Parathyroid hormone receptors coupled to cyclic adenosine monophosphate formation in an established renal cell line.
Parathyroid hormone receptors coupled to cyclic adenosine monophosphate formation in an established renal cell line.
复制标题
DOI：
10.1210/endo-114-3-980
复制
发表时间：
1984-03
期刊：
影响因子：
4.8
通讯作者：
A. Teitelbaum;G. Strewler
中科院分区：
文献类型：
--
作者：
A. Teitelbaum;G. Strewler
我们在负鼠肾细胞系（OK）中检测了PTH结合与刺激cAMP形成之间的关系。在异丁基甲基黄嘌呤（1mm）的存在下，牛甲状旁腺激素（1-34）[bPTH(1-34)] （244 nM）刺激cAMP在融合培养中的积累高达40倍；这种对甲状旁腺激素的反应在35代内是稳定的。将细胞cAMP水平提高一半所需的bPTH（1-34）浓度为5-12 nM。[125I]bPTH（1-34）与OK细胞的结合是饱和的；竞争结合数据的Scatchard分析得到解离常数(KD) = 6 +/- 2 nM，结合位点为1.0 pmol /mg细胞蛋白。在稳定的结合条件下，89%的标记甲状旁腺激素仍可被10%的三氯乙酸沉淀，这表明该激素的代谢最低。PTH拮抗剂（8Nle, 18Nle, 34Tyr）bPTH（3-34）酰胺竞争[125I]bPTH（1-34）结合位点，抑制bPTH（1-34）的作用，提高cAMP水平。完整的PTH分子bPTH（1-84）和Brewer合成的弱激动剂hPTH（1-34）在结合和cAMP生成方面的效力均低于bPTH(1-34)（分别为6倍和30倍）。降钙素和精氨酸加压素不与甲状旁腺激素受体结合，但在OK细胞培养中分别使cAMP水平升高3倍和10倍；胰高血糖素和ACTH（1-24）均不影响PTH与cAMP在OK细胞中的结合。改变培养液中细胞外钙浓度对基底或甲状旁腺激素刺激的cAMP生成没有影响。这些数据表明，OK细胞中的甲状旁腺激素受体具有高亲和力，对甲状旁腺激素具有选择性，并且与腺苷酸环化酶偶联。这种建立的上皮细胞系为研究甲状旁腺素在肾脏中的作用机制提供了一个模型。
We examined the relationship between PTH binding and stimulation of cAMP formation in a cell line derived from opossum kidney (OK). In the presence of isobutylmethylxanthine (1 mM) bovine PTH(1-34) [bPTH(1-34)] (244 nM) stimulated cAMP accumulation in confluent cultures up to 40-fold over basal; this response to PTH was stable for 35 passages. The concentration of bPTH(1-34) required to raise cell cAMP levels half-maximally was 5-12 nM. Binding of [125I]bPTH(1-34) to OK cells was saturable; Scatchard analysis of competitive binding data yielded a dissociation constant (KD) = 6 +/- 2 nM, with 1.0 pmol binding sites/mg cell protein. Under steady state binding conditions 89% of labeled PTH remained precipitable by 10% trichloroacetic acid, suggesting minimal metabolism of the hormone. The PTH antagonist (8Nle, 18Nle, 34Tyr)bPTH(3-34)amide competed for [125I]bPTH(1-34) binding sites and inhibited the action of bPTH(1-34) to raise cAMP levels. The intact PTH molecule, bPTH(1-84), and the weak agonist hPTH(1-34) synthesized by Brewer were both less potent than bPTH(1-34) (6 times and 30 times, respectively) with regard to binding and cAMP production. Calcitonin and arginine vasopressin did not bind to PTH receptors but raised cAMP levels in OK cell cultures 3- and 10-fold, respectively; neither glucagon nor ACTH(1-24) influenced PTH binding of cAMP in OK cells. Varying the extracellular calcium concentration in the medium bathing cells did not influence basal or PTH-stimulated cAMP generation. These data suggest that PTH receptors in OK cells are of high affinity, are selective for PTH, and are coupled to adenylate cyclase. This established epithelial cell line provides a model in which to study the mechanism of action of PTH in the kidney.