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John Cuppoletti, Jayati Chakrabarti, Danuta H Malinowska; Unoprostone Isopropyl and M1 Protect Against ER Stress in a Model of Retinitis Pigmentosa via an Intracellular BK Channel. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5432.
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© ARVO (1962-2015); The Authors (2016-present)
Class II mutations in rhodopsin (Rho) that cause unfolded rod cell proteins are responsible for autosomal dominant retinitis pigmentosa. We have previously shown that HEK293 cells with a tetracycline induction system for Rho was a useful system to study effects of P23HRho expression and unoprostone isopropyl (uno) had protective effects (Cuppoletti et al FASEB J April 2014 28:LB832). The present studies examined effects of M1 (active uno metabolite), trans-uno (stereoisomer), latanoprost free acid (lat) and fluprostenol (flu) on ER stress indicators: ROS, [Ca2+]i, changes in mitochondrial membrane potential (ΔµH), apoptosis (caspase activity), WTRho & P23HRho production and cleaved ATF6 and phosphoelf2α levels to determine if uno and M1 effects involved ER stress relief.<br />
Cell counts in the presence and absence of 10nM & 100 nM uno or M1 and 100 nM trans-uno, lat or flu were carried out. Effects of 1 nM iberiotoxin (IbTX) or 100 nM of membrane permeant BK channel inhibitor verruculogen on uno protection were measured. Effects of uno on WTRho and P23HRho ER stress response indicators were determined using fluorescent probes for ROS, [Ca2+]i, ΔµH and caspase activity and using western blots for Rho, cleaved ATF6 and phosphoelf2α.<br />
WTRho cell numbers increased by 40% in 72 h; uno and M1 had no effect. P23HRho cell numbers decreased to 60% in 72 h but remained at 95% with uno and M1. Verruculogen, but not IbTX prevented uno’s protective effects, while trans-uno,lat and flu had no effect. ROS, [Ca2+]i and caspase activity were greatly increased in P23H cells, and uno and M1 greatly reduced these levels. ΔµH was greatly reduced in P23HRho cells and this loss was largely reversed by uno. Rho levels were similar in WTRho and P23HRho cells and uno had no effect. Phosphoelf2α and cleaved ATF6 levels were similar and reduced by uno.
In this cell model system, cells expressing P23HRho resulted in cell death through ER stress. Uno and M1, but not trans-uno, lat or flu reduced cell death and changes in ER stress indicators. Uno’s site of action in protection against cell death appears to be an intracellular/mitochondrial BK channel. Uno and M1 reduce ER stress, a factor in numerous eye diseases and other conditions.
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