CELLULAR CALCIUM TRANSPORT IN URINARY EPITHELIA

我们工作的长期目标是实现一个完整的 了解钙的细胞机制和调节 通过肾小管上皮细胞转运。 虽然大多数钙 吸收在近端小管中进行，远端小管是 甲状旁腺激素对钙转运生理调节 (PTH)、降钙素和维生素D3。 目前的具体目标 建议是：1）表征钙进入的机制， 远曲小管（DCT）细胞顶端质膜; 2） 评价Na+/Ca 2+交换在 介导钙外流;和，3）定义激活的信号通路 通过对DCT细胞中PTH和降钙素的测定， 通过1，25（OH）2维生素D3调节PTH依赖性钙转运， 雌二醇 我们鉴定并部分表征了一种新的钙通道， 介导钙进入。 为了获得有关这些的更多信息， 通道，我们将：1）表征这些通道的属性， 考虑到它们的离子选择性、药理学灵敏度和电压- 依赖性; 2）通过以下方式确定这些钙通道的调节： 蛋白激酶; 3）确定G蛋白参与调节 钙离子进入通道; 4）鉴定和克隆钙离子通道 成绩单 钙离子跨基底外侧质膜的挤出 远曲小管的Na ~+/Ca ~（2+）交换和Ca ~（2+）-ATP酶介导的。 初步研究表明，Na+/Ca 2+交换是主要的外流 DCT单元中的机制。 我们将：1）检验噻嗪类化合物 利尿剂可以抑制Na+/Ca 2+交换;和2）表征 PTH对Na+/Ca ~（2+）交换的刺激作用。 钙转运 远端肾单位由PTH、降钙素和1，25（OH）2维生素D3调节。 以下研究将评估介导激素的机制 调节DCT细胞中的钙转运。 这些目标 实验是：1）表征信号通路， 特别关注负责激活PKA的磷脂酶 和蛋白激酶C和特征的时间序列，其中PKA和蛋白激酶C是 激活 我们将检验PTH和降钙素激活 PKC通过磷脂酶D。 2)确定PTH 激活C1通道，这是膜超极化的主要事件， 刺激DCT细胞中的钙转运。 我们将测试 假设这些氯离子通道是由PKA分支调控的， PTH激活的信号通路。 3)审查对 维生素D3和雌二醇通过促进PTH依赖性钙转运， 上调PTH受体表达。 具体目标将通过应用单细胞荧光来实现， 膜片钳，示踪剂通量测量，生化和分子 技术的DCT细胞系，我们开发的，它表达了一个 合适的表型。 在选定的研究中， 近端或远端小管细胞将用于验证结果， 转化细胞或作为对照。 所有程序都在我们的 实验室 实验结果将提供新的信息 肾钙转运的机制和调节 正常情况下，但也在钙消耗综合征，包括 甲状旁腺功能亢进，肾衰竭，骨质疏松症和恶性肿瘤- 相关的高钙血症。

The long-term objective of our work is directed toward a complete understanding of the cellular mechanisms and regulation of calcium transport by renal tubular epithelial cells. Although most calcium absorption proceeds in proximal tubules, distal tubules are the site of the physiological regulation of calcium transport by parathyroid hormone (PTH), calcitonin and vitamin D3. The specific aims of the present proposal are to: 1) characterize the mechanism of calcium entry across apical plasma membranes of distal convoluted tubule (DCT) cells; 2) evaluate the participation and regulation of Na+/Ca2+ exchange in mediating calcium efflux; and, 3) define the signaling pathways activated by PTH and calcitonin in DCT cells, and to examine the coordinate regulation of PTH-dependent calcium transport by 1,25(OH)2 vitamin D3 and estradiol. We identified and partially characterized a novel calcium channel that mediates calcium entry. To acquire additional information about these channels we will: 1) characterize the properties of these channels with regard to their ion selectivity, pharmacologic sensitivity and voltage- dependence; 2) determine the regulation of these calcium channels by protein kinases; 3) define the participation of G-proteins in regulating calcium entry channels; and, 4) identify and clone calcium channel transcripts. Extrusion of calcium across basolateral plasma membranes of distal tubules is mediated by Na+/Ca2+ exchange and by /ca2+-ATPase. Pilot studies suggest that Na+/Ca2+ exchange is the dominant efflux mechanism in DCT cells. We will: 1) Test the hypothesis that thiazide diuretics can inhibit Na+/Ca2+ exchange; and 2) Characterize the stimulatory effects of PTH on Na+/Ca2+ exchange. Calcium transport in distal nephrons is regulated by PTH, calcitonin and 1,25(OH)2 vitamin D3. The following studies will evaluate the mechanism mediating the hormonal regulation of calcium transport in DCT cells. The goals of these experiments are to: 1) Characterize the signaling pathways with particular regard to the phospholipases responsible for activating PKA and PKC and characterize the temporal sequence in which PKA and PKC are activated. We will test the hypothesis that PTH and calcitonin activate PKC via phospholipase D. 2) Identify the mechanism by which PTH activates C1- channels, a primary event in membrane hyperpolarization and stimulation of calcium transport in DCT cells. We will test the hypothesis that these chloride channels are regulated by the PKA limb of the PTH-activated signaling pathway. 3) Examine the regulation of vitamin D3 and estradiol accelerate PTH dependent calcium transport by up-regulating PTH receptor expression. The specific aims will be achieved by applying single cell fluorescence, patch clamp, tracer flux measurements, biochemical and molecular techniques to a DCT cell line that we developed, which expresses an appropriate phenotype. In selected studies, primary cell cultures of proximal or distal tubule cells will be used to verify results in transformed cells or as controls. All procedures are established in our lab. Results from the proposed experiments will provide new information on the mechanism and regulation of calcium transport in the kidney under normal conditions, but also in calcium-wasting syndromes including hyperparathyroidism, renal failure, osteoporosis and malignancy- associated hypercalcemia.