Department of Ophthalmology, Duke University, Durham, North Carolina, USA.
Invest Ophthalmol Vis Sci. 2012 Oct 1;53(11):7142-8. doi: 10.1167/iovs.12-9968.
To investigate the mechanisms for endogenous production of extracellular adenosine in trabecular meshwork (TM) cells and evaluate its physiological relevance to the regulation of aqueous humor outflow facility.
Extra-cellular levels of adenosine monophosphate (AMP) and adenosine in porcine trabecular meshwork (PTM) cells treated with adenosine triphosphate (ATP), AMP, cAMP or forskolin with or without specific inhibitors of phosphodiesterases (IBMX) and CD73 (AMPCP) were determined by high-pressure liquid chromatography fluorometry. Extracellular adenosine was also evaluated in cell cultures subjected to cyclic mechanical stress (CMS) (20% stretching; 1 Hz) and after disruption of lipid rafts with methyl-β-cyclodextrin. Expression of CD39 and CD73 in porcine TM cells and tissue were examined by Q-PCR and Western blot. The effect of inhibition of CD73 on outflow facility was evaluated in perfused living mouse eyes.
PTM cells generated extracellular adenosine from extracellular ATP and AMP but not from extracellular cAMP. Increased intracellular cAMP mediated by forskolin led to a significant increase in extracellular adenosine production that was not prevented by IBMX. Inhibition of CD73 resulted, in all cases, in a significant decrease in extracellular adenosine. CMS induced a significant activation of extracellular adenosine production. Inhibition of CD73 activity with AMPCP in living mouse eyes resulted in a significant decrease in outflow facility.
These results support the concept that the extracellular adenosine pathway might play an important role in the homeostatic regulation of outflow resistance in the TM, and suggest a novel mechanism by which pathologic alteration of the TM, such as increased tissue rigidity, could lead to abnormal elevation of IOP in glaucoma.
研究小梁网（TM）细胞内源性产生细胞外腺苷的机制，并评估其对房水流出阻力调节的生理相关性。
用高效液相色谱荧光法测定用三磷酸腺苷（ATP）、单磷酸腺苷（AMP）、环磷酸腺苷（cAMP）或佛司可林处理的猪 TM 细胞（PTM）中细胞外 AMP 和腺苷的浓度，并用磷酸二酯酶（IBMX）和 CD73（AMPCP）的特定抑制剂处理。还评估了细胞培养物在周期性机械应激（CMS）（20%拉伸；1 Hz）和用甲基-β-环糊精破坏脂筏后细胞外腺苷的浓度。通过 Q-PCR 和 Western blot 检测猪 TM 细胞和组织中 CD39 和 CD73 的表达。用 CD73 抑制剂评价其对灌流活体小鼠眼流出阻力的影响。
PTM 细胞从细胞外 ATP 和 AMP 生成细胞外腺苷，但不从细胞外 cAMP 生成。佛司可林介导的细胞内 cAMP 增加导致细胞外腺苷生成显著增加，但 IBMX 不能阻止。在所有情况下，CD73 抑制均导致细胞外腺苷显著减少。CMS 诱导细胞外腺苷生成显著激活。在活体小鼠眼中用 AMPCP 抑制 CD73 活性导致流出阻力显著降低。
这些结果支持细胞外腺苷途径可能在 TM 中对流出阻力的稳态调节中起重要作用的概念，并提示 TM 的病理改变，如组织刚性增加，可能导致青光眼眼压异常升高的新机制。
