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 P2X₇ receptor (P2X₇R), since it was mimicked by cell exposure to Bz-ATP, an agonist of P2X₇R, and blocked by cell pre-treatment with the P2X₇R 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）是嘌呤代谢中的一种关键酶，可将核糖核苷转化为嘌呤碱基，主要在神经胶质细胞内部发现。由于我们最近证明了PNP是从大鼠C6胶质瘤细胞中释放出来的，因此我们想知道这是否发生在正常的脑细胞中。使用小胶质细胞，星形胶质细胞和小脑颗粒神经元的大鼠原代培养物，我们发现在基础条件下，所有这些细胞都组成性地释放出具有Km值的代谢活性PNP，其Km值与C6胶质瘤细胞中测得的KNP值非常相似。但是，小胶质细胞或星形胶质细胞中的酶表达/释放比神经元中的更高。此外，我们将原发性脑细胞培养物暴露于促炎剂，例如单独或组合使用的脂多糖（LPS）或ATP。单独的LPS引起白细胞介素1β（IL-1β）分泌增加，主要来自小胶质细胞，PNP释放没有改变，甚至来自神经元也能增强细胞死亡。相反，以高微摩尔浓度单独向神经胶质细胞施用ATP可以在1 h内显着刺激PNP的释放，LPS的存在并不能改变这种作用，而仅在LPS引发2 h的细胞中，ATP可以刺激IL-1β分泌。在这两种情况下，ATP的作用都由P2X介导 而IL-1β分泌仅在LPS刺激2 h的细胞中受ATP刺激。在这两种情况下，ATP的作用都由P2X介导 而IL-1β分泌仅在LPS刺激2 h的细胞中受ATP刺激。在这两种情况下，ATP的作用都由P2X介导₇受体（P2X ₇ R），因为它被细胞暴露于B2-ATP（P2X ₇ R的激动剂）所模仿，并被P2X ₇ R拮抗剂A438079预处理。有趣的是，ATP诱导的PNP从神经胶质细胞释放的部分原因是溶酶体囊泡在细胞外培养基中的分泌。因此，在炎性脑事件期间，由细胞外ATP积累促进的PNP分泌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，嘌呤代谢级联反应中PNP下游酶的共有活性是否避免了细胞外嘌呤碱基的积累，而这些碱基可能会因反应性氧的异常形成而导致脑损伤。