Oxidative Damage to Connexins Precedes Cataract Formation in Lenses From Glutathione Peroxidase–1 Deficient Mice

Abstract

Abstract: : Purpose: A recent study of lenses from glutathione peroxidase–1 knockout mice (GPX^–/–) revealed age related oxidative damage to fiber cell membranes and crystallins, resulting in a nuclear cataract. We have looked at membrane transport properties of GPX^–/– lenses at an early age to see if we could detect changes that precede the cataract. Methods: Intact lenses were characterized using impedance studies. Isolated epithelial cells were studied using whole cell patch clamp to determined Na/K pump current (I_P). Impedance studies were performed on freshly dissected lenses from wild type and GPX^–/– mice (6–8 weeks old), and guinea pig lenses cultured for 5 days in control or elevated (100 µM) H₂O₂ solutions. I_P was measured in equatorial epithelial cells isolated from guinea pig lenses; we were unable to study I_P in mouse lens epithelial cells. Results: GPX^–/– lenses did not have any obvious reduction in transparency at this age, and their membrane conductances as well as resting voltage were not different from wild type. However, they had significantly reduced coupling conductance in both peripheral differentiating fibers (DF) and central mature fibers (MF): G_DF(WT)=0.81±0.07, n=23, G_DF(GPX^–/–)=0.60±0.09, n=8; G_MF (WT)=0.43±0.08, n=28, G_MF(GPX^–/–)=0.19±0.03, n=8 (S/cm²). Although coupling conductance was reduced in the GPX^–/– lenses, it had the same pH sensitivity as WT. H₂O₂ cultured guinea pig lenses did not have significant differences in coupling conductance when compared with control, however they had significantly reduced I_P: I_P(Con)=0.31±0.04, n=8; I_P(H₂O₂)= 0.19±0.06, n=9 (pA/pF). Conclusions: Short term oxidative stress caused a reduction in Na/K pump activity but did not affect gap junctional coupling. However, long term oxidative stress reduced coupling conductance well before cataract formation. Furthermore, the changes in coupling were similar to those produced by knocking out one Cx46 allele. Thus age related cataract could be the consequence of oxidative damage to Cx46 and compromise of the lens circulatory system. Although I_P was not measured in the GPX^–/– lenses, if it were also compromised, as in the guinea pig lenses, this would further compromise the lens circulation.

Keywords: gap junctions/coupling • oxidation/oxidative or free radical damage • transgenics/knock-outs