DEVELOPMENT AND USE OF METHODS TO MEASURE CELLULAR PH

调节pH值对所有活细胞的生命都是至关重要的。为 例如，胞液的pH值必须通过以下方法保持接近中性 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，才能增加水解酶的活性。水溶液的pH值 线粒体是碱性的。质子在这些细胞器中的运动是 生产三磷酸腺苷所必需的。 最近的证据表明，胞浆中pH值的变化可能 调节众多的细胞活动。可能需要改变酸碱度 启动静止的、不分裂的细胞的生长。中的更改 细胞内pH也可能决定所选择的分化途径 通过发育细胞。酸碱值的局部变化可能会导致 细胞的运动和分裂等。 已经开发了使用pH来测量pH的技术 荧光探针发射的荧光对已被 整合到活细胞中。微型荧光分光光度计可用于 连续记录个体局部区域的pH值及其变化 活细胞。 在本提案中，将首先使用微型荧光分光光度计来 确定荧光测量的准确度和精密度 青竹鱼卵和变形虫体内的细胞内pH 细胞黏菌，盘基网柄菌。酸碱度的变化 青冈蛋将被确定、本地化并与正在进行的 发育过程。胚胎发育过程中细胞内pH的变化 D.Discoideum将被详细分析，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的变化将通过使用特定的药理学方法进行评估 探员们。此外，在细胞运动过程中细胞内pH的变化 D.将对盘状突进行检查，以检验pH是一种 细胞质结构的调节器，以及细胞质的形状和运动 细胞。 细胞运动和分化是生命中的基本过程 所有脊椎动物。阐明pH在调节中的作用 这些过程将促进我们对基本机制的了解 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育 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.