SECOND MESSENGERS AND REGULATION OF CELLULAR FUNCTION BY PARATHYROID HORMONE

甲状旁腺激素调控钙和骨代谢 骨和肾脏中靶细胞的关键功能。这些行动 这种激素以前完全归因于腺苷的刺激。 环酶，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转导多个 细胞内信号，包括环磷酸腺苷、二酰甘油、 肌醇多磷酸和胞浆游离钙。终极的 这种信令多样性的功能相关性很少被理解， 然而，因为个体第二信使和PTH之间的联系 调控特定的远端生物反应，如生长、基因 转录，酶活性和离子转运，还不清楚 已经成立了。这些不同秒之间相互作用的重要性 信使；它们受其他激素、生长因子或 细胞因子；以及不同激素方案的可能影响 对荷尔蒙反应模式的给药也仍然存在 动荡不安。信号特异性甲状旁腺素的研究进展 类似物，加上天然和突变的PTH受体的可用性 CDNA，激发了对这些问题的新的实验方法。这 子项目将解决两个主要悬而未决的问题 甲状旁腺素的细胞作用：(1)个体第二信使是如何 荷尔蒙对靶区特异性远端功能的影响 细胞--即不同的信号是否主要调节不同的亚群 这些细胞的甲状旁腺素反应？以及(2)靶向PTH反应如何 受激素暴露时间模式影响的细胞-是 受调控的特定信号机制或生物反应 持续性与间歇性(或搏动性)甲状旁腺激素不同 行政部门？通过使用突变的甲状旁腺素配体、突变的甲状旁腺素受体和 突变的肾脏和成骨细胞靶细胞选择性激活，或 阻断体外特定的甲状旁腺素信号事件，之间的联系 个体第二信使和特定远端细胞的产生 荷尔蒙的作用将被定义。在成骨细胞的情况下， 将特别强调区分那些符合以下条件的信号 调节甲状旁腺素对骨骼的“合成代谢”和“分解代谢”效应。为了获得 间歇性和持续性甲状旁腺激素的发病机制 体内给药对骨骼的影响截然不同 将使用大规模灌流技术来比较 持续与间歇或脉冲性甲状旁腺素暴露对细胞的影响 表达正常的PTH/PTHrP受体或突变的受体 无法传递特定的信使信号。密钥的确认 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消融小鼠的原代骨细胞 甲状旁腺素受体缺乏症的纯合子和突变型甲状旁腺素受体 在体内或体外被“移位”。通过定义功能 甲状旁腺素受体转导的个体信号在靶区的后果 细胞，这些研究将提供Rational 具有新颖作用光谱的信号特异性PTH类似物的设计 肾脏和骨头。他们还可能提出新的药代动力学方法。 与这种新颖的PTH类似物一起，最终可能使 对骨转换和矿物质的高度选择性系统控制 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.