Abstract
Purpose :
Pannexin 1 is a non-specific ion channel that can mediate the release of ATP and activation of purinoreceptors. Our goals are to determine the role of pannexin 1 expression and localization after injury in C57Bl6/J wild type and in mice fed a high fat diet causing diet induced obesity (DiO) and to examine its role in cell communication at the wound edge.
Methods :
Epithelial debridement wounds were performed on wildtype (WT) C57Bl6/J and DiO mice corneas after 15 weeks of a high fat diet. Corneas from unwounded WT and DiO corneas were evaluated. Real-time PCR and immunohistochemical analyses were performed and imaged using confocal microscopy. Calcium mobilization after injury was imaged on corneal epithelial cultures in the presence of 10panx, an inhibitory peptide, or the scrambled control sequence using a Zeiss 880 and AIRYScan every 5 minutes for 12 hours. To examine cell shape and communication cells were loaded with SiR700 actin (1 uM) and Fluo-3AM (5 uM), injured and monitored every 5 minutes for 12 hours.
Results :
There was an increase in pannexin 1 after epithelial abrasion compared to unwounded WT mice. Pannexin 1 was localized apically in both WT and DiO unwounded corneal epithelium, but was diffuse in the DiO. After injury pannexin 1 was concentrated at the wound margin and decreased distal from the edge, while remained diffuse in the DiO corneas. Inhibition of pannexin 1 resulted in a slower migration. This was associated with a minimal change in cell shape over time compared to control. Inhibition also resulted in negligible communication between cells at the wound edge. Lamellipodial activity was present when calcium mobilization between cells was detected, and was not detected in the absence of communication. When control cells where cells stimulated with the agonist for P2Y2 receptors there was no change in the presence of 10panx, while a diminished response occurred when cells were stimulated with an agonist to the P2X7 receptor.
Conclusions :
While injury causes an increase in Pannexin 1 in the WT, the mice fed a HFD do not respond. The diffuse localization of Pannexin 1 indicates that the sensing role of the ion channel is altered. We predict that inhibition decreases transport of calcium and ATP, thereby inhibiting the activation of purinoreceptors.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.