| Summary: | The kidney represents a unique endocrine organ for the metabolism and action of vitamin D₃. Renal biosynthesis of 1,25-hydroxyvitamin D₃ (1,25(OH)₂D₃), and other vitamin D₃ metabolites, is regulated by ionic and hormonal factors. 1,25(OH)₂D₃ elicits a dramatic increase in 25-hydroxyvitamin D₃-24-hydroxylase (24-OHase) activity while concomitantly suppressing 25-hydroxyvitamin D₃-1α-hydroxylase (1-OHase) activity. To study this mechanism(s), biosynthesis and purification of the radiolabeled metabolites in question was necessary. Protocols for the selective induction of either 1-OHase or 24-OHase activity in chick kidney (in vivo) and the use of these kidneys (homogenates) for the biosynthesis of very high specific radioactivity metabolites, in vitro, are presented. A number of chromatographic systems including HPLC were developed and employed for the detection and purification of these biosynthetic metabolites (1,25(OH)₂[³H]D₃; 1,24,25(OH)₃[³H]D₃; 24,25(OH)₂[³H]D₃). Characterization of specific receptors for 1,25(OH)₂D₃ in the rat and chick kidney is presented and by comparison, these receptors appear similar to the chick intestinal component. 1,25(OH)₂D₃ receptors were also identified in a number of cultured mammalian cells. These receptors, irrespective of origin (tissue or species), selectively bind to immobilized DNA, a property which may indicate a physiologic function. Extensive metabolite binding studies indicate that 1,24,25(OH)₃D₃ is the second most active metabolite at the receptor level. To evaluate the role of the 1,25(OH)₂D₃ receptor in modulating the 1,25(OH)₂D₃-responsive activity of the renal 24-OHase enzyme, two renal cell lines (LLC-PK₁ and LLC-MK₂) were studied. A sensitive microassay for 24-OHase activity in intact cells was developed to monitor the enzyme responses. Although both cell lines contain 1,25(OH)₂D₃-responsive 24-OHase activity, only the LLC-PK₁ cells contain receptors for 1,25(OH)₂D₃. It is concluded that 1,25(OH)₂D₃ can rapidly and specifically regulate (induce) the activity of the 24-OHase enzyme in the absence of its specific receptor protein and this induction is inhibited by actinomycin D. Hence, although 1,25(OH)₂D₃ may act in renal cells to induce enzymes for the metabolism of vitamin D via its receptor component, this mechanism is not obligatory.
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