Cysteinyl Leukotriene Receptor 2 Modulates The Differentiation Of Rat Glioma C6 Cells, And Screening For Its SiRNA And New Antagonists

Objective:In rat glioma C6 cells, cysteinyl leukotriene receptor 2 (CysLT2 receptor) is main subtype of the CysLT receptors. The properties of responses of the CysLT2 receptor in C6 cells are unclear. To investigate the response properties of the CysLTs receptor in C6 cells, in this study we investigated the followings:(1) whether CysLT2 receptor regulates C6 cell differentiation; (2) screening siRNA interference lentiviral vector for CysLT2 receptor for further studies; (3) primarily screening compound with CysLT2 receptor-antagonizing activity from a series of synthetic compounds using the agonist LTD4-enhanced intracellular Ca2+ as an indicator.Methods:CysLT2 receptor mRNA expression was detected by RT-PCR; the distribution of CysLT2 receptor in C6 cells was observed by Immunocytochemical staining. To determine the effects of LTD4 and CysLT1 receptor antagonists, C6 cell morphology in bright field was observed by microphotography; GFAP expression was detected by Western blotting analysis. To screen CysLT2 receptor siRNA, lentivirus vector infection was observed by fluorescence microscopy; CysLT2 receptor mRNA or protein expression was observed by RT-PCR and real-time PCR, or Western blotting analysis;CysLT2 receptor-mediated changes in intracellular Ca2+ was measured by confocal laser microscopy. To screen the compounds with CysLT2 receptor-antagonizing activity, the agonist LTD4-enhanced intracellular Ca2+ was used as an indicator. Bay u9773, a CysLT1/CysLT2 receptor non-selective antagonist, and AP-100984, a CysLT2 receptor antagonist, were used as controls.Results:RT-PCR analysis showed a higher expression of CysLT2 receptor in C6 cells, and the CysLT2 receptor was mainly distributed in the cytoplasm of C6 cells. The agonist LTD4 alone did not induce morphological changes and GFAP protein expression in C6 cells. The CysLT1 receptor antagonist montelukast alone did not induce differentiation of C6 cells as well. However, combination of LTD4 with montelukast induced prolongation and increased numbers of processes in C6 cell, indicating cell differentiation. The lentiviral interference vector (SH3) for CysLT2 receptor siRNA was successfully screened. SH3 inhibited the expression of CysLT2 receptor mRNA and protein, and LTC4-enhanced intracellular Ca2+. LTD4 at 1μmol/L significantly increased intracellular calcium in C6 cells, and the maximal effect was about 37.5% of ATP, a positive stimulus. Thus, these conditions were used as the indicator for screening the compounds with CysLT2 receptor- antagonizing activity. LTD4-induced increase of intracellular Ca2+ was blocked by the CysLT2 receptor antagonist AP-100984, but not by CysLT1 receptor antagonists. Among the synthetic compounds, Dxw-1,2,13,23,29 and 30 inhibited LTD4-induced increase of intracellular Ca2+.Conclusion:(1) C6 cells mainly express CysLT2 receptor. The agonist LTD4 or CysLT1 receptor antagonists alone can not induce C6 cell differentiation, while their combination induces the differentiation. This suggests that CysLT2 receptor may promote C6 cell differentiation. (2) The lentiviral interference vector for CysLT2 receptor siRNA has been screened successfully, and has been confirmed by changes in the expression of CysLT2 receptor mRNA and protein as well as intracellular Ca2+ signals. This will provide a tool for further studies of CysLT2 receptor. (3) Using LTD4-enhanced intracellular Ca2+ as the indicator, a series of compounds have been screened; the compounds, Dxw-1,2,13,23,29 and 30, possess antagonist activity for CysLT2 receptor. This is a cue for further discovery of CysLT2 receptor antagonists.