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V. E. Trinkaus-Randall, D. Oswald; Purinergic Receptor Signaling in Trigeminal Ganglion Neurons and Their Role in Corneal Wound Response. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3468.
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Upon corneal wounding, there is a collaborative system of communication between the remaining cells surrounding the wound, inducing migration. We have shown that epithelial disruption results in nucleotide triphosphate release by cells lining the periphery of the wound. The nucleotides bind to and activate the P2X and P2Y subfamilies of purinergic receptors. We hypothesize that injury to trigeminal ganglion neurons, stimulates P2Y receptor receptors and up-regulates intracellular pathways that promote cell survivability and induces regeneration of the corneal epithelium. Our goal is to characterize the response of trigeminal neurons to purinergic agonists.
Trigeminal ganglia were dissected from neonatal rats, triturated, and the cells cultured in neuronal medium. Mixed cultures were lysed, mRNA extracted, and reverse transcription (RT) PCR performed using primers designed for transcripts of P2Y and P2X receptors. Cultures grown on coverslips were incubated with the Ca2+ indicator dye, Fluo-3 AM and mounted on a Zeiss LSM510 confocal microscope equipped with a flow-through system. Agonists of varying concentrations were flowed through the incubation chamber, and fluorescence measured.
RT-PCR results indicate the presence of P2X1,3,4,5,7 and P2Y1,2,4,6&12. Using specific agonists, live-cell confocal microscopy substantiated the RT-PCR results. Cells exposed to ATP, UTP, and ADP exhibited a concentration-dependent Ca2+ release, with waves propagating between cells. In contrast, UDP elicited a negligible response. Cells pre-incubated with MFA, a gap-junction inhibitor, showed a 20% reduction in response to stimuli, while cells in a Ca2+-free buffer displayed a 48% reduction. FRAP analysis showed refilling between specific cells of the trigeminal culture.
Trigeminal cultures express a similar array of purinergic receptors to epithelial cells, which may influence the wound response, and upon activation display the propagation of a Ca2+ wave. In addition, a complex P2Y and P2X signaling event occurs, including the release of nucleotides and gap-junction coupling between cells.
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