Carl J. Nielsen

Glucocorticoid receptor inactivation and activation by phosphorylation mechanisms.

The specific glucocorticoid binding capacity of cytosol preparations is rapidly lost on incubation at 25 degrees in the absence of ligand. We have examined this process in cell-free preparations from rat thymus, rat liver and mouse fibroblasts (L 929 cells), and we have found that the unoccupied receptor is inactivated by endogenous enzymes to a form that does not bind steroids. The inactivation can be prevented by inhibitors of phosphatase action such as molybdate, fluoride and glucose-1-phosphate. On the basis of this type of evidence we propose that the receptor activity of cytosol can be rendered inactive by a dephosphorylation process. We have now been able to partially reactivate the receptor in both L cell and rat thymus cytosols in an ATP dependent manner. If fibroblast (L cell) cytosol is preincubated to permit receptor inactivation by endogenous enzyme, further inactivation can be prevented by the addition of 10 mM molybdate and reactivation of the binding capacity can be obtained by adding 5 to 10 mM ATP in addition to molybdate. ATP dependent activation is prevented with EDTA and this block is overcome by added magnesium. ADP, CTP, GTP, and UTP are inactive. After inactivating the glucocorticoid binding capacity of rat thymocyte cytosol by incubation for 45 minutes at 25 degrees, considerable reactivation is obtained by addition of dithiothreitol and ATP. This system does not absolutely require the presence of a phosphatase inhibitor in order to show activation. Thymocyte cytosol can also be activated to a steroid binding state by addition of DTT and heat-treated (90 degrees for 15 min.) cytosol from a variety of cell types. The heat-treated cytosol contains ATP, reducing equivalents, and a relatively small molecular weight heat-stable activator(s) that potentiates the reactivation process. Maximum receptor activation is obtained by adding dithiothreitol, heat-stable factor, ATP, and molybdate to the inactivated thymocyte cytosol.

Inability of Potassium Canrenoate to Convert Experimentally Induced Ouabain Arrhythmias in the Canine Heart

The antiarrhythmic effects of potassium canrenoate were examined in 20 closed-chest, pentobarbital-anesthetized dogs with ouabain-induced ventricular tachycardia. In a group of 8 dogs, a mean dose of ouabain of 64.4 ± 3.7 μg/kg induced sustained ventricular tachycardia. Subsequent administration of potassium canrenoate (30 mg/kg, iv) caused ventricular fibrillation in 1 dog and sinus tachycardia in 3 dogs. Stimulation of the distal end of the cut right vagus in the latter 3 dogs slowed the sinus rhythm enough to permit the ventricular focus to become the dominant pacemaker. In the other 4 dogs, potassium canrenoate did not alter the ouabain-induced ventricular tachycardia. In a second group of 12 dogs, ventricular tachycardia was also induced by administering a toxic dose of ouabain (65.5 ± 2.9 μg/kg, iv). After ouabain intoxication, atrial overdrive suppression of the ventricular rhythm was initiated by electrically pacing the right atrium at a mean frequency of 184.5 ± 7.9 beats/min. Subsequent administration of potassium canrenoate (30 mg/kg, iv) to these dogs restored sinus rhythm in 4 of the dogs; however, stimulation of the distal end of the cut right vagus caused a reappearance of the ventricular ectopic focus. After potassium canrenoate administration, the minimum atrial pacing rate required to capture the ventricular rhythm was 160.9 ± 9.4 beats/min. This rate represents a significant reduction (P < 0.005) in the rate of the ouabain-induced ventricular ectopic focus, but in no instance was the focus directly suppressed by potassium canrenoate. Propranolol, in contrast to potassium canrenoate, restored normal sinus rhythm in all 20 ouabain-intoxicated dogs and suppressed the ouabain-induced ectopic pacemaker during the period of vagally induced sinoatrial arrest. The results of this study show that potassium canrenoate fails to exert an antiarrhythmic effect against digitalis-induced arrhythmias and that any apparent restoration of sinus rhythm in response to potassium canrenoate is due to overdrive suppression of the ouabain-induced ventricular ectopic pacemaker.