Signaling Mechanisms Of PGD₂-and L-PGDS-promoted ANP Secretion In Beating Rat Atria

It is well known that activated phospholipase A2(PLA2)leading to an accumulation of unesterified arachidonic acid(AA)in the heart,which could be converted into prostaglandins(PGs).All kinds of the PGs bind to different G protein coupled receptors(GPCR)with different affinities and to participate in the regulation of cardiac hypertrophy,myocardial fibrosis,myocardial infarction and other cardiac diseases.Prostaglandin D2(PGD2)is a member of the PGs that it was found by Hamberg in 1973.Hematopoietic PGD synthase(H-PGDS)and lipocalin-type PGD synthase(L-PGDS)are two enzymes that converte prostaglandin H2(PGH2)into PGD2.PGD2 may play both pro-and anti-inflammatory roles through two distinct receptors,the D-type prostanoid receptor(DPi)and the chemoattractant receptor-homologous molecule expressed on Th2 cells(CRTH2,also named DP2).Studies shown that PGD2 may activates peroxisome proliferator-activated receptor gamma(PPARy)by its metabolite,15-deoxy-?12,14-PGJ2(15d-PGJ2),and to play important roles in anti-inflammation,anti-apoptosis and stabilization of the atherosclerosis.The protective role on the ischemic/hypoxic heart has also been demonstrated.As an endocrine gland,cadiac atrium synthesize and secrete atrial natriuretic peptide(ANP).There are many factors may regulate atrial ANP secretion,while increased body fluids-or blood volume-induced atrial wall stretch has been recognized an important stimuli for ANP secretion.It has been suggested that effects of atrial stretch,ischemia,and endothelin on the regulation of ANP secretion were related with AA metabolism and to production of prostaglandins(PGs),such as PGF2a and PGE2 stimulated the ANP synthesis and secretion in cultured rat atrial myocytes;PGF2?,PGE2,and PGI2 stimulated the ANP release from cultured neonatal cardiomyocytes in a dose-dependent manner;PGE2 increased the ANP secretion in perfused rabbit atria or in beating neonatal ventricular cardiomyocytes.In addition,it was reported that PGD2 significantly increased atrial ANP secretion and play a positive inotropy in isolated beating rat atria.However,the mechanism by which PGD2-increased atrial ANP secretion is not well known.Therefore,the purpose of the present study was to investigated the mechanism of PGD2-increased atrial ANP secretion and its mechanical dynamics in isolated perfused beating rat atria.The roles of PPARy,mitogen-activated protein kinase(MAPK)/extracellular signal-regulated kinase(ERK)and phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt)signaling on the regulation of PGD2-induced atrial ANP secretion were also been determined in the present study.Results of the present study are as follows:1.Different doses of PGD2(0.1,1.0 and 10.0 ?mol/L)significantly increased atrial ANP secretion and mechanical dynamics(all of P<0.05 vs.control period respectively).PGD2(1.0?mol/L)induced atrial ANP secretion and mechanical dynamics were showed time-dependent manner(P<0.05 vs.control period).2.PGD2-increased atrial ANP secretion and mechanical dynamics were completely blocked by AH 6809(1.0 ?mol/L)and AL 8810(1.0 ?mol/L),antagonists of PGD2 and PGF2a receptors(all of P>0.05 vs.control period respectively).3.15d-PGJ2(0.1 ?mol/L),a PGD2 metabolite,dramatically increased atrial secretion of ANP(P<0.05 vs.control period)and mimicked the effect of PGD2 on the regulation of ANP secretion;but 15d-PGJ2 clearly inhibited the atrial mechanical dynamics(P<0.05 vs.control period).In addition,15d-PGJ2-induced atrial ANP secretion and mechanical dynamics were also blocked by AH 6809(all of P>0.05 vs.control period respectively).4.PGD2 significantly upregulated the expression of atrial PPAR?(P<0.05 vs.control group)and this was abolished by an antagonist of PPARy GW 9662(P<0.05 vs.PGD2 group).In addition,PGD2-and 15d-PGJ2-induced atrial ANP secretion and mechanical dynamics were also blocked by GW 9662(all of P>0.05 vs.control period respectively).5.PD 98059(10.0 ?mol/L),an antagonist of MAPK/ERK,obviously attenuated PGD2-induced atrial ANP secretion(P<0.05 vs.control period),and completely blocked the positive inotropy induced by PGD2.(P>0.05 vs.control period).The PI3K/Akt antagonist LY 294002(1.0 ?mol/L)also significantly decreased PGD2-induced atrial ANP secretion(P<0.05 vs.control period)without changes of PGD2-induced positive inotropy(P<0.05 vs.control period).These results indicate that:1.PGD2 promotes atrial ANP secretion and to play a positive inotropy by activating of PPARy signaling in beating rat atria;2.MAPK/ERK and PI3K/Akt signaling are partially involved in regulation of PGD2-induced atrial ANP secretion.Lipocalin-type prostaglandin D synthase(L-PGDS)is a low-molecular mass glycoprotein.Recently,L-PGDS is found to be expressed in the heart and being advocated as a novel biomarker for cardiovascular risk prediction.Hypoxia-inducible factor-1?(HIF-1?),a heterodimeric transcription factor,activates hypoxia responsive genes.It has been demonstrated that hypoxia strongly stimulates the secretion of ANP,leading to cellular adaptation in hypoxia and protection of the ischemic heart.Recently,it was reported that expression both of HIF-1? and peroxisome proliferator-activate receptor ?(PPAR?)were upregulated in human and mouse cardiac hypertrophy.HIF-1? directly activated PPAR? transcription and which was shown to be a key downstream effector.Moreover,L-PGDS converts prostaglandin H2(PGH2)into prostaglandin D2(PGD2)and its metabolite,15-deoxy-?12,14-PGJ2(15d-PGJ2),is an endogenous ligand for PPAR?,protecting the heart against ischemia-reperfusion injury.Nevertheless,the effect of HIF-1?-PPAR? signaling on the regulation of hypoxia-induced ANP secretion in hypoxic atria is not clear.In addition,the role of L-PGDS on the regulation of HIF-1?-induced PPARy transcription in atria is not yet to be define.It is hypothesis that HIF-1? activates PPAR? transcription through controlling the L-PGDS activity and to promotes ANP secretion in hypoxic atria.Purpose of the present study,therefore,to confirm this hypothesis in isolated perfused beating rat acute hypoxic atria by using radioimmunoassay,Western blot technique,the physiology and pharmacology methods.The results of the present study as follows:1.Hypoxia potently stimulated ANP secretion and showed a time-dependent manner in isolated beating rat atria.The peak of hypoxia-induced ANP secretion was reached at the end of hypoxic fourth cycle(about 48 min after hypoxia)(P<0.05 vs.control period),concomitant with decreased atrial pulse pressure(P<0.05 vs.control period).2.Hypoxia significantly increased atrial HIF-la protein level(P<0.05 vs.control group)concomitant with obviously increased atrial ANP secretion(P<0.05 vs.control period).While hypoxia-increased atrial HIF-1? level was completely abolished by a HIF-la antagonist rotenone(0.5 ?mol/L)(P<0.05 vs.hypoxia group)concomitant with dramatically attenuation of hypoxia-induced ANP secretion(all of P<0.05 vs.control and fourth cycle of hypoxic period).3.Hypoxia markedly increased atrial L-PGDS protein level(P<0.05 vs.control period)and which was substantially attenuated by AT 56(5.0 ?mol/L)as well as HQL 49(5.0 ?mol/L),antagonists of L-PGDS(both of P<0.05 vs.control period;both of P<0.05 vs.hypoxia group respectively).Hypoxia-induced atrial ANP secretion was also dramatically attenuated by AT 56 and HQL 49 pretreatment.4.The PPARy antagonist GW9662(0.1 ?mol/L)mimicked the inhibitory roles of rotenone and AT 56 on hypoxia-induced atrial ANP secretion,i.e.it was also significantly attenuated hypoxia-induced atrial ANP secretion(P<0.05 vs.control;P<0.05 vs.hypoxia;P>0.05 vs.rotenone or AT 56,respectively).5.Hypoxia notably increased atrial PPARy protein level(P<0.05 vs.control)and it was also completely abolished by rotenone concomitantly with down-regulation of atrial L-PGDS protein level(P>0.05 vs.control;P<0.05 vs.hypoxia group).Moreover,the inhibitory effect of rotenone plus AT 56 on the hypoxia-induced atrial PPARy protein level was not showed significant differences compared with rotenone or AT 56 alone respectively(all of P>0.05 vs.rotenone or AT 56).These results indicate that:1.HIF-1? significantly increases PPARy transcription activity by activation of L-PGDS in isolated perfused beating rat hypoxic atria.2.HIF-la-L-PGDS-PPAR? signaling is one of the important mechanism,at least in part in,by which hypoxia-promoted secretion of ANP in rat atria.