Faculty Collaboration Database

Evidence that stimulation of 1,25(OH)2D3 production in primary cultures of mouse kidney cells by cyclic AMP requires new protein synthesis. J Bone Miner Res 1987 Dec;2(6):517-24

Date

12/01/1987

Pubmed ID

2458676

DOI

10.1002/jbmr.5650020608

Scopus ID

2-s2.0-0023506622 (requires institutional sign-in at Scopus site)   28 Citations

Abstract

When primary culture of C75BL6 mouse cortical kidney cells in serum-free medium were incubated with unlabeled 25(OH)D3, they produced a metabolite which co-migrated with authentic 1,25(OH)2D3 and which could be measured by competitive receptor assay. A metabolite co-migrating with authentic 10-oxo-19-nor-25-OH-D3 was also produced. However, when cultures were incubated with 25(OH)D3 for 1 hour or longer, 10-oxo-19-nor-25-OH-D accounted for less than 15% of the total 3H-1,25(OH)2D3 displacement activity. Production of 1,25(OH)2D3 increased with increasing content of the culture, with time of incubation, and with substrate concentration. The apparent Km was 1.4 +/- 0.6 microM and Vmax 2.6 +/- 0.4 pM/mg protein/hr. These cultures possessed a very high level of phosphodiesterase activity, as indicated by their high cyclic AMP (cAMP) response to IBMX. This high phosphodiesterase activity may have been responsible for the lack of stimulation of 1,25(OH)2D3 production by physiologic or near physiologic concentrations of parathyroid hormone (PTH) in the absence of IBMX. However, when IBMX 10(-6) M was present, bPTH 10(-9) M significantly increased production of both cAMP and 1,25(OH)2D3. There was a close correlation between 1,25(OH)2D3 production and cAMP content of the cultures (basal or stimulated). An incubation time of at least 4 hours was required for cAMP to increase 1,25(OH)2D3 production and was inhibited in the presence of cycloheximide and actinomycin D. This study further documents the regulation of renal 1,25(OH)2D3 synthesis by PTH in mammalian kidney and provides evidence for cAMP as a possibly important second messenger in this effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Author List

Korkor AB, Gray RW, Henry HL, Kleinman JG, Blumenthal SS, Garancis JC

Author

Samuel S. Blumenthal MD Professor in the Medicine department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

1-Methyl-3-isobutylxanthine
Animals
Calcitriol
Cells, Cultured
Cyclic AMP
Cycloheximide
Dactinomycin
In Vitro Techniques
Kidney
Mice
Mice, Inbred C57BL
Parathyroid Hormone
Protein Synthesis Inhibitors