Measurement of the cytoplasmic pH of Dictyostelium discoideum using a low light level microspectrofluorometer.
Measurement of the cytoplasmic pH of Dictyostelium discoideum using a low light level microspectrofluorometer.
复制标题
使用低光水平微光谱计测量迪斯特尔迪斯特尔的细胞质pH值。
DOI：
10.1083/jcb.107.6.2541
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发表时间：
1988-12
期刊：
影响因子：
--
通讯作者：
Fechheimer M
中科院分区：
文献类型：
--
作者：
Furukawa R;Wampler JE;Fechheimer M
吡啶是一种灵敏的pH指示剂，用于微光显微荧光光谱仪。吡嗪460/410 nm荧光激发比随pH值变化的体内、体外标准曲线相同。因此，pyranine对Dictyostelium细胞质pH特别敏感。本文首次测定了变形虫盘状盘齿钢属（Dictyostelium disideum amoebae）群体中单细胞的细胞质pH值。营养阿米巴的胞质pH中位数为7.19。羰基氰化物间氯苯腙是线粒体解偶联剂和质子载体，当胞外pH为6.1时，胞质pH中位数降至6.12。这一结果与羰基氰间氯苯腙的质子团活性一致。细胞质酸化促进柄前细胞的发育，而细胞质碱化有利于孢子前途径，这一理论极大地激发了人们对Dictyostelium细胞质pH值的兴趣（Gross， J. D.， J. Bradbury， R. R. Kay， M. J. Peacey. 1983）。自然(Lond)。303:244 - 245)。该理论认为，二乙基己烯雌酚（DES）是一种茎细胞分化诱导剂和质膜质子转运atp酶抑制剂，应引起细胞质溶胶的酸化。先前使用31P核磁共振测量包括DES在内的茎细胞诱导剂对细胞内pH值的影响的测量未能证实该理论的预测，并表明可能需要对模型进行重大修改。以pyranine作为pH指标，我们发现10微米DES处理的细胞细胞质pH中位数从7.19下降到6.02。这种作用与DES的药理作用一致，也与DES作为茎细胞诱导剂应使细胞质酸化的建议一致。这些结果直接支持了细胞质pH是盘齿柱发育途径的重要调节因子的理论。
Pyranine was employed as a sensitive pH indicator in a low light level microspectrofluorometer. The in vivo and in vitro standard curves of the 460/410-nm fluorescence excitation ratio of pyranine as a function of pH are identical. Therefore, pyranine is specifically sensitive to cytoplasmic pH in Dictyostelium. The cytoplasmic pH of single cells in a population of Dictyostelium discoideum amoebae was obtained for the first time. The median cytoplasmic pH of vegetative amoebae was 7.19. Carbonyl cyanide m-chlorophenylhydrazone, a mitochondrial uncoupler and a protonophore, lowered the median cytoplasmic pH to 6.12 when the extracellular pH was 6.1. This result is in accord with the protonophore activity of carbonyl cyanide m-chlorophenylhydrazone. Interest in the cytoplasmic pH of Dictyostelium has been greatly stimulated by the theory that cytoplasmic acidification promotes development of pre-stalk cells, while cytoplasmic alkalinization favors the pre-spore pathway (Gross, J. D., J. Bradbury, R. R. Kay, M. J. Peacey. 1983. Nature (Lond.). 303:244-245). The theory postulates that diethylstilbestrol (DES), an inducer of stalk cell differentiation and a plasma membrane proton translocating ATPase inhibitor, should cause acidification of the cytosol. Previous measurements of the effects of stalk cell inducers including DES on intracellular pH using 31P nuclear magnetic resonance measurements have failed to confirm the predictions of the theory, and have suggested that significant modification of the model may be required. Using pyranine as the pH indicator, we find that the median cytoplasmic pH in cells treated with 10 microM DES dropped from 7.19 to pH 6.02. This effect is consistent with the pharmacological action of DES and with the proposal that DES, a stalk cell inducer, should acidify the cytosol. These results provide direct support for the theory that cytoplasmic pH is an essential regulator of the developmental pathway in Dictyostelium.