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MT Coroneo, S Li, A Agar, N Agarwal, M Hill; The Two Pore Domain Mechano-gated K+ Channel Opener Arachidonic Acid Induces Apoptosis in Rgc5 and Differentiated Pc12 Neuronal Cell Lines . Invest. Ophthalmol. Vis. Sci. 2002;43(13):752.
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© ARVO (1962-2015); The Authors (2016-present)
The two pore domain K+ channel family includes mechano-gated K+ channels TRAAK, TREK1 and TREK2. These channels can be opened by mechanical forces, such as membrane stretch and pressure, and by polyunsaturated fatty acids like Arachidonic Acid (AA). The specific role of these channels is still controversial, with different studies having shown either neuron protection or neuron death following their activation. Purpose:To determine the distribution of TRAAK channels on RGC5 and PC12 neurons, and investigate the effect of Arachidonic Acid on their survival in-vitro. Methods: The RGC-5 Retinal Ganglion Cell line is derived from primary rat retinal cultures. PC12 cells were rendered post-mitotic by NGF and cAMP treatment. A range of AA concentrations from 0.1 to 100 uM were added to the culture medium. Cultures were incubated in a 5% CO2 and air mixture at 370C for 24 hours. Control cultures were treated identically except for the addition of AA. Apoptosis was detected by morphology and specific markers including TUNEL. Automated quantitative analysis of apoptotsis was performed by Laser Scaning Cytometry (LSC- Compucyte, MA). TRAAK channel distribution on RGC5 and PC12 was detected by immunocytochemistry using anti-TRAAK antibody. Results: Apoptosis was confirmed by fluorescent microscopy with no significant necrosis seen. LSC analysis of fluorescent marker showed 1 uM of AA induced significant apoptosis in both cell lines (P<0.05, n=9), which was highly significant over 10 uM (P<0.01, n=9). TRAAK channels were highly expressed on the membranes of both cell lines. Conclusion: We have found that mechanically-sensitive K+ channels are distributed in the RGC-5 and PC12 cell lines. AA induced significant neuronal apoptosis from 1 uM in-vitro. This suggests that neuronal loss in pressure related diseases such as glaucoma may be associated with the opening of mechano-gated K+ channels, with subsequent K+ efflux affecting neuronal viability.
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