Department of Pharmacy and Bio-Technology, University of Bologna, Via Selmi 3, 40126 Bologna, Italy.
Department of Medical, Oral and Biotechnology Sciences, University of Chieti-Pescara, Via dei Vestini 29, 66100 Chieti, Italy; Aging Research Center and Translational Medicine (CeSI-MeT), University of Chieti-Pescara, Via dei Vestini 29, 66100 Chieti, Italy.
Neurochem Int. 2018 May;115:37-49. doi: 10.1016/j.neuint.2017.10.010. Epub 2017 Oct 20.
Purine nucleoside phosphorylase (PNP), a crucial enzyme in purine metabolism which converts ribonucleosides into purine bases, has mainly been found inside glial cells. Since we recently demonstrated that PNP is released from rat C6 glioma cells, we then wondered whether this occurs in normal brain cells. Using rat primary cultures of microglia, astrocytes and cerebellar granule neurons, we found that in basal condition all these cells constitutively released a metabolically active PNP with Km values very similar to those measured in C6 glioma cells. However, the enzyme expression/release was greater in microglia or astrocytes that in neurons. Moreover, we exposed primary brain cell cultures to pro-inflammatory agents such as lipopolysaccharide (LPS) or ATP alone or in combination. LPS alone caused an increased interleukin-1β (IL-1β) secretion mainly from microglia and no modification in the PNP release, even from neurons in which it enhanced cell death. In contrast, ATP administered alone to glial cells at high micromolar concentrations significantly stimulated the release of PNP within 1 h, an effect not modified by LPS presence, whereas IL-1β secretion was stimulated by ATP only in cells primed for 2 h with LPS. In both cases ATP effect was mediated by P2X receptor (P2XR), since it was mimicked by cell exposure to Bz-ATP, an agonist of P2XR, and blocked by cell pre-treatment with the P2XR antagonist A438079. Interestingly, ATP-induced PNP release from glial cells partly occurred through the secretion of lysosomal vesicles in the extracellular medium. Thus, during inflammatory cerebral events PNP secretion promoted by extracellular ATP accumulation might concur to control extracellular purine signals. Further studies could elucidate whether, in these conditions, a consensual activity of enzymes downstream of PNP in the purine metabolic cascade avoids accumulation of extracellular purine bases that might concur to brain injury by unusual formation of reactive oxygen species.
嘌呤核苷磷酸化酶（PNP）是嘌呤代谢中的关键酶，可将核糖核苷转化为嘌呤碱基，主要存在于神经胶质细胞中。由于我们最近证明 PNP 可从大鼠 C6 神经胶质瘤细胞中释放，我们想知道这种情况是否发生在正常脑细胞中。使用大鼠原代小胶质细胞、星形胶质细胞和小脑颗粒神经元培养物，我们发现，在基础条件下，所有这些细胞都持续释放具有代谢活性的 PNP，其 Km 值与在 C6 神经胶质瘤细胞中测量的值非常相似。然而，在小胶质细胞或星形胶质细胞中，酶的表达/释放比神经元中更大。此外，我们将原代脑细胞培养物暴露于促炎剂，如单独的脂多糖（LPS）或三磷酸腺苷（ATP）或两者的组合。LPS 单独作用会导致白细胞介素-1β（IL-1β）的分泌增加，主要来自小胶质细胞，而不会改变 PNP 的释放，即使在神经元中，它也会增强细胞死亡。相反，单独用高微摩尔浓度的 ATP 处理胶质细胞会在 1 小时内显著刺激 PNP 的释放，这种作用不受 LPS 存在的影响，而只有在预先用 LPS 处理 2 小时的细胞中，IL-1β 的分泌才会被 ATP 刺激。在这两种情况下，ATP 的作用都是由 P2X 受体（P2XR）介导的，因为用 Bz-ATP （P2XR 的激动剂）处理细胞可模拟该作用，并用 P2XR 拮抗剂 A438079 预处理细胞可阻断该作用。有趣的是，ATP 诱导的胶质细胞 PNP 释放部分通过细胞外基质中溶酶体囊泡的分泌发生。因此，在炎症性脑事件中，细胞外 ATP 积累促进的 PNP 释放可能有助于控制细胞外嘌呤信号。进一步的研究可以阐明，在这些条件下，PNP 代谢级联下游酶的一致活性是否可以避免细胞外嘌呤碱基的积累，而这些嘌呤碱基可能通过异常形成活性氧而导致脑损伤。
