Nongenomic activation of the calcium message system by vitamin D metabolites in osteoblast-like cells

1,25-dihydroxycholecalciferol (1,25(OH)₂D₃) rapidly affects calcium (Ca²⁺) transport in several cell systems, suggesting physiological actions independent of genomic activation. To test this hypothesis, we studied immediate to early effects (0.5-300 sec) of 1,25(OH)₂D₃ on cytosolic Ca²⁺ [Ca²⁺]_i in single osteogenic sarcoma ROS 17/2.8 cells loaded with fura-2. An acute rise in [Ca²⁺]ⁱ was observed in 40% of the cells following addition of 1,25(OH)₂D₃, with a threshold concentration of 10^-11 M. In most cases, the [Ca²⁺]_i rise was transient, with return to baseline within 1 min; less frequently a more prolonged effect was observed, with variable recovery times. 25-hydroxycholecalciferol (25(OH)D₃) reproduced the effect of 1,25(OH)₂D₃ on [Ca²⁺]_i, with equal potency and similar responses, whereas 24,25-dihydroxycholecalciferol, 1α-hydroxycholecalciferol, and 22 oxa-1,25(OH)₂D₃ were not effective. 1,25(OH)₂D₃ also increased [Ca²⁺]_i in ROS 24/1 cells, which are defective of receptors for the vitamin D metabolites. At high doses (10^-8-10^-7 M) of 1,25(OH)₂D₃ the [Ca²⁺]_i rise in ROS 17/2.8 cells was due to both influx of extracellular Ca²⁺ and release of Ca²⁺ from intracellular stores, as the effect was only partially inhibited by Ca²⁺-channel blockade by nifedipine. At low doses (10^-9-10^-10 M), the effect was entirely dependent on extracellular Ca²⁺. 1,25(OH)₂D₃ also increased the production of inositol 1,4,5 trisphosphate (Ins(1, 4, 5)P₃) and diacylglycerol, at a threshold dose of 10^-9 M, indicating activation of phospholipase C (PLC). In two thirds of the cells studied, a second addition of 1,25(OH)₂D₃ within 5 min to cells prestimulated with equimolar doses of the vitamin D metabolite resulted in a [Ca²⁺]_i transient of higher amplitude than the first, a phenomenon occurring at all doses of the hormone, and associated with production of Ins(1, 4, 5)P₃. This response amplification was not produced by 25(OH)D₃, and pretreatment with 1α(OH)D₃ did not significantly enhance 1,25(OH)₂D₃-induced production of Ins(1, 4, 5)P₃. In conclusion, activation of the Ca²⁺ message system by vitamin D metabolites is a rapid, nongenomic effect; 1,25(OH)₂D₃ specifically activates both PLC and dihydropyridine-sensitive Ca²⁺ channels, and "primes" the cells to respond with an enhanced [Ca²⁺]_i rise to a subsequent homologous stimulation; the presence of both the 1α and 25 hydroxyl groups is necessary to express the full hormonal action of vitamin D on [Ca²⁺]_i.