Decoding the Substrate Supply to Human Neuronal Nitric Oxide Synthase

Nitric oxide, produced by the neuronal nitric oxide synthase (nNOS) from L-arginine is an important second messenger molecule in the central nervous system: It influences the synthesis and release of neurotransmitters and plays an important role in long-term potentiation, long-term depression and neuroendocrine secretion. However, under certain pathological conditions such as Alzheimer’s or Parkinson’s disease, stroke and multiple sclerosis, excessive NO production can lead to tissue damage. It is thus desirable to control NO production in these situations. So far, little is known about the substrate supply to human nNOS as a determinant of its activity. Measuring bioactive NO via cGMP formation in reporter cells, we demonstrate here that nNOS in both, human A673 neuroepithelioma and TGW-nu-I neuroblastoma cells can be fast and efficiently nourished by extracellular arginine that enters the cells via membrane transporters (pool I that is freely exchangeable with the extracellular space). When this pool was depleted, NO synthesis was partially sustained by intracellular arginine sources not freely exchangeable with the extracellular space (pool II). Protein breakdown made up by far the largest part of pool II in both cell types. In contrast, citrulline to arginine conversion maintained NO synthesis only in TGW-nu-I neuroblastoma, but not A673 neuroepithelioma cells. Histidine mimicked the effect of protease inhibitors causing an almost complete nNOS inhibition in cells incubated additionally in lysine that depletes the exchangeable arginine pool. Our results identify new ways to modulate nNOS activity by modifying its substrate supply.神经元型一氧化氮合酶（neuronal nitric oxide synthase, nNOS）以L-精氨酸（L-arginine）为底物合成的一氧化氮（nitric oxide, NO）是中枢神经系统中重要的第二信使分子：它可影响神经递质的合成与释放，并在长时程增强（long-term potentiation）、长时程抑制（long-term depression）和神经内分泌分泌过程中发挥关键作用。然而在阿尔茨海默病（Alzheimer’s disease）、帕金森病（Parkinson’s disease）、脑卒中（stroke）以及多发性硬化（multiple sclerosis）等特定病理条件下，过量的NO生成会引发组织损伤，因此在此类情境下调控NO的合成具有重要意义。迄今为止，关于人类nNOS的底物供给如何作为其活性的决定因素，相关研究仍较为匮乏。本研究通过报告细胞（reporter cells）中的cGMP生成量检测具有生物活性的NO，证实人类A673神经上皮瘤细胞与TGW-nu-I神经母细胞瘤细胞中的nNOS均可通过细胞膜转运蛋白（membrane transporters）摄取胞外精氨酸，该精氨酸池可与胞外空间自由交换（记为池I），从而实现快速且高效的底物供给。当该池被耗尽后，NO的合成可部分由无法与胞外空间自由交换的胞内精氨酸来源（池II）维持，在两种细胞系中，蛋白质降解（protein breakdown）均是池II最主要的组成部分。与之相反，瓜氨酸（citrulline）向精氨酸的转化仅能在TGW-nu-I神经母细胞瘤细胞中维持NO的合成，而对A673神经上皮瘤细胞无此效果。在额外添加赖氨酸（lysine）以耗尽可交换精氨酸池的细胞中，组氨酸（histidine）可模拟蛋白酶抑制剂（protease inhibitors）的作用，实现nNOS的近乎完全抑制。本研究结果揭示了通过调控nNOS的底物供给来调节其活性的全新途径。