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

1.用Ca^<2+>成像系统观察缺乏酶原颗粒的大鼠腮腺腺泡细胞中Ca^<2 +>动员的模式。Ca^<2+>波的波形与对照组基本相似.免疫印迹结果未显示IP_3受体存在于腮腺酶原颗粒中。腮腺腺泡细胞中未检测到ryanodine受体.利用Ca^2+成像系统观察了受体激动剂诱导的大鼠下颌下腺导管细胞Ca^2+信号传导。用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几乎没有影响。用荧光比率成像系统分析了ATP诱导的HSY细胞[Ca^<2+>]I的振荡变化。上述结果支持了Ca 2+对IP 3受体活性的正反馈和负反馈作用在振荡产生中起重要作用的假说.我们构建了一个含有全长大鼠3型IP 3R连接到GFP（GFP-IP 3R 3）的质粒载体，用于在HSY细胞中表达。荧光共聚焦显微镜显示GFP-IP 3R 3的荧光分布于ER样网状结构中.我们表达了与GFP融合的PKCα（PKCα-GFP），并观察了其在HSY细胞中的易位。结果表明，在PKCα转位的调节中，Ca^<2+>、甘油二酯和磷酸化之间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IP 3R膜结构域，并基于荧光共振能量转移（FRET）分析了HSY细胞中IP_3的细胞内形成. SNARE蛋白，包括VAMP-2，syntaxin-4和SNAP-23，在活细胞中表达为GFP标记的融合蛋白。我们研究了SNARE蛋白的定位、蛋白质-蛋白质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.