Turner R T, Bottemiller B L, Howard G A, Baylink D J
Proc Natl Acad Sci U S A. 1980 Mar;77(3):1537-40. doi: 10.1073/pnas.77.3.1537.
Cells were dispersed from rat kidney after enzymatic digestion of the extracellular matrix. When the cells were suspended in a serum-free medium and incubated with (3)H-labeled 25-hydroxyvitamin D(3) (25-OH-D(3)) several polar metabolites, including 1,25-(OH)(2)[(3)H]D(3) and 24,25-(OH)(2)[(3)H]D(3) were produced. The specific activities of the 25-OH-D(3):1- and 24-hydroxylases in isolated rat kidney cells were 10-100 times greater than in avian kidney homogenates. The rates of production of 1,25-(OH)(2)D(3) and 24,25-(OH)(2)D(3) were linear over a wide range in cell densities (0.65-5.0 x 10(6) cells per ml) and substrate concentrations (3.5-70 nM). The rate of production of 24,25-(OH)(2)[(3)H]D(3) from 25-OH-[(3)H]D(3) by cells isolated from rats fed control diet was linear with time for up to 30 min, while the synthesis of 1,25-(OH)(2)[(3)H]D(3) was linear for over 90 min. The specific activity of the 25-OH-D(3):1-hydroxylase was increased in kidney cells from vitamin D-deficient rats (11.5 fmol/min per 10(6) cells) as well as calcium-deficient rats (8.1 fmol/min per 10(6) cells) when compared to cells from rats fed the control diet (2.0 fmol/min per 10(6) cells). Also, the specific activity of the 25-OH-D(3):24-hydroxylase was reduced in cells from the vitamin D-deficient rats (<0.2 fmol/min per 10(6) cells) and calcium-deficient rats (5.1 fmol/min per 10(6) cells) compared to the controls (15.2 fmol/min per 10(6) cells). On the basis of these results, as well as previous in vivo studies, we conclude that the metabolism of 25-OH-D(3) by freshly isolated rat kidney cells reflects the in vivo activities of the renal vitamin D-metabolizing enzymes and may prove useful as an assay.
通过对细胞外基质进行酶消化后，从大鼠肾脏中分离出细胞。当将这些细胞悬浮于无血清培养基中，并与³H标记的25-羟基维生素D₃（25-OH-D₃）一起孵育时，会产生几种极性代谢产物，包括1,25-(OH)₂[³H]D₃和24,25-(OH)₂[³H]D₃。分离出的大鼠肾细胞中25-OH-D₃:1-羟化酶和24-羟化酶的比活性比禽肾匀浆中的高10 - 100倍。在较宽的细胞密度范围（每毫升0.65 - 5.0×10⁶个细胞）和底物浓度范围（3.5 - 70 nM）内，1,25-(OH)₂D₃和24,25-(OH)₂D₃的生成速率呈线性。由喂食对照饮食的大鼠分离出的细胞将25-OH-[³H]D₃转化为24,25-(OH)₂[³H]D₃的生成速率在长达30分钟的时间内与时间呈线性关系，而1,25-(OH)₂[³H]D₃的合成在90多分钟内呈线性关系。与喂食对照饮食的大鼠的细胞（每10⁶个细胞2.0 fmol/分钟）相比，维生素D缺乏大鼠的肾细胞（每10⁶个细胞11.5 fmol/分钟）以及钙缺乏大鼠的肾细胞（每10⁶个细胞8.1 fmol/分钟）中25-OH-D₃:1-羟化酶的比活性增加。此外，与对照组（每10⁶个细胞15.2 fmol/分钟）相比，维生素D缺乏大鼠的细胞（每10⁶个细胞<0.2 fmol/分钟）和钙缺乏大鼠的细胞（每10⁶个细胞5.1 fmol/分钟）中25-OH-D₃:24-羟化酶的比活性降低。基于这些结果以及先前的体内研究，我们得出结论，新鲜分离的大鼠肾细胞对25-OH-D₃的代谢反映了肾脏维生素D代谢酶的体内活性，并且可能被证明可作为一种检测方法。
