明，质膜转运不太可能成为一氧化氮合酶抑制剂药物开发中的限制因素。
