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Evidence that stimulation of 1,25(OH) 2 D 3 production in primary cultures of mouse kidney cells by cyclic AMP requires new protein synthesis
Author(s) -
Korkor Adel B.,
Gray Richard W.,
Henry Helen L.,
Kleinman Jack G.,
Blumenthal Samuel S.,
Garancis John C.
Publication year - 1987
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1002/jbmr.5650020608
Subject(s) - ibmx , stimulation , incubation , metabolite , medicine , endocrinology , phosphodiesterase , phosphodiesterase inhibitor , parathyroid hormone , chemistry , kidney , biology , biochemistry , calcium , enzyme , forskolin
When primary culture of C75BL6 mouse cortical kidney cells in serum‐free medium were incubated with unlabeled 25(OH)D 3 , they produced a metabolite which co‐migrated with authentic 1,25(OH) 2 D 3 and which could be measured by competitive receptor assay. A metabolite co‐migrating with authentic 10‐oxo‐19‐nor‐25‐OH‐D 3 was also produced. However, when cultures were incubated with 25(OH)D 3 for 1 hour or longer, 10‐oxo‐19‐nor‐25‐OH‐D accounted for less than 15% of the total 3 H‐1,25(OH) 2 D 3 displacement activity. Production of 1,25(OH) 2 D 3 increased with increasing protein content of the cultures, with time of incubation, and with substrate concentration. The apparent Km was 1.4 ± 0.6 μM and V max 2.6 ± 0.4 p M /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) 2 D 3 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) 2 D 3 . There was a close correlation between 1,25(OH) 2 D 3 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) 2 D 3 production and was inhibited in the presence of cycloheximide and actinomycin D. This study further documents the regulation of renal 1,25(OH) 2 D 3 synthesis by PTH in mammalian kidney and provides evidence for cAMP as a possibly important second messenger in this effect. The synthesis of 1,25(OH) 2 D 3 in response to cAMP appears to depend upon the ability of the cell to synthesize new protein.