1,25-Dihydroxycholecalciferol (calcitriol) modifies uptake and release of 25-hydroxycholecalciferol in skeletal muscle cells in culture
Introduction
Our knowledge about the role of vitamin D continues to expand beyond its classical roles in calcium and phosphate homeostasis. The concentration of 25-hydroxycholecalciferol (25(OH)D) in blood is usually taken as an index of vitamin D status. However, it gives no indication of the total body pool size of 25(OH)D.
There is now evidence that skeletal muscle cells contain a mobile pool of 25(OH)D which accumulates from and returns to the extracellular environment. We have previously shown in cultures of mature muscle cells that 25(OH)D, is taken up and retained in the cells by binding to vitamin D binding protein (DBP), which had been internalized via membrane megalin and then binds to actin in the cytoplasm [1]. We postulated that if the capacity to hold 25(OH)D out of the circulation in skeletal muscle were high, when vitamin D status was falling in winter, it would be protected from wasteful uptake and destruction in the liver. This would increase the residence time of circulating 25(OH)D and thus would maintain adequate status during the months when vitamin D supply was low. If this muscle retention mechanism for 25(OH)D were important in increasing its half-life in blood, the process may be regulated by calciotropic hormones, such as parathyroid hormone and 1,25-dihydroxyvitamin D3 (calcitriol). We have shown that net muscle uptake or release of 25(OH)D is indeed modulated by parathyroid hormone [2] and have reported a short term effect of calcitriol on muscle uptake of 25(OH)D over short incubation periods [3].
In the current study, we tested whether longer incubations with calcitriol would have similar or different effects on 25(OH)D uptake or release. 4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) was used as an inhibitor of chloride channel opening, which has been used to investigate non-genomic actions of calcitriol [4].
Section snippets
Materials
Reagents were purchased from Sigma Aldrich (MO, USA) unless otherwise indicated. Calcitriol was purchased from Cayman, USA, dissolved in spectroscopic grade ethanol and then diluted in the differentiation medium to concentrations of 10−10, 10−9, and 10−8M. 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) was purchased from Sigma Aldrich (MO, USA) and dissolved in dimethyl sulfoxide (DMSO). The vitamin D receptor antibody, clone D6, used for immunohistochemistry and western blots and the
Immunohistochemistry: VDR detection in different cell types
Immunofluorescent studies of freshly isolated FDB myofibers showed immunoreactivity to the VDR (D6) antibody (Fig. 1A). Isotype staining was negligible for all cell types (Figs. 1b,d,f,h,). We previously reported that VDR staining was not detected in muscle fibers from VDR −/- mice [3]. VDR staining was detected throughout differentiated C2 myotubes (Fig. 1c). Normal skin fibroblasts showed dense staining distributed along the cell and in the perinuclear regions (Fig. 1e) but no staining was
Discussion
This study examined the concentration- and time-dependent effects of calcitriol on the capacity of muscle cells to take up and release 25(OH)D. We have previously reported that when C2 cells were differentiated into myotubes, the time-dependent uptake of labelled-25(OH)D is 2–3 times higher than in undifferentiated myoblasts and osteoblasts. Our previous experiments also showed that C2 myotubes released only 32% of the previously accumulated 25(OH)D after 4 h compared to 60% for osteoblasts [1].
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