Effect of hydrogen peroxide on lens transparency, intracellular pH, gap junction coupling, hydrostatic pressure and membrane water permeability

Abstract

Clouding of the eye lens or cataract is an age-related anomaly that affects middle-aged humans. Exploration of the etiology points to a great extent to oxidative stress due to different forms of reactive oxygen species/metabolites such as Hydrogen peroxide (H₂O₂) that are generated due to intracellular metabolism and environmental factors like radiation. If accumulated and left unchecked, the imbalance between the production and degradation of H₂O₂ in the lens could lead to cataracts. Our objective was to explore ex vivo the effects of H₂O₂ on lens physiology. We investigated transparency, intracellular pH (pH_i), intercellular gap junction coupling (GJC), hydrostatic pressure (HP) and membrane water permeability after subjecting two-month-old C57 wild-type (WT) mouse lenses for 3 h or 8 h in lens saline containing 50 μM H₂O₂; the results were compared with control lenses incubated in the saline without H₂O₂. There was a significant decrease in lens transparency in H₂O₂-treated lenses. In control lenses, pH_i decreases from ∼7.34 in the surface fiber cells to 6.64 in the center. Experimental lenses exposed to H₂O₂ for 8 h showed a significant decrease in surface pH (from 7.34 to 6.86) and central pH (from 6.64 to 6.56), compared to the controls. There was a significant increase in GJC resistance in the differentiating (12-fold) and mature (1.4-fold) fiber cells compared to the control. Experimental lenses also showed a significant increase in HP which was ∼2-fold higher at the junction between the differentiating and mature fiber cells and ∼1.5-fold higher at the center compared to these locations in control lenses; HP at the surface was 0 mm Hg in either type lens. Fiber cell membrane water permeability significantly increased in H₂O₂-exposed lenses compared to controls. Our data demonstrate that elevated levels of lens intracellular H₂O₂ caused a decrease in intracellular pH and led to acidosis which most likely uncoupled GJs, and increased AQP0-dependent membrane water permeability causing a consequent rise in HP. We infer that an abnormal increase in intracellular H₂O₂ could induce acidosis, cause oxidative stress, alter lens microcirculation, and lead to the development of accelerated lens opacity and age-related cataracts.

Keywords: Aquaporin; Connexin; Gap junction coupling; Hydrogen peroxide; Hydrostatic pressure; Intracellular pH; Lens; Membrane water permeability.

Fig. 1.

Schematic sagittal section of a mammalian lens, depicting the expression pattern of GJ channels (Cx43, Cx46 and Cx50), and aquaporins (AQP0, AQP1, AQP3 and AQP5). The pH gradient from the periphery to the center of the lens is also shown. Cxs and AQPs expressed in the differentiating fiber cells in the outer cortex have intact pH regulatory domains. Their C-terminal regulatory domains gradually cleave off during post-translational modifications in the inner cortex and outer nuclear regions. Therefore, the inner cortex and outer nuclear regions contain a mixture of matured (lost the pH regulatory domains) as well as maturing (undergoing the process of losing pH regulatory domains) fiber cells (Korlimbinis et al., 2009; Gutierrez et al., 2011; Sindhu Kumari and Varadaraj, 2014; Slavi et al., 2016; Gong et al., 2021) whereas the inner nuclear region has only completely matured fiber cells. Note: The inner cortex, outer nucleus and inner nucleus together are referred to as the MF region.

Fig. 2.

Depth-dependent calibration curves for H⁺ were generated using a pH-sensitive dye BCECF (2′−7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein). The distance from the surface of the lens to its center was divided lengthwise into seven equal sections (shown in the Figure as a,b). A small volume of 2 mM BCECF solution was injected into the fiber cells at different depths into the lens (c,d). Images were acquired for calculating fluorescence ratios. The pipette fluorescence ratio between excitations at 495 nm (a) and 436 nm (b) was determined for each section of pipettes having different pH values; the ratio of the former to the latter was calculated for each section. Then, ratios versus pH were plotted and fit with Eq. pH = pK – Log [(Rmax – R)/(R – Rmin)] (e). pH values were determined by curve fitting.

Fig. 3.

Lens transparency evaluation and quantification. (a) Upper Panel: Comparison of the dark-field images of 2-month-old C57 wild-type control mouse lenses with lenses exposed to H₂O₂ for 3 h or 8 h. Lower Panel: Focusing of a copper grid by the respective lenses shown in the Upper Panel. (b) Quantification of pixel brightness intensity to assess transparency in control, and H₂O₂ exposed lenses (3 h or 8 h). The higher the pixel brightness intensity, the lower would be the lens transparency. There was a statistically significant difference in transparency in the lenses exposed to H₂O₂ for 3 h (P < 0.01), or for 8 h (P < 0.00001) compared to that of the respective control lenses. Eight lenses each were used for controls and experimentals. Error bars represent Standard Deviations.

Fig. 4.

The intracellular pH (pHi) of 2-month-old C57 WT control (n = 11) and WT experimental lenses (n = 6; exposed to H₂O₂ for 8 h). pHi of the lens as a function of the distance from the lens center (r/a), where ‘r’ (cm) is the actual distance and ‘a’ (cm) is the lens radius. LC - Lens Center; LS - Lens Surface. P < 0.00001. n = number of lenses used.

Fig. 5.

Impedance analyses of C57 WT control and experimental lenses. Lenses exposed to H₂O₂ showed a significant decrease in gap junctional conductance. Series Resistance (R_S) of WT control lenses (no H₂O₂ exposure) and those exposed to H₂O₂ for 3 h (a) or 8 h (b) as a function of distance from lens center (r/a), where ‘r’ (cm) is actual distance and ‘a’ (cm) is lens radius. The higher the Series Resistance, the lower would be GJ coupling conductance. Lenses exposed to H₂O₂ (both 3h and 8h) showed a significant increase in Series Resistance in DF (Inset in (a) shows expanded data from DF), hence a decrease in conductance in DF compared with the Resistance in similar areas in WT lenses not exposed to H₂O₂. Between MF of 3h and 8h H₂O₂ exposed lenses, only 8h H₂O₂ exposed lenses showed a significant decrease in conductance. DF - Differentiating fiber cells; MF - mature fibers. LC - Center of the lens; LS - Surface of the lens.

Fig. 6.

Intracellular HP ((p_p) _i) in the lenses of C57 WT control and H₂O₂ exposed experimental lenses (n = 8) for 3 h (a) or 8 h (b) as a function of normalized distance from the lens center (r/a), where ‘r’ (cm) is actual distance and ‘a’ (cm) is lens radius. H₂O₂-exposed (3 h or 8 h) experimental lenses showed a significant increase in HP compared to WT lenses not exposed to H₂O₂. Inset in (a): Expanded data from DF.

Fig. 7.

Alteration in lens fiber cell membrane water permeability (P_w) due to H₂O₂ exposure. Mouse lenses were dissected out and exposed to H₂O₂ for 3 or 8 h under dark. Membrane vesicles were prepared and P_w studies were conducted. n = number of FCMVs tested for each.