CONTROL OF ACID-BASE TRANSPORTERS BY GROWTH FACTORS & HORMONES IN MESANGIAL CELLS

肾小球系膜细胞(MC)在肾小球生物学中起着关键作用，两者 在健康和疾病方面。这些血管-平滑肌-的收缩- 类细胞被认为是调节GFR的细胞。增殖和分泌 MC对基质物质的作用在MCs的发展中起着核心作用 多种形式的慢性肾小球疾病。监管 细胞内pH(Phi)，对多种细胞 功能(酶活性、离子电导等)，预计 对MC来说尤其重要。这是因为肌肉紧张度和 细胞增殖对pH非常敏感。这项建议是 旨在继续深入研究大鼠的pH(I)生理学 系膜细胞原代培养(第3~8代)。我们的终极 目标是了解在受控的MC中pH(I)是如何被调节的 条件，以及在短期和长期暴露于增长因素之后 它们通过不同的信号转导途径发挥作用。在以前的 我们的工作表明，MC使用三种转运蛋白来调节pH(I)： Na-H交换器，依赖于Na+的Cl-HCO3交换器(这两者都会导致 PH(I)增加)，以及不依赖于Na+的Cl-HCO3交换剂(这导致 PH(I)降低)。此外，在对细胞群体的研究中，我们 已经表明(当在单一pH(I)下测定时)这些 转运蛋白被有丝分裂原激活，但激活的程度 是依赖时间的。此外，我们还开发了一种方法来 准确评估有丝分裂原对细胞pH(I)依赖性的影响 Na-H交换器。我们提出了三个主要目标：第一，刻画 有丝分裂原和细胞收缩对pH(I)依赖性的影响 三个传送者。这些实验将在人群中进行。 负载了一种pH敏感的荧光染料的MC，并在双... 光束荧光分光光度计。我们的方法将是确定 PH(I)从酸或碱负荷中回收，在没有和存在 特定的抑制剂，并使用这些数据来计算pH(I)依赖关系 由三个转运体各自调节的通量。第二，使用 PH(I)调节的数学模型确定动力学 对运输者的描述(在第一个目标中得出)说明了 PH9I0与时间记录(也是在第一个目标中获得的)。第三，到 确定有丝分裂原的长期影响，以及进展到 有丝分裂，与三种转运蛋白的活性有关。这些 实验将在多个单独的MC上进行，这些MC负载了一种pH- 灵敏的荧光染料，并用显微镜数码进行研究 荧光成像系统。我们将同步我们的手机，后自组织， 根据有丝分裂的时间；这将允许确定 转运蛋白活动如何在有丝分裂期间和附近发生变化，其中 以前从未有人尝试过。拟议的工作将导致 对pH(I)调节及其控制的最全面的描述 任何细胞中的有丝分裂原。此外，结合实验工作与 该模型将提供对以下重要因素的新见解 PH(I)调节。扩大我们对巨噬细胞pH(I)调节的理解 无论是在控制条件下还是在有丝分裂原刺激下，都可以 提高对肾小球疾病发病机制的认识。

Mesangial cells (MCs) play a critical role in glomerular biology, both in health and disease. The contraction of these vascular-smooth-muscle- like cells is believed to modulate GFR. The proliferation and secretion of matrix substances by MCs plays a central role in the development of many forms of chronic glomerular disease. The regulation of intracellular pH (pHi), important for a wide variety of cellular functions (enzyme activities, ion conductances, etc.), is expected to be especially critical for MCs. That is because both muscle tension and cell proliferation are exquisitely pH sensitive. This proposal is designed to continue an intensive study of the pH (i) physiologies of rat mesangial cells in primary culture (3rd - 8th passage). Our ultimate goal is to understand how pH(i) is regulated in MCs under control conditions, and after short- and long-term exposure to growth factors that act through different signal-transduction pathways. In previous work, we have shown that MCs use three transporters to regulate pH(i): a Na-H exchanger, a Na+-dependent Cl-HCO3 exchanger (both of which cause pH(i) to increase), and a Na+-independent Cl-HCO3 exchanger (which causes pH(i) to decrease). Furthermore, in work on populations of cells, we have shown that (when assayed at a single pH(i) each of these transporters is activated by mitogens, but that the degree of activation is time dependent. Furthermore, we have developed an approach for accurately assessing the effects of mitogens on the pH(i) dependence of the Na-H exchanger. We propose three major aims: First, to characterie the effects of mitogens and cell shrinkage on the pH(i) dependencies of the three transporters. These experiments will be done on populations of MCs loaded with a pH-sensitive fluorescent dye, and studied in a dual- beam spectrofluorometer. Our approach will be to determine the rates of pH(i) recovery from acid or alkali loads, in the absence and presence of specific inhibitors, and use these data to compute the pH(i) dependencies of the fluxes mediated by each of the three transporters. Second, to use a matematical model of pH(i) regulation to determine if the kinetic descriptions of the transporters (derived in the 1st Aim) account for the pH9I0 vs time records (also obtained in the 1st Aim). Third, to determine the long-range effects of mitogens, and the progression to mitosis, on the activities of each of the three transporters. These experiments will be done on multiple individual MCs loaded with a pH- sensitive fluorescent dye, and studied using a microscope-based digital fluorescence imaging system. We will synchronize our cells, post hoc, based on the time of mitosis; this will allow to determine the time course of how transporter activities change at and near mitosis, which has never before been attempted. The proposed work would lead to the most comprehensive description of pH(i) regulation, and its control by mitogens, in any cell. Moreover, combining the experimental work with the modeling will provide new insights into the factors important for pH(i) regulation. Extending our understanding of pH(i) regulation in MCs both under control conditions and after stimulation by mitogens, could improve our insight into the pathogenesis of glomerular disease.