Furukawa R, Wampler J E, Fechheimer M
Department of Biochemistry, University of Georgia, Athens 30602.
J Cell Biol. 1988 Dec;107(6 Pt 2):2541-9. doi: 10.1083/jcb.107.6.2541.
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.
吡喃荧光素被用作微光水平显微分光荧光计中的一种灵敏pH指示剂。吡喃荧光素460/410纳米荧光激发比作为pH的函数的体内和体外标准曲线是相同的。因此，吡喃荧光素对盘基网柄菌中的细胞质pH具有特异性敏感性。首次获得了盘基网柄菌变形虫群体中单个细胞的细胞质pH。营养变形虫的细胞质pH中位数为7.19。羰基氰化物间氯苯腙，一种线粒体解偶联剂和质子载体，当细胞外pH为6.1时，将细胞质pH中位数降至6.12。该结果与羰基氰化物间氯苯腙的质子载体活性一致。细胞质酸化促进柄前细胞发育，而细胞质碱化有利于孢子前途径的理论（格罗斯，J.D.，J.布拉德伯里，R.R.凯，M.J.皮西。1983年。《自然》（伦敦）。303:244 - 245）极大地激发了人们对盘基网柄菌细胞质pH的兴趣。该理论假设，己烯雌酚（DES），一种柄细胞分化诱导剂和质膜质子转运ATP酶抑制剂，应导致细胞质酸化。先前使用31P核磁共振测量对包括DES在内的柄细胞诱导剂对细胞内pH的影响进行测量时，未能证实该理论预测，并表明可能需要对模型进行重大修改。使用吡喃荧光素作为pH指示剂，我们发现用10微摩尔己烯雌酚处理的细胞中细胞质pH中位数从7.19降至pH6.02。该效应与己烯雌酚的药理作用一致，也与己烯雌酚作为柄细胞诱导剂应使细胞质酸化的提议一致。这些结果为细胞质pH是盘基网柄菌发育途径的重要调节因子这一理论提供了直接支持。
