ION CHANNELS CONTROLLING BONE REMODELING

描述（改编自申请人的摘要）： 这项研究是为了确定信号背后的分子机制， 成骨细胞中的信号转导途径，并阐明成骨细胞的生理学机制。 钙离子通道在骨中的作用。具体目标是：1）测试 假设通过VDR定量预测总[3H]-1，25 D3结合 信息，RO置换质膜（或胞质）结合位点， 而EO取代了核标记。利用受体结合技术 用[~3H]-1，25D_3研究了RO和EO（一种药剂）的药理和分布 其作用于nVDR以刺激转录，但在 低于10 nM的Ca+通道激活）将被定量并关联 在表达不同水平VDR信息的细胞系中; 2）确定 是否其他类固醇激素的核受体的信息预测 类固醇激素增强Ca+通道。ROS 17/2.8细胞显示 添加0.1 nM睾酮可增强Ca+电流，但剂量 高达100 nM的17B雌二醇引起最小的影响。校长 研究者提出检验转染正常细胞的假设 雌激素受体进入"雌激素无反应"的ROS 17/2.8细胞，会引起 低剂量17 B雌二醇可快速增强Ca+通道。的 非基因组应答的灵敏度将定量至 类固醇受体信息的表达; 3）确定Ca+通道亚型 在ROS 17/2.8细胞和成熟大鼠骨中， 对类固醇激素的反应回答存在哪些通道亚型 在成熟大鼠骨或ROS细胞培养物中， 主要的L型Ca+通道亚型，敲除技术将用于 具体定义哪种通道类型被类固醇受体激活。 将克隆和表达类固醇激活的Ca+通道亚型。CA + 通道参与骨基质产生和骨吸收， 对它们药理学的研究可能会产生新的药物治疗方法 治疗骨病

DESCRIPTION (Adapted from the Applicant's Abstract): The long-term goal of this research is to determine the molecular mechanisms underlying the signal transduction pathways in osteoblasts and to elucidate the physiological functions of Ca+ channels in bone. The Specific Aims are to: 1) Test the hypothesis that total [3H]-1,25D3 binding is predicted quantitatively by VDR message, the RO displaces plasma membrane (or cytosolic) binding sites, whereas EO displaces nuclear labeling. Using receptor binding techniques with [3H]-1,25D3, the pharmacology and distribution of RO and EO (an agent which acts at the nVDR to stimulate transcription but is not effective at activation of Ca+ channels below 10 nM) will be quantitated and correlated in cells lines which express different levels of VDR message; 2) Determine whether message of nuclear receptors of other steroid hormones predict the steroid hormone potentiation of Ca+ channels. ROS 17/2.8 cells show potentiation of Ca+ currents with addition of 0.1 nM testosterone, but doses of up to 100 nM 17B estradiol cause minimal effects. The principal investigator proposes to test the hypothesis that transfection of the normal estrogen receptor into "estrogen unresponsive" ROS 17/2.8 cells, will cause a rapid potentiation of Ca+ channels by low doses of 17B estradiol. The sensitivity of the non-genomic response will be quantitated to the level of expression of steroid receptor message; 3) Determine the Ca+ channel isoform in ROS 17/2.8 cells and mature rat bone which is associated with the response to steroid hormones. To answer which channel isoforms are present in mature rat bone or ROS cell cultures, oligonucleotides specific to the major L-type Ca+ channel isoforms, knockout technology will be used to specifically define which channel type is activated by steroid receptors. The steroid-activated Ca+ channel isoform will be cloned and expressed. Ca+ channels are involved in bone matrix production and bone resorption, and investigation of their pharmacology may yield new methods of drug treatment for bone disease.