The Effect Of NADPH Oxidase 4 On Hypoxia-induced Atrial Natriuretic Peptide Secretion

Nicotinamide adenine dinucleotide phosphate oxidases(NOXs)are the major enzymatic source of reactive oxygen species(ROS)production in the cardiovascular system.NOXs are transmembrane oxidases with multiple subunits,so far,seven isoforms of NOXs namely NOX1-NOX5,dual oxidase 1(DUOX1)and DUOX2 have been identified.Two NOXs isoforms,NOX2 and NOX4,are expressed in the heart.Studies have shown that NOXs are involved in the processes of cardiac hypertrophy,fibrosis,cell apoptosis,and induce protective effects in the heart under chronic stress by generating ROS.In addition,G protein-coupled receptor agonists,such as endothelin-1(ET-1)trigger the generation of ROS through activation of NOXs and mediate the pathological processes in the cardiovascular system.However,the effect of NOXs on the regulation of atrial natriuretic peptide(ANP)secretion that play an important roles in the anti-inflammation,anti-oxidant,adaption to hypoxia and protects cells,is not clear especially under hypoxic conditions.This study,therefore,is to investigate the effects of NOXs on the regulation of ANP secretion during hypoxia in isolated perfused beating rat atria.The results of this study showed that:1.Hypoxia significantly promoted ET-1 release and upregulated the expression of ET receptor type A and B(ETRA&ETRB)concomitantly with strongly stimulated the secretion of ANP and inhibition of pulse pressure in isolated perfused beating rat atria.Hypoxia-induced ANP secretion was attenuated by BQ123(0.3 ?mol/L)and BQ788(0.3 ?mol/L),antagonists of ETRA and ETRB.Moreover,hypoxia also obviously upregulated expression of NOX4 but not NOX2 and increased the production of H2O2,which were blocked by antagonists of ETRs and NOX4,GLX351322(35.0 ?mol/L),respectively.GLX351322 and an antioxidant,nacetyl cysteine(NAC,15.0 mmol/L),mimicked the inhibitory effects of BQ123,BQ788 and GLX351322 on ANP secretion during hypoxia.2.Exogenous ET-1 clearly increased expression of NOX4 and phosphorylation of cytosolic phospholipase A2(cPLA2)in beating atria under normoxic conditions.The expression of NOX4 induced by ET-1 was abolished by antagonists of ETRs.Similarly,an antagonist of secreted PLA2(sPLA2),varespladib(5.0 ?mol/L),not only inhibited the phosphorylation of cPLA2,but also eliminated expression of NOX4 induced by ET-1.An antagonist of cPLA2,CAY10650(0.12 ?mol/L),may also mimicked the inhibitory effects of ETRs and sPLA2 on ET-1-induced NOX4 expression3.Hypoxia notably increased expression of tyrosine kinase Src,this was repealed by BQ123,BQ788 and GLX351322.The NAC imitated the preventive role of ETRs on hypoxia-induced Src expression.Furthermore,exogenous ET-1 also upregulated Src expression under normoxic conditions and this was revoked by NAC too.4.Hypoxia markedly enhanced phosphorylation of extracellular signal-regulated kinase 1/2(ERK1/2)and Akt(protein kinase B)concomitantly with upregulated expression of GATA4.The hypoxia-induced ERK1/2 and Akt phosphorylation were abrogated by antagonists of ETRs and Src,Src inhibitor 1(1.0 ?mol/L).The expression of GATA4 induced by hypoxia was also completely prevented by ERK1/2 and Akt blockers PD98059(10.0 ?mol/L)and LY294002(10.0 ?mol/L)accompanying by a significant attenuation of ANP secretion during hypoxia.5.Exogenous ET-1 remarkably increased atrial pulse pressure and upregulated expression of silent information regulator 1(sirtuin 1/Sirtl),p-Akt,p62,Keap1 and nuclear factor erythroid-2-related factor 2(Nrf2)under normoxic conditions,which was blocked by GLX351322.An antagonist of Sirtl,EX527(0.25 ?mol/L),also abolished the effects ET-1 on p-Akt,p62,Keapl and Nrf2 expression.These results were also displayed in the hypoxic atria.6.Atrial activating transcription factor(ATF)3 and 4 protein levels were evidently increased not only by exogenous ET-1 under normoxia but also by hypoxia,and these effects were abolished by an antagonist of Nrf2,ML385(10.0 ?mol/L).7.The expression of T cell factor(TCF)3 as well as 4 and lymphoid enhancer factor 1(LEF1)were also upregulated by exogenous ET-1 under normoxia or by hypoxia concomitantly with stimulating of ANP secretion.The exogenous ET-1-or hypoxia-induced expression of TCFs and LEF1 were abolished by ML385 simultaneously by a dramatic attenuating of ANP secretion.These results indicated that:1.NOX4-Src modulated by endogenous ET-1 regulates ANP secretion by activating ERK1/2-and Akt-GATA4 signaling in isolated beating rat hypoxic atria.2.NOX4-Sirt1-Nrf2 is also participated in the regulation of ANP secretion induced by hypoxia through activation of TCF3/and TCF4/LEF1 via ATF3 and ATF4 signaling.