Center for Commercialization of Fluorescence Technologies, Department of Molecular Biology and Immunology, University of North Texas, Fort Worth, TX, USA.
Purinergic Signal. 2012 Mar;8(1):59-70. doi: 10.1007/s11302-011-9259-2. Epub 2011 Sep 1.
Nucleotide release constitutes the first step of the purinergic signaling cascade, but its underlying mechanisms remain incompletely understood. In alveolar A549 cells much of the experimental data is consistent with Ca(2+)-regulated vesicular exocytosis, but definitive evidence for such a release mechanism is missing, and alternative pathways have been proposed. In this study, we examined ATP secretion from A549 cells by total internal reflection fluorescence microscopy to directly visualize ATP-loaded vesicles and their fusion with the plasma membrane. A549 cells were labeled with quinacrine or Bodipy-ATP, fluorescent markers of intracellular ATP storage sites, and time-lapse imaging of vesicles present in the evanescent field was undertaken. Under basal conditions, individual vesicles showed occasional quasi-instantaneous loss of fluorescence, as expected from spontaneous vesicle fusion with the plasma membrane and dispersal of its fluorescent cargo. Hypo-osmotic stress stimulation (osmolality reduction from 316 to 160 mOsm) resulted in a transient, several-fold increment of exocytotic event frequency. Lowering the temperature from 37°C to 20°C dramatically diminished the fraction of vesicles that underwent exocytosis during the 2-min stimulation, from ~40% to ≤1%, respectively. Parallel ATP efflux experiments with luciferase bioluminescence assay revealed that pharmacological interference with vesicular transport (brefeldin, monensin), or disruption of the cytoskeleton (nocodazole, cytochalasin), significantly suppressed ATP release (by up to ~80%), whereas it was completely blocked by N-ethylmaleimide. Collectively, our data demonstrate that regulated exocytosis of ATP-loaded vesicles likely constitutes a major pathway of hypotonic stress-induced ATP secretion from A549 cells.
核苷酸释放构成了嘌呤能信号级联的第一步，但它的潜在机制仍不完全清楚。在肺泡 A549 细胞中，大量的实验数据与 Ca(2+)调节的囊泡胞吐作用一致，但缺乏这种释放机制的明确证据，并且提出了替代途径。在这项研究中，我们通过全内反射荧光显微镜检查 A549 细胞中的 ATP 分泌，以直接观察 ATP 装载的囊泡及其与质膜的融合。用吖啶橙或 Bodipy-ATP 标记 A549 细胞，这是细胞内 ATP 储存位点的荧光标记物，并对消失场中的囊泡进行延时成像。在基础条件下，单个囊泡偶尔会出现荧光的准瞬时丧失，这与囊泡与质膜融合并分散其荧光货物的自发融合相符。低渗应激刺激（渗透压从 316 降低至 160 mOsm）导致胞吐事件频率短暂增加几倍。将温度从 37°C 降低至 20°C 时，在 2 分钟的刺激过程中经历胞吐作用的囊泡比例从约 40%急剧降至≤1%。用荧光素生物发光测定法进行平行的 ATP 外排实验表明，囊泡转运的药理学干扰（布雷菲德菌素、莫能菌素）或细胞骨架的破坏（诺考达唑、细胞松弛素）显著抑制了 ATP 的释放（高达约 80%），而 N-乙基马来酰亚胺则完全阻断了 ATP 的释放。总的来说，我们的数据表明，受调控的 ATP 装载囊泡的胞吐作用可能是低渗应激诱导 A549 细胞 ATP 分泌的主要途径。
