May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
A Novel Drug, PQ1, Protects Retinal Cells From Hypoxia-Induced Apoptosis Through Gap Junction Inhibition
Author Affiliations & Notes
  • S. Das
    Kansas State University, Manhattan, Kansas
    Biochemistry,
  • D. Lin
    Kansas State University, Manhattan, Kansas
    Biochemistry,
  • S. Jena
    Kansas State University, Manhattan, Kansas
    Biochemistry,
  • M. Katz
    Kansas State University, Manhattan, Kansas
    Biochemistry,
  • D. Hua
    Kansas State University, Manhattan, Kansas
    Chemistry,
  • A. Shi
    Kansas State University, Manhattan, Kansas
    Chemistry,
  • S. Battina
    Kansas State University, Manhattan, Kansas
    Chemistry,
  • K. Lou
    Kansas State University, Manhattan, Kansas
    Chemistry,
  • D. Takemoto
    Kansas State University, Manhattan, Kansas
    Biochemistry,
  • Footnotes
    Commercial Relationships  S. Das, None; D. Lin, None; S. Jena, None; M. Katz, None; D. Hua, None; A. Shi, None; S. Battina, None; K. Lou, None; D. Takemoto, None.
  • Footnotes
    Support  NIH Grant EY-13421 to DJT, Grants CHE-0555341, NSF43529, NIH-R01AG025500, and AHA-0750115Z to DHH
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 4549. doi:https://doi.org/
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      S. Das, D. Lin, S. Jena, M. Katz, D. Hua, A. Shi, S. Battina, K. Lou, D. Takemoto; A Novel Drug, PQ1, Protects Retinal Cells From Hypoxia-Induced Apoptosis Through Gap Junction Inhibition. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4549. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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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 
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