Department of Physiology and Biophysics, Miller School of Medicine, University of Miami, Miami, Florida 33136, USA.
J Neurosci. 2011 Sep 21;31(38):13654-61. doi: 10.1523/JNEUROSCI.3356-11.2011.
In response to gustatory stimulation, taste bud cells release a transmitter, ATP, that activates P2X2 and P2X3 receptors on gustatory afferent fibers. Taste behavior and gustatory neural responses are largely abolished in mice lacking P2X2 and P2X3 receptors [P2X2 and P2X3 double knock-out (DKO) mice]. The assumption has been that eliminating P2X2 and P2X3 receptors only removes postsynaptic targets but that transmitter secretion in mice is normal. Using functional imaging, ATP biosensor cells, and a cell-free assay for ATP, we tested this assumption. Surprisingly, although gustatory stimulation mobilizes Ca(2+) in taste Receptor (Type II) cells from DKO mice, as from wild-type (WT) mice, taste cells from DKO mice fail to release ATP when stimulated with tastants. ATP release could be elicited by depolarizing DKO Receptor cells with KCl, suggesting that ATP-release machinery remains functional in DKO taste buds. To explore the difference in ATP release across genotypes, we used reverse transcriptase (RT)-PCR, immunostaining, and histochemistry for key proteins underlying ATP secretion and degradation: Pannexin1, TRPM5, and NTPDase2 (ecto-ATPase) are indistinguishable between WT and DKO mice. The ultrastructure of contacts between taste cells and nerve fibers is also normal in the DKO mice. Finally, quantitative RT-PCR show that P2X4 and P2X7, potential modulators of ATP secretion, are similarly expressed in taste buds in WT and DKO taste buds. Importantly, we find that P2X2 is expressed in WT taste buds and appears to function as an autocrine, positive feedback signal to amplify taste-evoked ATP secretion.
针对味觉刺激，味蕾细胞释放一种递质，ATP，激活味觉传入纤维上的 P2X2 和 P2X3 受体。缺乏 P2X2 和 P2X3 受体的小鼠[P2X2 和 P2X3 双敲除（DKO）小鼠]的味觉行为和味觉神经反应基本消失。人们一直认为，消除 P2X2 和 P2X3 受体只会消除突触后靶标，但小鼠的递质分泌是正常的。使用功能成像、ATP 生物传感器细胞和无细胞 ATP 测定法，我们测试了这一假设。令人惊讶的是，尽管味觉刺激会动员 DKO 小鼠的味觉感受器（II 型）细胞中的 Ca(2+)，但与野生型（WT）小鼠一样，当用味觉刺激物刺激时，DKO 小鼠的味觉细胞无法释放 ATP。用 KCl 去极化 DKO 感受器细胞可以引发 ATP 释放，这表明 DKO 味觉感受器中的 ATP 释放机制仍然具有功能。为了探究不同基因型之间 ATP 释放的差异，我们使用逆转录酶（RT）-PCR、免疫染色和关键蛋白的组织化学染色来研究 ATP 分泌和降解的基础：Pannexin1、TRPM5 和 NTPDase2（外切 ATP 酶）在 WT 和 DKO 小鼠之间没有区别。DKO 小鼠中味觉细胞与神经纤维之间的接触超微结构也正常。最后，定量 RT-PCR 显示 P2X4 和 P2X7，ATP 分泌的潜在调节剂，在 WT 和 DKO 味觉感受器中表达相似。重要的是，我们发现 P2X2 在 WT 味觉感受器中表达，并似乎作为一种自分泌的正反馈信号来放大味觉诱导的 ATP 分泌。
