ROLE OF CALBINDIN-D-9K IN BUFFERING CYTOSOLIC-FREE CA2+ IONS IN PIG DUODENAL ENTEROCYTES

1. The aim of the present study was to test whether the vitamin D-depe ndent Ca2+-binding protein calbindin-D-9k could function as an importa nt cytosolic Ca2+ buffer in duodenal enterocytes while facilitating tr ansepithelial active transport of Ca2+ ions. For the investigations Iv e used dual-wavelength, fluorescence ratio imaging, with fura-2 as the Ca2+-sensitive dye, to measure changes in cytosolic concentrations of free Ca2+ ions ([C2+](i)) in isolated pig duodenal enterocytes affect ed by different cytosolic calbindin-D-9k concentrations.2. Epithelial cells were obtained from weaned piglets with normal calbindin-D-9k con centrations (con-piglets), from piglets with low calbindin-D-9k levels due to inherited calcitriol deficiency caused by defective renal 25-h ydroxycholecalciferol D-3-1 alpha-hydroxylase activity (def-piglets), and from piglets with reconstituted calbindin-D-9k concentrations, i.e . def-animals treated with high doses of vitamin D-3 which elevated pl asma calcitriol levels by extrarenal production (def-D-3-piglets). Bas al levels of [Ca2+](i) ranged between 170 and 205 nM and did not diffe r significantly between the groups. 3. After addition of 5 mnl theophy lline, the [Ca2+](i) in enterocytes from con-piglets doubled during th e 10 min incubation. This effect, however, was three times higher in e nterocytes from def-piglets compared with those from con-piglets. Simi lar results were obtained after 4 min incubation of enterocytes from c on- and def-piglets in the presence of 1 mu M ionomycin. In preparatio ns from def-D-3-piglets, ionomycin-induced increases in [Ca2+](i) were significantly lower compared with enterocytes from def-piglets and we re not different from the control values. 4. From the results, substan tial support is given for the hypothesis that one of the major functio ns of mucosal calbindin-D-9k is the effective buffering of Ca2+ ions.