Is there a Glucocorticoid Receptor in the Bovine Lens?

Abstract

Prolonged glucocorticoid therapy is a risk factor for cataract development. The mechanism remains unknown. If cataract results from the direct effect of steroids on lens function, a glucocorticoid receptor is required. In order to determine whether such a receptor was present in the bovine lens, metabolic and steroid binding experiments were undertaken. Cultured bovine lens epithelial cells were exposed to 10−4and 10−8M dexamethasone or prednisolone and the uptake and incorporation of14C leucine,14C glucose and3H thymidine, examined. Neither glucocorticoid affected cell protein synthesis or glucose uptake. Both dexamethasone concentrations and the lower concentration of prednisolone had no effect on thymidine uptake or incorporation, however, the 10−4M prednisolone exposure reduced these by 15±5%. This regulation is thought to be due to membrane fluidity changes and not the action of the glucocorticoid receptor. As the glucocorticoid receptor is very heat labile in vitro, the effects of increasing temperature on dexamethasone binding by proteins from lens epithelium, lens nucleus and liver were examined. At 0°C, lens epithelial extract bound nine-fold more dexamethasone than liver extract. After exposure to 37°C, liver binding decreased by 66% whereas that for lens epithelium increased by 18%. For both lens extracts, steroid binding increased with temperature up to 50°C. Scatchard analysis of the steroid binding kinetics showed there to be no high affinity sites in lens epithelial extract, with the binding best described as a non-specific partitioning event. Western blotting with a specific glucocorticoid receptor antibody revealed protein bands of approximately 94 and 79kDa in liver, which is known to contain significant levels of receptor. No immunoreactivity was observed for lens epithelial extract. Therefore, within the limits of detection, these results suggest the bovine lens does not contain a glucocorticoid receptor. This raises questions about the validity of receptor-mediated mechanisms proposed for cataract development.