USDA-Agricultural Research Service Children's Nutrition Research Center.
Department of Obstetrics and Gynecology.
J Nutr. 2018 Jun 1;148(6):844-850. doi: 10.1093/jn/nxy065.
The recycling of citrulline by argininosuccinate synthase 1 (ASS1) and argininosuccinate lyase (ASL) is crucial to maintain arginine availability and nitric oxide (NO) production. Pegylated arginine deiminase (ADI-PEG20) is a bacterial enzyme used to deplete circulating arginine.
The goal of this research was to test the hypothesis that citrulline is able to sustain intracellular arginine availability for NO production in ADI-PEG20 arginine-depleted mice.
Six- to 8-wk-old male C57BL/6J mice injected with ADI-PEG20 (5 IU) or saline (control) were used in 4 different studies. Arginine, citrulline, and NO kinetics were determined by using stable isotopes in unchallenged (study 1) and endotoxin-challenged (study 2) mice. Blood pressure was determined by telemetry for 6 d after ADI-PEG20 administration (study 3), and vasomotor activity and ASS1 and ASL gene expression were determined in mesenteric arteries collected from additional mice (study 4).
ADI-PEG20 administration resulted in arginine depletion (<1 compared with 111 ± 37 µmol/L) but in greater plasma citrulline concentrations (900 ± 123 compared with 76 ± 8 µmol/L; P < 0.001) and fluxes (402 ± 17 compared with 126 ± 4 µmol ⋅ kg-1 ⋅ h-1; P < 0.001) compared with controls. Endotoxin-challenged ADI-PEG20-treated mice produced less NO than controls (13 ± 1 compared with 27 ± 2 µmol ⋅ kg-1 ⋅ h-1; P < 0.001). No differences (P > 0.50) were observed for cardiovascular variables (heart rate, blood pressure) between ADI-PEG20-treated and control mice. Furthermore, no ex vivo vasomotor differences were observed between the 2 treatments. ADI-PEG20 administration resulted in greater gene expression of ASS1 (∼3-fold) but lower expression of ASL (-30%).
ADI-PEG20 successfully depleted circulating arginine without any effect on cardiovascular endpoints in healthy mice but limited NO production after endotoxin challenge. Therefore, the citrulline recycling pathway can sustain local arginine availability independently from circulating arginine, satisfying the demand of arginine for endothelial NO production; however, it is unable to do so when a high demand for arginine is elicited by endotoxin.
精氨酸代琥珀酸合成酶 1（ASS1）和精氨酸代琥珀酸裂解酶（ASL）对瓜氨酸的再循环对于维持精氨酸的可用性和一氧化氮（NO）的产生至关重要。聚乙二醇化精氨酸脱亚氨酶（ADI-PEG20）是一种用于耗尽循环精氨酸的细菌酶。
本研究旨在测试以下假设，即在 ADI-PEG20 耗尽精氨酸的小鼠中，瓜氨酸能够维持细胞内精氨酸的可用性以产生 NO。
使用在未受到挑战（研究 1）和内毒素挑战（研究 2）的小鼠中使用稳定同位素测定的 6 至 8 周龄雄性 C57BL/6J 小鼠，注射 ADI-PEG20（5IU）或生理盐水（对照）。在 ADI-PEG20 给药后 6 天通过遥测法测定血压（研究 3），并从其他小鼠中收集肠系膜动脉以测定血管舒缩活性和 ASS1 和 ASL 基因表达（研究 4）。
ADI-PEG20 给药导致精氨酸耗竭（<1 与 111±37µmol/L 相比），但血浆瓜氨酸浓度更高（900±123 与 76±8µmol/L 相比；P<0.001），通量更大（402±17 与 126±4µmol ⋅ kg-1 ⋅ h-1 相比；P<0.001）与对照组相比。内毒素挑战的 ADI-PEG20 治疗的小鼠产生的 NO 少于对照组（13±1 与 27±2µmol ⋅ kg-1 ⋅ h-1 相比；P<0.001）。ADI-PEG20 治疗和对照组之间的心血管变量（心率、血压）没有差异（P>0.50）。此外，两种治疗方法之间没有观察到体外血管舒缩差异。ADI-PEG20 给药导致 ASS1 的基因表达增加（约 3 倍），但 ASL 的表达降低（-30%）。
ADI-PEG20 在健康小鼠中成功耗尽了循环精氨酸，而对心血管终点没有影响，但在内毒素挑战后限制了 NO 的产生。因此，瓜氨酸再循环途径可以独立于循环精氨酸维持局部精氨酸的可用性，满足内皮细胞 NO 产生对精氨酸的需求；然而，当内毒素引起精氨酸的高需求时，它就无法做到这一点。
