Liang C T, Barnes J, Cheng L, Balakir R, Sacktor B
Am J Physiol. 1982 May;242(5):C312-8. doi: 10.1152/ajpcell.1982.242.5.C312.
Renal cells from Vitamin D-deficient and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-repleted chicks were isolated by a collagenase-hyaluronidase procedure. Exclusion of trypan blue and respiratory measurements indicate that the cells were functionally intact and metabolically active. The uptakes of phosphate and alpha-methylglucoside were stimulated markedly by Na+ in the extracellular medium. Phosphate uptake in the presence of Na+ was saturable with respect to phosphate concentration; half-maximal activity was obtained with approximately 0.2 mM. Three hours after 1,25-(OH)2D3 was injected into vitamin D-deficient chicks the Na+-dependent phosphate uptake by the isolated cells had increased about 40%, i.e., 2.00 compared with 1.44 nmol.min-1.mg protein-1. Phosphate uptake in the presence of K+ in the extracellular medium and alpha-methylglucoside uptake in the presence or absence of Na+ were unchanged. In a secondary response found 17 h after 1,25-(OH)2D3 injection, Na+-dependent phosphate uptake decreased. Serum concentrations of phosphorus and calcium were not measurably changed in the 3-h repleted bird, but both levels were increased 17 h after treatment. Administration of phosphate into vitamin D-deficient chicks, so that the serum concentration of phosphorus was raised to that of the 17-h 1,25-(OH)2D3 repleted animal, effected a comparable decrease in phosphate uptake. Serum calcium levels were not altered by this treatment. The actions of parathyroid hormone in stimulating adenylate cyclase and in inhibiting phosphate uptake were notably blunted in the vitamin D-deficient chick. Sensitivity to parathyroid hormone was not restored until several days after 1,25-(OH)2D3 repletion. These findings suggest that the initial response to 1,25-(OH)2D3, to increase renal phosphate uptake, and the secondary response, to decrease phosphate uptake, were by parathyroid hormone-independent processes. The results also indicate that the isolated renal cell represents an excellent model for studying the mechanism by which 1,25-(OH)2D3 regulates phosphate transport in the kidney.
采用胶原酶-透明质酸酶法分离维生素D缺乏及1,25-二羟基维生素D3 [1,25-(OH)2D3]补充后的雏鸡肾细胞。台盼蓝排斥试验及呼吸测定表明细胞功能完整且代谢活跃。细胞外培养基中的Na+显著刺激磷酸盐和α-甲基葡萄糖苷的摄取。在存在Na+的情况下，磷酸盐摄取相对于磷酸盐浓度呈饱和状态；约0.2 mM时达到半数最大活性。向维生素D缺乏的雏鸡注射1,25-(OH)2D3三小时后，分离细胞对Na+依赖的磷酸盐摄取增加约40%，即从1.44 nmol·min-1·mg蛋白-1增至2.00 nmol·min-1·mg蛋白-1。细胞外培养基中存在K+时的磷酸盐摄取以及存在或不存在Na+时的α-甲基葡萄糖苷摄取均未改变。在注射1,25-(OH)2D3 17小时后出现的二次反应中，Na+依赖的磷酸盐摄取减少。在补充1,25-(OH)2D3 3小时的雏鸡中，血清磷和钙浓度无明显变化，但治疗17小时后两者水平均升高。向维生素D缺乏的雏鸡给予磷酸盐，使血清磷浓度升至1,25-(OH)2D3补充17小时动物的水平，可使磷酸盐摄取出现类似程度的降低。此处理未改变血清钙水平。在维生素D缺乏的雏鸡中，甲状旁腺激素刺激腺苷酸环化酶及抑制磷酸盐摄取的作用明显减弱。直至1,25-(OH)2D3补充数天后，对甲状旁腺激素的敏感性才得以恢复。这些发现提示，1,25-(OH)2D3增加肾磷酸盐摄取的初始反应及减少磷酸盐摄取的二次反应是通过甲状旁腺激素非依赖过程实现的。结果还表明，分离的肾细胞是研究1,25-(OH)2D3调节肾磷酸盐转运机制的极佳模型。
