Endocrinology, Vol 130, 276-281, Copyright © 1992 by Endocrine Society

ARTICLES

Binding and metabolism of 1,25-dihydroxyvitamin D3 in cultured bovine parathyroid cells

AJ Brown, M Berkoben, CS Ritter and E Slatopolsky
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.

Several laboratories, including ours, have reported that receptors for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] are decreased in parathyroid glands of uremic animals and patients. To elucidate the factors involved in receptor regulation in this tissue, we have characterized the receptor in primary cultures of bovine parathyroid cells. Extracts from these cells contain a single binding component that binds 1,25- (OH)2D3 with a Kd of 58 pM and sediments in sucrose density gradients at 3.4S, indicating the continued expression of the vitamin D receptor in these cells. Labeling of the intact parathyroid cells with tritiated 1,25-(OH)2D3 was maximal by 2 h, and binding affinity by this method was estimated to be 22 pM. Longer incubation of the cells with tritiated 1,25-(OH)2D3 resulted in a loss of specific binding to 10% maximal by 12 h. The decrease in binding correlated temporally with degradation of 1,25-(OH)2D3 in the medium. This metabolic activity was absent in vitamin D-deficient cells and was first detectable 3-4 h after the addition of 1,25-(OH)2D3, indicating that 1,25-(OH)2D3 induces its own metabolism in parathyroid cells. Replenishment of the cultures after 12 h with fresh tritiated 1,25-(OH)2D3 restored maximal binding, demonstrating that the loss of binding was not due to down- regulation of receptor. Inclusion of the cytochrome P450 inhibitor ketoconazole did not alter maximal binding at 2 h, but blocked both the metabolism of 1,25-(OH)2D3 and the decrease in binding after 3 h. In contrast to other cell types, such as osteosarcoma cells, no homologous up-regulation was seen in cultured parathyroid cells even after 12 h in the presence of 0.5 nM 1,25-(OH)2D3. Furthermore, receptor levels in preparations from cells treated for 20 h with unlabeled 1,25-(OH)2D3 at concentrations of 0.1, 1.0, and 10 nM were not different from controls. Thus, it appears that the vitamin D receptors in parathyroid cell cultures are not up-regulated by their ligand.