Abstract
Demonstrating 1,25(OH)~2~D~3~‐stimulated calcium uptake in isolated chick intestinal epithelial cells has been complicated by simultaneous enhancement of both uptake and efflux. We now report that in intestinal cells of adult birds, or those of young birds cultured for 72 h, 1,25(OH)~2~D~3~‐stimulates ^45^Ca uptake to greater than 140% of corresponding controls within 3 min of addition. Such cells have lost hormone‐stimulated protein kinase C (PKC) activity, believed to mediate calcium efflux. To further test this hypothesis, freshly isolated cells were preincubated with calphostin C, and calcium uptake monitored in the presence or absence of steroid. Only cells treated with the PKC inhibitor demonstrated a significant increase in ^45^Ca uptake in response to 1,25(OH)~2~D~3~, relative to corresponding controls. In addition, phorbol ester was shown to stimulate efflux, while forskolin stimulated uptake. To further investigate the mechanisms involved in calcium uptake, we assessed the role of TRPV6 and its activation by β‐glucuronidase. β‐Glucuronidase secretion from isolated intestinal epithelial cells was significantly increased by treatment with 1,25(OH)~2~D~3~, PTH, or forskolin, but not by phorbol ester. Treatment of cells with β‐glucuronidase, in turn, stimulated ^45^Ca uptake. Finally, transfection of cells with siRNA to either β‐glucuronidase or TRPV6 abolished 1,25(OH)~2~D~3~‐enhanced calcium uptake relative to controls transfected with scrambled siRNA. Confocal microscopy further indicated rapid redistribution of enzyme and calcium channel after steroid. 1,25(OH)~2~D~3~ and PTH increase calcium uptake by stimulating the PKA pathway to release β‐glucuronidase, which in turn activates TRPV6. 1,25(OH)~2~D~3~‐enahnced calcium efflux is mediated by the PKC pathway. J. Cell. Biochem. 105: 1109–1116, 2008. © 2008 Wiley‐Liss, Inc.