Center for Molecular Physiology Research, Children's National Medical Center, George Washington University, Washington, DC 20010, USA.
Free Radic Biol Med. 2012 Aug 1;53(3):437-46. doi: 10.1016/j.freeradbiomed.2012.05.015. Epub 2012 May 23.
The dopamine D(2) receptor (D(2)R) regulates renal reactive oxygen species (ROS) production, and impaired D(2)R function results in ROS-dependent hypertension. Paraoxonase 2 (PON2), which belongs to the paraoxonase gene family, is expressed in various tissues, acting to protect against cellular oxidative stress. We hypothesized that PON2 may be involved in preventing excessive renal ROS production and thus may contribute to maintenance of normal blood pressure. Moreover, D(2)R may decrease ROS production, in part, through regulation of PON2. D(2)R colocalized with PON2 in the brush border of mouse renal proximal tubules. Renal PON2 protein was decreased (-33±6%) in D(2)(-/-) relative to D(2)(+/+) mice. Renal subcapsular infusion of PON2 siRNA decreased PON2 protein expression (-55%), increased renal oxidative stress (2.2-fold), associated with increased renal NADPH oxidase expression (Nox1, 1.9-fold; Nox2, 2.9-fold; and Nox4, 1.6-fold) and activity (1.9-fold), and elevated arterial blood pressure (systolic, 134±5 vs 93±6mmHg; diastolic, 97±4 vs 65±7mmHg; mean 113±4 vs 75±7mmHg). To determine the relevance of the PON2 and D(2)R interaction in humans, we studied human renal proximal tubule cells. Both D(2)R and PON2 were found in nonlipid and lipid rafts and physically interacted with each other. Treatment of these cells with the D(2)R/D(3)R agonist quinpirole (1μM, 24h) decreased ROS production (-35±6%), associated with decreased NADPH oxidase activity (-32±3%) and expression of Nox2 (-41±7%) and Nox4 (-47±8%) protein, and increased expression of PON2 mRNA (2.1-fold) and protein (1.6-fold) at 24h. Silencing PON2 (siRNA, 10nM, 48h) not only partially prevented the quinpirole-induced decrease in ROS production by 36%, but also increased basal ROS production (1.3-fold), which was associated with an increase in NADPH oxidase activity (1.4-fold) and expression of Nox2 (2.1-fold) and Nox4 (1.8-fold) protein. Inhibition of NADPH oxidase with diphenylene iodonium (10μM/30 min) inhibited the increase in ROS production caused by PON2 silencing. Our results suggest that renal PON2 is involved in the inhibition of renal NADPH oxidase activity and ROS production and contributes to the maintenance of normal blood pressure. PON2 is positively regulated by D(2)R and may, in part, mediate the inhibitory effect of renal D(2)R on NADPH oxidase activity and ROS production.
多巴胺 D(2) 受体 (D(2)R) 调节肾脏活性氧 (ROS) 的产生，而 D(2)R 功能受损会导致 ROS 依赖性高血压。对氧磷酶 2 (PON2) 属于对氧磷酶基因家族，在各种组织中表达，可起到保护细胞免受氧化应激的作用。我们假设 PON2 可能参与防止肾脏 ROS 过度产生，从而有助于维持正常血压。此外，D(2)R 可能通过调节 PON2 来减少 ROS 的产生。D(2)R 与小鼠肾脏近端肾小管刷状缘的 PON2 共定位。与 D(2)(+/+) 小鼠相比，D(2)(-/-) 小鼠的肾脏 PON2 蛋白减少了 (-33±6%)。肾脏被膜下输注 PON2 siRNA 会降低 PON2 蛋白表达 (-55%)，增加肾脏氧化应激 (2.2 倍)，同时增加肾脏 NADPH 氧化酶表达 (Nox1，1.9 倍；Nox2，2.9 倍；和 Nox4，1.6 倍) 和活性 (1.9 倍)，并升高动脉血压 (收缩压，134±5 对 93±6mmHg；舒张压，97±4 对 65±7mmHg；平均 113±4 对 75±7mmHg)。为了确定 PON2 和 D(2)R 相互作用在人类中的相关性，我们研究了人类肾脏近端肾小管细胞。D(2)R 和 PON2 均存在于非脂筏和脂筏中，并相互物理作用。用 D(2)R/D(3)R 激动剂喹吡罗 (1μM，24 小时) 处理这些细胞会减少 ROS 产生 (-35±6%)，同时降低 NADPH 氧化酶活性 (-32±3%)和 Nox2 (-41±7%) 和 Nox4 (-47±8%) 蛋白的表达，并增加 PON2 mRNA (2.1 倍) 和蛋白 (1.6 倍) 的表达，持续 24 小时。PON2 沉默 (siRNA，10nM，48 小时) 不仅部分阻止了喹吡罗诱导的 ROS 产生减少 36%，而且还增加了基础 ROS 产生 (1.3 倍)，这与 NADPH 氧化酶活性 (1.4 倍) 和 Nox2 (2.1 倍) 和 Nox4 (1.8 倍) 蛋白的表达增加有关。用二苯基碘鎓 (10μM/30 分钟) 抑制 NADPH 氧化酶会抑制 PON2 沉默引起的 ROS 产生增加。我们的结果表明，肾脏 PON2 参与抑制肾脏 NADPH 氧化酶活性和 ROS 产生，并有助于维持正常血压。PON2 受到 D(2)R 的正向调节，可能部分介导了肾脏 D(2)R 对 NADPH 氧化酶活性和 ROS 产生的抑制作用。
