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

1.通过多光子显微镜观察唾液腺导管细胞中的Ca^<2+>信号。结果表明，导管含有一定的细胞亚群，这些细胞对这些Ca^2+动员激动剂表现出特别高的敏感性。低浓度的苯丙酮酸、卡巴胆碱和ATP激活不同亚群的导管细胞.为了分析唾液细胞中IP_3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LIBRA，我们将FRET受体EYFP替换为Venus，一种更明亮且pH稳定的突变体。接下来，我们优化了ECFP与IP_3结合结构域之间以及Venus与IP_3结合结构域之间的接头。此外，我们将Venus和ECFP替换为循环排列类型。我们将LIBRA的变体表达到培养的细胞中，并测量皂苷透化的细胞中LIBRA荧光的变化。使用改进的LIBRA，变化率增加到140%左右.采用酶消化法制备大鼠腮腺腺泡细胞，在DMEM-F12培养基中培养。在分散后6 h，培养的腺泡细胞显示出与分散后立即的细胞相似的形态。极性似乎至少维持6 h。分散48 h后，大部分细胞的极性消失，但也有部分细胞保持了极性。在负载fura-2的细胞中测定细胞内Ca^2+浓度（[Ca^2+]i）。用激动剂刺激甚至在培养48小时的细胞中诱导[Ca^<2+>]i的显著增加。用GFP和PKC-GFP的cDNA转染分散的大鼠腮腺腺泡细胞。用不同的试剂和培养基对转染的最佳条件进行了研究。采用Lipofectoamine 2000和Opti MEM转染效果最好。用离子霉素刺激诱导PKC-GFP从细胞质易位到质膜。

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.