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The dynamic equilibrium between in vivo occupied and unoccupied 1,25-dihydroxyvitamin D(3)[1,25(OH)(2)D(3)] receptors of the chick intestinal mucosa was investigated by the exchange assay previously reported [(1980). J. Biol. Chem.255: 9534-9537]. These parameters and their correlation to biological response, i.e., the levels of intestinal vitamin D-dependent calcium binding protein (CaBP), were assessed under different physiological conditions. After a single 1,25(OH)(2)D(3) injection (3.25 nmol), occupied receptor levels increased sharply to a maximum between 1 and 2 h, followed by a rapid decline. A single dose of 1alpha-hydroxy-vitamin D(3) [1alpha(OH)D(3)], an analog that requires 25-hydroxylation for biological activity, resulted in a protracted, albeit lower, response with maximal receptor occupancy at 6 h and half maximal levels 24 h after injection. The intestinal receptor occupancy patterns mirrored the serum 1,25(OH)(2)D(3) levels after either 1,25(OH)(2)D(3) or 1alpha(OH)D(3) treatment. Additionally, time-course (half-life) of blood disappearance of 1,25(OH)(2)D(3) and occupied receptor levels were similar (1.9 and 2.3 h, respectively), suggesting that the amount of occupied 1,25(OH)(2)D(3) receptor is determined by a simple equilibrium between serum 1,25(OH)(2)D(3) and unoccupied receptors. A dose-response study after intramuscular 1,25(OH)(2)D(3) injection yielded a hyperbolic curve with an apparent plateau at 70% receptor occupancy, corresponding to 5 nmol 1,25(OH)(2)D(3) injected. Half-maximal occupancy was reached after a dose of 1 nmol 1,25(OH)(2)D(3), corresponding to 1.5 ng 1,25(OH)(2)D(3)/ml serum. From this value the apparent K(d) in vivo is 3.7 nM, which is similar to that determined in vitro. A 10-fold increase in the 1alpha(OH)D(3) dose resulted in less than a doubling of the levels of serum 1,25(OH)(2)D(3), occupied 1,25(OH)(2)D(3) receptors, or CaBP. Under all experimental conditions, there was a positive correlation between occupied receptor and CaBP levels; however, the slope of the lines depended on the times chosen for the assays due in part to the lag period for CaBP induction and its accumulation within the cell. Conversely, the correlation between serum 1,25-(OH)(2)D(3) levels and occupied receptor levels yielded a single regression line independent of the observation time. Short and long-term treatment with different vitamin D metabolites, estrogen, progesterone, or cortisol did not affect the levels of total intestinal 1,25(OH)(2)D(3) receptor. Under normal physiological conditions, only 10-15% of the total 1,25(OH)(2)D(3) receptor population was occupied by ligand. These studies provide a basis for further investigations of physiological and biochemical parameters of the vitamin D endocrine system and their clinical applications.

Effect of Vitamin D Status on the Equilibrium between Occupied and Unoccupied 1,25-Dihydroxyvitamin D Intestinal Receptors in the Chick