A Novel Drug, PQ1, Protects Retinal Cells From Hypoxia-Induced Apoptosis Through Gap Junction Inhibition

Abstract

Purpose: : The purpose was to find out whether inhibition of gap junctions can protect the retinal precursor cell line R28 from CoCl₂ induced chemical hypoxia.

Methods: : A class of substituted quinolines (PQs) was synthesized and their effect on gap junction intercellular communication (GJIC) was measured by scrape-loading and dye transfer in R28 cells. For chemical hypoxia, 70% confluent R28 cells were pre-incubated with PQ1 (10 µM, 30 min) followed by CoCl₂ treatments at 10 µM for 24 hr in a cell culture chamber (5 % CO₂, room air, 37° C). Hypoxia induction was confirmed by testing the hypoxia-inducible factor 1-α (HIF1α) expression levels by Western blotting. The expression of caspase-3 levels was also measured by western blotting. To check the ability of PQ1 to prevent the damage caused by CoCl₂ induced hypoxia in vivo, B6129PF2/J 10093 mice were treated for 30 min by intravitreal injection with 2µL of 100 µM PQ1, followed by injection of 2 µL of 500 nM CoCl2. Mice were sacrificed after 48 hr or 1 week. After this time period eyes were removed and fixed for microscopy.

Results: : PQ1 was found to be the most potent blocker of GJIC in R28 cells. Gap junction dye transfer activity analyses demonstrated that PQ1 treatment at 10 µM for 40 minutes caused significant decrease in dye transfer activity in R28 cells. Treatment of 70% confluent R28 cells with 10 µM CoCl₂ resulted in significant increase in the expression of HIF1α. This incubation with CoCl₂ also resulted in the activation of caspase-3 indicating that CoCl₂ induced hypoxic apoptosis. We pre-incubated R28 cells with PQ1 at 10 µM for 30 min, and then co-incubated with CoCl₂ for additional 24 hours. Western blotting demonstrated that pre-incubation of PQ1 alone or with CoCl₂ did not cause activation of HIF1α and caspase-3. These data suggested that inhibition of gap junctions protects cells from CoCl₂- induced ischemic apoptosis. Mice injected with CoCl₂ had damaged photoreceptors and this was ameliorated by PQ1.

Conclusions: : PQ1 was found to be a potent inhibitor of GJIC. Treatment of R28 cells with CoCl₂ results in chemical hypoxia which leads to cellular apoptosis as confirmed by the activation of Caspase-3. Inhibition of the GJIC by the treatment with PQ1 prevented apoptosis caused by chemical hypoxia. In vivo studies showed that treatment with PQ1 prior to the injection of CoCl₂ helped maintain the regular structure in the outer segment (OS) of retina whereas the OS was damaged in the mice injected only with CoCl₂.

Keywords: apoptosis/cell death • gap junctions/coupling • retinal degenerations: cell biology