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

Abstract

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.