title: Membrane receptor-initiated signaling in 1,25(OH)2D3-stimulated calcium uptake in intestinal epithelial cells.
abstract: Demonstrating 1,25(OH)2D3-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)2D3-stimulates 45Ca 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 45Ca uptake in response to 1,25(OH)2D3, 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 beta-glucuronidase. beta-Glucuronidase secretion from isolated intestinal epithelial cells was significantly increased by treatment with 1,25(OH)2D3, PTH, or forskolin, but not by phorbol ester. Treatment of cells with beta-glucuronidase, in turn, stimulated 45Ca uptake. Finally, transfection of cells with siRNA to either beta-glucuronidase or TRPV6 abolished 1,25(OH)2D3-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)2D3 and PTH increase calcium uptake by stimulating the PKA pathway to release beta-glucuronidase, which in turn activates TRPV6. 1,25(OH)2D3-enhanced calcium efflux is mediated by the PKC pathway.
authors.affiliation: Department of Nutrition and Food Sciences and Center for Integrated BioSystems, Utah State University, Logan, Utah 84322-8700, USA.
authors.forename: Ramesh C
authors.initials: RC
authors.lastname: Khanal
authors.forename: Tremaine M Sterling
authors.initials: TM
authors.lastname: Peters
authors.forename: Nathan M
authors.initials: NM
authors.lastname: Smith
authors.forename: Ilka
authors.initials: I
authors.lastname: Nemere
mesh_terms: D000818:Animals; D002117:Calcitriol / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D004847:Epithelial Cells / Q000378:metabolism*; D005966:Glucuronidase / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000166:cytology*; D011493:Protein Kinase C / Q000172:deficiency; D021381:Protein Transport; D015398:Signal Transduction; D013256:Steroids / Q000494:pharmacology; D050916:TRPV Cation Channels / Q000378:metabolism*
chemical_list: D013256:Steroids; D050916:TRPV Cation Channels; C120776:TRPV6 channel; D011493:Protein Kinase C; D005966:Glucuronidase; D002117:Calcitriol; D002118:Calcium