Research Of Lung Injury Caused By Activation Of NMDA Receptors And The Related Mechanisms

Chapter 1Activation of N-methyl-D-aspartate receptorinduced acute lung injury in miceBACKGROUNDThe NMDA receptor (NMDAR) is an ionotropic receptor forglutamate. NMDA receptor-dependent neurotoxicity as a final commonpathway for neurologic disorders plays an important role in the acute andchronic brain diseases. Recently, sufficient evidences have beendemonstrated for the presence of NMDA receptor in the lungs, while thefunction is largely unknown.Acute respiratory distress syndrome (ARDS) is a common clinicalsyndrome. The exact pathogenesis of ARDS is undefined up to now. Theneutrophils sequestration and the activation of neutrophils and alveolarmacrophages play key roles in the development of ARDS. Theneutrophils and alveolar macrophages release glutamate when stimulatedaccording to reports. The glutamate concentrations in pulmonary veins ofrat model septicemia are higher than in the pulmonary artery. Thoseevidences suggest that NMDAR could be overactivated in lung tissue of patients suffering from ARDS or some other diseases. The NMDARagonist NMDA induced excitotoxic lung injury in perfused, ventilated ratlungs. The acute lung injury resulted from glutamate in vivo was nearlyabolished by MK801 (dizocilpine), a NMDAR antagonist. These findingsraised the possibility for the participation of signaling via NMDAR in thepathogenesis of acute lung injury. Recognition of the NMDA effect invivo on the lungs and related mechanism could lead to the introduction ofnovel therapeutic approaches.METHODSThe ratios of lung wet/dry weight (W/D) were determined asmeasures of the severity of pulmonary edema, as well as pathologyexamination.Biochemical analysis of myeloperoxidase (MPO) in lung tissue wascarried out using commercial kits, reflecting neutrophil sequestration.The percentages of neutrophils in blood samples got from venacaudalis were obtained to reflect the depletion of neutrophils.The protein of proSP-C and CCTαin lung tissue were analyzed byWestern blot to indicate the function of the alveolar typeⅡcells (ATⅡ).RESULTSNMDA (50 mg/kg, ip) induced the increase of lung W/D. The inflammation caused by NMDA was confirmed in pathology examination,which was attenuated by pretreatment with MK-801 (0.1 mg/kg, ip), theblocker of NMDAR.NMDA (50 mg/kg, ip) caused enhancement of myeloperoxidaseactivity in lung tissue, which was nearly abolished by pretreatment withMK-801.Pretreatment with vinblastine (5 mg/kg, iv) for 4 d leaded todepletion of neutrophils, and resulted in relief of increase of W/D causedby NMDA.NMDA(50 mg/kg, ip)decreased proSP-C and CCTαin lung tissue,and these changes were converted by pretreatment with MK-801.CONCLUSIONNMDA in vivo caused acute lung injury, which was associated withneutrophils accumulation and dysfunction of alveolar typeⅡcells. Chapter 2Expression of N-methyl-D-aspartate receptorin alveolar typeⅡcellsBACKGROUNDOur previous study in chapter 1 demonstrated firstly that NMDA invivo caused acute lung injury, but the mechanism is not completelyunderstood. Alveolar typeⅡcells (ATⅡ) produce and secrete pulmonarysurfactant (PS), a heterogeneous complex of lipid and protein thatstabilizes alveoli at the end of expiration. Pulmonarysurfactant-associated protein C (SP-C) is synthesized only in ATⅡ. Thepro SP-C, a predecessor of SP-C, is a mark of ATⅡ. CTP:phosphocholine cytidylyltransferaseα(CCTα) is the rate-limitingenzyme and regulator of biosynthesis of phosphatidylcholine (PC), amajor component of lipids of PS. NMDA in vivo leaded to dysfunction ofATⅡas evidenced by decrease of pro SP-C and CCTαin lung tissue.Whether NMDA in vitro leads injury to ATⅡis waiting for furtherresearch.NR1 is a necessary component of NMDA receptor. It's reported thatNR1 was expressed in all lung regions (peripheral, mid lung, andmainstem). Additionally, receptor autoradiography with radiolabelledMK801 as the ligand for NMDA receptor sites showed binding sites in rat peripheral lung including alveolar walls. But there is no exact evidencefor the presence of NR1 on ATⅡ.METHODSExpression of NR1 in paraffin sections of human lung wasanalyzed by SABC or double stained immunohistologic methods.ATⅡwere isolated from rat lungs, purified by immunobindingwith IgG coated on the plates, and identified by staining for alkalinephosphatase.Expression of NR1 on ATⅡwas detected by RT-PCR, Western blotand immunocytologic methods.RESULTSNR1 was expressed on ATⅡin paraffin sections of human lung.The mRNA and protein of NR1 were detected in primary ATⅡ.CONCLUSIONNR1 was expressed on ATⅡ. Chapter 3Effect of N-methyl-D-aspartate on pulmonary surfactantsynthesisBACKGROUNDAlveolar typeⅡcells as the major component of alveolar walls,produce and secrete pulmonary surfactant (PS). Surfactantsdeficiency or dysfunction is associated with occurrence anddevelopment of many pulmonary diseases, such as acute respiratorydistress syndrome (ARDS) and pulmonary fibrosis.CTP: phosphocholine cytidylyltransferaseα(CCTα) is therate-limiting enzyme and regulator of biosynthesis ofphosphatidylcholine (PC), a major component of lipids of PS.NR1 was expressed in ATⅡas stated on chapter 3. NMDA invivo leaded to decrease of CCTαin lung tissue. The activation ofNMDA receptor in Lung explants was proved to result in inhibitionof synthesis of PC. Whether NMDA in vitro influences synthesis ofPS waited for further research.METHODSThe quantity of [~3H] choline incorporation was determined byliquid scintillation counting, as measurement of synthesis of PC. CCTαmRNA and protein in microsome were analyzed byRT-PCR and Western blot respectively to indicate the molecularmechanism of changed synthesis of PC.The changes of mRNA of CCTα,SP-A,SP-B,SP-C and SP-Dwere analyzed by Real time PCR.RESULTSThe treatment with NMDA(30μmol/L～100μmol/L) of A549cells leaded to decrease of [~3H] choline incorporation in dosedependent manner.NMDA(10μmol/L～100μmol/L) leaded to decrease of CCTαmRNA and protein in microsome in cultured A549 cells in dosedependent manner.NMDA(100μmol/L) resulted in the decrease of mRNA of CCTα, SP-B, SP-C, and SP-D.CONCLUSIONNMDA in vitro inhibited synthesis of PC through inhibition ofCCTαtranscription and decrease of CCTαprotein in themicrosome in ATⅡ.NMDA in vitro inhibited transcription of SP-B, SP-C, and SP-Din ATⅡ. Chapter 4Signal pathway of inhibition Effect of N-methyl-D-aspartateon CTP: phosphocholine cytidylyltransferaseBACKGROUNDPulmonary surfactants (PS), which is produced mainly, in thealveolar typeⅡcells (ATⅡ) plays a critical role in the respiration.CTP: phosphocholine cytidylyltransferaseα(CCTα) is therate-limiting enzyme and regulator of biosynthesis ofphosphatidylcholine (PC), a major component of lipids of PS.Research of the murine CCT gene (Ctpct) reveals the 5'-terminal～200 bp sequence proximal promoter contains consensus elements forsome nuclear factors including NFκB, indicating potentiallyregulation of the Ctpct gene. But the transcriptional regulation ofCtpct gene by NFκB has not yet been established.Nitric oxide (NO), the product of conversion of L-arginine toL-citrulline, which is catalyzed by the enzyme NO synthase (NOS),functions as an upper stream signaling molecule leading to theactivation of NFκB in the excitotoxity. The activation of NOS playsa key role in the acute lung injury induced by NMDA in perfused,ventilated rat lungs and in vivo. Our previous data in chapter 3 indicated that NMDA inhibitedtranscription of Ctpct gene in cultured ATⅡ. We speculate thatNMDA induced activation of NOS is one of the upper stream eventsin the activation of NFκB as the signaling pathways of excitotoxity,and that activation of NFκB down-regulate the expression of CCTα.METHODSThe detection of p65 nuclear translocation reflecting NFκBactivation and assay of CCTαwere analyzed by Western blot.Biochemical analysis of NOS and NO was carried out usingcommercial kits.RESULTSThe treatment with NMDA(100μmol/L) of A549 cells for 2 h or8 h leaded to activation of NFκB.The pretreatment with SN50 (360μmol/L) of A549 cells for 2 habolished the activation of NFκB caused by NMDA, and protectedCCTαfrom decrease.The treatment with NMDA(100μmol/L) for 7 h inducedactivation of NOS in cultured A549 cells and NO released into themedium. The pretreatment with L-NNA (2.5 m mol/L) for 3 h abolishedthe activation of NFκB resulted from 8 h exposure to NMDA.CONCLUSIONNMDA inhibited expression of CCTαthrough activation ofNFκB. The activation of NOS is an upper stream event of activationof NFκB.