Diurnal changes in specific binding of cortisol to cytosolic and nuclear fractions from equine peripheral blood mononuclear cells

Recent research suggests that the subcellular location and nuclear concentration of glucocorticoid receptors may have a significant impact on cell sensitivity to stimuli acting through cyclic AMP or protein kinase C second messengers. The present study was performed in order to determine whether there may be diurnal fluctuations in the cytosolic and/or nuclear glucocorticoid receptors in equine peripheral blood mononuclear cells (PBMC). Jugular blood samples were collected by venipuncture from four resting unconditioned horses. Samples were collected at 8 AM, 2 PM, 8 PM and 2 AM. The PBMC were harvested from Ficoll gradients. Cytosolic and nuclear fractions were recovered by centrifugation from homogenized PBMC. Protein concentrations of the resulting fractions was determined and serial dilutions incubated with 3H-cortisol in the presence or absence of excess unlabeled dexametha- sone. Saturable specific binding was linear for 75 to 200 µg of protein which was recovered from 10xl0⁵ to 100xl0⁶ equine PBMCs. This binding was eliminated in heat-treated samples. Further, specific binding of cortisol to cytosolic fractions was reduced (P<.05) at 8AM as compared to binding at 2 PM and 2 AM. Nuclear receptor binding was high during daylight hours and undetectable at 2 AM. Total cellular receptor was highest at 2 PM and lowest at 2 AM. Results from this experiment suggest that diurnal fluctuations in equine cortisol concentrations are related to changes in the subcdlular distribution ofglucocorticoid receptors in PBMCs. Changes in the total amount of receptor also suggests that there are diurnal changes in glucocorticoid receptor synthesis and/ or recycling. Thus, diurnal variations in glucocorticoid interactions with second messenger pathways may be of importance in the equine species.