EXOCYTOSIS & RECYCLING IN RBL 2H3 MUCOSAL MAST CELLS

黏膜肥大细胞系RBL-2H3分泌组胺、5-羟色胺 (5-羟色胺，5-羟色胺)和其他炎症介质 对外用过敏原的反应。以往的研究表明，5-羟色胺 5-羟色胺负载的RBL-2H3细胞中的分布可以用三种方法成像 5-羟色胺自体荧光的光子激发(3PE)(Maiti等人，1997， 科学275：530)。在多光子显微镜的这一应用中， 用红外(740 Nm)、超快(脉冲)激光激发； 300 nm 5-羟色胺电子跃迁，减轻色差 以及与深紫外光波长相关的严重光损伤。我们 正在进行RBL-2H3细胞分泌的3PE成像测量。在.期间 对细胞进行成像时，可以观察到典型的褶皱和扩散。 颗粒状5-羟色胺自发荧光消失，检测不到 颗粒运动。单个细胞表现出高度的异质性 释放动力学，通常具有准周期性爆发。邻近的 颗粒消失通常是相关的，其中任何一种都是 空间局域信号或颗粒-颗粒相互作用。之后 个别颗粒释放事件，微弱残留荧光模式 保留最初的颗粒形状通常是可见的，这表明 残留的5-羟色胺仍与颗粒基质结合。双色的 实验中，我们正在使用一种溶质传感器染料同时记录 颗粒分泌时的酸碱史。使用这种染料，pH值会跳跃 我们目前正在研究它们之间的时间关系 细胞受到刺激时也会发生的钙振荡。

The mucosal mast cell line RBL-2H3 secrete histamine, serotonin (5-hydroxytryptamine, 5-HT) and other inflammatory mediators in response to applied allergen. Previously it has been shown that 5-HT distributions can be imaged in 5-HT loaded RBL-2H3 cells using three photon excitation (3PE) of 5-HT autofluorescence (Maiti et al., 1997, Science 275:530). In this application of multiphoton microscopy, an infrared (740 nm), ultrafast (pulsed) laser was used to excite the < 300 nm 5-HT electronic transition, alleviating chromatic aberrations and the severe photodamage associated with deep UV wavelengths. We are making 3PE imaging measurements of RBL-2H3 cell secretion. During imaging the cells are observed to characteristically ruffle and spread as granular 5-HT autofluorescence disappears with no detectable granule movement. Individual cells exhibit highly heterogeneous release kinetics, often with quasi periodic bursts. Neighboring granule disappearances are generally correlated, indic ative of either spatially localized signaling or granule-granule interactions. After individual granule release events, weak residual fluorescence patterns retaining the initial granule shapes are often visible, suggesting leftover 5-HT still bound to the granule matrix. In two-color experiments, we are using a LysoSensor dye to simultaneously record the pH history of granules as they secrete. Using this dye pH jumps are observed and we are presently examining their temporal relation to calcium oscillations that also occur upon cell stimulation.