Hammermann R, Dreissig M D, Mössner J, Fuhrmann M, Berrino L, Göthert M, Racké K
Institute of Pharmacology and Toxicology, Rheinische Friedrich Wilhelms University, Bonn, Germany.
Mol Pharmacol. 2000 Dec;58(6):1294-302.
The connection between the regulation of L-arginine transport and nitric oxide (NO) synthesis was studied in rat alveolar macrophages. Lipopolysaccharides (LPSs) and interferon-gamma stimulated in the same concentration- and time-dependent manner NO synthesis (measured by nitrite accumulation) and L-[(3)H]arginine uptake. This correlated with an increased mRNA expression for iNOS and the cationic amino acid transporter CAT-2B (analyzed by reverse transcription-polymerase chain reaction), with the same kinetics observed for the up-regulation of both mRNAs. Because nuclear factor-kappaB (NF-kappaB) is essential for induction of iNOS its role for the regulation of CAT-2B expression and L-arginine transport was investigated. The NF-kappaB inhibitors pyrrolidine dithiocarbamate and N(alpha)-p-tosyl-L-lysine chloromethyl ketone abrogated LPS- and interferon-gamma-induced increase of nitrite accumulation and L-[(3)H]arginine uptake as well as up-regulation of iNOS and CAT-2B mRNA. LPS-induced increase in iNOS and CAT-2B mRNA was also suppressed by specific NF-kappaB decoy oligodesoxynucleotides, confirming the essential role of NF-kappaB for iNOS and CAT-2B expression. Dexamethasone did not affect the initial (5 h) LPS-induced increase of iNOS and CAT-2B mRNA, but down-regulated both mRNAs after prolonged (20 h) exposure and this was accompanied by partial inhibition of LPS-stimulated nitrite accumulation and L-[(3)H]arginine uptake. These findings demonstrate parallel regulation of the expression of iNOS and CAT-2B, and of NO synthesis and L-arginine uptake in rat alveolar macrophages. NF-kappaB is an essential transcription factor not only for the induction of iNOS, but also for the up-regulation of CAT-2B. The simultaneous up-regulation of CAT-2B with iNOS is considered as a mechanism to ensure a high substrate supply for iNOS.
在大鼠肺泡巨噬细胞中研究了L-精氨酸转运调节与一氧化氮(NO)合成之间的联系。脂多糖(LPS)和干扰素-γ以相同的浓度和时间依赖性方式刺激NO合成(通过亚硝酸盐积累来测定)以及L-[³H]精氨酸摄取。这与诱导型一氧化氮合酶(iNOS)和阳离子氨基酸转运体CAT-2B的mRNA表达增加相关(通过逆转录-聚合酶链反应分析),两种mRNA上调观察到相同的动力学。因为核因子-κB(NF-κB)对于iNOS的诱导至关重要,所以研究了其对CAT-2B表达和L-精氨酸转运调节的作用。NF-κB抑制剂吡咯烷二硫代氨基甲酸盐和N-α-对甲苯磺酰-L-赖氨酸氯甲基酮消除了LPS和干扰素-γ诱导的亚硝酸盐积累增加、L-[³H]精氨酸摄取增加以及iNOS和CAT-2B mRNA的上调。LPS诱导的iNOS和CAT-2B mRNA增加也被特异性NF-κB诱饵寡脱氧核苷酸所抑制,证实了NF-κB对iNOS和CAT-2B表达的重要作用。地塞米松不影响LPS诱导的iNOS和CAT-2B mRNA的初始(5小时)增加,但在长时间(20小时)暴露后下调了两种mRNA,并且这伴随着LPS刺激的亚硝酸盐积累和L-[³H]精氨酸摄取的部分抑制。这些发现表明在大鼠肺泡巨噬细胞中iNOS和CAT-2B的表达、NO合成和L-精氨酸摄取存在平行调节。NF-κB不仅是iNOS诱导的必需转录因子,也是CAT-2B上调的必需转录因子。CAT-2B与iNOS同时上调被认为是确保iNOS有高底物供应的一种机制。