title: Cultured osteoblasts from normal and hypophosphatemic mice: calcitriol receptors and biological response to the hormone.
abstract: The content and affinity of calcitriol receptors were analyzed in cultured osteoblasts from normal and hypophosphatemic mice. Hypertonic cell extracts were prepared by sonication followed by centrifugation at 200,000 g x 30 min. Analysis, at saturating levels of labeled 1,25(OH)2D3, revealed that binding of the hormone was dependent on the density of the cells plated and on the length of time in culture. It reached a maximum at 5 days of culture when 1.0 x 10(6) cells were plated. Under those conditions the binding capacity of Hyp osteoblasts was 6306 +/- 1267 sites/ng protein (mean +/- SEM) not different from N cells (7594 +/- 1713). The dissociation constant (Kd) was 18.3 +/- 5.4 and 20.0 +/- 5.7 pM for mutant and normal mouse osteoblasts respectively (NS). In both genotypes, a single peak for specific binding, migrating at approximately 3.0-3.5 S was observed by sucrose gradient centrifugation. 25-hydroxycholecalciferol-24-hydroxylase (24-OHase) was induced at 1 and 10 nM 1,25(OH)2D3 in a dose-dependent fashion. However, the induction was higher in mutant than in normal cells when the medium contained 1 mM and 2 mM phosphate salts. The difference vanished when cells were incubated in the presence of 3 and 4 mM phosphate salts. The effect of calcitriol on cultured osteoblasts was also analyzed in terms of collagen synthesis and alkaline phosphatase activity. In the range of 10(-10) M to 10(-7) M, 1,25(OH)2D3 was found to inhibit collagen synthesis in a dose-dependent fashion. At physiological levels, 1,25(OH)2D3 (10(-11)M-10(-10)M), stimulated alkaline phosphatase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Genetics Unit, Shriners Hospital, Montreal, Quebec, Canada.
authors.forename: E E
authors.initials: EE
authors.lastname: Delvin
authors.forename: P
authors.initials: P
authors.lastname: Richard
authors.forename: M
authors.initials: M
authors.lastname: Desbarats
authors.forename: B
authors.initials: B
authors.lastname: Ecarot-Charrier
authors.forename: F H
authors.initials: FH
authors.lastname: Glorieux
mesh_terms: D000469:Alkaline Phosphatase / Q000502:physiology; D000818:Animals; D002117:Calcitriol / Q000378:metabolism*; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D003094:Collagen / Q000096:biosynthesis; D003577:Cytochrome P-450 Enzyme System; D004790:Enzyme Induction; D051379:Mice; D010006:Osteoblasts / Q000378:metabolism*; D010710:Phosphates / Q000097:blood*; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000378:metabolism*; D013250:Steroid Hydroxylases / Q000096:biosynthesis; D013997:Time Factors; D065668:Vitamin D3 24-Hydroxylase
chemical_list: D010710:Phosphates; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D003094:Collagen; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D000469:Alkaline Phosphatase; D002117:Calcitriol