The Role Of Cystine/glutamate Transporter In Lipopolysaccharide Induced Glutamate Releasing In The Lungs And The Primary Investigation On Its Mechanism

Charpter1 The roles of cystine/glutamate transporter in Glutamate release from different lung cells induced by lipopolysaccharideObjective:To investigate the diverse expression of cystine/glutamate transporter(Xc^-) on the different lung cells and its role in Glutamate(Glu) release induced by lipopolysaccharide(LPS).Methods:1.Human bronchial epithelial cell line(HBEc),human lung adenocarcinoma cell line(A549) and human embryonic lung fibroblast cell line(HLF-02) were selected as bronchial epithelial cells,alveolar epithelial cells and fibroblasts,respectively.And the mRNA of Xc^-expressions in these cells were detected by RT-PCR.2.Glu concentration and lactate dehydrogenase(LDH) activity in culture medium were detected by using colorimery method respectively. Both treatment of L-HCA,an inhibitor of Xc^-,and culture medium without cystine were applied to clarify the role of Xc^- in Glu release from lung cells induced by LPS.The glutathione(GSH) contents were also measured to further confirm the Xc^- -blocking- effect in A549 cell line.3.The in vivo effect of L-HCA on LPS-induced Glu release in the lungs were investigated.Bronchoalveolar lavage fluid(BALF) in mice were collected and the Glu concentrations were measured as above.Results:1.Glu release from A549 cell line were significantly increased by the treament of LPS for 8h.This effect was dose dependent between 0.3ng/ml and 100.0ng/ml,and the maximum increasement in Glu release from A549 cell line was reached after the 1.0ng/ml LPS treatment for 8h.2.LPS(1.0ng/ml) treatment for 8h increased the Glu release in A549, HBEc and HLF-02 cell line,and the increement of Glu release from A549 cell line was the most significant(reached to 391.5%compared with control group).3.LDH concentration in the culture medium of these cell lines was also determined and showed no difference between the LPS treatment and control group,which indicated that the concentration of LPS and the treatment duration used in this experiment would not cause injury to these cells,thereby not driving a non-specific intracellular Glu release.4.The mRNA expressions of both xCT,the light chain of Xc^- and 4F2hc,the heavy chain of Xc^-,were positive in A549,HBEc and HLF-02 cell line as confirmed by RT-PCR.In normal condition,the mRNA expression of xCT in HLF-02 and that of 4F2hc in HBEc were most obvious.However,after the treatment by LPS,mRNA expressions of xCT and 4F2hc were raised especially in A549 cell line.5.In A549,HBEc and HLF-02 cells,pre-incubation with L-HCA (0.9mg/ml) for 30min did not change the Glu release from the cells but abolished the Glu release response to 8h treatment of LPS.This data implicated that Xc^- mediates LPS-induced Glu release from these lung cells.To further investigate that Glu release from lung cells-induced by LPS was dependent on Xc^-,both treatment of L-HCA,an inhibitor of Xc^-, and culture medium without cystine were applied in A549 cells.The results showed that LPS could promote synthesis of GSH in A549 cells which was inhibited by L-HCA interference;the promotion of Glu release and GSH synthesis induced by LPS were no longer occurrence after removal of extracellular cystine.6.Glu concentration was notably augmented in BALF in mice injected intraperitoneally by LPS(10.0mg/kg) for 6h.However, pretreatment with L-HCA(4.5mg/kg) for 30min significantly attenuated the LPS induced increment of Glu concentration.Conclusions:1.LPS stimulated Glu release from different lung cell lines in a dose-dependent and time-dependent manner,which indicates that Glu release from various kinds of lung cells might involve in the pathogenesis of LPS-induced acute lung injury(ALI). 2.The mRNA expressions of xCT and 4F2hc in HBEc,A549 and HLF-02 cells suggested that Xc^- was widespread expression in different lung cells.3.This study confirmed firstly that Glu release from lung cells-induced by LPS was mediated by Xc^-.Charpter2The mechanism of Glutamate release from A549 cells-induced by LPSObjective:To preliminarily investigate the mechanism of Glu release from A549 cells-induced by LPS so that to provide a new target for ALI treatment.Methods:1.A549 cells were selected as research object.And The concentration of Glu released from A549 cells induced by LPS were determined in culture medium after pre-incubation with Ca²⁺ chelator(EGTA), actinomycin D(Act D) and NOS inhibitor(L-NAME) respectively.2.The activities of glutamic oxaloacetic transaminase(GOT) and glutamic pyruvic transaminase(GPT) as well as the content of L-glutamate dehydrogenase(L-GDH) in A549 cells -treated by LPS were measured to investigate the changes of intracellular enzymes-related to Glu biosynthesis.The concentration of extracellular NO were determined by NO assay kit.3.The mRNA expressions of xCT,4F2hc,GOT and L-GDH in A549 cells were detected by real time-PCR.Results:1.Pretreatment with ActD(3.5μg/ml) for 30min significantly inhibited LPS(1.0ng/ml) induced Glu release from A549 cells.While incubation with EGTA(18.7mg/ml) or L-NAME(0.3mg/ml) for 30 min did not change the Glu release or the Glu release response to LPS.2.To further study enzymes associated with Glu synthesis in lung cells,the activities of GOT and GPT as well as the content of L-GDH were measured in LPS challenged A549.The results showed that LPS enhance GOT activity and increase L-GDH content,but not affect GPT activity and NO production.3.The mRNA expressions of xCT,4F2hc,GOT and L-GDH in A549 cells were up-regulated by LPS.Conclusions:1.The results that the mRNA expressions of xCT,4F2hc,L-GDH and GOT were elevated by LPS and that ActD inhibiting Glu release from A549 cells induced by LPS indicated that LPS-promoted Glu release from A549 cells might be regulated by gene transcription.2.LPS-induced Glu release might be involved in the elevation of both the activity of GOT and the content of L-GDH.3.It is the first time to confirm that extracellular Ca²⁺ was not related with Glu release from A549 cells induced by LPS.4.This experiment preliminarily verified that NO did not play a part in the process of LPS-induced Glu release from A549,which manifested that the expression of Xc^- gene was mediated by LPS of the low concentration in present study through other signal pathway.