ION CHANNELS CONTROLLING BONE REMODELING

描述(改编自申请人的摘要)：长期目标 这项研究是为了确定信号背后的分子机制 成骨细胞中的转导途径及其生理意义 钙离子通道在骨骼中的作用。具体目标是：1)测试 VDR定量预测[~3H]-1，25D3结合总量的假设 消息，RO取代质膜(或胞质)结合部位， 而EO取代了核标记。使用受体结合技术 在[~3H]-1，25D3的作用下，RO和EO的药理和分布 它作用于nVDR来刺激转录，但在 10 nM以下的钙离子通道的激活)将被量化和关联 在表达不同级别VDR消息的细胞系中；2)确定 其他类固醇激素核受体的信息是否可以预测 类固醇激素对钙通道的增强作用。ROS 17/2.8细胞显示 添加0.1 nM睾酮对钙电流的增强作用，但剂量 高达100毫微米的17B雌二醇造成的影响微乎其微。校长 研究人员建议检验这样一种假设，即正常细胞的转染率 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17B雌二醇对钙通道的快速增强作用这个 非基因组反应的敏感性将被量化到 类固醇受体信息的表达；3)钙通道亚型的测定 在ROS 17/2.8细胞和成熟的大鼠骨中，这与 对类固醇激素的反应。要回答存在哪些通道异构体 在成熟的大鼠骨或ROS细胞培养中，针对 主要采用L型钙通道亚型，基因敲除技术将用于 明确定义哪种通道类型由类固醇受体激活。 类固醇激活的钙通道亚型将被克隆和表达。CA+ 通道参与骨基质的产生和骨吸收，以及 对它们的药理研究可能会产生新的药物治疗方法 用来治疗骨病。

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.