Besterman J M, Airhart J A, Woodworth R C, Low R B
J Cell Biol. 1981 Dec;91(3 Pt 1):716-27. doi: 10.1083/jcb.91.3.716.
The uptake and fate of pinocytosed fluid were investigated in monolayers of pulmonary alveolar macrophages and fetal lung fibroblasts using the fluid-phase marker, [14C]sucrose. Initial experiments revealed that cellular accumulation of chromatographically repurified [14C]sucrose was not linear with incubation time. Deviation from linearity was shown to be due to constant exocytosis of accumulating marker. Chromatographic analysis revealed that the cells were unable to metabolize sucrose and were releasing it intact by a process that was temperature-sensitive but not dependent on extracellular calcium and magnesium. A detailed analysis of the kinetics of exocytosis was undertaken by preloading cells with [14C]sucrose for various lengths of time and then monitoring the appearance of radioactivity into isotope-free medium. Results indicated that modeling the process of fluid-phase pinocytosis and subsequent exocytosis required at least two intracellular compartments in series, one compartment being of small size and turning over very rapidly (t1/2 = 5 min in macrophages, 6--8 min in fibroblasts) and the other compartment being apparently larger in size and turning over very slowly (t1/2 = 180 min in macrophages, 430--620 min in fibroblasts). Computer-simulation based on this model confirmed that the kinetics of efflux faithfully reflected the kinetics of influx and that the rate of efflux completely accounted for the deviation from linearity of accumulation kinetics. Moreover, the sizes of the compartments and magnitude of the intercompartment fluxes were such that the majority of fluid internalized in pinocytic vesicles was rapidly returned to the extracellular space via exocytosis. This result provides direct experimental evidence for a process previously thought necessary based solely on morphological and theoretical considerations. Furthermore, the turnover of pinocytosed fluid was so dynamic that accumulation deviated from linearity even within the first few minutes of incubation. We were able to show that the kinetics of exocytosis allowed calculation of the actual pinocytic rate, a rate that was nearly 50% greater than the apparent initial rate obtained from the slope of the uptake curve over the first 10 min.
利用液相标记物[¹⁴C]蔗糖，对肺泡巨噬细胞和胎儿肺成纤维细胞单层中胞饮摄入的液体的摄取和去向进行了研究。初步实验表明，经色谱法重新纯化的[¹⁴C]蔗糖在细胞内的积累与孵育时间并非呈线性关系。结果表明，这种线性偏差是由于积累的标记物不断发生胞吐作用所致。色谱分析显示，细胞无法代谢蔗糖，而是通过一个对温度敏感但不依赖细胞外钙和镁的过程将其完整释放。通过用[¹⁴C]蔗糖预加载细胞不同时长，然后监测放射性物质在无同位素培养基中的出现情况，对胞吐作用的动力学进行了详细分析。结果表明，对液相胞饮作用及随后的胞吐作用过程进行建模至少需要两个串联的细胞内区室，一个区室体积小且周转非常迅速（巨噬细胞中t1/2 = 5分钟，成纤维细胞中t1/2 = 6 - 8分钟），另一个区室体积明显较大且周转非常缓慢（巨噬细胞中t1/2 = 180分钟，成纤维细胞中t1/2 = 430 - 620分钟）。基于该模型的计算机模拟证实，流出动力学忠实地反映了流入动力学，且流出速率完全解释了积累动力学与线性关系的偏差。此外，区室大小和区室间通量的大小使得胞饮小泡内化的大部分液体通过胞吐作用迅速返回细胞外空间。这一结果为一个此前仅基于形态学和理论考虑而认为必要的过程提供了直接的实验证据。此外，胞饮摄入液体周转非常活跃，以至于即使在孵育的最初几分钟内积累就偏离了线性关系。我们能够证明，胞吐作用的动力学使得能够计算实际的胞饮速率，该速率比从最初10分钟摄取曲线斜率获得的表观初始速率高出近50%。
