Imaging of the signal molecules mediating secretory responses in salivary gland cells

1. 利用Ca^<2+>成像系统观察缺乏酶原颗粒的大鼠颈腺泡细胞Ca^<2+>的动员模式。Ca^<2+>波的模式与对照细胞基本相似。免疫印迹数据未提供IP_3受体存在于腮腺酶原颗粒中的证据。此外，腮腺腺泡细胞中未检出良胺受体。应用Ca^<2+>显像系统观察了受体激动剂诱导的大鼠颌下腺导管细胞Ca^<2+>信号通路。肾上腺素能和毒蕈碱激动剂刺激导致导管[Ca^<2+>]I显著增加，但P物质对[Ca^<2+>]I的影响很小或没有影响。利用荧光比值成像系统分析了atp诱导的HSY细胞[Ca^<2+>]I的振荡变化。所得结果支持了Ca^<2+>本身对IP_3受体活性的正负反馈效应在振荡产生中起重要作用的假设。我们构建了一个包含全长大鼠3型IP3R与GFP连接的质粒载体（GFP- ip3r3），用于在HSY细胞中表达。荧光共聚焦显微镜显示GFP-IP3R3的荧光呈er样网状分布。我们表达PKCα与GFP融合（PKCα-GFP），并观察其在HSY细胞中的易位。结果表明Ca^<2+>、二酰基甘油和磷酸化在PKCα.7易位调控中存在复杂的相互作用。我们在HSY细胞中表达了与CFP和YFP融合的IP3R膜结构域，并基于荧光共振能量转移（FRET）分析了IP_3在HSY细胞内的形成。SNARE蛋白，包括VAMP-2、syntaxin-4和SNAP-23，在活细胞中以gfp标记的融合蛋白表达。我们研究了SNARE蛋白的定位、蛋白相互作用和细胞内运输。

1. The pattern of Ca^<2+> mobilization in rat perotid acinar cells lacking zymogen granules was visualized using a Ca^<2+> imaging system. The pattern of the Ca^<2+> wave was essentially similar to that in control cells.2. The immunoblotting data did not provide evidence that IP_3 receptors are present in parotid zymogen granules. In addition, ryanodine receptors were not detected in parotid acinar cells.3. The Ca^<2+> signaling induced by receptor agonists was observed in rat submandibular gland ductal cells using a Ca^<2+> imaging system. Stimulation with adrenergic and muscarinic agonists resulted in significant increases in ductal [Ca^<2+>]I, but substance P had little or no effect on [Ca^<2+>]i.4. The ATP-induced oscillatory changes in [Ca^<2+>]I were analyzed in HSY cells using a fluorescence ratio imaging system. The obtained results support the hypothesis that the positive and negative feedback effects of Ca^<2+> itself on IP_3 receptor activity play an important role in the generation of oscillations.5. We constructed a plasmid vector containing full-length rat type 3 IP3R linked to GFP (GFP-IP3R3) for expression in HSY cells. Fluorescence confocal microscopy showed that the fluorescence of GFP-IP3R3 was distributed to an ER-like reticular network.6. We expressed PKCα fused to GFP (PKCα-GFP) and visualized its translocation in HSY cells. The results suggest a complex interplay between Ca^<2+>, diacylglycerol, and phosphorylation in the regulation of the translocation of PKCα.7. We expressed the IP3R membrane domain fused to CFP and YFP in HSY cells and analyzed the intracellular formation of IP_3 in HSY cells based on the fluorescence resonance energy transfer (FRET).8. SNARE proteins, including VAMP-2, syntaxin-4, and SNAP-23, were expressed as GFP-tagged fusion proteins in living cells. We examined the localization, protein-protein interaction, and intracellular trafficking of the SNARE proteins.