The steroid hormone Ia,25-dihydroxyvitamin D3 has been shown to exert rapid effects (1 5 s to 5 min) in osteoblasts. These effects occur in osteoblast-like cells lacking the nuclear vitamin D receptor, ROS 24/1, suggesting that a separate signalling system mediates the rapid actions. These non-genomic actions include rapid activation of phospholipase C and opening of calcium channels, pointing to a membrane localization of this signalling system.
Previous studies have shown that the 1 p epimer of Ia,25-dihydroxyvitmina D3 can block these rapid actions, indicating that the 1p epimer may bind to the receptor responsible for the rapid actions in a competitive manner. We have assessed the displacement of 3H-1 a,25-dihydroxyvitamin D3 by vitamin D compounds, as well as the apparent dissociation constant of la,25-dihydroxyvitamin D3 and its 1 p epimer for the membrane receptor in membrane preparations from ROS 24/1 cells. Increasing concentrations of Ia,25-dihydroxyvitamin D3, 7.25 nM to 725 nM, displaced 3H-la,25-dihydroxyvitamin D3 from the membranes with 725 nM of the hormone displacing 40-49% of the radioactivity. Similarly, 1 p,25-dihydroxyvitamin D3, 7.25 nM and 72.5 nM, displaced 1 a,25-dihydroxyvitamin D3 binding while 25-hydroxyvitamin D3, 72.5 nM and 725 nM, did not. The apparent dissociation constant (KD) for Ia,25-dihydroxyvitamin D3 was determined from displacement of 3H-1 a,25-dihydroxyvitamin D3 yielding a value of 8.1 x 1 O-' M by Scatchard analysis. The KD for the 1 p epimer determined from displacement of 3 H -l p,25dihydroxyvitamin D3 was 4.8 x lo-' M. The data suggest the presence of a receptor on the membranes of ROS 24/1 cells that recognizes Ia,25-dihydroxyvitamin D3 and its 1 p epimer, but not 25-hydroxyvitamin D3. Its ability to recognize the 1 p epimer which appears to be a specific antagonist of the rapid effects of the hormone suggests that these studies may be the initial steps in the isolation and characterization of the signalling system mediating the rapid actions of vitamin D. IZ 1994Wiley-Liss, Inc.