Parathyroid hormone modulation of 25-hydroxyvitamin D3 metabolism by cultured chick kidney cells is mimicked and enhanced by forskolin.

In order to determine whether cAMP mediates the effects of PTH on the metabolism of 25-hydroxyvitamin D3 (25-OH-D3) on chick kidney cells in primary culture, the effect of forskolin on the production of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] was assessed. In 4-h incubations with [3H]25-OH-D3 and forskolin, (1-10 microM) [3H]1,25-(OH)2D3 accumulation was increased 50-100%, and that of [3H]24,25-(OH)2D3 was decreased 30-60%. PTH (1-10 ng/ml) brought about identical changes. Similar results were observed when cultures were preincubated with nonradioactive 25-OH-D3 for 4 h in the presence of PTH and forskolin, followed by a 30-min incubation with radioactive substrate. At a low concentration (0.05 microM), forskolin alone had no effect on the metabolism of [3H]25-OH-D3 but markedly enhanced that of PTH. At maximal concentrations of PTH (10 ng/ml) and forskolin (10 microM), the effects of the two on 25-OH-D3 metabolism were not additive. Both PTH and forskolin decreased the further metabolism of [3H]1,25-(OH)2D3, probably by inhibiting its 24-hydroxylation, but there are also cycloheximide-sensitive steps in the metabolism of 1,25-(OH)2D3 that are not affected by PTH and forskolin. In time course experiments, increased [3H]1,25-(OH)2D3 accumulation could be observed before the detection of 24-hydroxylase activity suggesting that the primary effect of PTH and forskolin is on the production of [3H] 1,25-(OH)2D3 rather than its catabolism. Raising the calcium concentration of the medium to 2.5 mM from the normal 1.8 mM or lowering it to 0.5 mM for 24 h in serum-free medium did not alter the response of 25-OH-D3 metabolism to these agents. The results of these studies indicate that the effects of PTH on the metabolism of 25-OH-D3 by chick kidney cells are mediated by cAMP, since they can be enhanced and mimicked by forskolin, that they are exerted at the level of both 1- and 24-hydroxylase activity, and that they are not dependent on the calcium concentration of the medium.