Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA, USA.
Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA, USA; Faculty of Medicine, University of Oslo, Oslo, Norway; Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway.
Ocul Surf. 2020 Jul;18(3):470-482. doi: 10.1016/j.jtos.2020.04.011. Epub 2020 Apr 30.
Specialized pro-resolving lipid mediator resolvin (Rv) E1 stimulates secretion including mucins from conjunctival goblet cells. RvE1 can use both its ChemR23 receptor and the LTB receptor BLT1 to increase [Ca]. The purpose of this study was to determine the expression of ChemR23 and BLT1 and receptors on conjunctival goblet cells and the respective roles these two receptors play in goblet cell responses to RvE1.
Goblet cells were cultured from male rat or human conjunctiva from both sexes. Western blotting analysis, reverse transcription PCR and immunofluorescence microscopy were used to demonstrate the expression of ChemR23 and BLT1 in conjunctival goblet cells. High molecular weight glycoprotein secretion was determined using an enzyme-linked lectin assay. Signaling pathways were studied by measuring the increase in [Ca] using fura 2/AM.
ChemR23 and BLT1 and receptors were present on both rat and human conjunctival goblet cells. The BLT1 inhibitors LY293111 and U75302 significantly blocked RvE1-and LTB-stimulated [Ca] increase. RvE1-and LTB-stimulated [Ca] and secretion increases were blocked by BLT1-targeted siRNA. RvE1-stimulated [Ca] and secretion increases were also blocked by ChemR23-targeted siRNA. Addition of RvE1 2 min before or simultaneously with LTB desensitized the LTB [Ca] response. Addition of RvE1 and LTB simultaneously caused secretion that was decreased compared to either response alone.
RvE1, in addition to the ChemR23 receptor, uses the BLT1 receptor to increase [Ca] and stimulate secretion in both rat and human cultured conjunctival goblet cells.
专门的促解决脂质介质分辨素（Rv）E1 刺激包括结膜杯状细胞分泌粘蛋白。RvE1 可以使用其 ChemR23 受体和 LTB 受体 BLT1 来增加 [Ca]。本研究的目的是确定结膜杯状细胞上 ChemR23 和 BLT1 及其各自受体的表达，以及这两个受体在 RvE1 刺激杯状细胞反应中的作用。
从雄性大鼠或来自两性的人结膜中培养杯状细胞。使用 Western 印迹分析、逆转录 PCR 和免疫荧光显微镜来证明结膜杯状细胞中 ChemR23 和 BLT1 的表达。使用酶联凝集素测定法测定高分子量糖蛋白的分泌。通过使用 fura 2/AM 测量 [Ca] 的增加来研究信号通路。
ChemR23 和 BLT1 及其受体存在于大鼠和人结膜杯状细胞上。BLT1 抑制剂 LY293111 和 U75302 显著阻断了 RvE1 和 LTB 刺激的 [Ca] 增加。BLT1 靶向 siRNA 阻断了 RvE1 和 LTB 刺激的 [Ca] 和分泌增加。RvE1 刺激的 [Ca] 和分泌增加也被 ChemR23 靶向 siRNA 阻断。在 LTB 之前或同时加入 RvE1 可使 LTB [Ca] 反应脱敏。同时加入 RvE1 和 LTB 会导致与任一反应单独相比分泌减少。
除了 ChemR23 受体外，RvE1 还使用 BLT1 受体增加 [Ca] 并刺激大鼠和人培养的结膜杯状细胞分泌。
