Echevarria M, Verkman A S
Department of Medicine, Cardiovascular Research Institute, University of California, San Francisco 94143-0532.
J Gen Physiol. 1992 Apr;99(4):573-89. doi: 10.1085/jgp.99.4.573.
Methodology was developed to measure osmotic water permeability in monolayer cultured cells and applied to examine the proposed role of glucose transporters in the water pathway (1989. Proc. Natl. Acad. Sci. USA. 86:8397-8401). J774 macrophages were grown on glass coverslips and mounted in a channel-type perfusion chamber for rapid fluid exchange without cell detachment. Relative cell volume was measured by 45 degrees light scattering using an inverted microscope; measurement accuracy was validated by confocal imaging microscopy. The time required for greater than 90% fluid exchange was less than 1 s. In response to a decrease in perfusate osmolality from 300 to 210 mosM, cells swelled without lag at an initial rate of 4.5%/s, corresponding to a water permeability coefficient of (6.3 +/- 0.4) x 10(-3) cm/s (SE, n = 20, 23 degrees C), assuming a cell surface-to-volume ratio of 4,400 cm-1. The initial rate of cell swelling was proportional to osmotic gradient size, independent of perfusate viscosity, and increased by amphotericin B (25 micrograms/ml), and had an activation energy of 10.0 +/- 1 kcal/mol (12-39 degrees C). The compounds phloretin (20 microM) and cytochalasin B (2.5 micrograms/ml) inhibited glucose transport by greater than 85% but did not influence Pf in paired experiments in which Pf was measured before and after inhibitor addition. The mercurials HgCl2 (0.1 mM) and p-chloromercuribenzoate (1 mM) did not inhibit Pf. A stopped-flow light scattering technique was used to measure Pf independently in J774 macrophages grown in suspension culture. Pf in suspended cells was (4.4 +/- 0.3) x 10(-3) cm/s (assuming a surface-to-volume ratio of 8,800 cm-1), increased more than threefold by amphotericin B, and not inhibited by phloretin and cytochalasin B under conditions of strong inhibition of glucose transport. The glucose reflection coefficient was 0.98 +/- 0.03 as measured by induced osmosis, assuming a unity reflection coefficient for sucrose. These results establish a quantitative method for measurement of osmotic water transport in adherent cultured cells and provide evidence that glucose transporters are not involved in the water transporting pathway.
已开发出一种方法来测量单层培养细胞中的渗透水渗透率，并用于检验葡萄糖转运蛋白在水转运途径中所提出的作用（1989年。美国国家科学院院刊。86:8397 - 8401）。J774巨噬细胞生长在玻璃盖玻片上，并安装在通道型灌注室中，以便在不使细胞脱离的情况下进行快速液体交换。使用倒置显微镜通过45度光散射测量相对细胞体积；通过共聚焦成像显微镜验证了测量准确性。大于90%的液体交换所需时间小于1秒。响应于灌注液渗透压从300降至210 mosM，细胞立即以4.5%/秒的初始速率肿胀，假设细胞表面与体积之比为4400 cm-1，则对应的水渗透系数为(6.3±0.4)×10(-3) cm/秒（标准误，n = 20，23℃）。细胞肿胀的初始速率与渗透梯度大小成正比，与灌注液粘度无关，并因两性霉素B（25微克/毫升）而增加，其活化能为10.0±1千卡/摩尔（12 - 39℃）。根皮素（20微摩尔）和细胞松弛素B（2.5微克/毫升）化合物使葡萄糖转运抑制超过85%，但在添加抑制剂前后测量Pf的配对实验中不影响Pf。汞剂HgCl2（0.1毫摩尔）和对氯汞苯甲酸（1毫摩尔）不抑制Pf。使用停流光散射技术独立测量悬浮培养的J774巨噬细胞中的Pf。悬浮细胞中的Pf为(4.4±0.3)×10(-3) cm/秒（假设表面与体积之比为8800 cm-1），因两性霉素B增加了三倍多，并且在葡萄糖转运受到强烈抑制的条件下不受根皮素和细胞松弛素B的抑制。通过诱导渗透测量的葡萄糖反射系数为0.98±0.03，假设蔗糖的反射系数为1。这些结果建立了一种定量方法来测量贴壁培养细胞中的渗透水转运，并提供证据表明葡萄糖转运蛋白不参与水转运途径。
