Differential behaviour of 25-hydroxyvitamin D3 24-hydroxylase and 1-hydroxylase in response to protein synthesis inhibitors and cytochrome P-450 inhibitors.
To characterize 25-hydroxyvitamin D3 24-hydroxylase and 25-hydroxyvitamin D3 1-hydroxylase, the activities of the two enzymes were measured in the presence of two types of inhibitors. The effect of protein synthesis inhibitors on 25-hydroxyvitamin D3-stimulated 24-hydroxylase activity in 1-hydroxylating rat kidneys perfused in vitro was tested. Actinomycin D (4 microM) and cycloheximide (10 microM) each abolished 25-hydroxyvitamin D3 24-hydroxylase synthesis when added at the start of perfusion but not when added 4 h later; they did not affect 25-hydroxyvitamin D3 1-hydroxylase activity. The effects of cytochrome P-450 inhibitors on the two enzyme activities were then studied in vivo. Metyrapone and SKF-525A (50 mg/kg body weight) each inhibited 25-hydroxyvitamin D3 24-hydroxylase at 6 and 24 h; in contrast 1-hydroxylase increased and was 5 times the control value at 24 h. Finally, the in vitro effects of six cytochrome P-450 inhibitors at concentrations ranging from 10(-7) to 10(-3) M on enzyme activities in renal mitochondrial preparations were compared. Both enzymes were inhibited by all of the inhibitors, but inhibition of 25-hydroxyvitamin D3 24-hydroxylase was consistently greater than that of 25-hydroxyvitamin D3 1-hydroxylase. These studies demonstrate that 24-hydroxylation and 1-hydroxylation respond differently to protein synthesis inhibitors and to cytochrome P-450 inhibitors. The findings are consistent with the hypothesis that the two enzyme activities are associated with different cytochrome P-450 moieties.
Institute of Medical Science, University of Toronto, Ont., Canada.
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M
Kung
S W
SW
Kooh
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W
Paek
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D
Fraser
D000818:Animals; D053493:Cholestanetriol 26-Monooxygenase; D003513:Cycloheximide / Q000494:pharmacology; D065607:Cytochrome P-450 Enzyme Inhibitors; D003609:Dactinomycin / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology / Q000648:ultrastructure; D008297:Male; D008797:Metyrapone / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D013250:Steroid Hydroxylases / Q000378:metabolism*; D065668:Vitamin D3 24-Hydroxylase
D065607:Cytochrome P-450 Enzyme Inhibitors; D011500:Protein Synthesis Inhibitors; D003609:Dactinomycin; D003513:Cycloheximide; D013250:Steroid Hydroxylases; D053493:Cholestanetriol 26-Monooxygenase; D065668:Vitamin D3 24-Hydroxylase; D008797:Metyrapone