SECOND MESSENGERS AND REGULATION OF CELLULAR FUNCTION BY PARATHYROID HORMONE

甲状旁腺激素通过控制钙和骨代谢来调节 骨和肾中靶细胞的关键功能。 的这些行动 这些激素以前完全归因于腺苷酸刺激 环化酶，但最近克隆的PTH/PTHrp的功能分析 受体已经证实，这种单一的受体可以抑制多种 细胞内信号，包括环AMP，甘油二酯， 肌醇多磷酸和胞质游离钙。 最终 这种信号多样性的功能相关性知之甚少， 然而，由于个体第二信使和PTH之间的联系， 调节特定的远端生物反应，如生长、基因 转录，酶活性和离子转运，尚未清楚 确立了习 这些不同的第二次之间的相互作用的重要性 信使;它们通过其他激素、生长因子或 细胞因子;以及不同激素时间表的可能影响 根据激素反应性的模式给药也仍然存在 不安 信号特异性甲状旁腺激素的研究进展 类似物，再加上天然和突变体PTH受体的可用性 cDNAs，启发了新的实验方法来解决这些问题。 这 分项目将解决两个尚未解决的主要问题， 甲状旁腺素的细胞作用：（1）如何个别第二信使 由激素引起的影响靶点的特定远端功能 细胞-即不同的信号主要调节不同的子集 PTH的反应吗和（2）PTH反应如何在靶 受激素暴露时间模式影响的细胞- 特定的信号传导机制或生物反应调节 根据连续性与间歇性（或脉冲性）PTH的不同 行政管理？ 通过使用突变型PTH配体、突变型PTH受体和 选择性激活突变的肾和成骨细胞靶细胞，或 阻断，体外特定PTH信号传导事件， 单个第二信使和特定远端细胞的产生 激素的影响将被定义。 对于成骨细胞， 将特别强调区分那些 介导PTH对骨的“合成代谢”与“分解代谢”作用。 获得 了解间歇性和持续性PTH 体内给药导致对骨的显著不同的作用 将使用大量灌注技术来比较 连续vs.间歇或脉冲式PTH暴露于细胞 表达正常PTH/PTHrp受体或突变体受体， 不能识别特定的信使信号。 密钥确认 在转化的骨肉瘤细胞模型中的发现将使用 来自正常小鼠或来自基因切除小鼠的原代骨细胞， 对于PTH受体缺乏纯合子，并且其中突变型PTH受体 已经在体内或体外被“置换”。 通过定义函数 PTH受体在靶细胞中转导的单个信号的结果 这些研究将提供合理的信息， 具有新的作用谱的信号特异性PTH类似物的设计 肾和骨头 他们还可能提出新的药代动力学方法 与这种新型PTH类似物一起， 骨转换和矿物质高选择性系统控制 新陈代谢.

Parathyroid hormone regulates calcium and bone metabolism by controlling critical functions of target cells in bone and kidney. These actions of the hormone were formerly attributed entirely to stimulation of adenylate cyclase, but functional analysis of the recently cloned PTH/PTHrp receptor has confirmed that this single receptor can transduce multiple intracellular signals, including increases in cyclic AMP, diacylglycerol, inositol polyphosphates and cytosolic free calcium. The ultimate functional correlates of this signalling diversity are poorly understood, however, because the links between individual second messengers and PTH regulation of specific distal biologic responses, such as growth, gene transcription, enzyme activity and ion transport, have not been clearly established. The importance of interactions between these various second messengers; their modulation by other hormones, growth factors or cytokines; and the possible influence of different schedules of hormone administration upon the patterns of hormonal responsiveness also remain unsettled. Recent advances in the development of signal-specific PTH analogs, coupled with the availability of native and mutant PTH receptor cDNAs, have inspired new experimental approaches to these issues. This Subproject will address two major unresolved questions concerning the cellular actions of PTH: (1) how do individual second messengers elicited by the hormone influence specific distal functions of target cells - i.e. do different signals principally regulate distinct subsets of PTH responses in these cells? and (2) how are PTH responses in target cells influenced by the temporal pattern of hormone exposure - are specific signalling mechanisms or biologic responses regulated differently by continuous vs. intermittent (or pulsatile) PTH administration? by using mutant PTH ligands, mutant PTH receptors and mutant renal and osteoblastic target cells to selectively activate, or block, specific PTH signalling events in vitro, the links between generation of individual second messengers and specific distal cellular effects of the hormone will be defined. In the case of osteoblasts, particular emphasis will be placed upon distinguishing those signals that mediate "anabolic" vs. "catabolic" effects of PTH on bone. To gain insight into the mechanisms whereby intermittent and continuous PTH administration in vivo lead to dramatically different effects upon bone mass perifusion techniques will be used to compare the effects of continuous vs. intermittent or pulsatile PTH exposure upon cells expressing either normal PTH/PTHrp receptors or mutant receptors that cannot transduce specific messenger signals. Confirmation of key findings in transformed osteosarcoma-cell models will be sought using primary bone cells from normal mice or from gene-ablated mice that are homozygous for PTH receptor deficiency and in which mutant PTH receptors have been "transplaced" in vivo or in vitro. By defining the functional consequences of individual signals transduced by PTH receptors in target cells, these studies will provide the information needed for rational design of signal-specific PTH analogs with novel spectra of action in kidney and bone. They may also suggest new pharmacokinetic approaches that, together with such novel PTH analogs, could ultimately enable highly selective systemic control of bone turnover and mineral metabolism.