Departments of Biochemistry and Ophthalmology, Boston University School of Medicine, Boston, Massachusetts, United States of America.
PLoS One. 2012;7(9):e44574. doi: 10.1371/journal.pone.0044574. Epub 2012 Sep 7.
Previously, we demonstrated that nucleotides released upon mechanical injury to corneal epithelium activate purinergic (P2) receptors resulting in mobilization of a Ca(2+) wave. However, the tissue is extensively innervated and communication between epithelium and neurons is critical and not well understood. Therefore, we developed a co-culture of primary trigeminal neurons and human corneal limbal epithelial cells. We demonstrated that trigeminal neurons expressed a repertoire of P2Yand P2X receptor transcripts and responded to P2 agonists in a concentration-dependent manner. Mechanical injuries to epithelia in the co-cultures elicited a Ca(2+) wave that mobilized to neurons and was attenuated by Apyrase, an ectonucleotidase. To elucidate the role of factors released from each cell type, epithelial and neuronal cells were cultured, injured, and the wound media from one cell type was collected and added to the other cell type. Epithelial wound media generated a rapid Ca(2+) mobilization in neuronal cells that was abrogated in the presence of Apyrase, while neuronal wound media elicited a complex response in epithelial cells. The rapid Ca(2+) mobilization was detected, which was abrogated with Apyrase, but it was followed by Ca(2+) waves that occurred in cell clusters. When neuronal wound media was preincubated with a cocktail of N-methyl-D-aspartate (NMDA) receptor inhibitors, the secondary response in epithelia was diminished. Glutamate was detected in the neuronal wound media and epithelial expression of NMDA receptor subunit transcripts was demonstrated. Our results indicate that corneal epithelia and neurons communicate via purinergic and NMDA receptors that mediate the wound response in a highly orchestrated manner.
先前，我们已经证实角膜上皮细胞在机械损伤时释放的核苷酸激活了嘌呤能（P2）受体，导致钙波的动员。然而，该组织广泛受神经支配，上皮细胞与神经元之间的通讯至关重要，但目前尚不完全清楚。因此，我们构建了原代三叉神经神经元和人角膜缘上皮细胞的共培养物。我们证实三叉神经神经元表达了一系列 P2Y 和 P2X 受体转录本，并以浓度依赖性方式对 P2 激动剂作出反应。在共培养物中对上皮细胞进行机械损伤会引发钙波，钙波会向神经元动员，并被外核苷酸酶 Apyrase 减弱。为了阐明来自每种细胞类型的释放因子的作用，培养上皮细胞和神经元，损伤其中一种细胞类型，并收集其伤口培养基并添加到另一种细胞类型中。上皮细胞的伤口培养基在上皮细胞中迅速引发钙动员，而 Apyrase 存在时则消除了这种钙动员，而神经元的伤口培养基在上皮细胞中引发了复杂的反应。检测到快速的钙动员，Apyrase 可消除该钙动员，但随后会发生在细胞群中的钙波。当将神经元的伤口培养基与 N-甲基-D-天冬氨酸（NMDA）受体抑制剂鸡尾酒预先孵育时，上皮细胞中的次级反应减弱。在神经元的伤口培养基中检测到谷氨酸，并且证明了上皮细胞表达 NMDA 受体亚基转录本。我们的结果表明，角膜上皮细胞和神经元通过嘌呤能和 NMDA 受体进行通讯，这些受体以高度协调的方式介导伤口反应。
