Visualization of spatio-temporal pattern of IP3/Ca2+ signaling in salivary glands

1.用多光子显微镜观察涎腺导管细胞内的钙信号。结果表明，导管含有一定的细胞亚群，对这些钙动员激动剂表现出特别高的敏感性。此外，低浓度的苯肾上腺素、氨基甲胆碱和三磷酸腺苷激活了不同亚群的导管细胞。为了分析唾液细胞中IP3的产生，构建了基于FRET的生物传感器Libra的变体。为了获得pH稳定的天秤座，我们将FRET受体EYFP替换为Venus，一个更明亮和pH稳定的突变体。接下来，我们优化了ECFP和IP_3结合域以及Venus和IP_3结合域之间的连接子。此外，我们将金星和ECFP替换为它们的循环置换类型。我们在培养细胞中表达了Libra的变异体，并测量了皂苷通透性细胞中Libra荧光的变化。使用改进的天秤座，这一比例的变化增加到约140%。用酶消化法制备分散的大鼠腮腺腺泡细胞，在DMEM-F12培养液中培养。分散后6h，培养的腺泡细胞形态与分散后即刻细胞形态相似。在分散后48h，大部分细胞的极性消失，但也有部分细胞维持了极性。用Fura-2负载细胞测定细胞内[Ca~(2+)]~(2+)浓度。激动剂的刺激使培养48h的细胞内[Ca~(2+)]i显著升高。将GFP和PKC-GFP基因分别导入分散的大鼠腮腺腺泡细胞。用不同试剂和不同的培养基对转化条件进行了研究。脂质体2000和OPTI MEM的转导效果最好。经离子霉素刺激后，PKC-GFP由胞浆移位至质膜。

1. The Ca^<2+> signaling in salivary ductal cells was visualized by multi-photon microscopy. The results indicated that ducts contain a certain subpopulation of cells, which exhibits particularly high sensitivity to these Ca^<2+>-mobilizing agonists. Also, low concentrations of phenylephrine, carbachol and ATP activated different subpopulations of ductal cells.2. To analyze IP_3 production in salivary cells, variants of the FRET-based biosensor LIBRA were constructed. To obtain the pH-stable LIBRA, we replaced the FRET acceptor EYFP to Venus, a more bright and pH-stable mutant. Next, we optimized the linkers between ECFP and IP_3-binding domain and between Venus and IP_3-binding domain. Furthermore, we replaced Venus and ECFP to their circularly permuted types.3. We expressed the variants of LIBRA to cultured cells and measured the changes in LIBRA fluorescence in saponin- permeabilized cells. Using the improved LIBRA, change in the ratio increased to about 140%.4. Dispersed rat parotid acinar cells were prepared by enzymatic digestion and cultured in DMEM-F12 medium. At 6 h after dispersion, the cultured acinar cells showed a similar morphology to that of the cells immediately after dispersion. The polarity seemed to maintain at least for 6 h. At 48 h after dispersion, the polarity was lost in most of the cells, but some cells maintained it. Intracellular Ca^<2+> concentrations ([Ca^<2+>]i) were measured in fura-2-loaded cells. Stimulation with agonists induced significant increases in [Ca^<2+>]i even in the cells cultured for 48 h.5. Dispersed rat parotid acinar cells were transfected with cDNA for GFP and PKC-GFP. The optimal condition for tranfection was examined using various reagents and medium. The used of Lipofectoamine 2000 and Opti MEM was most effective for tranfection. Stimulation with ionomycin induced translocation of PKC-GFP from cytoplasm to plasma membrane.