Swanson J A, Yirinec B D, Silverstein S C
J Cell Biol. 1985 Mar;100(3):851-9. doi: 10.1083/jcb.100.3.851.
Lucifer Yellow CH (LY) is an excellent probe for fluid-phase pinocytosis. It accumulates within the macrophage vacuolar system, is not degraded, and is not toxic at concentrations of 6.0 mg/ml. Its uptake is inhibited at 0 degree C. Thioglycollate-elicited mouse peritoneal macrophages were found to exhibit curvilinear uptake kinetics of LY. Upon addition of LY to the medium, there was a brief period of very rapid cellular accumulation of the dye (1,400 ng of LY/mg protein per h at 1 mg/ml LY). This rate of accumulation most closely approximates the rate of fluid influx by pinocytosis. Within 60 min, the rate of LY accumulation slowed to a steady-state rate of 250 ng/mg protein per h which then continued for up to 18 h. Pulse-chase experiments revealed that the reduced rate of accumulation under steady-state conditions was due to efflux of LY. Only 20% of LY taken into the cells was retained; the remainder was released back into the medium. Efflux has two components, rapid and slow; each can be characterized kinetically as a first-order reaction. The kinetics are similar to those described by Besterman et al. (Besterman, J. M., J. A. Airhart, R. C. Woodworth, and R. B. Low, 1981, J. Cell Biol. 91:716-727) who interpret fluid-phase pinocytosis as involving at least two compartments, one small, rapidly turning over compartment and another apparently larger one which fills and empties slowly. To search for processes that control intracellular fluid traffic, we studied pinocytosis after treatment of macrophages with horseradish peroxidase (HRP) or with the tumor promoter phorbol myristate acetate (PMA). HRP, often used as a marker for fluid-phase pinocytosis, was observed to stimulate the rate of LY accumulation in macrophages. PMA caused an immediate four- to sevenfold increase in the rate of LY accumulation. Both HRP and PMA increased LY accumulation by stimulating influx and reducing the percentage of internalized fluid that is rapidly recycled. A greater proportion of endocytosed fluid passes into the slowly emptying compartment (presumed lysosomes). These experiments demonstrate that because of the considerable efflux by cells, measurement of marker accumulation inaccurately estimates the rate of fluid pinocytosis. Moreover, pinocytic flow of water and solutes through cytoplasm is subject to regulation at points beyond the formation of pinosomes.
路西法黄CH（LY）是一种用于液相胞饮作用的优良探针。它在巨噬细胞液泡系统中积累，不会被降解，在浓度为6.0mg/ml时无毒。其摄取在0℃时受到抑制。发现用巯基乙酸诱导的小鼠腹腔巨噬细胞对LY表现出曲线摄取动力学。向培养基中加入LY后，染料会有一段非常快速的细胞积累期（在1mg/ml LY时为每小时1400ng LY/毫克蛋白质）。这种积累速率最接近通过胞饮作用的液体流入速率。在60分钟内，LY积累速率减慢至每小时250ng/毫克蛋白质的稳态速率，然后持续长达18小时。脉冲追踪实验表明稳态条件下积累速率降低是由于LY的流出。进入细胞的LY只有20%被保留；其余的被释放回培养基中。流出有快速和缓慢两个成分；每个成分在动力学上都可表征为一级反应。其动力学与Besterman等人描述的相似（Besterman, J. M., J. A. Airhart, R. C. Woodworth, and R. B. Low, 1981, J. Cell Biol. 91:716 - 727），他们将液相胞饮作用解释为至少涉及两个区室，一个小的、快速周转的区室和另一个明显较大的、填充和排空缓慢的区室。为了寻找控制细胞内液体运输的过程，我们在用辣根过氧化物酶（HRP）或肿瘤启动子佛波醇肉豆蔻酸酯乙酸酯（PMA）处理巨噬细胞后研究了胞饮作用。HRP常被用作液相胞饮作用的标志物，观察到它能刺激巨噬细胞中LY的积累速率。PMA使LY积累速率立即增加4到7倍。HRP和PMA都通过刺激流入和减少快速再循环的内化液体百分比来增加LY积累。更大比例的内吞液体进入缓慢排空的区室（推测为溶酶体）。这些实验表明，由于细胞有相当大的流出，标记物积累的测量不准确地估计了液体胞饮作用的速率。此外，水和溶质通过细胞质的胞饮流在胞饮体形成之外的点受到调节。
