PAR1 activation induces Glutamate release from RPE cells that is Ca⁺² and PKC-dependent

Abstract

Purpose : Clinical studies have shown that glutamate (GLU) concentration as well as thrombin activity are significantly increased in the vitreous humour of patients suffering from retinal detachment. In the present study we analyzed in vitro the activation of thrombin receptor PAR1 on GLU release from rat RPE cells. We have previously shown that PAR1 activation induces RPE cell proliferation by increasing the expression of the cyclin D1 gene. Moreover, GLU also induces RPE cell proliferation through the activation of signaling pathways involving type I metabotropic glutamate receptors and NMDA receptor-mediated calcium increase. On this line, we analyzed intracellular Ca⁺², and the effect of inhibition of Phospholipase C and PKC.

Methods : Primary cultures of rat RPE cells were pre-loaded with 1 µCi/ml ³H-Glutamate in Krebs Ringer Bicarbonate (KRB) buffer for 30 minutes at 37 ^oC. Cells were rinsed and superperfused for 15 minutes. On the 8^th minute, stimuli were added. Perfusion was monitored every minute for 15 minutes. Inhibitors were included 30 minutes prior to superfusion. For [Ca⁺²]i imaging cells were incubated with 3 μM of Ca⁺² indicator fluo-4 AM for 45 min in KRB buffer. Images were acquired using a FlexStation 3 (Molecular Devices). Cells were stimulated for 1 min. Change in fluorescence was monitored for 8 min. The area under the curves (AUC) was calculated from the basal point up to the 7th minute.

Results : Under physiological conditions, the activation of PAR1 by thrombin or by PAR-1 agonist peptide stimulates GLU release from RPE cells in a specific, dose-dependent manner. This effect was prevented by the chelation of intracellular calcium, by BAPTA-AM. Phospholipase C inhibitor U73122, also blocks GLU release, further supporting the notion that intracellular calcium pools play a critical role in this process. On this line, PKC is also involved in PAR1 GLU release. Additionally, we analyzed the release mechanisms by inhibiting vesicular traffic, GLU transporter and cell swelling.

Conclusions : Together with our previous findings, these results suggest that thrombin and glutamate might exert a synergistic effect on the promotion of RPE cell proliferation in fibroproliferative diseases derived from the disruption of the BRB, such as PVR.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.