Tonnessen B H, Severson S R, Hurt R D, Miller V M
Department of Surgery, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
J Pharmacol Exp Ther. 2000 Nov;295(2):601-6.
Effects of nicotine on arterial endothelium-dependent relaxations mediated by nitric oxide are controversial. Experiments were designed to test the hypothesis that nicotine can directly alter activity of endothelial nitric-oxide synthase (eNOS). NOS from aortic endothelial cells of untreated dogs and recombinant eNOS, neuronal NOS, and inducible NOS were used for these experiments. NOS activity was determined as conversion of L-[(3)H]arginine to L-[(3)H]citrulline in the absence or presence of nicotine (10(-7)-10(-3) M) in vitro. In separate assays, concentrations of cofactors NADPH, FAD, and tetrahydrobioprotein were reduced by half to assess for possible interaction with nicotine. With enzyme from aortic endothelial cells, total and calcium-dependent accumulation of citrulline increased by 30% in the presence of 10(-5) M nicotine. Nicotine dose dependently also increased citrulline accumulation by recombinant eNOS and neuronal NOS but not inducible NOS. Effects of nicotine on accumulation of citrulline by isolated eNOS and recombinant eNOS were further modulated by changes in the concentration of NADPH in the incubation solution. Our data demonstrate a significant effect of nicotine on eNOS-mediated citrulline accumulation. These results suggest that effects of nicotine on production of nitric oxide may depend on NADPH or oxygen radical interactions with NOS and thus may explain, in part, inconsistent findings of changes in production of endothelium-derived nitric oxide with nicotine administration.
尼古丁对由一氧化氮介导的动脉内皮依赖性舒张的影响存在争议。本实验旨在验证尼古丁可直接改变内皮型一氧化氮合酶（eNOS）活性这一假说。实验采用未处理犬主动脉内皮细胞的一氧化氮合酶以及重组eNOS、神经元型一氧化氮合酶和诱导型一氧化氮合酶。体外实验中，在有无尼古丁（10⁻⁷ - 10⁻³ M）的情况下，通过测定L-[(³)H]精氨酸向L-[(³)H]瓜氨酸的转化来确定一氧化氮合酶的活性。在单独的检测中，将辅因子NADPH、FAD和四氢生物蝶呤的浓度减半，以评估与尼古丁可能的相互作用。对于主动脉内皮细胞的酶，在10⁻⁵ M尼古丁存在的情况下，瓜氨酸的总量和钙依赖性积累增加了30%。尼古丁剂量依赖性地也增加了重组eNOS和神经元型一氧化氮合酶瓜氨酸的积累，但对诱导型一氧化氮合酶没有影响。孵育溶液中NADPH浓度的变化进一步调节了尼古丁对分离的eNOS和重组eNOS瓜氨酸积累的影响。我们的数据表明尼古丁对eNOS介导的瓜氨酸积累有显著影响。这些结果表明，尼古丁对一氧化氮产生的影响可能取决于NADPH或氧自由基与一氧化氮合酶的相互作用，因此可能部分解释了尼古丁给药后内皮源性一氧化氮产生变化的不一致结果。
