DOWN-REGULATION OF PARATHYROID (PTH) PTH-RELATED PEPTIDE RECEPTOR IMMUNOREACTIVITY AND PTH BINDING IN OPOSSUM KIDNEY-CELLS BY PTH AND DEXAMETHASONE
DOWN-REGULATION OF PARATHYROID (PTH) PTH-RELATED PEPTIDE RECEPTOR IMMUNOREACTIVITY AND PTH BINDING IN OPOSSUM KIDNEY-CELLS BY PTH AND DEXAMETHASONE
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DOI：
10.1210/en.135.6.2588
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发表时间：
1994-12-01
期刊：
影响因子：
4.8
通讯作者：
SEGRE, GV
中科院分区：
文献类型：
--
作者：
ABOUSAMRA, AB;GOLDSMITH, PK;SEGRE, GV
最近的数据表明，PTH下调其受体对负鼠肾（OK）细胞是不相关的PTH/PTH相关肽（PTHrP）受体信使RNA的稳态水平的任何变化。为了分析OK细胞中PTH/PTHrP受体的下调，本工作使用了特异性受体抗血清SR-2，这是有用的检测和定量PTH/PTHrP受体免疫反应性的完整细胞轴承负鼠PTH/PTHrP受体。SR-2与瞬时表达负鼠PTH/PTHrP受体互补DNA（OK-O）的COS-7细胞、稳定表达重组负鼠PTH/PTHrP受体的LLCPK 1细胞（AOK细胞）和表达内源性PTH/PTHrP受体的OK细胞特异性结合，但不与模拟转染的COS-7细胞或未转染的LLCPK 1细胞结合。在转染不同量OK-O质粒DNA的COS-7细胞中，SR-2结合与PTH结合也呈线性相关。用PTH（100 nM）处理4和6 h并没有显著下调PTH/PTHrP受体免疫反应性，尽管PTH结合分别降低至对照的51%和49%，PTH刺激的cAMP积累分别降低至对照的27%和28%。PTH（100 nM）治疗24和48小时显着降低PTH结合到51%和60%的控制和降低PTH/PTHrP受体免疫反应性分别为68%和58%的控制。OK细胞与0.1 nM至1 μ M PTH孵育4小时没有下调PTH/PTHrP受体免疫反应性，尽管PTH结合显着降低。Scatchard印迹分析显示，在用PTH处理4小时的OK细胞中，结合亲和力降低了7倍，而受体数量没有变化。相反，用PTH处理OK细胞24小时导致受体数量和受体免疫反应性平行减少，而受体结合亲和力无任何变化。用地塞米松（0.1 nM至1 μ M）处理OK细胞对PTH结合或PTH/PTHrP受体免疫反应性没有影响。然而，与单独用PTH处理的细胞相比，用地塞米松（1 μ M）和PTH（0.1 nM至1 μ M）孵育OK细胞引起PTH结合和PTH/PTHrP受体免疫反应性的显著下调。这些数据表明，在OK细胞暴露于PTH的第一个4小时期间，PTH/PTHrP受体保留在细胞表面上，但具有降低的结合配体的亲和力，并且地塞米松增强了PTH对OK细胞中PTH/PTHrP受体下调的作用。
Recent data have shown that PTH down-regulation of its receptor on opossum kidney (OK) cells is not associated with any change in the steady state level of the PTH/PTH-related peptide (PTHrP) receptor messenger RNA. For analysis of down-regulation of the PTH/PTHrP receptor in OK cells, the present work uses a specific receptor antiserum, SR-2, that is useful for detection and quantification of PTH/PTHrP receptor immunoreactivity on intact cells bearing the opossum PTH/PTHrP receptor. SR-2 specifically binds to COS-7 cells transiently expressing the opossum PTH/PTHrP receptor complementary DNA (OK-O), to LLCPK1 cells stably expressing the recombinant opossum PTH/PTHrP receptor (AOK cells), and to OK cells expressing endogenous PTH/PTHrP receptors, but not to mock-transfected COS-7 cells or untransfected LLCPK1 cells. SR-2 binding was also linearly correlated with PTH binding in COS-7 cells transfected with different amounts of OK-O plasmid DNA. Treatment with PTH (100 nM) for 4 and 6 h did not significantly down-regulate the PTH/PTHrP receptor immunoreactivity, although PTH binding was decreased to 51% and 49% of control, respectively, and PTH-stimulated cAMP accumulation was decreased to 27% and 28% of control, respectively. Treatment with PTH (100 nM) for 24 and 48 h significantly decreased PTH binding to 51% and 60% of control and decreased PTH/PTHrP receptor immunoreactivity to 68% and 58% of control, respectively. Incubation of OK cells with 0.1 nM to 1 mu M PTH for 4 h did not downregulate the PTH/PTHrP receptor immunoreactivity, although PTH binding was decreased dramatically. Scatchard blot analysis revealed that the binding affinity was decreased by 7-fold in OK cells treated with PTH for 4 h without change in receptor number. Conversely, treatment of OK cells with PTH for 24 h resulted in a parallel decrease in both receptor number and receptor immunoreactivity without any change in receptor binding affinity. Treatment of OK cells with dexamethasone (0.1 nM to 1 mu M) had no effect on PTH binding or PTH/PTHrP receptor immunoreactivity. Incubation of OK cells with both dexamethasone (1 mu M) and PTH (0.1 nM to 1 mu M), however, caused a significantly greater down-regulation of both PTH binding and PTH/PTHrP receptor immunoreactivity than in cells treated with PTH alone. These data indicate that during the first 4 h of exposure of OK cells to PTH, PTH/PTHrP receptors remain on the cell surface but have lowered affinity to bind the ligand and that dexamethasone potentiates the effect of PTH on PTH/PTHrP receptor down-regulation in OK cells.