PARATHYROID HORMONE RECEPTORS IN KIDNEY AND BONE

甲状旁腺激素(PTH)对骨骼产生分解代谢和合成代谢作用， 但人们对其骨骼活动的分子基础知之甚少。在……里面 除了增加cAMP水平外，PTH还增加了靶细胞中的Ca2+， 有时伴随着磷脂酶C(PL-C介导的)的效应 肌醇磷酸盐(IP)的积累。在UMR 106-H5细胞中，PTH和 凝血酶引起CaI2+的相应增加，但只有后者增加 IP积累。这项提案的长期目标是定义 甲状旁腺激素激活这对偶联蛋白的分子基础及意义 信号通路。具体目标是：1)确定是否 PI代谢/IP3途径以外的机制参与甲状旁腺激素的诱导 Ca2+的动员。载药UMR 106-H5细胞的显微荧光测定 钙指示剂INDO-1将用于监测单细胞Ca2+。 细胞将被显微注射有望破坏PL-1的药物- C/IP3/Ca2+信号通路包括1，4，5-IP3(耗尽IP3- 反应性钙池)、肝素(阻断IP3受体)和PIP2 抗体(以防止PL-C介导的水解)。这些因素的影响 关于单细胞Ca2+信令的代理。循环AMP信令将是 用表达载体导入UMR 106-H5细胞扩增 编码野生型G-S-α，刺激性GTP结合成分 腺苷环化酶。在第二种方法中，营地信号将被阻止 通过用编码酵母cAMP的表达质粒急性转染- 磷酸二酯酶(PDE)。甲状旁腺素对Ca2+信号转导的影响 3)评估甲状旁腺激素在细胞中的表达。 Ca2+诱导的增量对腺苷环化酶/cAMP/PK-A的调控 路径。在这些研究中，cAMP途径的甲状旁腺素激活将是 比较对照UMR 106-H5细胞和Ca2+ 对甲状旁腺激素的反应是使用细胞内钙螯合剂缓冲的；4) 利用一系列新的甲状旁腺素(1-34)类似物获得药理学证据 赞成或反对不同的甲状旁腺素受体偶联到 CAMP和Ca2+信号通路；5)评估突变型UMR中PTH的作用 106个细胞系在cAMP信号通路中存在缺陷。UMR 106-H5细胞 将酵母cAMP-PDE质粒稳定地转入细胞系 CAMP积累缺陷将被评估为甲状旁腺素依赖 生物效应。成功完成这些研究将提供 对分子机制和生理学的重要见解 甲状旁腺激素激活双细胞信号通路的相关性。

Parathyroid hormone (PTH) produces catabolic and anabolic effects on bone, but the molecular bases of its skeletal actions are poorly understood. In addition to increasing cAMP levels, PTH also raises Cai2+ in target cells, an effect that is sometimes accompanied by phospholipase C (PL-C-mediated accumulation of inositol phosphates (IPs). In UMR 106-H5 cells, PTH and thrombin elicit comparable increases in cai2+ but only the latter augments IP accumulation. The long-term goal of this proposal is to define the molecular basis and significance of the activation by PTH of these dual signalling pathways. The specific objectives are to: 1) determine whether mechanisms other than the PI turnover/IP3 pathway contribute to PTH-induced mobilization of Cai2+. Microfluorometry of UMR 106-H5 cells loaded with the calcium indicator indo-1 will be used to monitor single-cell Cai2+. Cells will be microinjected with agents expected to disrupt the PL- C/IP3/Cai2+ signalling pathway including 1,4,5-IP3 (to deplete IP3- responsive calcium pools), heparin (to block IP3 receptors), and PIP2 antibodies (to prevent PL-C mediated hydrolysis). The effects of these agents on the single-cell Cai2+ signalling. Cyclic AMP signalling will be amplified by transfecting UMR 106-H5 cells with an expression plasmid encoding wild-type G-s-alpha, the stimulatory GTP-binding component of adenylate cyclase. In a second approach, cAMP signalling will be blocked by acute transfection with an expression plasmid encoding a yeast cAMP- phosphodiesterase (PDE). The influence of PTH on the Cai2+ signalling pathway in transfected cells will be evaluated; 3) assess whether PTH- induced increments in Cai2+ modulate the adenylate cyclase/cAMP/PK-A pathway. In these studies, PTH-activation of the cAMP pathway will be compared in control UMR 106-H5 cells and in cells in which the Cai2+ response to PTH is buffered out using an intracellular calcium chelator; 4) use a novel series of PTH(1-34) analogs to obtain pharmacological evidence for or against the notion that distinct PTH receptors are coupled to the cAMP and Cai2+ signalling pathways; and 5) assess PTH action in mutant UMR 106 cell lines defective in the cAMP signalling pathway. UMR 106-H5 cells will be stably transfected with the yeast cAMP-PDE plasmid, and cell lines defective in accumulating cAMP will be evaluated for PTH-dependent biological effects. Successful completion of these studies will provide important insights into the molecular mechanisms and physiological relevance of the activation by PTH of dual cell signalling pathways.