Abstract 363: Urinary Free Cortisol Variations in Essential Hypertension are Associated with Altered Glucocorticoid Receptor Binding in ex vivo Human Neutrophils
The role of cortisol in essential hypertension (HTN) is yet unclear. Urinary free cortisol (UFC) levels have been shown to be bimodally distributed among hypertensives with about 30% of individuals with HTN showing high levels of UFC (high mode) and significant familial aggregation of this trait. These data suggest that UFC is a genetically determined intermediate phenotype of HTN. The mechanisms underlying this UFC variation in HTN are unknown. Prior studies have shown that despite higher UFC, the high mode group has less sodium sensitivity of blood pressure and no clinical manifestations of high cortisol, suggesting an element of glucocorticoid resistance. We evaluated the glucocorticoid receptor (GR) binding characteristics in neutrophils obtained from hypertensives with high mode versus normal/low mode UFC by immunomagnetic isolation of untouched circulating human blood cells following density gradient sedimentation with Polymorphprep. Flow cytometric analyses showed >97% of these cells were positive for CD45, CD16 and CD66B. These results were consistent with manual and automated white blood cell differential counts. Our isolation procedure produced >96% live cells as estimated by Live/Dead fluorescent viability assays. Dexamethasone (DEXA), a synthetic cortisol that binds and activates GR, showed increases in mitochondrial membrane potential but no effect on superoxide production or degranulation in these cells. In contrast, increases in superoxide production and degranulation (P<0.02 vs vehicle; n=6) were clearly observed when ex vivo cells were incubated with either 100nM fMLP or 10nM PMA, two well-described neutrophil activators. We determined DEXA binding parameters in neutrophils isolated from hypertensives with high mode and low mode UFC using 3H-DEXA. Both the Kd (85±16 vs 199±51 nM, n=5, P<0.030) and the Bmax (20.3±5 vs 61.2±20 fmol/106 cells, n=5, P<0.040) for DEXA were lower in cells from high mode subjects compared to low mode. We confirmed binding to GR as 3H-DEXA was displaced by >90% with RU-486, a GR antagonist. Thus our results suggest that hypertensives with high mode UFC have altered GR binding, which may drive the higher UFC levels in vivo and may have important clinical implications that warrant further investigation.
- © 2012 by American Heart Association, Inc.