STRUCTURE AND REGULATION OF OSTEOBLAST CALCIUM CHANNELS

成骨细胞钙通道的结构和调节

• 批准号: 6381074
• 负责人: ELIZABETH L BARRY
• 金额: $ 12.47万
• 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/6381074
• 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（根据申请人的摘要改编）： 该研究建议是了解表达方式的变化或 成骨细胞中钙通道的调节会影响骨代谢。 最近的研究表明，钙通道激活 成骨细胞是许多骨调节因素的共同特征，表明 细胞内钙信号传导的重要作用 重塑。 但是，分子结构和细胞调节 成骨细胞钙通道在很大程度上保持不确定。 先前的研究已有 证明钙通道的三种同工型在 成骨细胞样骨肉瘤细胞（alpha1s，alpha1c和alpha1d），并且 甲状旁腺激素有选择地激活α1D同工型。 这 这项研究的目的是更充分地表征结构， 钙通道α1D同工型的功能和调节 成骨细胞。 具体目的是：1）确定分子 α1D同工型的结构； 2）检查时间和激素 在成骨细胞分化过程中的α1D基因表达的调节； 3） 定义甲状旁腺激素激活α1D的机制； 4） 确定由alpha1d信号介导的特定功能效应 途径； 5）检查其他激素对α1D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 评估（使用荧光探针）来测量 细胞内钙，以及放射性同位素的摄取测定法 钙涌入。 特定的钙通道拮抗剂以及反义 技术将用于检查功能作用和激素 调节成骨细胞钙通道。 建议结果 拟议的研究将提供新颖而重要的信息 关于成骨细胞钙的分子结构和细胞调节 渠道及其在骨骼代谢中的作用，并提供洞察力 进入骨调节激素作用的细胞机制。 这些 见解对于理解调节骨骼的事件至关重要 重塑，最终为开发有效的治疗性 治疗骨骼疾病的方法，包括骨质疏松症。