Arthritis Res Ther. 2013 Oct 17;15(5):R154. doi: 10.1186/ar4337.
Extracellular ATP (eATP) is released by articular chondrocytes under physiological and pathological conditions. High eATP levels cause pathologic calcification, damage cartilage, and mediate pain. We recently showed that stable over-expression of the progressive ankylosis gene product, ANK, increased chondrocyte eATP levels, but the mechanisms of this effect remained unexplored. The purpose of this work was to further investigate mechanisms of eATP efflux in primary articular chondrocytes and to better define the role of ANK in this process.
We measured eATP levels using a bioluminescence-based assay in adult porcine articular chondrocyte media with or without a 10 minute exposure to hypotonic stress. siRNAs for known ATP membrane transporters and pharmacologic inhibitors of ATP egress pathways were used to identify participants involved in chondrocyte eATP release.
eATP levels increased after exposure to hypotonic media in a calcium-dependent manner in monolayer and 3-dimensional agarose gel cultures (p < 0.001). A potent transient receptor potential vanilloid 4 (TRPV4) agonist mimicked the effects of hypotonic media. ANK siRNA suppressed basal (p < 0.01) and hypotonically-stressed (p < 0.001) ATP levels. This effect was not mediated by altered extracellular pyrophosphate (ePPi) levels, and was mimicked by the ANK inhibitor, probenecid (p < 0.001). The P2X7/4 receptor inhibitor Brilliant Blue G also suppressed eATP efflux induced by hypotonic media (p < 0.001), while ivermectin, a P2X4 receptor stimulant, increased eATP levels (p < 0.001). Pharmacologic inhibitors of hemichannels, maxianion channels and other volume-sensitive eATP efflux pathways did not suppress eATP levels.
These findings implicate ANK and P2X7/4 receptors in chondrocyte eATP efflux. Understanding the mechanisms of eATP efflux may result in novel therapies for calcium crystal arthritis and osteoarthritis.
细胞外三磷酸腺苷（eATP）在生理和病理条件下由关节软骨细胞释放。高浓度的 eATP 会导致病理性钙化、软骨损伤，并介导疼痛。我们最近发现，进行性强直基因产物 ANK 的稳定过表达会增加软骨细胞的 eATP 水平，但这种效应的机制仍未得到探索。本研究的目的是进一步研究原代关节软骨细胞中 eATP 外排的机制，并更好地定义 ANK 在这一过程中的作用。
我们使用生物发光法检测成年猪关节软骨细胞培养基中的 eATP 水平，在培养基中加入或不加入低渗应激 10 分钟。使用已知的 ATP 膜转运体的 siRNA 和 ATP 外排途径的药理学抑制剂来鉴定参与软骨细胞 eATP 释放的参与者。
在单层和 3 维琼脂糖凝胶培养物中，eATP 水平在暴露于低渗培养基后以钙离子依赖的方式增加（p < 0.001）。一种有效的瞬时受体电位香草酸 4（TRPV4）激动剂模拟了低渗培养基的作用。ANK siRNA 抑制基础（p < 0.01）和低渗应激（p < 0.001）ATP 水平。这种作用不是通过改变细胞外焦磷酸盐（ePPi）水平介导的，并且可以被 ANK 抑制剂丙磺舒模拟（p < 0.001）。P2X7/4 受体抑制剂 Brilliant Blue G 也抑制了低渗培养基诱导的 eATP 外排（p < 0.001），而 P2X4 受体刺激剂伊维菌素增加了 eATP 水平（p < 0.001）。其他的半通道、最大阴离子通道和其他体积敏感的 eATP 外排途径的药理学抑制剂并未抑制 eATP 水平。
这些发现表明 ANK 和 P2X7/4 受体参与了软骨细胞的 eATP 外排。了解 eATP 外排的机制可能会为钙晶体关节炎和骨关节炎的新型治疗方法提供依据。
