The Roles And Mechanisms Of Alpha1Adrenergic Receptors And Calcium Desensitization In Vascular Hyporeactivity Mediated By Interleukin-1Beta Following Sepsis

Objective:Sepsis is one of the oldest and most elusive syndromes in clinic. Despite recentadvances in drugs and treatment for sepsis therapy, the mortality from sepsis remains veryhigh. So it is very important to further elucidate the pathogenesis of sepsis, and find newand specific targets for its therapy.Vascular hyporeactivity to vasoconstrictors or vasodilators following sepsis plays animportant role in the development of refractory hypotension and high mortality of sepsis.Current opinions about the occurrence of vascular hyporeactivity might be related todesensitization of receptors (downregulation of expression or affinity); membraneoushyperpolarization of vascular smooth muscle cells (VSMCs) which is induced byoveractivation of potassium channels of VSMCs, and desensitization of calcium of VSMCswhich was raised by our lab.As one of the key cytokines generated in sepsis, in vivo and in vitro studies showedthat IL-1βcould mediate the desensitization of vascularα1adrenergic receptor (α1ARs)which is one of vital factors modulate vascular hyporeactivity. Further studies showed thatIL-1βmediated desensitization ofα1ARs by downregulation of the expression but not theaffinity ofα1ARs, however, the mechanism that how IL-1βmodulatesα1ARs expressionincluding which pathway and which transcription factors involved in are still unknown.α1ARs were classified into three subtypes includingα1A, α1B, α1DAR, their distributions,expressions and mechanisms of expression regulation are species and tissues dependent.It’s well known that IL-1βregulates gene expression mainly through nuclear factor-κB(NF-κB), p38mitogen-activated protein kinase (p38MAPK) and c-Jun Nterminus proteinkinase (JNK) pathway. Also, IL-1βcould active other pathways such as extracellular signal-regulated kinase (ERK)1/2，Janus kinase–signal transducer and activator oftranscription (JAK–STAT) etc. to regulate gene expression in different cells. The promotersofα1ARs in VSMCs of rat had been cloned:α1AAR expression are mainly regulated bySp1，AP1，AP2，NFκB，CTF/NF1，NF/IL-6;α1BAR expression are mainly regulated bySp1，AP2，NF1，CP1，HNF-1/5，CREB;α1DAR expression are mainly regulated by Sp1，AP2. The common transcription factors are Sp1and AP2. Previous results showed thatIL-1βregulated all three subtypes ofα1ARs expression in VSMCs of rat. WhetherIL-1βregulates all three subtypes ofα1ARs expression in VSMCs of septic rat through Sp1and AP2or not? Need investigation.Just like hemorrhagic shock, our pilot study found that there was also vascular calciumdesensitization following sepsis in rabbits, PKC and Rho kinase played a crucial role in theoccurrence of calcium desensitization, and Rho kinase seemed more important than PKC.Basic researches show that the activity of Rho kinase is regulated by RhoA, RhoA isregulated by RhoGEFs, RhoGEFs are regulated by Gαq/11and Gα12/13(VSMCs). Themain family members of RhoGEFs exsit in VSMCs are p115RhoGEF，PDZ-RhoGEF，LARG and p63RhoGEF. The activities of p115RhoGEF，PDZ-RhoGEFand LARG areregulated by Gα12/13, and the activities of LARG and p63RhoGEF are regulated byGαq/11. The questions are presented: Whether IL-1βis involved in vascular calciumdesensitization following sepsis, or not? And how does IL-1βregulate it? They are notknown untilnow.In order to find the answers to these questions, using a LPS-induced septic rabbit and aCLP-induced septic rat model, their SMAs and VSMCs as research objects, and a lot ofexperimental methods as tools, we pursued the molecular mechanisms underlying the effectof IL-1βon vascularα1ARs desensitization and calcium desensitization in the presentstudy.Methods:Part I. To identify whether IL-1βmediates vascularα1adrenergic receptors andcalcium desensitization following sepsis1. To identify whether IL-1βmediates vascularα1adrenergic receptors and calciumdesensitization following sepsis in rabbit(1) Septic rabbit model was induced by LPS intravenously infusion. At0,0.5,1,2,4h after LPS administration, the superior mesenteric arteries (SMAs) with intact endotheliumwere adopted to exam theα1ARs and Ca2+sensitivity,α1ARs expression and serous IL-1βlevels were assayed, the correlations of IL-1βlevels,α1ARs and calcium sensitivity, andα1ARs expression were analyzed.(2) IL-1ra was intravenously given to LPS-induced septic rabbit, and its effects onα1ARs sensitivity, Ca2+sensitivity andα1ARs expression were observed.(3) Under sterile condition, the SMAs with intact endothelium from normal rabbitswere incubated with different doses of IL-1β. Then theα1ARs sensitivity, Ca2+sensitivityandα1ARs expression of SMAs were measured.2. To identify whether IL-1βmediates vascularα1adrenergic receptors and calciumdesensitization following sepsis in rat(1) Septic rat model was induced by cecal ligation and puncture (CLP). At0,3,6,12hafter CLP process, the superior mesenteric arteries (SMAs) with intact endothelium wereadopted to exam the vascular sensitivity ofα1ARs and calcium. What′s more, theexpression ofα1ARs and plasma IL-1βlevels were assayed, the correlations ofIL-1βlevels,α1ARs and calcium reactivity, andα1ARs expression were analyzed.(2) Rat vascular smooth muscle cells (VSMCs) were cultured primarily by tissue blockadhering wall method. VSMCs were incubated with different doses(0,1,10,100ng/ml) ofIL-1βto observe its effect on expression ofα1ARs and phosphorylation of MLC20ofVSMCs.Part II. To explore the regulatory mechanism of IL-1βon vascular calciumdesensitization following sepsis1. To explore the regulatory mechanism of IL-1βon vascular calciumdesensitization following sepsis in rabbitIncubated SMAs of rabbit with IL-1βas above illustrated, to observe(1) Effects of the specific agonists and antagonists of PKC and Rho kinase on thecalcium sensitivity of the SMAs incubated with IL-1β.(2) Effect of IL-1βon the phosphorylation of MLC20in SMAs.(3) Effects of IL-1βon the activities of PKC and Rho kinase in SMAs.2. To explore the regulatory mechanism of IL-1βon vascular calcium desensitizationfollowing sepsis in rat Incubated VSMCs of rat with IL-1βas above illustrated, to observe(1) Effect of IL-1βon the activity of Rho kinase of VSMCs.(2) Effects of IL-1βon the expression of Gα11, Gαq, Gα12, Gα13of VSMCs.(3) Effects of IL-1βon the activities of total RhoGEF, PDZ-RhoGEF and p63RhoGEFof VSMCs.(4) Effect of NF-κB, p38MAPK and JNK pathway inhibitors on expression of Gα11and Gα12of the VSMCs incubated with IL-1β.(5) Effect of IL-1βon the expression of AP2αof VSMCs, and effect of AP2αonGα12expression of VSMCs by RNAi.Part III. To explore the regulatory mechanism of IL-1βon the vasculardesensitization ofα1adrenergic receptor following sepsis1. To explore the regulatory mechanism of IL-1βon the vascular desensitization ofα1adrenergic receptor following sepsis in rabbit(1) AG490, a specific inhibitor of JAK2-STAT3pathway was used to observe theeffect of JAK2-STAT3pathway onα1ARs reactivity,α1ARs expression of the SMAsincubated with IL-1β.(2) Effect of IL-1βand AG490on DNA binding ability of STAT3of SMAs.2. To explore the regulatory mechanism of IL-1βon the vascular desensitization ofα1adrenergic receptor following sepsis in ratIncubated VSMCs of rat with IL-1βas above illustrated, to observe(1) Effect of NF-κB, p38MAPK and JNK pathway inhibitors on expression ofα1ARsin the VSMCs incubated with IL-1β. In addition, RNAi was applied to find out whichpathway was involved in effect of IL-1βon expression ofα1ARs in VSMCs.(2) Effect of IL-1βon mRNA stability ofα1ARs by using actinomycin D, and effectof IL-1βon activity ofα1DAR promoter. In order to find out IL-1βregulates expressionofα1ARs in VSMCs through transcription or mRNA stability ofα1ARs.(3) Effects of IL-1βon expressions of Sp1, Sp3and AP2(mainly AP2αand AP2γ) innucleus; Effects of IL-1βon DNA binding abilities of Sp1, AP2and NF-κB by EMSA;Effects of Sp1and AP2on expression ofα1ARs by RNAi. In order to find out that whetherIL-1βregulates expression ofα1ARs in VSMCs through transcription factors Sp1and AP2.(4) Effects of NF-κB, p38MAPK and JNK pathway inhibitors on expression of Sp1 and AP2; Effects of IL-1βon interactions of Sp1, AP2, NF-κB p50and NF-κB p65byco-IP; Effect of NF-κB on DNA binding abilities of Sp1, AP2by RNAi and EMSA. Inorder to find out the interactions between pathways and transcription factors in rat VSMCsincubated with IL-1β.Results:Part I. IL-1βinduces vascular hyporeactivity following sepsis by mediating vascularα1adrenergic receptors and calcium desensitization.1. IL-1βinduces vascular hyporeactivity following sepsis in rabbit by mediatingvascularα1adrenergic receptors and calcium desensitization.(1) The vascular sensitivity ofα1adrenergic receptors and calcium, and theexpression ofα1adrenergic receptors in SMAs of LPS-induced septic rabbit were markedlydecreased. However, the serous IL-1βlevel was significantly increased. The change ofIL-1βlevel significantly and negatively correlated with sensitivity ofα1adrenergicreceptors and calcium, expression ofα1ARs in SMAs. In addition, IL-1ra could markedlyincrease sensitivity ofα1adrenergic receptors and calcium, expression ofα1ARs in SMAsof LPS-induced septic rabbit. These data hint that IL-1βmediates vascularα1adrenergicreceptors and calcium desensitization following sepsis in rabbit.(2) Vascular sensitivity ofα1ARs and calcium, expression ofα1ARs in SMAs weresignificantly decreased by IL-1βin vitro. This data verified the effect of IL-1βon vasculardesensitization ofα1ARs and calcium.2. IL-1βinduces vascular hyporeactivity following sepsis in rat by mediating vascularα1adrenergic receptors and calcium desensitization.(1) The vascular sensitivity ofα1ARs and calcium, and the expression ofα1ARs inSMAs of CLP-induced septic rat were markedly decreased. However, the plasmaIL-1βlevel was significantly increased. The change of IL-1βlevel significantly andnegatively correlated with sensitivity ofα1ARs and calcium, expression ofα1ARs inSMAs. These data hint that IL-1βmay mediate vascularα1adrenergic receptors andcalcium desensitization following sepsis in rat.(2) The expression ofα1ARs and phosphorylation of MLC20in VSMCs weredose-dependently decreased by IL-1βin vitro. These data indicate that IL-1βcould mediateα1adrenergic receptors and calcium desensitization in VSMCs of rat. Part II. The possible mechanisms of IL-1βacts on inducing vascular calciumdesensitization following sepsis.1. The possible mechanisms of IL-1βacts on inducing vascular calciumdesensitization following sepsis in rabbit.(1) The agonists of PKC and Rho kinase could significantly increase the calciumsensitivity of the SMAs incubated with IL-1β, the antagonist of Rho kinase could markedlydecrease the calcium sensitivity, however, the antagonist of PKC could not markedlydecrease the calcium sensitivity. These data hint that PKC and Rho kinase may involved invascular calcium desensitization mediated by IL-1β.(2) The phosphorylation of MLC20of SMAs was significantly decreased by IL-1β.These data verify the effect of IL-1βon mediating vascular calcium desensitization.(3) The activities of PKC and Rho kinase of SMAs were significantly decreased byIL-1β. These data indicate that IL-1βmediates vascular calcium desensitization throughdownregulation of activities of PKC and Rho kinase.2. The possible mechanisms of IL-1βacts on inducing vascular calciumdesensitization following sepsis in rat.(1) The activity of Rho kinase of VSMCs was significantly decreased by IL-1β. Thishint that Rho kinase is involved in calcium desensitization of VSMCs mediated by IL-1β.(2) The expression of Gα11of VSMCs was significantly increased by IL-1β,however, the expression of Gα12was significantly decreased by IL-1β. The expressions ofGα13and Gαq were not significantly changed by IL-1β. These data indicate that the effectof IL-1βon expression of G protein is distinct for different G protein.(3) The activities of total RhoGEF and PDZ-RhoGEF were significantly decreasedby IL-1β, however, the activity of p63RhoGEF was significantly increased by IL-1β. Thesedata hint that the change of activity of RhoGEF is as same as the change of itscorresponding upstream G protein expression, the change of activity of G protein is as sameas its expression. In addition, because the total RhoGEF activity is decreased by IL-1β,itmeans that the effect of Gα12on regulation of activity of RhoGEF is stronger than Gα11inthe present condition.(4) The expression of Gα11of the VSMCs incubated with IL-1βwas significantlydecreased by NF-κB inhibitor; however, p38MAPK and JNK inhibitor could not significantly changed expression of Gα11or Gα12of VSMCs; NF-κB inhibitor could notsignificantly changed expression of Gα12of VSMCs. These data mean that NF-κBpathway is involved in upregulation of expression of Gα11of the VSMCs incubated withIL-1β.(5) The expression of AP2αof VSMCs was significantly decreased by IL-1β. Theexpression of Gα12of VSMCs was significantly decreased by AP2αsiRNA.These dataindicate that the expression of Gα12of VSMCs is downregulated by IL-1βthroughdownregulation of expression of AP2α.Part III. The possible mechanisms of IL-1βacts on inducing the vasculardesensitization ofα1adrenergic receptor following sepsis.1. The possible mechanisms of IL-1βacts on inducing the vascular desensitization ofα1adrenergic receptor following sepsis in rabbit.(1) The expression and sensitivity ofα1ARs of the SMAs incubated with IL-1βweresignificantly increased by AG490(JAK2-STAT3pathway inhibitor). This means thatIL-1βcould downregulate expression of vascularα1ARs through JAK2-STAT3pathway.(2) The DNA binding ability of STAT3of SMAs was dose-dependently increased byIL-1β, but was significantly decreased by AG490or unlabeled STAT3consensus sequence(cold competitor). These data may indicate that IL-1βdownregulates expression of vascularα1ARs through transcription factor STAT3.2. The possible mechanisms of IL-1βacts on inducing the vascular desensitization ofα1adrenergic receptor following sepsis in rat.(1) The expression ofα1ARs of the VSMCs incubated with IL-1βwas significantlyincreased by NF-κB inhibitor, but was not significantly changed by JNK or p38MAPKinhibitor. NF-κB siRNA showed the same effect on expression ofα1ARs as NF-κBinhibitor. In addition, the expression and DNA binding ability of trscription factor NF-κBwas significantly increased by IL-1β.These data indicate that IL-1βdownregulatesexpression ofα1ARs of VSMCs through activing NF-κB pathway.(2) The mRNA stability ofα1ARs of VSMCs was not significantly changed byIL-1β. Nevertheless, the activity ofα1DAR promoter of VSMCs was significantlydecreased by IL-1β.These data indicate that IL-1βdownregulates expression ofα1ARs ofVSMCs through transcription but not mRNA stability ofα1ARs. (3) The expressions and DNA binding abilities of Sp1and AP2(mainly AP2αandAP2γ)in nucleus of VSMCs were significantly decreased by IL-1β.but the expression ofSp3was not significantly changed. In addition, the expression ofα1ARs of the VSMCsincubated with IL-1βwas significantly decreased by Sp1, AP2αand AP2γsiRNA. Thesedata hint that IL-1βdownregulates expression ofα1ARs of VSMCs throughdownregulation of expressions of transcription factors Sp1and AP2.(4) The expressions of Sp1and AP2of the VSMCs incubated with IL-1βwere notsignificantly changed by NF-κB or JNK or p38MAPK inhibitor. But the DNA bindingabilities of Sp1and AP2of the VSMCs incubated with IL-1βwere significantly increasedby NF-κB siRNA. What is more, co-IP results showed that the interactions between NF-κBp50with NF-κB p65or Sp1or AP2were enhanced by IL-1β; the interactions betweenNF-κB p65with Sp1or AP2, or between Sp1and AP2were negative. These data indicatethat IL-1βcould enhance the interaction between NF-κB pathway and transcription factorsSp1and AP2to downregulate expression ofα1ARs of VSMCs.Conclusion:1. IL-1βmediates vascular hyporeactivity through down-regulation of the vascularcalcium andα1adrenergic receptors sensitivity following sepsis.2. IL-1βmay induce vascular calcium desensitization following sepsis throughdown-regulation of activities of PKC and Rho kinase and phosphorylation of MLC20.Further studies show that IL-1βmay induce vascular calcium desensitization followingsepsis through following mechanisms: on one hand, down-regulating transcription factorAP2αexpression which induces downregulation of expression of Gα12and activity ofpDZ-RhoGEF; on the other hand, activing NF-κB pathway to upregulation of expression ofGα11and activity of p63RhoGEF, final effect of the two hand is downregulation of totalRhoGEF which induces downregulation of activity of Rho kinase and phosphorylation ofMLC20.3. IL-1βmay induce vascularα1adrenergic receptors desensitization following sepsisthrough activing JAK2-STAT3pathway and enhancing DNA binding ability of STAT3todownregulate expression ofα1adrenergic receptors.4. IL-1βmay induce vascularα1adrenergic receptors desensitization following sepsisthrough following mechanisms: on one hand, through activing NF-κB pathway and enhancing interaction between transcription factors NF-κB and Sp1or AP2; on the otherhand, through downregulation expressions of Sp1and AP2, both hands throughdownregulation of the binding abilities of Sp1and AP2to promoter regions ofα1ARs todownregulate expression ofα1ARs at transcription level.