CELLULAR REGULATION OF PHOSPHATE TRANSPORT IN KIDNEY

肾脏磷酸盐转运的细胞调节

• 批准号: 2138507
• 负责人: THOMAS P DOUSA
• 金额: $ 19.95万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2138507
• 关键词: biological signal transduction; brush border membrane; calcium flux; cyclic AMP; enzyme induction /repression; gluconeogenesis; hormone regulation /control mechanism; intracellular transport; ion transport; laboratory rabbit; laboratory rat; membrane transport proteins; microtubule associated protein; microtubules; nicotinamide adenine dinucleotide; nucleotide analog; phosphates; protein kinase A; purinergic receptor; renal tubular transport; ribose phosphate; sodium

The major goal of this research project is to elucidate intracellular signal transduction pathways by which metabolic changes modulate and hormones regulate the Na+-Pi symport across brush border membranes (BBM). The studies are based on the following hypothesis. While hormones such as PTH or dopamine regulate Na+-Pi symport via cAMP-PKA and/or pathway(s), the change in metabolic states namely gluconeogenesis (GNG) of proximal tubular (PT) modulate the Na+ Pi symport via a novel pathway involving NAD+, cyclic adenosine diphosphoribose (CADPR), and Ca2+ release. The final steps of the cADPR signalling pathway is regulation of microtubule (MT) disassembly and assembly by microtubule associated proteins (MAP2), which are phosphorylated by Ca2+ dependent and cAMP- dependent protein kinases. Specific objective include: 1. To determine the characteristics of cADPR system in PT cells and OK cells. This includes subcellular localization and enzymatic properties of ADP-cyclase, cADRP-glucohydrolase (cADPR-GH), and dynamics of cellular cADPR levels. 2. To examine binding of cADPR onto specific cADPR-receptor and its characteristic (KD, Bmax, specificity, pH optimum, etc) and relationship between cADPR binding and release of Ca2+ from the intracellular Ca2+ stores. To determine whether multifunctional Ca2+/CaM protein kinases-II (Ca/CaM-PK-II) is activated by Ca2+ released in response to cADPR, and whether it phosphorylates microtubule associated proteins (MAP2), and whether phosphorylated MAP2 promote disassembly of MT in PT cells. Further, to test whether PKA also phosphorylates MAP2 and whether the effects of both protein kinases are additive. 3. To determine whether the cADPR signalling system operates in intact cells as integral pathway and modulates Na+-Pi symport across BBM in response to change in PT metabolism, namely GNG. 4. To delineate intracellular mechanisms by mutual "crosstalk" between the hormonal signals acting via cAMP-PKA pathway and metabolic signals acting via cADPR-Ca2+ pathway in PT cells. We will also test the hypothesis which states that the degree of MT assembly in cytoplasm of PT cells determines the extent of internalization and reinsertion of Na+- Pi symporters into BBM, and thereby regulates the Na+-Pi reabsorption. We propose that MAP2 phosphorylation by the two kinases is the focal point of additivity or synergism.

该研究项目的主要目标是阐明细胞内 信号转导途径，代谢变化调节和 激素调节跨刷边框膜（BBM）的Na+-PI同步。 研究基于以下假设。 而激素也是如此 作为PTH或多巴胺通过CAMP-PKA和/或 途径，代谢状态的变化，即糖异生（GNG） 近端管状（PT）通过新途径调节Na+ Pi的同步 涉及NAD+，环状腺苷二磷蛋白（CADPR）和Ca2+ 发布。 CADPR信号通路的最后一步是调节 微管（MT）拆卸和组装的微管 蛋白质（MAP2），由Ca2+依赖性和cAMP-磷酸化 依赖性蛋白激酶。 具体目标包括： 1。确定PT细胞中CADPR系统的特征和OK 细胞。 这包括亚细胞定位和酶特性 ADP - 周期酶，CADRP-葡萄糖水解酶（CADPR-GH）和细胞动力学 CADPR水平。 2。检查CADPR与特定CADPR受体及其的结合 特征（KD，BMAX，特异性，pH最佳等）和关系 在CADPR结合和从细胞内Ca2+的Ca2+释放之间 商店。 确定多功能CA2+/CAM蛋白激酶II是否是否 （CA/CAM-PK-II）被响应CADPR释放的Ca2+激活，并且 它是否磷酸化微管相关蛋白（MAP2）和 磷酸化的MAP2是否促进PT细胞中MT的拆卸。 此外，要测试PKA是否还磷酸化MAP2以及是否磷酸 两种蛋白激酶的作用都是加性的。 3。确定CADPR信号系统是否完整运行 单元格为积分途径，并在BBM中调节Na+-PI在 对PT代谢的变化的反应，即GNG。 4。通过相互的“串扰”来描绘细胞内机制 通过CAMP-PKA途径和代谢信号作用的激素信号 通过PT细胞中的CADPR-CA2+途径作用。 我们还将测试 假设指出，细胞质中MT组装程度 PT细胞确定Na+ - 内在化和重新插入的程度 PI分类器进入BBM，从而调节Na+-PI的重吸收。 我们提出，两个激酶的MAP2磷酸化是焦点 添加性或协同作用。