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.
Schröder
B
B
Department of Veterinary-Physiology, Justus-Liebig-University, Giessen, Germany.
Schlumbohm
C
C
Kaune
R
R
Breves
G
G
D000818:Animals; D001693:Biological Transport, Active; D064026:Calbindins; D002117:Calcitriol / Q000097:blood / Q000172:deficiency; D002118:Calcium / Q000378:metabolism*; D002470:Cell Survival; D002762:Cholecalciferol / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism*; D004195:Disease Models, Animal; D004386:Duodenum / Q000378:metabolism*; D005260:Female; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008297:Male; D008871:Microvilli / Q000378:metabolism; D064030:S100 Calcium Binding Protein G / Q000494:pharmacology*; D013552:Swine; D013806:Theophylline / Q000494:pharmacology; D014808:Vitamin D Deficiency / Q000378:metabolism
D064026:Calbindins; D007476:Ionophores; D064030:S100 Calcium Binding Protein G; D002762:Cholecalciferol; D015759:Ionomycin; D013806:Theophylline; D002117:Calcitriol; D002118:Calcium