【作者】 杨光明；

【导师】 刘良明；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 在严重创伤/休克以及临床许多重症都存在血管对血管活性药物治疗的反应减弱甚至不反应的现象（血管低反应性）,这严重影响着这些疾病的救治。精氨酸血管加压素（AVP）是下丘脑合成的九肽神经垂体激素,本室既往研究已证实休克后血管平滑肌细胞存在钙失敏,钙失敏在休克血管低反应性的发生中起重要作用,AVP对休克后血管低反应性和钙失敏有一定的恢复作用,但具体机制尚未完全阐明。本实验室前期研究表明,蛋白激酶C（PKC）和Rho激酶是重要的调节休克后血管反应性和血管平滑肌细胞钙敏感性的激酶分子;并且Rho激酶参与了AVP的抗休克作用,但是我们发现用Rho激酶的抑制剂Fasudil和Y-27632预处理仅部分拮抗了AVP调节休克后血管反应性和钙敏感性的作用,提示可能有其它机制参与AVP的作用。PKC是一类由多种同工酶组成的丝氨酸/苏氨酸蛋白激酶家族,存在多种组织分布和功能不同的亚型。而AVP是否通过一种或几种PKC亚型来发挥恢复血管平滑肌细胞钙敏感性和血管反应性的作用,具体调节机制如何,尚不清楚。据此,我们利用大鼠失血性休克模型和缺氧损伤体外培养血管平滑肌细胞（VSMC）模型,研究AVP对休克血管反应性和血管平滑肌细胞钙敏感性的调节作用,并从钙敏感性调节分子PKC途径入手研究其机制。主要内容包括三部分:①进一步明确AVP恢复失血性休克血管低反应性的作用;②AVP调节休克血管反应性和钙敏感性的作用与PKCα、δ、ε亚型的关系;③AVP激活PKC的机制,包括AVP激活PKC亚型与V_1a、V₂受体的关系,以及PLC、PLD和PLA2在AVP这一信号传导途径中的作用。主要实验方法:第一部分,进一步明确AVP对失血性休克血管反应性的恢复作用1.整体实验,采用失血性休克大鼠模型（30 mmHg,2 h,下同）,观察大鼠失血性休克后AVP（0.04、0.1、0.4 U/kg）对去甲肾上腺素（NE）诱导肠系膜上动脉（SMA）血管收缩反应和升压效应的影响。2.离体血管环实验,取失血性休克大鼠肠系膜上动脉,利用离体血管环张力测定技术,观察AVP(5×10^-11、5×10^-10、5×10^-9 mol/L)对休克大鼠SMA血管反应性的影响（用血管环对梯度浓度NE的收缩反应反映血管反应性）。3. VSMC实验,贴块法取大鼠SMA血管平滑肌细胞原代培养,传代至第3-5代接种于Transwell小室建立VSMC双室培养模型,观察缺氧1.5 h后VSMC对NE的收缩反应变化以及AVP对缺氧VSMC收缩反应性的影响（通过观察荧光素标记的牛血清白蛋白在Transwell小室中的渗透率变化反映VSMC的收缩反应性）。第二部分,AVP改善失血性休克血管反应性和血管平滑肌细胞钙敏感性的作用与PKCα、δ和ε亚型的关系1.血管环实验,取失血性休克大鼠SMA,观察AVP处理后,休克血管环反应性和钙敏感性的变化,以及特异性的PKCα、δ、ε亚型抑制剂预处理对AVP这一作用的影响(以去极化状态下[120 mmol/L K⁺]血管环对梯度浓度Ca²⁺的收缩反应来反映钙敏感性)。同时取肠系膜动脉检测血管平滑肌肌球蛋白轻链(MLC₂₀)磷酸化的变化,分析PKCα、δ和ε亚型在AVP调节失血性休克血管反应性和钙敏感性中的作用及其与MLC₂₀之间的关系。2. VSMC实验,采用培养的大鼠肠系膜动脉VSMC,观察在各PKC亚型（α、δ、ε）抑制剂预处理缺氧培养的VSMC下,AVP对VSMC收缩反应性的影响;同时观察AVP对缺氧VSMC胞浆和胞膜成分中PKCα、δ、ε亚型蛋白表达的影响,研究AVP是通过哪一种或几种PKC亚型来调节休克血管反应性。观察AVP和PKC亚型抑制剂对缺氧VSMC肌球蛋白轻链激酶（MLCK）和肌球蛋白轻链磷酸酶（MLCP）活性的影响,探讨AVP恢复血管反应性和血管平滑肌细胞钙敏感性与MLCK/MLCP的关系以及PKC亚型在其中的作用。第三部分,研究AVP激活PKC的机制1.血管环实验,取失血性休克大鼠SMA,观察V_1a、V₂受体拮抗剂预处理对AVP改善休克血管反应性和钙敏感性的影响,同时检测肠系膜动脉平滑肌MLC₂₀磷酸化的变化,研究AVP诱导PKC激活与V_1a和V₂受体的关系及其机制。2. VSMC实验,包括:①采用培养的大鼠肠系膜动脉VSMC,观察V_1a、V₂受体拮抗剂对AVP调节PKCα、δ、ε亚型表达的影响,同时观察VSMC收缩反应性以及MLCK/MLCP活性的变化,分析AVP诱导PKC激活与V_1a和V₂受体的关系及其机制;②观察缺氧后血管平滑肌细胞三种磷脂酶（PLC、PLD、PLA₂）的活性变化以及AVP对其的作用,并进一步观察V_1a、V₂受体拮抗剂预处理对AVP调节PLC、PLD、PLA₂活性的影响,探讨PLC、PLD、PLA₂是否参与了休克后AVP诱导PKC激活调节钙敏感性的信号传导过程。主要结果:一、AVP对失血性休克血管反应性的恢复作用失血性休克后大鼠对NE的升压反应和肠系膜上动脉对NE诱导的收缩反应明显降低（P<0.01）,三个剂量的AVP（0.04、0.1、0.4 U/kg）处理均使失血性休克大鼠对NE的升压反应和肠系膜上动脉对NE的收缩反应升高（P<0.05～0.01）;在离体血管环实验中,AVP(5×10^-11、5×10^-10、5×10^-9 mol/L)处理后可明显恢复休克诱导的血管反应性降低,使SMA对NE的量-效曲线明显左移,Emax升高（P<0.01）;缺氧处理使VSMC对NE的收缩反应明显降低,AVP可升高VSMC对NE的收缩反应性,其荧光标记物累计渗透率分别在给NE后的45 min、60 min、75 min明显高于缺氧1.5 h组（P<0.05～P<0.01）。结果提示,AVP对严重失血性休克/缺氧处理的整体动物、离体血管和血管平滑肌细胞对NE的反应性均有明显的改善作用。二、AVP改善失血性休克血管反应性和血管平滑肌细胞钙敏感性的作用与PKCα、δ和ε亚型的关系1. AVP(5×10^-10 mol/L)可显著升高休克大鼠血管环对NE的反应性和钙敏感性,使NE和Ca2+的量-效曲线明显左移,Emax升高（P<0.01）。特异性的PKCα、δ和ε亚型抑制剂或抑制肽可拮抗AVP对休克后血管反应性和钙敏感性的恢复作用,其中PKCε亚型抑制肽表现出较强的拮抗作用。结果初步证明,PKCα、δ和ε亚型都参与AVP对休克后血管反应性和钙敏感性的改善作用,但它们的作用强度并不相同。2.缺氧1.5 h可明显降低VSMC对NE的收缩反应性,AVP可显著升高缺氧处理VSMC对NE的收缩反应性（P<0.05^P<0.01）,PKCα亚型的特异性抑制剂G? 6976和PKCε亚型的抑制肽预处理可明显抑制AVP对缺氧处理VSMC收缩反应的恢复作用（P<0.01）;PKCδ亚型的特异性拮抗剂Rottlerin预处理也部分抑制AVP的这一作用（P<0.05）。缺氧后VMSC胞膜成分中PKCα和ε亚型的表达明显升高,而胞浆成分中蛋白表达降低。AVP处理进一步升高胞膜PKCα和ε亚型的表达,与缺氧组相比有显著差异（P<0.05^P<0.01）;但AVP对胞浆PKCα和ε的表达量无明显影响。而胞浆和胞膜PKCδ亚型显示了相似的变化趋势,但无显著差异。结果显示,AVP可能通过促进VSMC胞浆中的PKC向胞膜转位而激活,来调节失血性休克大鼠血管平滑肌细胞的钙敏感性和血管反应性,其主要亚型为PKCα和ε亚型。3.休克大鼠肠系膜动脉血管平滑肌的MLC₂₀磷酸化水平明显低于正常对照组（P<0.01）,AVP预处理可显著升高MLC₂₀磷酸化水平（P<0.01）,PKCα和ε亚型的抑制剂对AVP诱导MLC₂₀磷酸化水平的升高有显著抑制作用（P<0.01）,而PKCδ亚型抑制剂无明显抑制作用。结果提示,AVP可通过调节血管平滑肌的MLC₂₀磷酸化水平来改善休克后血管反应性和钙敏感性,PKCα和ε亚型在其中发挥了重要作用。4.缺氧处理VSMC MLCP活性明显升高,MLCK活性明显降低（P<0.01）,AVP处理可抑制缺氧VSMC的MLCP活性的升高（P<0.05）,使MLCK活性略有升高,但与缺氧组相比无显著差异;PKCα亚型的抑制剂G? 6976和PKCε亚型的抑制肽预处理可拮抗由AVP引起的MLCP活性的降低（P<0.05）,PKCδ亚型的抑制剂Rottlerin预处理使AVP诱导的MLCP活性降低有所升高,但无显著差异（P>0.05）;三种抑制剂对细胞MLCK活性变化无明显影响。结果提示AVP激活PKC后,主要通过抑制MLCP活性,升高MLC₂₀磷酸化水平,升高VSMC的钙敏感性发挥改善休克血管反应性的作用。三、AVP激活PKC的机制1.血管加压素V_1a受体拮抗剂[d（CH2）5-Tyr2（Me）]AVP预处理可明显抑制AVP诱导的SMA对NE和Ca2+的反应性升高（P<0.01）,使NE和Ca2+的量-效曲线明显右移,Emax降低（P<0.01）;V₂受体拮抗剂[d（CH2）（d-Ile2Abu4）]AVP预处理也部分抑制了AVP的作用（P<0.05^P<0.01）。在缺氧VSMC收缩反应性的变化中也显示了相似的趋势,V_1a受体拮抗剂明显拮抗AVP升高缺氧VSMC收缩反应性的作用（P<0.01）,V₂受体拮抗剂预处理组的细胞反应性也有轻度降低,但无显著差异。结果说明,AVP恢复失血性休克动物血管反应性和钙敏感性的机制与其V_1a和V₂受体有关,其中V_1a受体可能发挥着更为重要的作用。2.缺氧处理VMSC胞膜中PKCα和ε亚型的蛋白表达量明显升高,AVP进一步升高胞膜PKCα和ε的表达,V_1a受体拮抗剂预处理可明显拮抗AVP诱导的胞膜PKCα和ε亚型的蛋白表达升高（P<0.05^P<0.01）,而V₂受体拮抗剂无明显作用。而胞浆成分和胞膜成分中PKCδ亚型的蛋白表达也显示了相似的变化趋势,但各组间无统计学差异。V_1a受体拮抗剂预处理可拮抗由AVP引起的缺氧处理VSMC MLCP活性的降低和休克血管平滑肌MLC₂₀磷酸化水平升高（P<0.05～0.01）,而V₂受体拮抗剂无明显作用; AVP及其V_1a、V₂受体拮抗剂对缺氧细胞MLCK活性无明显影响。结果提示,AVP通过激活PKC激活来调节血管反应性和血管平滑肌细胞钙敏感性的作用可能是通过V_1a受体介导的,V₂受体在这一信号传导途径中可能并不起主要作用。3.缺氧后PLC和PLD活性升高,AVP处理可使PLC和PLD活性进一步升高,明显高于缺氧组（P<0.05）。V_1a受体拮抗剂预处理可明显拮抗AVP诱导PLC和PLD活性升高的作用（P<0.01）,V₂受体拮抗剂无明显作用。各组PLA₂活性均无明显差异。结果提示,PLC和PLD参与了休克后AVP通过V_1a受体介导的PKC激活过程。结论:1.失血性休克后血管反应性显著降低,AVP对休克后全身和局部的血管反应性以及缺氧血管平滑肌细胞的收缩反应性都有较好的恢复作用。2. AVP可通过激活PKCα和ε亚型,来调节失血性休克大鼠血管平滑肌细胞的钙敏感性和血管反应性,其机制是AVP首先促进血管平滑肌细胞胞浆中无活性的PKC向胞膜转位、并激活,进而抑制MLCP活性,使MLC₂₀磷酸化水平升高,最后增加血管平滑肌细胞的钙敏感性,升高血管反应性。3. V_1a受体拮抗剂预处理可抑制AVP诱导的胞膜PKCα和ε的蛋白表达升高、MCLP活性降低、MLC₂₀磷酸化水平升高和血管反应性升高,同时也可抑制AVP诱导的PLC和PLD活性升高。提示AVP诱导PKC激活的机制可能与V_1a受体-PLC/PLD途径有关。更多还原

【Abstract】 The occurrence of vascular hyporeactivity after severe trauma or shock has been shown to have important roles in the incidence, development, and the outcome of shock and interfered with the therapy of shock. Arginine vasopressin （AVP） is a 9-amino-acid neurohypophysial peptide hormone synthesized in the hypothalamus. Our previous studies have demonstrated that hemorrhagic shock could also cause calcium desensitization of blood vessels, which played important roles in vascular hyporeactivity following hemorrhagic shock, and protein kinase C （PKC） and Rho-kinase can regulate the calcium sensitivity of vascular smooth muscle cell （VSMC） by induced the inhibition of myosin light chain phosphatase （MLCP）. And recent observations from our laboratory showed that AVP is beneficial to hemorrhagic shock, which may be related to AVP-induce increase of vascular reactivity and calcium sensitivity of vascular smooth muscle cell via the activation of Rho-kinase. However, Rho-kinase antagonist, Fasudil and Y-27632 only partially blocked this effect of AVP, it’s suggested that there may be other mechanisms involved in the effect of AVP. PKC was lipid-regulated serine/threonine kinases, included multiple isozymes with distinct tissue distributions and regulatory properties. However, little is known about which isoforms of PKC might be involved in modulating vascular reactivity and calcium sensitivity of vascular smooth muscle after hemorrhagic shock induced by AVP and the precise mechanisms. So with hemorrhagic shock model of rats and hypoxia-treated VSMC, we observed the effects of AVP on vascular reactivity amd calcium sensitivity following hemorrhagic shock in rats and explore its relations withα,δandεisoforms of PKC.Methods:The experiments were conducted in three parts. Part I. The effects of AVP on vascular reactivity following hemorrhagic shock in rats1. In vivo—The hemorrhagic shock （30 mmHg for 2 hours） model of rats was adopted to observe the effects of AVP（0.04、0.1、0.4 U/kg） on the contractile response of SMA to norepinephrine（NE） and pressor effect of NE.2. In isolated SMA—Superior mesenteric artery （SMA） rings from rats were used to observe the effects of AVP(5×10^-11、5×10^-10、5×10^-9 mol/L) on vascular reactivity of SMA with isolated organ perfusion system （The vascular reactivity was observed by measuring the contraction initiated by accumulative NE）.3. In VSMC—Primary cultures of VSMCs were obtained from the mesenteric artery of Wistar rats by an explant technique and the third to fifth passage cells were used in the present study. The contractile response of cultured VSMC to NE at different time after 1.5 hours hypoxia and the effects of AVP were observed （The contractile response of VSMC was measured by the ratio of accumulative infiltration of fluorescent isothiocyanate-conjugated bovine serum albumin with transwell）.Part II. The effects of AVP on vascular reactivity and calcium sensitivity of vascular smooth muscle and its relationship to PKCα,δandεisoforms1. In isolated SMA—With isolated SMA rings from hemorrhagic shock rats, the effects of AVP on vascular reactivity and calcium sensitivity of SMA from hemorrhagic shock rats and and the effects of PKCα,δ,εinhibitor were observed （The calcium sensitivity of SMA were observed by measuring the contraction initiated by accumulative calcium under depolarizing condition （120 mM K+） with an isolated organ perfusion system）. And the myosin light chain （MLC20） phosphorylation of mesenteric artery smooth muscle was detected by Western blotting.2. In VSMC—With hypoxia-treated VSMCs, the contractile response of VSMC to NE after 1.5 h hypoxia and the effects of AVP and PKCα,δ,εinhibitor were observed. And the effect of AVP on the expression of PKC-α,δandεisoforms in the cytosol and particulate fractions of VSMCs, the activity of MLCP and myosin light chain kinase （MLCK） of VSMC were also observed.Part III. The mechanism of AVP induced the PKC isozymes activation 1. In isolated SMA—The effects of V_1a and V₂ receptor inhibitor on AVP improving vascular reactivity and calcium sensitivity of SMA from hemorrhagic shock rats and its relationship to the phosphorylation of MLC₂₀ were observed.2. In VSMC—With hypoxia-treated VSMCs, we observe the effects of V_1a and V₂ receptor inhibitor on AVP regulating the expression of PKC-α,δandεisoforms in the cytosol and particulate fractions of VSMCs. At the same time, the activity of MLCP/MLCK and the contractile response of VSMC to NE were assayed. In addition, the activity of phospholipase C （PLC）, phospholipase D （PLD）, phospholipase A₂ （PLA₂） and the effects of V_1a and V₂ receptor inhibitor were observed.Results:1. Effects of AVP on vascular reactivity following hemorrhagic shock in ratsIn vivo NE-induced pressor response and vasoconstriction of SMA following hemorrhagic shock was significantly decreased （P<0.01）, AVP （0.04, 0.1 and 0.4 U/kg） significantly increased the pressor effect of NE and vasoconstriction of SMA to NE in hemorrhagic shock rats （P<0.05⁰.01）. In vitro, AVP （5×10-11, 5×10-10, 5×10-9 mol/L） pretreatment also improved reactivity of SMA rings to NE following hemorrhagic shock and made the cumulative dose-response curve of NE shift to the left, Emax was increased significantly （P<0.01）. After hypoxia for 1.5 h, the contractile response of VSMC to NE significant decreased, AVP pretreatment increased the contractile response of VSMCs, its ratio of accumulative infiltration of fluorescent isothiocyanate-conjugated BSA was significantly increased at 45, 60 and 75 min after NE administration （P<0.05^P<0.01）. It was suggested that AVP significantly improved the hypoxia or hemorrhagic shock-induced decrease in contractile reactivity of SMA and VSMC.2. Effects of AVP on calcium sensitivity of vascular smooth muscle and its relationship with PKCα,δandεisoforms（1） AVP markedly restored the decreased sensitivity of SMA to NE and Ca2+ following hemorrhagic shock, the concentration-response curve of NE and Ca2+ was shifted to the left and Emax was significantly increased （P<0.01）. G? 6976 （the specific PKCαisoform inhibitor）, Rottlerin （the specific PKCδisoform inhibitor） and PKCεinhibitor peptide antagonized AVP-induced increase of vascular reactivity and calcium sensitivity of SMA following hemorrhagic shock （P<0.05～P<0.01）, in which PKCεinhibitor peptide showed a stronger antagonistic effectiveness than the others. The data suggested that PKCα,δandεisoforms participated in the regulation of AVP on calcium sensitivity and vascular reactivity after shock with different importance.（2） AVP pretreatment significant increased the contractile response of VSMC to NE （P<0.05～P<0.01）, the effects of AVP was significantly blocked by G? 6976 and PKCεinhibitor peptide （P<0.01）, while partly inhibited by Rottlerin （P<0.05）. The expression of particulate PKCαandεincreased in response to hypoxia, with a concomitant decrease in cytosolic fractions. AVP treatment further increased expression of PKCαandεin the particulate fractions （P<0.05^P<0.01）, but the PKCαandεlevels in the cytosolic fractions were not significant changes. While PKCδshowed a similar changes in either the particulate or the cytosolic fractions during the process, but there were no statistical differences among the groups. It was suggested that AVP improved vascular reactivity and calcium sensitivity following hemorrhagic shock through translocating PKCαandεisoforms from a cytosol to a particulate and activation.（3） The MLC20 phosphorylation of SMA following hemorrhagic shock were significantly decreased （P<0.01）, AVP treatment resulted in an increase in MLC20 phosphorylation （P<0.01）, which could be inhibited by G? 6976 and PKCεinhibitor peptide, not Rottlerin. The results suggested that PKCαandεisoforms played an important role in AVP regulating vascular reactivity and calcium sensitivity through MLC20 phosphorylation.（4） Treatment with hypoxia for 1.5 h caused a significant increase in MLCP activity, with a decrease in MLCK activity of VSMC, AVP treatment resulted in an inhibition of MLCP activity （P<0.05）, G（o|¨）6976 and PKCεinhibitor peptide （P<0.01）, not Rottlerin, abolished AVP-induced decrease of MLCP activity. But there was no significant influence on MLCK activity.. The results of the present study suggested that AVP restores the vascular reactivity and calcium sensitivity of vascular smooth muscle following hemorrhagic shock via an activation of PKCαandεisoforms, and its mechanisms may be related to induce decrease of MLCP activity and MLC20 phosphorylation.3. Mechanism of AVP induced the PKC isozymes activation（1） [d（CH2）5-Tyr2（Me）]AVP, the V_1a receptor inhibitor, significantly antagonized AVP-induced increase of vascular reactivity and calcium sensitivity of SMA following hemorrhagic shock and V₂ receptor inhibitor （[d（CH₂）（d-Ile²Abu⁴）]AVP） also partly inhibit this effect （P<0.05）. And V_1a receptor inhibitor also blocked AVP-induced increase of the contractile response of VSMCs to NE （P<0.01）, while V₂ receptor inhibitor only slightly inhibited this effect of AVP. The results showed that AVP may restore the decreased vascular reactivity and calcium sensitivity of vascular smooth muscle after hemorrhagic shock through V_1a and V₂ receptor, and V_1a receptor may play more important roles than V₂ receptor.（2） The expression of particulate PKCαandεincreased in response to hypoxia, AVP treatment further increased expression of PKCαandεin the particulate fractions, V_1a receptor inhibitor significantly antagonized this effect of AVP （P<0.05^P<0.01）, but V₂ receptor inhibitor had no effect. While PKCδshowed a similar changes, but there were no statistical differences. AVP treatment resulted in an increase in MLC20 phosphorylation of SMA （P<0.01） and decreased in MLCP activity of VSMC （P<0.05）, which could be inhibited by V_1a receptor inhibitor, not V₂ receptor inhibitor. MLCK activity of VSMC were significantly decreased after hypoxia （P<0.01）, but AVP and V_1a/V₂ receptor inhibitor had no effect on it. It was suggested that AVP regulating the calcium sensitivity was mainly related to V_1a receptor, not V₂ receptor.（3） Treatment with hypoxia for 1.5 h caused a significant increase in PLC and PLD activity, AVP further increased the activity of PLC and PLD （P<0.05）. V_1a receptor inhibitor significantly antagonized AVP-induced increased in PLC and PLD activity （P<0.01）. The PLA₂ activity had no significant changes among the groups. The results suggested that PLC and PLD took part in the V_1a receptor-mediated effects of AVP.Conclusions:1. AVP can restore the decreased vascular reactivity in hemorrhagic shock rats including the systemic responsiveness and local vascular reactivity, and increase the contractile response of vascular smooth muscle cell to NE.2. AVP restores the vascular reactivity and calcium sensitivity of vascular smooth muscle following hemorrhagic shock via activation of PKCαandεisoforms, and its mechanisms is that AVP firstly induceαandεisoforms of PKC translocation from a cytosol to a particulate and activation, and then inhibits the activity of MLCP and increases MLC20 phosphorylation, followed by improves the calcium sensitivity of VSMC and at last enhances the vascular reactivity.3. V_1a receptor inhibitor antagonized AVP-induced increase expression of PKCαandεin the particulate fractions, decrease in MLCP activity and increase in MLC₂₀ phosphorylation of SMA following hemorrhagic shock. At the same time, V_1a receptor inhibitor also decreased the increased PLC/PLD activity induced by AVP. These data suggested that AVP induced translocation/activation of PKC in vascular smooth muscle through a V_1a receptor dependent mechanism, and PLC or PLD may participate in the signal transduction pathway.更多还原