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)₂D₃on cytosolic Ca²⁺[Ca²⁺h in single osteogenic sarcoma ROS 17/2.8 cells loaded with fura-2. An acute rise in [Ca²⁺]_iwas observed in 40% of the cells following addition of 1, 25(OH)₂D₃, with a threshold concentration of 10^-11M. In most cases, the [Ca²⁺]_irise 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)₂D₃on [Ca²⁺]_i(with equal potency and similar responses, whereas 24, 25- dihydroxycholecalciferol, la-hydroxycholecalciferol, and 22 oxa- 1, 25(OH)₂D₃were not effective. 1, 25(OH)₂D₃also increased [Ca²⁺]_iin ROS 24/1 cells, which are defective of receptors for the vitamin D metabolites. At high doses (10^-8-10^-7M) of 1, 25(OH)₂D₃the [Ca²⁺]_jrise 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^-10M), the effect was entirely dependent on extracellular Ca²⁺. 1, 25(OH)₂D₃also increased the production of inositol 1, 4, 5 trisphosphate (Ins(l, 4, 5)P3) and diacylglycerol, at a threshold dose of 10^-9M, indicating activation of phospholipase C (PLC). In two thirds of the cells studied, a second addition of 1, 25(OH)₂D₃within 5 min to cells prestimulated with equimolar doses of the vitamin D metabolite resulted in a [Ca²⁺]; transient of higher amplitude than the first, a phenomenon occurring at all doses of the hormone, and associated with production of Ins(l, 4, 5)P₃. This response amplification was not produced by 25(OH)D₃, and pretreatment with la(0H)D₃did not significantly enhance l, 25(OH)₂D₃-induced production of Ins(l, 4, 5)P₃. In conclusion, activation of the Ca²⁺message system by vitamin D metabolites is a rapid, nongenomic effect; 1, 25(OH)₂D₃specifically activates both PLC and dihydropyridine-sensitive Ca²⁺channels, and “primes” the cells to respond with an enhanced [Ca²⁺]_irise to a subsequent homologous stimulation; the presence of both the la and 25 hydroxyl groups is necessary to express the full hormonal action of vitamin D on [Ca²⁺]_i.