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K. Cusato, J. Zakevicius, D.C. Spray, H. Ripps; Gap Junctions Remain Open During Cell Death . Invest. Ophthalmol. Vis. Sci. 2004;45(13):784.
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Purpose: During both normal development and degenerative diseases, apoptosis leads to significant retinal cell loss. Gap junctions may be responsible for the spread of cell death in retinitis pigmentosa (Ripps, Exp. Eye Res., 2002), and we showed recently that during development gap junctions mediate the spread of cell death in the inner retina (Cusato et al, J. Neurosci., 2003). However, it remains unknown how gap junctions participate in this process. In the present study we have investigated the dynamics of electrical coupling changes between pairs of oocytes following induction of death in one cell. Methods: Stage VI Xenopus oocytes, which express endogenously connexin38 (Cx38) were harvested, and either injected with Cx43 cRNA together with an antisense oligonucleotides to Cx38, or uninjected. The vitelline membrane was removed, and cells were paired at the vegetal pole for 1–2 days. Gap junctional conductance was measured by holding cells in voltage clamp at –40 mV, and applying 1 sec, 20 mV depolarizing pulses to each cell with a 30 sec interval between pulses. Two measurements were taken 10 min apart to establish a baseline before cytochrome c (Cc; 40 µM) was injected into one cell to induce apoptosis, and coupling was measured at 10 min intervals. Gap junctional conductance (Gj) was plotted as the ratio after Cc injection to baseline. Results: In both Cx38 and Cx43 pairs, Gj was stable before Cc injection. In Cx38 pairs conductance increased at least 4 fold by 40 min post–injection; for Cx43 pairs, coupling increased more than 2 fold at 30 min post–injection. No increase in coupling occurred following vehicle injection. Cell death was typically observed in the uninjected cell when pairs were well–coupled. Conclusions: These results indicate that gap junctions remain open and that electrical coupling increases during Cc–mediated cell death. This continued coupling may allow the passage of death messages (i.e. passage of toxic metabolites from the dying cell or depletion of metabolites from the healthy cell) between a dying cell and it’s coupled neighbors ("bystanders"). Further studies are aimed at determining the timing and nature of the death signal.
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