Stimulation of the hexose monophosphate shunt in rabbit lens in response to the oxidation of glutathione

Abstract

The stimulation of hexose monophosphate shunt (HMS) activity in rabbit lens has been investigated during exposure to t-butyl hydroperoxide (TBHP), an oxidant for glutathione (GHS). Exposure of lenses to 1·0 m m -TBHP produced maximum stimulation of HMS activity as measured by the oxidation of 14C-labeled glucose, labeled in either the C-1, C-2 or C-6 position. A significant increase in the oxidation of both C-1 and C-2 labeled glucose was observed. Removal of TBHP from the medium resulted in a rapid decrease in the rate of oxidation. The maximum rate of liberation of CO2 from the HMS pathway was estimated to be 0·75 μmol/g wet wt/hr, which was 7·7 times the control value. The generation of NADPH at maximum stimulation was estimated to be 1·5 μmol/g wet wt/hr which corresponded to a complete turnover of NADP+ to NADPH in the lens every 48 sec. This rate of generation of NADPH was sufficient to result in a reduction of oxidized glutathione (GSSG) at the rate of 26% of total glutathione in the lens per hour. Exposure of lenses to 0·10 m m -TBHP stimulated shunt activity by three times the control. However, under these conditions only minimal accumulation of GSSG was detected in the lens and no effect was observed on the uptake or efflux of 86Rb. Higher levels of TBHP resulted in a significant accumulation of GSSG in the lens and impaired cation transport. The distribution of the activity of the HMS pathway was determined in homogenates of various regions of rabbit lens. It was found that 65% of the total activity was present in the equatorial cortex, which made up 45% of the total weight. The results indicate that a direct link exists between the rate of formation of GSSG in the lens and the stimulation of HMS activity. In addition, the findings exemplify the ability of the lens to protect itself against oxidative damage through an interrelationship of the shunt, glutathione peroxidase, GSH, NADPH and glutathione reductase.