Oscillations in inositol 1,4,5-trisphosphate and diacyglycerol induced by vitamin D3 metabolites in confluent mouse osteoblasts

Abstract

For the last 5 years, attention has focused on the nongenomic effects of 1,25‐(OH)₂D₃, but considerably less is known about the mechanisms of the nonnuclear actions of 24,25‐(OH)₂D₃. The present study examines and compares the rapid (5–90 s) effects of 100 pM to 10 nM 24,25‐(OH)₂D₃, 10 pM to 1 nM 1,25‐(OH)₂D₃, and 1–100 nM 25‐OHD₃ on the formation of inositol phosphates and lipids in confluent mouse osteoblasts. 24,25‐(OH)₂D₃ and 25‐OHD₃ effects were dose dependent; those of 1,25‐(OH)₂D₃, were dose dependent in a bell‐shaped manner. The two dihydroxylated metabolites induced a multiphasic response in inositol 1,4,5‐trisphosphate (IP₃) formation with three stimulation peaks; the IP₃ response to 25‐OHD₃ was monophasic. The amplitude of the IP₃ response to 24,25‐(OH)₂D₃ was greater and its oscillation period was slower than that induced by 1,25‐(OH)₂D₃. The diacylglycerol (DAG) responses to secosteroids showed two stimulation peaks that appeared at different times depending on the secosteroid used. Pretreatment with neomycin totally inhibited the first DAG response; neomycin had no effect on the second peak of DAG induced by 25‐OHD₃, whereas it partially blocked the second response of DAG to 24,25‐(OH)₂D₃ and 1,25‐(OH)₂D₃. These data show for the first time that 24,25‐(OH)₂D₃ can modulate phospholipid metabolism in confluent mouse osteoblasts as early as 5–10 s. The first pathway used by all three secosteroids is that of the hydrolysis of phosphatidylinositol 4,5‐bisphosphate via phospholipase C activation, leading to the formation of the two second messengers, IP₃ and DAG, since neomycin totally blocked the response. Thus, the action of these secosteroids on the osteoblast membrane may also implicate several steps of the phosphatidylcholine cycle, according to the metabolite tested. Finally, these data point to a direct interaction of vitamin D metabolites with specific membrane recognition moieties.