Effects And Mechanism Of Exendin-4on NO Production In The Endothelium

PART ⅠEFFECTS OF EXENDIN-4ONNO PRODUCTION IN ENDOTHELIUMObjectiveTo investigate the effects of Exendin-4（Ex-4）on nitric oxide（NO）produced by endothelium cells, and investigate their dose and timedependent relationships.MethodsHUVEC and PIEC were cultured separately in dulbecco’s modifiedeagle medium (DMEM) and Roswell Park Memorial Institute1640(RPMI1640). GLP-1receptor（GLP-1R）was studied using immunofluorescence.HUVEC and PIEC treated with various concentrations of Ex-4. NOproduction measured using Griess Reaction in cell culture supernatants, andmeasured using DAF-FM DA as Fluorescence probe.Results1.GLP-1R was expressed in the HUVEC and PIEC, mainly inmembrane and cytoplasm．2. PIEC cultured with DMEM and RPMI1640, NO production induced by1μg/ml and10μg/ml Ex-4was higher than other groups, NOproduction induced by10μg/ml Ex-4was highest (all P <0.05); NOproduction induced by10μg/ml Ex-4in calcium-free medium (RPMI1640) was higher than normal medium(DMEM)(P <0.01).3. In PIEC, NO production induced by8μg/ml Ex-4was higher thanother groups, NO production induced by8μg/ml Ex-4in calcium-freemedium (RPMI1640) was higher than normal medium(DMEM)(P <0.01).4. NO production at2,4,8,12,24h time point, induced by8μg/mlEx-4in PIEC, was higher than control group. NO production reach the peakat8h (P <0.05).5. Experiments in HUVEC with Ex-4get similar results.Conclusions1. GLP-1R was expressed in endothelial cells, mainly in membraneand cytoplasm.2. Ex-4increased the NO production in a dose and time dependentmanner in endothelial cells. PART ⅡRELATIONSHIP OF ENOS AND NO PRODUCTIONINDUCED BY EXENDIN-4IN ENDOTHELIUMObjectiveTo explore eNOS pathway, investigate the generating mechanism ofNO induced by Ex-4in the endothelial cells.MethodsCreate L-NAME (eNOS inhibitor) pretreatment group and the controlgroup, measure and compare NO production induced by Ex-4in endothelialcells. Representative Western blot analysis demonstrating expression ofphosphorylated eNOS at Ser1177in HUVEC.Results1. Both normal and calcium-free culture medium group showed:1,10,100μmol/l L-NAME pretreatment, NO production induced by Ex-4inendothelial cells significantly reduced compared with the control group (P<0.05).2. Ex-4significantly enhanced phosphorylation of eNOS-Ser1177inHUVEC.ConclusionsEx-4increased NO production in endothelial cells maybe througheNOS pathway, caused eNOS-Ser1177phosphorylation and activated eNOS, resulted in NO production increasing. PART ⅢRELATIONSHIP OF INTRACELLULAR CALCIUM ANDNO PRODUCTION INDUCED BY EXENDIN-4INENDOTHELIUMObjectiveMeasure intracellular calcium concentration, analysis relationship ofcalcium concentration and NO production, to investigate the generatingmechanism of NO production induced by Ex-4in endothelial cells.MethodsCreate eNOS inhibitor L-NAME pretreatment group，Ex-4treatmentgroup and control group，PIEC cultured with RPMI1640. Using DAF-FMDA as fluorescent probe to monitor the NO production, using Fluo-3AMand Frua-2AM as fluorescent probe to monitor the intracellular calcium,using flow cytometry, multifunctional microplate reader and confocalmicroscope measure intracellular calcium concentration and NO production.Analyze relationship of intracellular calcium and NO production inducedby Ex-4in endothelial cells.Results1. Intracellular NO concentration and intracellular calciumconcentration of Ex-4treatment groups was higher compared with thecontrol group (P <0.05). There was no significant change in intracellular NO concentration and intracellular calcium concentration of L-NAMEprtreatment groups.2. After Ex-4treated the endothelial cells, intracellular calciumconcentration changed with NO production increasing prophase. It tendedto increase during the first half of pro phase in NO production increasing (y=0.106x+1.326). The intracellular calcium concentration curve tended tobecome flat at first half of mid phase (y=0.003x+0.374) andlaterphase（y=-0.001x+0.324） in NO production increasing.ConclusionsNO production induced by Ex-4in endothelial cells, may be relevantto intracellular calcium release. Changes of intracellular calcium may playan important role in Ex-4induced endothelial cells NO production throughthe eNOS pathway.