Studies on the mechanism of action of calciferol. VIII. The effects of dietary vitamin D and the polyene antibiotic, filipin, in vitro, on the intestinal cellular uptake of calcium.

Intestinal transport and cellular uptake of calcium were studied in vitro in ileal segments obtained from calciferol (vitamin D)-deficient (minus D3) chicks or in calciferol-treated chicks (+ D3). When calcium flux (J) was measured in both the mucosal yields serosal (J-ms) and serosal yields mucosal (J-sm) directions it was found that calcium transport in the +D3 system was an active process. Calcium was not actively transported in the minus D3 system or by either the +D3 or minus D3 serosal tissues which underlie the intestinal mucosa tissue. It was also found that the serosal tissue was not the rate-limiting step in calcium translocation. When calcium uptake was measured at the mucosal or brush border surface it was found to be a cation-oriented, saturable process in both the +D3 and minus D3 systems and was enhanced by calciferol supplementation. The rate of uptake was found to exhibit a two component transport phenomenon, one a saturable process at low Ca2+ concentrations and the other a linear of diffusion process at higher Ca2+ concentrations (greater than 5 mM). The polyene antibiotic, filipin, was used to study the transport and uptake of calcium in both the +D and minusD chick ileum in vitro. Filipin (10 mug/ml), when added in vitro to solutions bathing the mucosal surface of the ileum, stimulated the calcium flux, J-ms, of minus D ileal tissue by 150 to 200%, but had little or no stimulatory effect on J-ms of +D3 ileal tissue. However, in contrast it stimulated calcium uptake across the mucosal membrane in both the +D3 and minus D3 ileal tissue (50 to 100%). The effect of filipin is specific for calcium uptake as compared to Rb+, P-i, SO4(2-), glycerol, thiourea, and urea uptake. Isolated brush borders from +D3 chicks were found to bind calcium to a greater extent than the minus D3 brush borders, but both membrane fractions had the same cholesterol content. The sum of this and other evidence suggests that of uptake of calcium is a calciferol-mediated event which is not the rate-limiting step in the total transport of this ion across the intestinal epithelial cell. The filipin effect appears to affect a structural reorganization of the brush border membrane in both the +D3 and minus D3 membranes in a manner specific for calcium translocation. It now appears that filipin treatment somehow makes both transport systems more efficient in the uptake of calcium, but in a manner independent of the mode of action of calciferol. However, these data suggest that other components associated with calcium uptake are calciferol-dependent in an as yet unknown fashion.