PMID- 4017943 OWN - NLM STAT- MEDLINE DCOM- 19850829 LR - 20191210 IS - 0013-7227 (Print) IS - 0013-7227 (Linking) VI - 117 IP - 2 DP - 1985 Aug TI - Bovine renal mitochondrial vitamin D3 hydroxylases: regulation of in vitro activities by inhibitors and antioxidants. PG - 447-56 AB - The regulation of bovine renal 1 alpha- and 24-hydroxylase activities was examined in primary bovine proximal tubule cell cultures. Maximal 1 alpha- and 24-hydroxylase activities in primary bovine proximal tubule cultures ranged from 1.5-1.8 and 2.0-2.7 pmol/min X 10(6) cells, respectively. The apparent Km was 795 nM for 1 alpha-hydroxylase activity and 1130 nM for 24-hydroxylase activity. 1 alpha- and 24-hydroxylase activities decreased in primary culture after cell plating. Activities decreased both as a function of cell number and as a function of the culture dish. 1 alpha-Hydroxylase activity decayed with a t1/2 of 37 h, while 24-hydroxylase activity decayed with a t1/2 of 45 h. Decreasing cell densities, at which cells were plated, increased the t1/2 for the decay of both activities [t1/2 = 21 h at 5,000 cells/cm vs. t1/2 = 37 h at 25,000 cells/cm for 1 alpha-hydroxylase (P greater than 0.001); t1/2 = 33 h at 5,000 cells/cm vs. t1/2 = 45 h at 25,000 cells/cm for 24-hydroxylase, (P greater than 0.0001)]. Direct addition of 0.25 mM metyrapone inhibited 1 alpha-hydroxylase activity by 33% and 24-hydroxylase activity by 51%. Long term incubation of cell cultures with 0.25 mM metyrapone resulted in a slowing in the loss of both hydroxylase activities, but did not stop the decay. 1 alpha-Hydroxylase activity in 4-day metyrapone-treated cultures was 35% higher than in 4-day untreated cultures. 24-Hydroxylase activity was increased 42% in treated cultures vs. that in untreated cell cultures. Both 1 alpha- and 24-hydroxylase activities were inhibited by direct addition of antioxidants. 1 alpha-Hydroxylase activity was directly inhibited 74% by the addition of 0.1 mM butylated hydroxyanisole (BHA), 69% by the addition of 0.1 mM butylated hydroxytoluene (BHT), and 56% by the addition of 0.05 mM benzyl sulfoxide (BS). 24-Hydroxylase activity was also directly inhibited 72% by 0.1 mM BHA, 55% by 0.1 mM BHT, and 73% 0.05 mM BS. There was no significant difference between the inhibition of either hydroxylase by each antioxidant. Antioxidant mixtures increased the inhibition of hydroxylase activities above that with single antioxidant. The addition of 0.1 mM BHA and 0.05 mM BS to cultures resulted in 100% inhibition of 24-hydroxylase activity and 95% inhibition of 1 alpha-hydroxylase activity. The results were very similar when 0.01 mM BS and 0.1 mM BHT were added to cultures, i.e. 100% and 91% inhibition of 24- and 1 alpha-hydroxylase activities.(ABSTRACT TRUNCATED AT 400 WORDS) FAU - Crivello, J F AU - Crivello JF LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't PL - United States TA - Endocrinology JT - Endocrinology JID - 0375040 RN - 0 (Antioxidants) RN - 9035-51-2 (Cytochrome P-450 Enzyme System) RN - EC 1.14.- (Steroid Hydroxylases) RN - EC 1.14.15.16 (Vitamin D3 24-Hydroxylase) RN - EC 1.14.15.18 (25-Hydroxyvitamin D3 1-alpha-Hydroxylase) RN - ZS9KD92H6V (Metyrapone) SB - IM MH - 25-Hydroxyvitamin D3 1-alpha-Hydroxylase/*metabolism MH - Animals MH - Antioxidants/pharmacology MH - Cattle MH - Cell Line MH - Culture Techniques/methods MH - *Cytochrome P-450 Enzyme System MH - Kidney Tubules, Proximal/drug effects/*enzymology MH - Kinetics MH - Metyrapone/pharmacology MH - Mice MH - Mitochondria/drug effects/*enzymology MH - Steroid Hydroxylases/*metabolism MH - Teratoma MH - Vitamin D3 24-Hydroxylase EDAT- 1985/08/01 00:00 MHDA- 1985/08/01 00:01 CRDT- 1985/08/01 00:00 PHST- 1985/08/01 00:00 [pubmed] PHST- 1985/08/01 00:01 [medline] PHST- 1985/08/01 00:00 [entrez] AID - 10.1210/endo-117-2-447 [doi] PST - ppublish SO - Endocrinology. 1985 Aug;117(2):447-56. doi: 10.1210/endo-117-2-447.