Studies on the mechanism of action of calciferol. IV. Interaction of the polyene antibiotic, filipin, with intestinal mucosal membranes from vitamin D-treated and vitamin D-deficient chicks

Abstract 1. 1. The polyene antibiotic, filipin, has been shown to be a specific tool with which to probe the biochemical function of vitamin D (calciferol)-stimulated calcium translocation, in vitro , in the chick intestinal ileum. Filipin treatment, in vitro (concentration about 2·10 -5 M of ileal segments obtained from vitamin D-deficient chicks increased the calcium flux, J ms (flux in the mucosal-to-serosal direction), by 150–250%. Filipin treatment, in vitro , of ileal segments from vitamin D-treated chicks had no effect on calcium flux, J ms . The effect of filipin on calcium was shown to exist only when the antibiotic was administered to the mucosal surface of an ileum. The filipin effect was specific for calcium translocation; little or no effect of filipin was noted on the flux, J ms , of phosphate, sulfate, serine or rubidium. Also the magnitude of the filipin-mediated increase in calcium flux was inversely proportional to the time after vitamin D administration, in vivo , to rachitic chicks, so that 60 h after vitamin D there was no filipin effect. 2. 2. Studies carried out on thiourea and water flux, J ms , in response to filipin treatment showed that the increase in specific calcium transport in ileal segments from vitamin D-deficient chicks is not mediated by a general increase in permeability. 3. 3. Filipin was shown to bind to a greater extent and a faster rate of ileal segments obtained from rachitic chicks than those from vitamin D-treated chicks. 4. 4. Electron micrographic studies showed that after prolonged exposure (80 min) to filipin on the mucosal side, in vitro , gross morphological changes could be detected in the microvillar region of the ileal segments obtained from vitamin D-deficient chicks but not in the segment obtained from vitamin D-treated chicks. There were no major morphological changes apparent in either system when ileal segments were exposed to filipin, in vitro , for periods equivalent to those used in studying the transport activities (20–40 min). 5. 5. Examination of the time-course of events noted after administration of filipin, in vitro , to ileal segments from vitamin D-deficient and vitamin D-treated chicks, supports the hypothesis that a fundamental difference exists in some structural aspect of the intestinal microvillar membranes of these two systems.