Department of Physiology, School of Medical Sciences.
The New Zealand National Eye Centre, the University of Auckland, New Zealand.
Mol Vis. 2022 Sep 4;28:245-256. eCollection 2022.
Purinergic signaling pathways activated by extracellular ATP have been implicated in the regulation of lens volume and transparency. In this study, we investigated the location of ATP release from whole rat lenses and the mechanism by which osmotic challenge alters such ATP release.
Three-week-old rat lenses were cultured for 1 h in isotonic artificial aqueous humor (AAH) with no extracellular Ca, hypotonic AAH, or hypertonic AAH. The hypotonic AAH-treated lenses were also cultured in the absence or presence of connexin hemichannels and the pannexin channel blockers carbenoxolone, probenecid, and flufenamic acid. The ATP concentration in the AAH was determined using a Luciferin/luciferase bioluminescence assay. To visualize sites of ATP release induced by hemichannel and/or pannexin opening, the lenses were cultured in different AAH solutions, as described above, and incubated in the presence of Lucifer yellow (MW = 456 Da) and Texas red-dextran (MW = 10 kDa) for 1 h. Then the lenses were fixed, cryosectioned, and imaged using confocal microscopy to visualize areas of dye uptake from the extracellular space.
The incubation of the rat lenses in the AAH that lacked Ca induced a significant increase in the extracellular ATP concentration. This was associated with an increased uptake of Lucifer yellow but not of Texas red-dextran in a discrete region of the outer cortex of the lens. Hypotonic stress caused a similar increase in ATP release and an increase in the uptake of Lucifer yellow in the outer cortex, which was significantly reduced by probenecid but not by carbenoxolone or flufenamic acid.
Our data suggest that in response to hypotonic stress, the intact rat lens is capable of releasing ATP. This seems to be mediated via the opening of pannexin channels in a specific zone of the outer cortex of the lens. Our results support the growing evidence that the lens actively regulates its volume and therefore, its optical properties, via puerinergic signaling pathways.
细胞外 ATP 激活的嘌呤能信号通路被认为参与了晶状体体积和透明度的调节。在这项研究中，我们研究了整个大鼠晶状体中 ATP 释放的位置，以及渗透压挑战改变这种 ATP 释放的机制。
将 3 周龄大鼠晶状体在不含细胞外 Ca 的等渗人工房水（AAH）、低渗 AAH 或高渗 AAH 中培养 1 小时。在无连接子半通道和嘌呤能通道阻滞剂 carbenoxolone、probenecid 和 flufenamic acid 的存在或不存在的情况下，对低渗 AAH 处理的晶状体进行培养。用荧光素/荧光素酶生物发光法测定 AAH 中的 ATP 浓度。为了可视化半通道和/或嘌呤能通道开放诱导的 ATP 释放部位，将晶状体如上所述在不同的 AAH 溶液中培养，并在 Lucifer yellow（MW = 456 Da）和 Texas red-dextran（MW = 10 kDa）存在下孵育 1 小时。然后将晶状体固定、冷冻切片，并使用共聚焦显微镜成像，以可视化从细胞外空间摄取的染料区域。
在缺乏 Ca 的 AAH 中孵育大鼠晶状体会导致细胞外 ATP 浓度显著增加。这与 Lucifer yellow 的摄取增加有关，但与 Texas red-dextran 在外晶状体的离散区域的摄取增加无关。低渗应激导致 ATP 释放增加，并在外晶状体的外皮层摄取 Lucifer yellow 增加，这一增加被 probenecid 显著减少，但 carbenoxolone 或 flufenamic acid 则没有。
我们的数据表明，在低渗应激下，完整的大鼠晶状体能够释放 ATP。这似乎是通过在晶状体外皮层的特定区域开放嘌呤能通道来介导的。我们的结果支持越来越多的证据表明，晶状体通过嘌呤能信号通路主动调节其体积，从而调节其光学特性。
