| When the cornea is injured, the diffusion of nucleotides from the damaged epithelial cells initiates an immediate signaling cascade by binding purinergic (P2) receptors. ATP binds to ionotropic P2X receptors, which allow the influx of Ca2+ from extracellular stores, resulting in the depolarization of sensory neurons. However, when nucleotides bind metabotropic P2Y receptors, the Galphaq protein signaling cascade is initiated, causing a Ca2+ release from intracellular stores, and upregulating survival and synaptic modulatory pathways. Since the cornea is a highly innervated tissue, our aims were to: examine the signaling interaction between epithelial cells and neurons during injury, determine the neuronal P2X and P2Y receptor expression profile, and elucidate the trophic factors released by neurons. We hypothesized that the initial wound cascade passes from the epithelium to neurons, and that the neuronal release of factors augments the injury response.;We developed a primary trigeminal nociceptive neuron and human corneal epithelium (HCLE) co-culture corneal model. Upon wounding the co-culture, a Ca2+ wave passed from the epithelium to the neurons, causing depolarizations. Further experiments showed that there was a P2X contribution to the Ca2+ wave that was reduced using Ca2+-free buffer. Connexin (Cx) hemichannel contributions were inhibited using an antagonist cocktail. Using RT-PCR we confirmed Cx26, 30, 37, 40, 43, and 45 mRNA expression in HCLEs, and immunocytochemistry substantiated Cx43 protein membrane localization. To examine P2 receptor transcript expression in neuronal cultures, RT-PCR and quantitative real-time PCR confirmed the presence of P2Y1,2,4,6&12 and P2X1,2,3,4,5,6&7, similar to that of whole trigeminal tissue. Treatment of neuronal cultures with varying concentrations of ATP, ADP, UTP, and UDP resulted in concentration-dependent intracellular Ca 2+ increases. Treatment with HCLE wound media incubated with apyrase ablated the neuronal nucleotide response; however, neuronal wound media initiated a unique secondary response in HCLEs, found to be caused by glutamate. N-methyl-D-aspartate (NMDA) treatment increased Ca2+ oscillations in HCLEs, and RT-PCR showed the presence of NMDA Receptor (NR)1, NR2A, NR2C, NR2D, NR3A, and NR3B subunit transcripts. These results show that through the release of nucleotides by the epithelium and the release of glutamate by neurons, corneal cells respond synergistically to promote wound closure. |
