STRUCTURE AND REGULATION OF OSTEOBLAST CALCIUM CHANNELS

成骨细胞钙通道的结构和调节

• 批准号: 2906149
• 负责人: ELIZABETH L BARRY
• 金额: $ 11.31万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2906149
• 关键词: RNase protection assay; bone metabolism; calcium channel; calcium channel blockers; cell differentiation; clone cells; fluorescent dye /probe; gel electrophoresis; gene expression; hormone regulation /control mechanism; human subject; messenger RNA; molecular cloning; northern blottings; nucleic acid sequence; osteoblasts; parathyroid hormones; physiologic bone resorption; polymerase chain reaction; protein isoforms; protein kinase; protein structure function; scintillation counter; statistics /biometry; tissue /cell culture

DESCRIPTION (Adapted from the Applicant's Abstract): The long-term goal of this research proposal is to understand how changes in the expression, or regulation of calcium channels in osteoblasts affect bone metabolism. Recent research indicating that the activation of calcium channels in osteoblasts is a common feature of many bone regulatory factors suggests a significant role for intracellular calcium signalling in the control of bone remodeling. However, the molecular structure and cellular regulation of osteoblast calcium channels remain largely undefined. Previous studies have demonstrated that three isoforms of calcium channels are expressed in osteoblast-like osteosarcoma cells (alpha1S, alpha1C and alpha1D), and that parathyroid hormone selectively activates the alpha1D isoform. The objective of this study is to more fully characterize the structure, function and regulation of the calcium channel alpha1D isoform in osteoblasts. The Specific Aims are to: 1) determine the molecular structure of the alpha1D isoform; 2) examine the temporal and hormonal regulation of alpha1D gene expression during osteoblast differentiation; 3) define the mechanism of alpha1D activation by parathyroid hormone; 4) determine the specific functional effects mediated by the alpha1D signaling pathway; and 5) examine the regulation of alpha1D activity by other hormones and drugs. Calcium channel cDNA clones will be isolated, sequenced and used for the quantitative analysis of mRNA levels. Calcium channel activity will be assessed (using a fluorescent probe) to measure the level of intracellular calcium, as well as a radioisotope uptake assay to measure calcium influx. Specific calcium channel antagonists, as well as antisense techniques, will be used to examine the functional roles and hormonal regulation of osteoblast calcium channels. It is suggested that the results of the proposed research study will provide novel and important information about the molecular structure and cellular regulation of osteoblast calcium channels and their role in bone metabolism, as well as providing insight into the cellular mechanisms of action of bone regulatory hormones. These insights are critical to an understanding of the events that regulate bone remodeling and, ultimately, for the development of effective therapeutic approaches for the treatment of bone diseases, including osteoporosis.

描述（改编自申请人的摘要）：长期目标 这项研究计划是为了了解表达方式的变化，或者 成骨细胞钙通道的调节影响骨代谢。 最近的研究表明，钙通道的激活 成骨细胞是许多骨调节因子的共同特征，表明 细胞内钙信号在骨控制中发挥重要作用 重塑。 然而，其分子结构和细胞调控 成骨细胞钙通道在很大程度上仍不明确。 之前的研究有 证明钙通道的三种亚型在 成骨细胞样骨肉瘤细胞（alpha1S、alpha1C 和 alpha1D） 甲状旁腺激素选择性激活 alpha1D 同工型。 这 这项研究的目的是更全面地表征结构， 钙通道α1D亚型的功能和调节 成骨细胞。 具体目标是： 1) 确定分子 alpha1D 同工型的结构； 2）检查时间和荷尔蒙 成骨细胞分化过程中α1D基因表达的调节； 3） 定义甲状旁腺激素激活 alpha1D 的机制； 4） 确定 alpha1D 信号传导介导的特定功能效应 途径； 5) 检查其他激素对 alpha1D 活性的调节 和毒品。 钙通道 cDNA 克隆将被分离、测序和使用 用于 mRNA 水平的定量分析。 钙通道活性将 进行评估（使用荧光探针）以测量 细胞内钙，以及放射性同位素摄取测定法来测量 钙流入。 特异性钙通道拮抗剂以及反义药物 技术，将用于检查功能作用和荷尔蒙 成骨细胞钙通道的调节。 建议将结果 拟议的研究将提供新颖且重要的信息 关于成骨细胞钙的分子结构和细胞调节 通道及其在骨代谢中的作用，并提供见解 深入研究骨调节激素的细胞作用机制。 这些 见解对于理解骨调节事件至关重要 重塑并最终开发有效的治疗方法 治疗骨疾病（包括骨质疏松症）的方法。