Bikle D D, Gee E
Department of Medicine, Veterans Administration Medical Center, San Francisco, California 94121.
Endocrinology. 1989 Feb;124(2):649-54. doi: 10.1210/endo-124-2-649.
Greater than 99% of the total circulating 1,25-dihydroxyvitamin D [1,25-(OH)2D] is bound to proteins such as the vitamin D-binding protein (DBP) and albumin; in the normal human only 0.4% of the circulating 1,25-(OH)2D is free. Although it is often assumed that only the free concentration of 1,25-(OH)2D is available to cells, this has not been demonstrated. In particular, it is not clear whether the DBPs facilitate 1,25-(OH)2D entry into target cells or serve only to transport these metabolites within the circulation. To address this question, we evaluated one of the best characterized target tissue responses to 1,25-(OH)2D, namely its ability to inhibit its own production and induce that of 24,25-(OH)2D, using one of the most sensitive cells, the human foreskin keratinocyte. We incubated keratinocytes in the presence of 1,25-(OH)2D (from 10(-11)-10(-8) M) in medium containing albumin (from 0.1-10%) or serum (from 0.1-10%) for 4 h [to inhibit the 25-hydroxyvitamin D (250HD) 1 alpha-hydroxylase] or 16 h (to induce the 250HD 24-hydroxylase) before evaluating [3H]250HD metabolism by these cells during a 1-h incubation in serum- and albumin-free medium. The free fraction of 1,25-(OH)2D was determined in each medium by centrifugal ultrafiltration and varied from 36% to 0.57% in direct proportion to the albumin or serum in the medium. Increasing the serum or albumin concentration in the medium increased the concentration of total 1,25-(OH)2D needed to inhibit its own production or stimulate that of 24,25-(OH)2D. In contrast, the concentration of free 1,25-(OH)2D needed to half-maximally inhibit its own production or induce 24,25-(OH)2D production remained constant at approximately 10(-11) M. We conclude that the free, not the total, 1,25-(OH)2D concentration regulates 250HD metabolism by keratinocytes, that DBPs do not facilitate 1,25-(OH)2D entry into the cell, and that these cells sense only free 1,25-(OH)2D.
循环中的1,25 - 二羟基维生素D[1,25 - (OH)₂D]总量的99%以上与维生素D结合蛋白(DBP)和白蛋白等蛋白质结合；在正常人体内，循环中的1,25 - (OH)₂D只有0.4%是游离的。尽管人们常常认为只有游离的1,25 - (OH)₂D能被细胞利用，但这一点尚未得到证实。特别是，尚不清楚DBP是促进1,25 - (OH)₂D进入靶细胞，还是仅在循环中运输这些代谢产物。为了解决这个问题，我们使用最敏感的细胞之一——人包皮角质形成细胞，评估了1,25 - (OH)₂D最具特征的靶组织反应之一，即其抑制自身产生并诱导24,25 - (OH)₂D产生的能力。我们在含有白蛋白(0.1% - 10%)或血清(0.1% - 10%)的培养基中，将角质形成细胞与1,25 - (OH)₂D(浓度为10⁻¹¹ - 10⁻⁸M)一起孵育4小时[以抑制25 - 羟基维生素D(250HD)1α - 羟化酶]或16小时(以诱导250HD 24 - 羟化酶)，然后在无血清和无白蛋白的培养基中孵育1小时，评估这些细胞对[³H]250HD的代谢。通过离心超滤法测定每种培养基中1,25 - (OH)₂D的游离部分，其在培养基中的含量从36%到0.57%不等，与培养基中的白蛋白或血清浓度成正比。增加培养基中的血清或白蛋白浓度会增加抑制自身产生或刺激24,25 - (OH)₂D产生所需的总1,25 - (OH)₂D浓度。相比之下，半最大抑制自身产生或诱导24,25 - (OH)₂D产生所需的游离1,25 - (OH)₂D浓度保持恒定，约为10⁻¹¹M。我们得出结论，调节角质形成细胞对250HD代谢作用的是游离的而非总的1,25 - (OH)₂D浓度，DBP不促进1,25 - (OH)₂D进入细胞，并且这些细胞仅感知游离的1,25 - (OH)₂D。
