Controversy remains regarding whether the seco-steroid hormone 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) enhances calcium and phosphate movement across the intestinal epithelial cell by facilitated diffusion or a vesicular transport mechanism. In this study we investigated whether membrane trafficking, as judged by confocal microscopy, was sufficiently rapid in comparison to hormone-stimulated uptake of phosphate (P-32). Primary cultures of chick intestinal cells were established overnight either in Petri dishes (uptake studies) or chambered coverslips (confocal microscopy). Addition of 130 pM 1,25(OH)(2)D-3 resulted in an apparent increase in P-32 uptake within 1 min, relative to controls, that was statistically significant from 3-10 min of incubation. Using the endocytic marker dye, FM1-43, confocal microscopy revealed a profound decrease in membrane-associated fluorescence (apical>basal) within 10 s of hormone treatment, a return of fluorescence at 15-65 S, followed by another round of decreasing and increasing fluorescence. Between 3-9 min of incubation, fluorescence intensity increased 50% (apical region) and 20% (basal region) over control conditions. An antibody (Ab 099) directed against a putative membrane receptor for 1,25(OH)(2)D-3 (1,25D(3)-MARRS) inhibited both P-32 uptake, and changes in fluorescence. In addition, the protein kinase C (PKC) inhibitor, calphostin C, inhibited both P-32 uptake and the observed 1,25(OH)(2)D-3-mediated changes in fluorescence. At the microscopic level, calphostin C pretreatment abolished the very rapid redistribution of the endocytic marker dye, although a slight increase in fluorescence was still observed. We conclude that 1,25(OH)(2)D-3-stimulated vesicular trafficking is mediated by the 1,25D(3)-MARRS protein, implicates a PKC signaling mechanism, and occurs in a time frame that is commensurate with a role in ion transport.
