Role of calbindin‐D9k in buffering cytosolic free Ca2+ ions in pig duodenal enterocytes.

1. The aim of the present study was to test whether the vitamin D‐dependent Ca(2+)‐binding protein calbindin‐D9k could function as an important cytosolic Ca2+ buffer in duodenal enterocytes while facilitating transepithelial active transport of Ca2+ ions. For the investigations we used dual‐wavelength, fluorescence ratio imaging, with fura‐2 as the Ca(2+)‐sensitive dye, to measure changes in cytosolic concentrations of free Ca2+ ions ([Ca2+]i) in isolated pig duodenal enterocytes affected by different cytosolic calbindin‐D9k concentrations. 2. Epithelial cells were obtained from weaned piglets with normal calbindin‐D9k concentrations (con‐piglets), from piglets with low calbindin‐D9k levels due to inherited calcitriol deficiency caused by defective renal 25‐hydroxycholecalciferol D3‐1 alpha‐hydroxylase activity (def‐piglets), and from piglets with reconstituted calbindin‐D9k concentrations, i.e. def‐animals treated with high doses of vitamin D3 which elevated plasma calcitriol levels by extrarenal production (def‐D3‐piglets). Basal levels of [Ca2+]i ranged between 170 and 205 nM and did not differ significantly between the groups. 3. After addition of 5 mM theophylline, the [Ca2+]i in enterocytes from con‐piglets doubled during the 10 min incubation. This effect, however, was three times higher in enterocytes from def‐piglets compared with those from con‐piglets. Similar results were obtained after 4 min incubation of enterocytes from con‐ and def‐piglets in the presence of 1 microM ionomycin. In preparations from def‐D3‐piglets, ionomycin‐induced increases in [Ca2+]i were significantly lower compared with enterocytes from def‐piglets and were not different from the control values. 4. From the results, substantial support is given for the hypothesis that one of the major functions of mucosal calbindin‐D9k is the effective buffering of Ca2+ ions.