Intermittent and continuous exposure to 1,25(OH)2D3 have different effects on growth plate chondrocytes in vitro

Abstract

Intermittent and continuous exposure to 1,25(OH)2D3 have different effects on growth plate chondrocytes in vitro. Intermittent 1,25(OH)2D3 administration is widely used to suppress parathyroid glands in secondary (renal) hyperparathyroidism. It is unknown whether the effects of continuous and intermittent 1,25(OH)2D3 differ on vitamin D target organs other than parathyroids. Using primary cultures of rat chondrocytes (tibia) we compared the effects of continuous versus intermittent exposure to physiologic concentrations of 1α,25(OH)2D3 on proliferation (radiothymidine incorporation), cell count, protein synthesis ([3H]leucine incorporation), alkaline phosphatase activity (as a marker of differentiation) and 1α,25(OH)2D3 receptor (VDR) regulation. Cells were synchronized and then exposed for variable periods to a medium containing 10% delipidated FCS and 10-8 M to 10-12 M 1α,25(OH)2D3 (or 1β,25(OH)2D3 as specifity control). Intermittent (8 hr exposure every 48 hr) as well as continuous (sham washing) administration of 1α,25(OH)2D3 had a biphasic effect on proliferation, that is, stimulation at low (10-12 M) and inhibition at high (10-8 M) concentrations. At 10-12 M intermittent 1α,25(OH)2D3 yielded higher cell counts than continuous 1,25(OH)2D3. This was seen in the log phase, which was day 3 (continuous 141 ± 2.3% of solvent control; intermittent 185 ± 2.0%) and in the plateau phase of growth, which was day 6 (128 ± 2.6 vs. 169 ± 2.7% of solvent control). Dependence on extracellular Ca is suggested by the effects of varying nominal Ca concentrations in the medium and of Ca channel blockers. Even two hours of exposure to 1α,25(OH)2D3 (10-12 M) yielded maximal activation of AP during postincubation. VDR more than doubled after 24 hours following brief (8 hr) or continuous exposure to 10-12 M 1α,25(OH)2D3 in the absence, but not in the presence of cycloheximide (5 μg/ml). Subsequently VDR declined with continuous 1α,25(OH)2D3, but not with intermittent 1α,25(OH)2D3. After 48 hours Kd was unchanged, but Nmax was significantly lower with continuous (2807 bound molecules/cell) than intermittent (5987 molecules/cell) 1α,25(OH)2D3. We conclude that intermittent exposure to 1,25(OH)2D3 in primary chondrocyte cultures is more effective in (i) stimulating cell proliferation and (ii) sustaining up-regulated VDR.