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 con centration of glucocorticoid receptors may have a significant impact o n cell sensitivity to stimuli acting through cyclic AMP or protein kin ase C second messengers. The present study was performed in order to d etermine whether there may be diurnal fluctuations in the cytosolic an d/or nuclear glucocorticoid receptors in equine peripheral blood monon uclear cells (PBMC). Jugular blood samples were collected by venipunct ure from four resting unconditioned horses. Samples were collected at 8 AM, 2 PM, 8 PM and 2 AM. The PBMC were harvested from Ficoll gradien ts. Cytosolic and nuclear fractions were recovered by centrifugation f rom homogenized PBMC. Protein concentrations of the resulting fraction s was determined and serial dilutions incubated With H-3-cortisol in t he presence or absence of excess unlabeled dexamethasone. Saturable sp ecific binding was linear for 75 to 200 mug of protein which was recov ered from 10x10(5) to 100x10(6) equine PBMCs. This binding was elimina ted 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 ho urs and undetectable at 2 AM. Total cellular receptor was highest at 2 PM and lowest at 2 AM. Results from this experiment suggest that diur nal fluctuations in equine cortisol concentrations are related to chan ges in the subcellular distribution of glucocorticoid receptors in PBM Cs. Changes in the total amount of receptor also suggests that there a re diurnal changes in glucocorticoid receptor synthesis and/or recycli ng. Thus, diurnal variations in glucocorticoid interactions with secon d messenger pathways may be of importance in the equine species .