Abstract
Cells obtained from male quail kidneys by digestion with collagenase and hyaluronidase were plated and maintained in a chemically defined, serum-free medium. Culture dishes (35 mm) were inoculated with 1.5 · 106 cells which became confluent in 5 days. The cells maintained an epithelial-like morphology over the entire culture period. During a 2 h incubation the cells metabolized 25-30% of the 10 nM 25-hydroxyvitamin D-3 (25-OH-D-3) provided. Seven metabolites were chromotographically separated on Sephadex LH-20. Three have been identified as 1α,25-dihydroxyvitamin D-3 (1,25(OH)2D-3), 24,25-dihydroxyvitamin D-3 (24,25(OH)2D-3) and 1α,24,25-trihhydroxyvitamin D-3 (1,24,25(OH)3D-3). The activities of the 25-OH-D-3:1α- and 24-hydroxylases increased eight times faster than the cell number in 5 days. Preincubation of the cells with 10 nM 25-OH-D-3 or 1,25(OH)2D-3 decreased 1,25(OH)2D-3 synthesis, and increased both 24,25(OH)2D-3 and metabolite IV synthesis. The decrease in 25-OH-D-3:1α-hydroxylase activity required a 2 h preincubation with 25-OH-D-3, while stimulation of 25-OH-D-3:24-hydroxylase activity and metabolite IV production required a 6 h preincubation. Incubations of cells for 1 h with parathyroid hormone resulted in a 30-fold increase in cyclic AMP in the medium. A 6 h preincubation with parathyroid hormone decreased 24,25-(OH)2D-3 synthesis 50% relative to control cells. These results demonstrate the amenability of this system for studying the regulation of 25-OH-D-3 metabolism, as well as its use for other in vitro studies on renal cell function in a chemically defined culture system.
| Original language | English |
|---|---|
| Pages (from-to) | 495-506 |
| Number of pages | 12 |
| Journal | BBA - General Subjects |
| Volume | 587 |
| Issue number | 4 |
| DOIs | |
| State | Published - Nov 1 1979 |
| Externally published | Yes |
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Keywords
- 25-Hydroxyvitamin D-3
- Kidney cells
- Metabolic regulation
- Serum-free culture
- Vitamin D-3
ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Molecular Biology
- Medicine(all)
Cite this
Serum-free culture of Japanese quail kidney cells regulation of vitamin D metabolism. / Howard, Guy; Turner, R. T.; Bottemiller, B. L.; Rader, J. I.
In: BBA - General Subjects, Vol. 587, No. 4, 01.11.1979, p. 495-506.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Serum-free culture of Japanese quail kidney cells regulation of vitamin D metabolism
AU - Howard, Guy
AU - Turner, R. T.
AU - Bottemiller, B. L.
AU - Rader, J. I.
PY - 1979/11/1
Y1 - 1979/11/1
N2 - Cells obtained from male quail kidneys by digestion with collagenase and hyaluronidase were plated and maintained in a chemically defined, serum-free medium. Culture dishes (35 mm) were inoculated with 1.5 · 106 cells which became confluent in 5 days. The cells maintained an epithelial-like morphology over the entire culture period. During a 2 h incubation the cells metabolized 25-30% of the 10 nM 25-hydroxyvitamin D-3 (25-OH-D-3) provided. Seven metabolites were chromotographically separated on Sephadex LH-20. Three have been identified as 1α,25-dihydroxyvitamin D-3 (1,25(OH)2D-3), 24,25-dihydroxyvitamin D-3 (24,25(OH)2D-3) and 1α,24,25-trihhydroxyvitamin D-3 (1,24,25(OH)3D-3). The activities of the 25-OH-D-3:1α- and 24-hydroxylases increased eight times faster than the cell number in 5 days. Preincubation of the cells with 10 nM 25-OH-D-3 or 1,25(OH)2D-3 decreased 1,25(OH)2D-3 synthesis, and increased both 24,25(OH)2D-3 and metabolite IV synthesis. The decrease in 25-OH-D-3:1α-hydroxylase activity required a 2 h preincubation with 25-OH-D-3, while stimulation of 25-OH-D-3:24-hydroxylase activity and metabolite IV production required a 6 h preincubation. Incubations of cells for 1 h with parathyroid hormone resulted in a 30-fold increase in cyclic AMP in the medium. A 6 h preincubation with parathyroid hormone decreased 24,25-(OH)2D-3 synthesis 50% relative to control cells. These results demonstrate the amenability of this system for studying the regulation of 25-OH-D-3 metabolism, as well as its use for other in vitro studies on renal cell function in a chemically defined culture system.
AB - Cells obtained from male quail kidneys by digestion with collagenase and hyaluronidase were plated and maintained in a chemically defined, serum-free medium. Culture dishes (35 mm) were inoculated with 1.5 · 106 cells which became confluent in 5 days. The cells maintained an epithelial-like morphology over the entire culture period. During a 2 h incubation the cells metabolized 25-30% of the 10 nM 25-hydroxyvitamin D-3 (25-OH-D-3) provided. Seven metabolites were chromotographically separated on Sephadex LH-20. Three have been identified as 1α,25-dihydroxyvitamin D-3 (1,25(OH)2D-3), 24,25-dihydroxyvitamin D-3 (24,25(OH)2D-3) and 1α,24,25-trihhydroxyvitamin D-3 (1,24,25(OH)3D-3). The activities of the 25-OH-D-3:1α- and 24-hydroxylases increased eight times faster than the cell number in 5 days. Preincubation of the cells with 10 nM 25-OH-D-3 or 1,25(OH)2D-3 decreased 1,25(OH)2D-3 synthesis, and increased both 24,25(OH)2D-3 and metabolite IV synthesis. The decrease in 25-OH-D-3:1α-hydroxylase activity required a 2 h preincubation with 25-OH-D-3, while stimulation of 25-OH-D-3:24-hydroxylase activity and metabolite IV production required a 6 h preincubation. Incubations of cells for 1 h with parathyroid hormone resulted in a 30-fold increase in cyclic AMP in the medium. A 6 h preincubation with parathyroid hormone decreased 24,25-(OH)2D-3 synthesis 50% relative to control cells. These results demonstrate the amenability of this system for studying the regulation of 25-OH-D-3 metabolism, as well as its use for other in vitro studies on renal cell function in a chemically defined culture system.
KW - 25-Hydroxyvitamin D-3
KW - Kidney cells
KW - Metabolic regulation
KW - Serum-free culture
KW - Vitamin D-3
UR - http://www.scopus.com/inward/record.url?scp=0018537713&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0018537713&partnerID=8YFLogxK
U2 - 10.1016/0304-4165(79)90003-5
DO - 10.1016/0304-4165(79)90003-5
M3 - Article
C2 - 228748
AN - SCOPUS:0018537713
VL - 587
SP - 495
EP - 506
JO - Biochimica et Biophysica Acta - General Subjects
JF - Biochimica et Biophysica Acta - General Subjects
SN - 0304-4165
IS - 4
ER -