Induction of calcium-binding protein before 1,25-dihydroxyvitamin D3 stimulation of duodenal calcium uptake.

Abstract

Protein synthesis occurring before the onset of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) stimulation of calcium uptake was examined in embryonic chick duodena by double label autoradiography. Duodena from 19-day-old embryos were cultured for 24 h. Duodena exposed to a saturating concentration of 1,25-(OH)2D3 during the last 2, 4, or 6 h were either cultured with [3H]leucine for the final 2 h or used for calcium uptake assays. Duodena not exposed to the hormone were either cultured with [14C]leucine for the final 2 h or used for calcium uptake assays. Calcium uptake by nonradiolabeled duodena was increased (p less than 0.05) only in tissues exposed to the hormone for 6 h. Tissue uptakes of [3H]leucine and [14C]leucine were identical and linear with time. Soluble proteins were extracted from 3H- and 14C-labeled tissues, mixed, and simultaneously resolved by two-dimensional polyacrylamide gel electrophoresis. Analysis of fluorographs and autoradiographs prepared from the double-labeled gels revealed a protein induced by 1,25-(OH)2D3 within 2 h of exposure to 1,25-(OH)2D3 and at lest 2 h before the calcium uptake response. The induced protein co-migrated with purified chick calcium-binding protein during two-dimensional electrophoresis. These results demonstrate that 1) this protein is the calcium-binding protein and 2) calcium-binding protein is clearly synthesized in the embryonic duodenum before the calcium uptake response to 1,25-(OH)2D3 occurs.