Estradiol attenuates mitochondrial depolarization inpolyol-stressed lens epithelial cells

[摘要] Purpose: This study examined the state of mitochondrial physiologysubsequent to exposing lens epithelium to high ambient galactose (Gal),which upon conversion to galactitol (GalOH) and resultant intracellularaccumulation thereof, leads to profound destabilization of mitochondrialmembrane potential (Δψm). Further, we determined whetherthe aldose reductase (AR) inhibitor, Sorbinil, or estrogen(17β-E2, and its isomer, 17α-E2, which exhibitsmarginal binding affinity for estrogen receptor), administered prior toand concomitant with Gal exposure might prevent or delay mitochondrialmembrane depolarization.Methods: Secondary cultures of bovine lens epithelial cells (BLECs),as well as a virally-transformed human lens epithelial cell line(HLE-B3), were maintained in 40 mM galactose (Gal) for up to seven daysin the presence and absence of Sorbinil, 17β-E2 or17α-E2. Endogenous accumulation of reactive oxygen species(ROS) was assessed by loading cells with H2DCF-DA, which uponoxidation in the presence of ROS transitions to the fluorescentcompound, DCF. To assess Δψm, confocal microscopy wasemployed in conjunction with the potentiometric dye, JC-1. Intracellularpolyol content was determined by gas chromatography. Cells weremonitored for apoptosis and necrosis as determined by annexinV-propidium iodide staining and visualized by confocal fluorescencemicroscopy.Results: BLECs, more so than HLE-B3 cells, accumulate highintracellular levels of GalOH upon exposure to high ambient Gal. BLECswere significantly depolarized while HLE-B3 cells showed littledepolarization over the same course of Gal exposure. The addition ofeither 17α-E2 or 17β-E2 to BLECs, over a dose rangeof 0.01 μM to 1.0 μM, prevented mitochondrial membranedepolarization as did the addition of 0.1 mM Sorbinil. The polyolcontent in BLECs after 3 days of exposure to Gal was 282 nmol/mgprotein. Co-addition of Sorbinil during the 3-day exposure periodprevented any significant accumulation of GalOH. Co-administration ofeither isoform of estrogen did not block GalOH synthesis and the levelof attained intracellular accumulation was similar to that of Gal alone.The observed accumulation of ROS from HLE-B3 cells subsequent to 3 daysof Gal exposure was negligible and consistent with that of control cellsmaintained in physiological medium. Intracellular accumulation of ROSwith 3-day, Gal-maintained BLECs, exhibited a marginal but statisticallysignificant increase over control cells maintained in physiologicalmedium (5.5 mM glucose) and similar levels of ROS were generatedirrespective of the presence of estrogen with Gal. Bolus addition of 100μM hydrogen peroxide to 3-day, Gal plus Sorbinil-maintained BLECsfailed to induce a change in mitochondrial membrane potential. Evidenceof apoptosis or necrosis was negligible through 7 days of sustainedexposure to high ambient Gal.Conclusions: Polyol accumulation promotes mitochondrial membranedepolarization and the decrease in Δψm is prevented byprior addition and co-administration of Sorbinil or estrogen with Gal.Unlike Sorbinil, estrogens' mode of action is not via the inhibition ofaldose reductase activity. The data supports the theory that with Galplus estradiol-treated cells, at a given intracellular polyol load, alarger portion of the mitochondrial population retainsΔψm, and hence continues to function relative toGal-treated cells. Results with 17α-E2 indicate thatmaintaining Δψm, in the face of chronic polyolaccumulation, is likely to be mediated via a nuclear estrogenreceptor-independent mechanism. The failure of supraphysiological levelsof hydrogen peroxide added to Gal plus Sorbinil-maintained BLECs todepolarize mitochondria indicates that polyol accumulation, not ROSgeneration, is the causative factor responsible for the loss ofmitochondrial membrane potential.

[学科分类] 生物化学/生物物理