DEVELOPMENT AND USE OF METHODS TO MEASURE CELLULAR PH

pH值的调节对所有活细胞的生命至关重要。 为 例如，细胞溶质的pH必须保持接近中性， 由于新陈代谢而产生的酸的排出。 溶酶体的pH值 必须是酸性的以增加水解酶的活性。 的pH 线粒体是碱性的。 质子在这些细胞器中的运动 生产ATP所必需的。 最近的证据表明，细胞质中pH值的变化可能 调节许多细胞活动。 可能需要改变pH值， 启动静止、非分裂细胞的生长。 的改变 细胞内pH值也可能决定所选择的分化途径 通过发展细胞。 pH的局部变化可能有助于 细胞，如运动和分裂。 已经开发了用于测量pH的技术，其采用pH测量方法。 荧光探针发射的荧光的依赖性， 融入活细胞中。 显微荧光分光光度计可用于 连续记录pH值及其变化在局部地区的单一 活细胞 在本提案中，显微荧光分光光度计将首先用于 确定荧光测量的准确度和精密度 细胞内的pH值在鸡蛋的青鳉鱼和变形虫在 细胞黏菌，盘基网柄菌。 pH值的变化， 青鳉卵将被确定、定位并与正在进行的 发展过程。 发育过程中细胞内pH的变化 D.将详细分析盘状突，以检验以下假设： pH的变化有助于细胞分化。 机制或监管 细胞内pH值将通过使用特定的药理学 剂. 此外，细胞运动过程中细胞内pH的变化， D.将检查盘状突，以测试pH值是一个 调节细胞质的结构，以及细胞的形状和运动。 细胞 细胞运动和分化是生命的基本过程， 所有的脊椎动物。 阐明pH在调节中的作用 这些过程将推进我们对基本机制的认识， 调节细胞活动，指导未来对这些现象的研究， 正常和病理状态，并可能最终有助于发展 诊断程序。

Regulation of pH is essential to the life of all living cells. For instance, the pH of the cytosol must be maintained near neutrality by extrusion of acid generated as a result of metabolism. The pH of lysosomes must be acidic to increase the activity of hydrolytic enzymes. The pH of mitochondria is alkaline. Movement of protons in these organelles are required for production of ATP. Recent evidence indicates that changes in the pH in the cytosol may regulate numerous cellular activities. Changes in pH may be required for initiation of growth of quiescent, non-dividing cells. Alterations in intracellular pH may also dictate the pathway of differentiation selected by developing cells. Local changes in pH may contribute to movements of cells, such as locomotion and division. Techniques for measurement of pH have been developed that employ the pH dependence of fluorescence emitted by fluorescent probes that have been incorporated into living cells. A microspectrofluorometer can be used to continuously record the pH and its changes in local regions of single living cells. In the present proposal, the microspectrofluorometer will first be used to determine the accuracy and precision of fluorometric measurements of intracellular pH in the egg of the Medaka fish and in amoeba in the cellular slime mold, Dictyostelium discoideum. The changes in pH in the Medaka egg will be determined, localized and correlated with ongoing developmental processes. Changes in intracellular pH during development in D. discoideum will be analyzed in detail to test the hypothesis that changes in pH contribute to cell differentiation. Mechanisms or regulation of intracellular pH will be assessed by use of specific pharmacologic agents. In addition, changes in intracellular pH during cell movement in D. discoideum will be examined in order to test the hypotesis that pH is a regulator of the structure of cytoplasm, and the shape and movements of cells. Cell movements and differentiation are fundamental processes in the life of all vertebrate organisms. Elucidation of the role of pH in regulating these processes will advance our knowledge of the fundamental mechanisms that regulate cell activities, direct future studies of these phenomena in normal and pathologic states, and may eventually contribute to development of diagnostic procedures.