Inactivation of hepatic glucocorticoid receptors by heparin

Heparin dramatically enhanced the rate of unbound glucocorticoid receptor inactivation in vitro in a concentration, time and temperature-dependent manner. Control specific binding decreased only about 25% after incubation for 6 h at 4 degrees C. However in the presence of heparin (40 micrograms per ml cytosol) receptor binding decreased about 75%. At 25 degrees C liver receptor specific binding was found to have a half-life of about 60 min in control cytosol. However, in the presence of heparin (40 micrograms per ml cytosol) the glucocorticoid receptor had a half-life of only 15 min at 25 degrees C. Interestingly, 10 mM molybdate (with or without 5 mM dithiothreitol) greatly inhibited heparin-dependent receptor inactivation at 4 degrees C. Dithiothreitol (alone) significantly stabilized receptor binding in control samples at 4 degrees C, but provided no protection from heparin-dependent receptor inactivation. Heparin had no apparent inactivating effect on prebound glucocorticoid receptor complexes at 4 degrees C. Interestingly however, heparin altered the sedimentation coefficient of prebound hepatic glucocorticoid-receptor complexes in low salt gradients from 7-8 S to about 3-4 S. When molybdate plus dithiothreitol were added with heparin, the sedimentation coefficient was found to be approx. 6-7 S. These results demonstrate that heparin, which is often used pharmacologically and which occurs naturally in animal tissues, has significant effects on liver glucocorticoid receptors in vitro.