EXOCYTOSIS & RECYCLING IN RBL 2H3 MUCOSAL MAST CELLS

粘膜肥大细胞系RBL-2 H3分泌组胺、血清素 （5-羟色胺，5-HT）和其他炎症介质， 对应用过敏原的反应。 以前已经表明，5-HT 在5-HT负载的RBL-2 H3细胞中，可以使用三种不同的成像方法对分布进行成像。 5-HT自发荧光的光子激发（3 PE）（Maiti等人，1997, Science 275：530）。 在多光子显微镜的这种应用中， 红外（740 nm），超快（脉冲）激光被用来激发< 300 nm 5-HT电子跃迁，缓解色差 以及与深紫外线波长相关的严重光损伤。 我们 正在进行RBL-2 H3细胞分泌的3 PE成像测量。 期间 观察到细胞的特征性褶皱和扩散 随着颗粒状5-HT自发荧光消失， 颗粒运动 单个细胞表现出高度异质性 释放动力学，通常具有准周期性爆发。 相邻 颗粒的消失通常是相关的， 空间定位信号或颗粒-颗粒相互作用。 后 单个颗粒释放事件，弱残留荧光模式 保留初始颗粒形状通常是可见的，这表明 剩余的5-HT仍然与颗粒基质结合。 双色 在实验中，我们使用LysoSensor染料同时记录 颗粒分泌时的pH历史。 使用这种染料pH值会跳变 我们目前正在研究它们的时间关系， 钙振荡也发生在细胞刺激时。

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.