Inosine is an endogenous nucleoside that is produced by metabolic deamination of adenosine. Inosine is metabolically more stable (half-life 15 h) than adenosine (half-life < 10 s). Inosine exerts anti-inflammatory and immunomodulatory effects similar to those observed with adenosine. These effects are mediated in part through the adenosine A_2A receptor (A_2AR). Relative to adenosine inosine exhibits a lower affinity towards the A_2AR. Therefore, it is generally believed that inosine is incapable of activating the A_2AR through direct engagement, but indirectly activates the A_2AR upon metabolic conversion to higher affinity adenosine. A handful of studies, however, have provided evidence for direct inosine engagement at the A_2AR leading to activation of downstream signaling events and inhibition of cytokine production. Here, we demonstrate that under conditions devoid of adenosine, inosine as well as an analog of inosine 6-S-[(4-Nitrophenyl)methyl]-6-thioinosine selectively and dose-dependently activated A_2AR-mediated cAMP production and ERK1/2 phosphorylation in CHO cells stably expressing the human A_2AR. Inosine also inhibited LPS-stimulated TNF-α, CCL3 and CCL4 production by splenic monocytes in an A_2AR-dependent manner. In addition, we demonstrate that a positive allosteric modulator (PAM) of the A_2AR enhanced inosine-mediated cAMP production, ERK1/2 phosphorylation and inhibition of pro-inflammatory cytokine and chemokine production. The cumulative effects of allosteric enhancement of adenosine-mediated and inosine-mediated A_2AR activation may be the basis for the sustained anti-inflammatory and immunomodulatory effects observed in vivo and thereby provide insights into potential therapeutic interventions for inflammation- and immune-mediated diseases.

肌苷是内源性核苷，通过腺苷的代谢脱氨作用产生。肌苷在代谢方面（半衰期为15小时）比腺苷（半衰期<10 s）更稳定。肌苷具有类似于用腺苷观察到的抗炎和免疫调节作用。这些作用部分通过腺苷A _2A受体（A _2A R）介导。相对于腺苷，肌苷对A _2A R的亲和力较低。因此，通常认为肌苷无法通过直接参与激活A _2A R，但间接激活A _2AR经代谢转化为更高亲和力的腺苷。然而，少数研究提供了肌苷直接参与A _2A R的证据，从而导致下游信号传导事件的激活和细胞因子产生的抑制。在这里，我们证明在没有腺苷的条件下，肌苷以及肌苷6-S-[（4-硝基苯基）甲基] -6-硫代肌苷的类似物选择性地且剂量依赖性地激活了A _2A R介导的cAMP产生和ERK1。稳定表达人A _2A R的CHO细胞中的/ 2磷酸化。肌苷还以A _2A R依赖的方式抑制脾单核细胞对LPS刺激的TNF-α，CCL3和CCL4的产生。此外，我们证明了A的正变构调节剂（PAM）_2A R增强肌苷介导的cAMP产生，ERK1 / 2磷酸化并抑制促炎性细胞因子和趋化因子的产生。腺苷介导和肌苷介导的A _2A R活化的变构增强作用的累积效应可能是体内观察到的持续抗炎和免疫调节作用的基础，从而为炎症和免疫介导的疾病的潜在治疗干预提供了见识。