Fusion of mouse alveolar macrophages induced by 1α,25‐dihydroxyvitamin D 3 involves extracellular, but not intracellular, calcium

We have reported that the active form of vitamin D 3 , 1α, 25‐dihydroxy‐vitamin D 3 [1α,25(OH) 2 D 3 ], directly induces the fusion of mouse alveolar macrophages (Abe et al: Proc. Natl. Acad. Sci. USA 80:5583–5587, 1983). The fusion process can be divided into two phases: the 1α,25(OH) 2 D 3 ‐dependent priming phase (0–18 hr) and the calcium‐dependent progression phase (18–72 hr) (Jin et al: Cell. Physiol. 137:110–116, 1988). In the present study, we examined the role of calcium in the progression phase of macrophage fusion induced by 1α,25(OH) 2 D 3 . Macrophages pretreated with 1α,25(OH) 2 D 3 for 48 hr in a lowcalcium (0.13 mM) medium began to fuse quickly 30 min after the culture medium was switched to a normal calcium (1.85 mM) medium. Of various cations tested, calcium was the most effective in inducing fusion, followed by strontium and manganese. Magnesium, potassium, and sodium had no effect. Calcium ionophores such as A23187 and ionomycin did not induce fusion in the lowcalcium medium, nor did they potentiate fusion in the media containing higher concentrations of calcium. The intracellular concentration of free Ca 2+ , measured by a fluorescent method using fura‐2 AM, was 116 ± 1 nM in the macrophages pretreated with 1α,25(OH) 2 D 3 for 48 hr in the low‐calcium medium. When calcium chloride was added to the assay system at a final concentration of 1.85 mM, the cytosolic free Ca 2+ concentration did not increase appreciably (from 116 to 144 nM). But the macrophages began to fuse quickly when CaCI 2 was added. In contrast, adding ionomycin increased cytosolic free Ca 2+ from 116 to 440 nM, but no fusion occurred. These results clearly indicate that the extracellular, but not the intracellular, calcium is involved in the progression phase of the fusion of mouse alveolar macrophages primed by 1α,25(OH) 2 D 3 .