A Na+/H+ exchanger and its relation to oxidative effects in plasma membrane vesicles from lens fibers

The existence of a Na+/H+ exchange mechanism and its role in the regulation of lens fiber cell intracellular pH, as well as the effect of oxidants and antioxidants, have been examined in vesicular preparations from spiny dogfish and bovine eyes. The fluorescent probes, SBFI (sodium-binding benzofuran isophthalate) for Na+ and SNARF-1 (seminaphthorhodafluor-1) for H+, were used to determine fluorescent ratios in a dual-wavelength spectrofluorimeter. The results show that: (1) the plasma membrane vesicles can be purified from lens fiber (52.5% of the vesicles were in the right-side out orientation for the fish eyes and 56.3% for the bovine eyes, show enrichment for the membrane marker activities and have an average size of 0.2-0.5 microns); (2) the influx of Na+ was dependent on an outwardly directed pH gradient with the uptake of Na+ sensitive to 500 microM amiloride but not affected by 50 microM valinomycin and 50 mM K gluconate; (3) when the pHo (extravesicular pH) of the vesicles was set at 8.1 and pHi (intravesicular pH) to 6.1, an inwardly directed Na+ gradient caused an increase in intravesicular pH by about 0.3 pH unit, and in bovine lens fiber vesicles the Na+ influx is highly dependent on the intravesicular pH (or on the pHo/pHi gradient); (4) 50 microM H2O2 increases the Na+/H+ exchange rate by 174% in the vesicles derived from fish lens fibers. Similarly, 100 microM H2O2 stimulates the Na+/H+ exchange rate by 194% in bovine eye fibers. This activation is prevented by preincubation with GSH (reduced glutathione) but not with GSSG (oxidizing glutathione). We conclude that a Na+/H+ exchange mechanism is present in the lens fiber membranes. This exchange possibly regulates intracellular pH and controls the intracellular Na+ concentration. The sensitivity of the Na+/H+ exchanger to the oxidant, hydrogen peroxide (H2O2) and the protection by the antioxidant GSH suggests a possible role for the Na+/H+ exchange mechanism in the formation of cataracts.