G蛋白耦联受体激动剂调节细胞膜PIP_2代谢及生理学意义的研究
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摘要
磷脂酰肌醇-4,5-二磷酸（Phosphatidylinositol-4,5-bisphosphate，PIP2）是分布在细胞膜内侧面的一种微量磷脂，其含量约占细胞膜磷脂总量的1%。虽然含量很低，但PIP2在细胞信号转导、锚定骨架蛋白以及激活或稳定膜蛋白等方面却起着举足轻重的作用。因此在不同的组织器官中PIP2代谢水平的改变可能与疾病的发生有关。
PIP2是磷脂酰肌醇（PI）在肌醇环4位和5位磷酸化的产物。一方面，在细胞信号转导中它可以被激活的磷脂酶C（PLC）水解生成二脂酰甘油（DAG）和肌醇1,4,5-三磷酸（IP3）；也可以被磷脂酰肌醇-3-激酶（PI3K）磷酸化成为磷脂酰肌醇-3,4,5-三磷酸（PIP3），后者是激活PI3K/Akt细胞生存通路的关键信号分子。另一方面，磷脂酰肌醇-4-激酶（PI4K）可以将细胞中的PI磷酸化成为磷脂酰肌醇-4-磷酸（PI4P），后者则会被细胞膜上的磷脂酰肌醇-4-磷酸-5-激酶（PIP5K）磷酸化从而完成PIP2的合成。
除酪氨酸激酶通路外，PLC的激活一般是通过Gq蛋白耦联受体（GqPCR）介导的。儿茶酚胺类化合物、血管紧张素II等激素能够激活细胞膜上的GqPCR。激活的Gq蛋白的α亚基与βγ亚基分离，GTP水解释放的能量将PLC激活，后者完成PIP2的水解。在较短时间内PIP2通过水解局部的再合成机制来维持自身含量水平的稳定，在加大刺激强度并延长刺激时间的情况下细胞中PIP2的水平则可能发生改变。与短时刺激造成的局部影响不同，在增大作用浓度或延长刺激时间的情况下，G蛋白耦联受体激动剂能够增加成年大鼠心肌细胞或脑组织中PIP2的含量。许多重要的体内活性物质都是G蛋白耦联受体激动剂，这些活性物质往往在疾病状态下发生改变，因此在疾病的发生过程中相应细胞中PIP2的代谢状态也可能发生改变。所以，研究在这些情况下PIP2的代谢特征对于探究疾病的发生机制以及寻找可能的治疗方法十分有意义。本研究首先针对去甲肾上腺素和血管紧张素II对心肌PIP2代谢的影响及机制，以及这些作用与心肌肥厚、心脏功能之间的关系进行了研究；此外还研究了去甲肾上腺素和5-羟色胺对脑PIP2相关代谢机制的影响及其与抑郁症的相关性。研究结果表明，在激动剂刺激的过程中PIP2合成酶PI4K与PIP5K的活性发生变化是PIP2含量改变的原因；而且，这种PIP2含量的改变可能是细胞在激动剂刺激下为维持自身功能正常而做出的一种适应性改变。具体的研究内容如下：
第一部分去甲肾上腺素和血管紧张素II对大鼠心肌细胞膜PIP2以及心肌功能的调节
目的：早在上个世纪研究者们就发现了Gq蛋白耦联受体激动剂对细胞膜磷脂酰肌醇代谢的调节作用，然而对其中具体的调节机制和生理意义却仍不清楚。本部分实验对Gq蛋白耦联受体激动剂去甲肾上腺素（NE）、血管紧张素II（Ang II）调节大鼠心肌细胞膜PIP2代谢的分子机制进行深入研究，并初步探讨了PIP2代谢改变对心肌细胞功能的影响及其与心肌肥厚发展不同阶段的相关性。
方法：分离大鼠心室肌细胞，使用薄层色谱（TLC）和脂-蛋白交叠分析（Lipid-protein overlay assay, LPOA）的方法来分析NE、Ang II对心肌细胞PIP2含量的影响；以Gq/PLC通路相关信号分子特异性抑制剂—U73122（抑制PLC活性）和bisindolylmaleimide-1（Bis-1）（抑制PKC活性）预处理细胞，以PIP2合成酶抑制剂wortmannin、PIK93以及腺苷（adenosine）预处理细胞，再按照前面所述的方法分析NE、Ang II刺激后心肌细胞中PIP2的含量变化；利用免疫共沉淀的方法检测激动剂刺激后PIP2合成酶PI4K与蛋白激酶C（PKC）之间的相互作用；使用TLC的实验方法测定NE、Ang II刺激后心肌细胞PIP2水平变化的时程曲线，20分钟到72小时之间取4-5个时间点进行研究；按照PIP2变化时程曲线的时间点，使用细胞动缘探测系统检测NE、Ang II刺激后心肌细胞收缩功能的变化；建立大鼠游泳致心肌肥厚以及异丙肾上腺素（isoprenaline）注射致心肌肥厚的实验模型，使用LPOA的实验方法测定在生理性心肌肥厚以及病理性心肌肥厚的不同条件下大鼠心室肌中PIP2总含量。
结果：（1）原代心肌细胞培养24小时后，再以NE（5μM）刺激2小时，或以Ang II（3μM）刺激24小时，经测定发现刺激后心肌细胞中PIP2的含量明显增加，使用α受体抑制剂—prazosin（3μM）或AT1受体抑制剂—losartan（3μM）处理细胞能够拮抗NE或Ang II对PIP2含量的增加作用；（2）以U73122（0.8μM）、U73343（U73122的同形无效物）、Bis-1（2μM）以及wortmannin（10μM）预处理细胞20-30分钟，再以NE（5μM，2h）、Ang II（3μM，24h）刺激细胞，经测定发现U73122、Bis-1和wortmannin均能够抑制NE或Ang II对心肌细胞PIP2以及PIP含量的增加作用；（3）以III型PI4K的抑制剂—PIK93（250nM）或II型PI4K的抑制剂—adenosine（2μM）提前30分钟处理细胞，再给予NE（5μM，2h）、Ang II（3μM，24h）孵育，经测定发现只有PIK93对NE或Ang II引起的PIP2含量增加具有明显的抑制作用并且显著降低了细胞中PIP的水平。单独给予细胞PIK93或adenosine，PIK93对心肌细胞中PIP2以及PIP的含量均无明显影响，但adenosine则显著下调了细胞中PIP2以及PIP的含量水平；（4）NE（5μM）、Ang I（I3μM）以及PKC激动剂佛波酯（PMA，0.5μM）均能够增加PI4KIIIβ与PKCα之间、PI4KIIIβ与PKCβ之间的相互作用，而在心肌细胞中PI4KIIIα与PKCs之间并无明显的相互作用；（5）当刺激心肌细胞24小时以后，NE或Ang II对PIP2含量的增加作用随刺激时间的延长而逐渐降低，甚至可能逆转为降低作用。与此同时，对应时间点上心肌细胞的收缩功能也出现与PIP2含量类似的变化；（6）使用LPOA的方法测定两种心肌肥厚动物模型心脏中PIP2的含量，相比正常动物，游泳致心肌肥厚大鼠心脏中PIP2的含量水平并无较大改变，而Iso注射组大鼠心脏中PIP2的含量却显著减少。
结论：NE和Ang II激动Gq蛋白耦联受体，并通过受体激动后的下游信号通路水解PIP2，同时激活PIP2的再合成。PKC与PI4KIIIβ之间的相互作用是连接PIP2水解与再合成通路的分子基础。通过对心肌细胞收缩功能以及两种不同心肌肥厚模型的研究我们发现，在应激刺激下PIP2含量的增加可能与心肌肥厚初期心脏的代偿功能有关。
第二部分PIP5Kγ在G蛋白耦联受体调节大鼠心肌细胞膜PIP2中的作用
目的：了解磷脂酰肌醇-4-磷酸-5-激酶（PIP5K）在去甲肾上腺素（NE）、血管紧张素II（Ang II）及异丙肾上腺素（Iso）等G蛋白耦联受体激动剂在调节心肌细胞PIP2代谢中的作用及细胞信号机制。
方法：利用western blot的方法检测PIP5Kγ的表达水平；使用TLC的实验方法测定心肌细胞中PIP2的含量水平，观察NE、Ang II及Iso对心肌细胞PIP2含量的影响；使用药理学手段了解上述激动剂影响心肌细胞中PIP5Kγ的表达以及PIP2的含量的信号机制；使用蛋白合成酶抑制剂放线菌酮（cycloheximide）处理细胞，通过测定激动剂刺激后心肌细胞PIP2含量的改变，并以此来分析激酶表达与PIP2合成之间的联系；利用免疫共沉淀的方法检测激动剂刺激后PIP2合成酶PIP5Kγ与其水解酶PLC之间的相互作用。
结果：（1）NE（5μM）、Ang II（3μM）、PMA（0.5μM）以及β受体激动剂isoprenaline（0.5μM）均能够增加心肌细胞中PIP5Kγ的表达量，但NE与Iso的增加作用要显著大于Ang II与PMA的作用；（2）使用propranolol（3μM）抑制β受体或使用H89（30μM）抑制Gs蛋白耦联受体信号通路下游效应分子PKA，均能够抑制激动剂对PIP5Kγ表达水平的上调作用，但prazosin和Bis-1却对此无效；（3）β受体拮抗剂propranolol预处理细胞30分钟能够抑制NE对PIP2的增加作用。与NE类似，Iso也能够增加心肌细胞中PIP2的含量，但对PIP的含量却无明显影响；（4）Cycloheximide不仅能够抑制NE引起的PIP5Kγ表达量的增加，也能够显著降低心肌细胞中PIP2的含量并抑制NE的作用；（5）NE（5μM）、Ang II（3μM），以及Iso（0.5μM）均能够增强PIP2合成酶PIP5Kγ与水解酶PLCγ之间的相互作用。
结论：NE、Iso激动Gs蛋白耦联受体，并通过提高PIP5Kγ的表达水平来增加细胞中PIP2的再合成。由于能够同时激活Gq/PLC/PKC与Gs/cAMP/PKA两条通路，NE引起的PIP2含量增加要明显快于Ang II的作用。
第三部分去甲肾上腺素和5-羟色胺对鼠脑PIP2代谢的调节以及与抑郁症的关系
目的：PIP2是突触囊泡释放以及回收过程中不可缺少的关键物质，它与突触可塑性之间存在关系，同时也可能影响突触间递质的释放。因此研究脑磷脂酰肌醇特别是PIP2的代谢对于了解疾病状态下如抑郁症的发病机制以及寻找新的治疗手段具有重要意义。去甲肾上腺素（NE）及5-羟色胺（5-HT）是重要的与抑郁症相关的单胺类神经递质。在本研究中我们将研究NE和5-HT对大鼠和小鼠脑PIP2代谢的影响，通过测定给药后PIP2合成酶PI4K表达水平以及突触中PIP2含量的变化，初步探究中脑PIP2代谢改变与抑郁症之间的联系。
方法：利用western blot的方法检测NE、5-HT孵育后鼠脑内PIP2合成酶—PI4K的表达水平；提取膜蛋白，观察NE、5-HT孵育后PKC在细胞膜中的表达情况；利用免疫共沉淀的方法检测NE、5-HT孵育后PI4K与PKC之间的相互作用是否发生变化；分离突触小体，利用LPOA的方法（ELISA试剂盒）测定其中PIP2的含量，并观察NE、5-HT孵育后PIP2水平是否发生改变；建立小鼠的慢性社交失败抑郁症模型，初步研究模拟精神疾病状态下鼠脑内PI4K的表达状况。
结果：（1）给予NE（10μM）、5-HT（30μM）17分钟后，大鼠中脑切片中总PI4KIIIβ以及膜PI4KIIIβ的表达水平均出现明显增高；（2）通过使用PKC阻断剂Bis-1（1.5μM）预处理中脑切片我们发现，NE、5-HT对PI4KIIIβ表达量的增加作用依赖于PKC的激活；（3）NE、5-HT能够增强大鼠中脑切片中PI4KIIIβ与PKCα以及PI4KIIIβ与PKCβ之间的相互作用并增加其中脑突触小体中PIP2的含量；（4）虽然对小鼠中脑区域PI4K和PKC在的相互作用并无明显影响，但NE、5-HT能够显著降低PI4K和PKC在小鼠前额叶皮层中的相互作用以及该区域突触小体中PIP2的水平；（5）社交失败模型小鼠脑内的PI4KIIIβ表达水平下调。
结论：NE和5-HT能够促进大鼠中脑PI4K、PIP5K的表达，增强大鼠中脑和前额皮层PI4K与PKC间的相互作用，升高大鼠中脑PIP2水平。相对于大鼠，NE和5-HT对小鼠有不同的作用：只降低前额皮层PI4K与PKC间的相互作用和PIP2水平，而没有其他上述对大鼠的作用。在小鼠社交失败抑郁模型中，小鼠中脑和前额皮层中PI4K的表达相对于对照小鼠均有降低。这些初步结果表明，与抑郁症相关的中枢单胺类神经递质可以影响以PIP2为代表的磷脂酰肌醇代谢通路，并可能以此影响抑郁症的发生。
Phosphatidylinositol-4,5-bisphosphate (PIP2), which is a majorphosphoinositide of the plasma membrane, comprises about1%of plasmamembrane phospholipids. PIP2is enriched in the inner leaflet of the plasmamembrane, where it serves as a precursor of the second messengers, a ‘lipidanchor’ that attaches the cytoskeleton to the plasma membrane, and stabilizesor activates many membrane proteins, such as ion channels and transporters.Thus, changes of membrane PIP2metabolism may have roles in pathologicalconditions.
One of the well established cellular events involved in PIP2dynamics ismediated by the activation of the Gqprotein-coupled receptor (GqPCR): thestimulation of GqPCR activates phospholipase C (PLC), which in turn,hydrolyzes PIP2into inositol1,4,5-trisphosphate and diacylglycerol.Meanwhile, both PI(4,5)P2generation by phosphatidylinositol4-phosphate5-kinases (PIP5K) and the subsequent generation of PI(3,4,5)P3by thephosphoinositide3-kinases are dependent on an initial phosphorylation ofphosphatidylinositol (PI) on the D4position by one of the four mammalian PI4-kinase enzymes synthesising phosphatidylinositol4-phosphate (PI4P). AndPIP3is also an important signaling molecule that can stimulate theAkt-mediated cellular survival pathways.
Catecholamines and angiotensin II are well established GqPCR agonists.After ligand binding, a conformational shift in the G protein complex occursresulting in activation of a number of effectors such as PLC, which hydrolyzesmembrane PIP2. Generally, depletion of local PIP2is followed by thephosphoinositide reproduction. The recruitment of PIP2induced by GqPCRactivation is essential for the maintaince of local ‘PIP2pool’ and stabilization of global PIP2. However, prolonged stimulation, such as treatment withGqPCR agonists, may change the total phosphoinositides level incardiomyocytes or brain. It is well established that many GqPCR agonists areinvolved in the pathophysiological processes of diseases and as such theseagonists-mediated modulation of membrane PIP2metabolism may well beinvolved in these dieases. In this study, we studied the modulation andmechanism of phosphoinositides turnover of cardiac mocytes understimulation of norepinephrine (NE) and angiotensin II (Ang II) at both thecellular and whole animal levels, and related the changes of membrane PIP2with the development of cardiac hypertrophy and cardiac function. Also westudied the modulation of brain membrane PIP2by NE and5-HT and relatedthese modulations with the development of major depressive disorder (MDD).We described an increase of PIP2levels in cardiomyocytes and cerebral slicesunder stimulation of NE, Ang II and5-HT. We also present a novelmechanism for these increases to be the result of the enhanced activity ofPI4KIIIβ, mediated by an up-regulated interaction between PI4KIIIβ and PKC;furthermore, an increased activity of phosphatidylinositol4-phosphate5-kinase γ was involved in NE-induced increase of cardiac PIP2. These GPCRagonists mediated modulation of phosphoinositides turnover was related to thecardiac hypertrophy and major depression.
Part1Molecular mechanisms for norepinephrine and angiotensinII-induced increase in PIP2and modulation of cardiac function
Objective: The seemly paradoxical GqPCR agonist-stimulatedphosphoinositide production has long been known, but the underlyingmechanism and its physiological significance are not known. In this study, westudied cardiac phosphoinositide levels in both cells and whole animals underthe stimulation of norepinephrine (NE), angiotensin II (Ang II), and otherphysiologically relevant interventions.
Methods:(1) The thin layer chromatography (TLC) and lipid-proteinoverlay assay (LPOA) were used to measure the change of PIP2in the primarycultured cardiomyocytes after treated with NE and Ang II.(2) The effects of NE and Ang II on PIP2of cardiomyocytes were studied in the presence ofPLC inhibitors (U73122), PKC inhibitors (Bis-1), or three kinds of PI4Kinhibitors (wortmannin, PIK93, and adenosine).(3) Immunoprecipitation wasused to detect the interaction between PI4K and PKC.(4) PIP2level incardiomyocytes was measured after treated with NE for the time of20min,1h,2h,24h and48h, and with Ang II for the time of2h,24h,48h and72h.(5) The systolic function of the cultured cardiomyocytes was assessed bymeasuring sarcomere shortening induced by an electrical stimulation, whichrepresents the amplitude of cardiomyocyte contraction.(6) Lipid-proteinoverlay assay was used to measure the changes of total PIP2from rat heartswith hypertrophy induced either with isopreternol or with swimming excise.
Results:(1) After incubation with NE (5μM,2h) or Ang II (3μM,24h),the total abundance of PIP2in cardiomyocytes, measured using TLC, wasfound to be significantly increased. We also used a lipid-protein overlay assay,which confirmed the TLC results. Specific antagonists of the α-adrenergicreceptor prazosin, or Ang II receptor losartan (3μM) abolished the effects ofNE and Ang II, respectively.(2) The PLC inhibitor U73122(0.8μM; inactiveanalog U73343,0.8μM, was used as a control), the PKC inhibitor Bis-1(2μM), and the PI4K inhibitor wortmannin (10μM) were used to assess the rolesof these molecules. Pretreatment of cardiomyocytes with U73122, Bis-1, andwortmannin prevented the NE-or Ang II-induced increase in PIP and PIP2.(3)Pretreatment of cardiomyocytes with PIK93(250nM) prevented the NE-orAng II-induced increase in PIP and PIP2. Adenosine (2μM,2h), but notPIK93(250nM,2h), reduced basal PIP2levels in primary culturedcardiomyocytes.(4) Co-immunoprecipitation results showed that both PKCisoforms of α and βII were able to interact with PI4KIIIβ, but not withPI4KIIIα.(5) When cardiomyocytes were incubated with NE (5μM) or Ang II(3μM) for up to72h, both NE and Ang II induced a gradual increase in PIP2levels within the first24h, but afterward, the effects of NE and Ang II beganto recede, and PIP2levels fell back to control or even lower levels. Furtherly,the agonists affected the contraction of cardiomyocytes with a pattern and time course similar to their effect on PIP2levels.(6) The PIP2levels in the rathearts of Iso-induced hypertrophy were significantly reduced compared withthe time matched controls; PIP2levels in the rat hearts of swimming-inducedhypertrophy were increased compared with control hearts, although theincrease did not reach statistical significance.
Conclusion: Stimulation of rat cardiomyocytes with NE and Ang IIincreased both cellular PIP and PIP2levels. The increased PIP and PIP2induced by NE and Ang II was the result of an enhanced activity of PI4KIIIβ,mediated by an up-regulated interaction between PI4KIIIβ and PKC. Thisenhanced PIP2turnover is correlated with maintained cardiac systolic functionin compensative cardiac hypertrophy.
Part2The role of PIP5Kγ plays in GPCR agonists-mediated regulation ofmembrane PIP2in rat cardiomyocytes
Objective: To investigate the role of PIP5Kγ plays in NE-, Ang II-andisoprenaline (Iso)-induced regulation of PIP2turnover in rat cardiac myocytesand the related signaling mechanism.
Methods:(1) Western blot was used to detect the expression of PIP5Kγ,a key enzyme of PIP2production, in the cardiomyocytes.(2) Pharmacologicaltools were used to dissect the signaling mechanism for receptoragonists-induced modulation of PIP5Kγ expression and PIP2level.(3)Cycloheximide (CHX), a protein synthesis inhibitor, was used to relate theturnover of PIP5Kγ expression with the increased PIP2level.(4)Immunoprecipitation method was used to detect the interaction betweenPIP5Kγ and PLC.
Results:(1) In rat cardiomyocytes, PIP5Kγ was found to be up-regulatedafter stimulation with NE (5μM,2h), Iso (0.5μM,2h), Ang II (3μM,2h),and PMA (0.5μM,20min). However, the effect of NE and Iso on PIP5Kγwas significantly greater than that of Ang II or PMA.(2) The enhancedexpression of PIP5Kγ by NE was abolished by propranolol (a β receptorblocker) and H89(a PKA blocker), but not by prazosin and Bis-1.(3)Propranolol abolished the NE-induced increase in PIP2, and Iso induced an increase in PIP2levels.(4) In the presence of CHX, the NE-induced increasein total PIP2and PIP5Kγ expression in cardiomyocytes was inhibited.(5) Theinteraction between PIP5Kγ and PLCγ was increased by NE (5μM), Ang II (3μM) and Iso (0.5μM).
Conclusion: NE and Iso, through activation of GsPCR and PKA pathwayincreased the expression and activity of PIP5Kγ which enhanced synthesis ofPIP2. This effect of NE, combined with its effect of enhanced activity of PI4Kdescribed in the first part may explain a faster and stronger modulation ofcellular PIP2by NE than Ang II.
Part3The modulation of cerebral PIP2turnover by norepinephrine and5-hydroxytryptamine and its possible role in major depressivedisorder
Objective: Phosphoinositide lipids regulate key cellular functionsincluding endocytosis, signaling and secretion. In neuronal systems,well-studied areas include the roles of PIP2in synaptic vesicle recycling.Recent evidence suggests that PI4K and PIP2have important neuronalfunctions and that dysfunction of PI4K may have neuropathologicalconsequences. NE and5-HT are important monoamines involved in thedevelopment of major depressive disorder (MDD). In this study, we studiedthe effects and the mechanism of NE and5-hydroxytryptamine (5-HT) oncerebral PIP2turnover, and investigated consequence of these effects on thedevelopment of major depressive disorder.
Methods:(1) Western blot was used to measure total PI4KIIIβ in rat andmice brain slices after incubation with NE or5-HT.(2) Ultracentrifugationwas performed to extract membrane protein.(3) Immunoprecipitation wasused to detect the interaction between PI4K and PKC.(4) The component ofsynaptosome was extracted, and the synaptosomal PIP2was measured byLPOA (ELISA) method.(5) Major depression model of mice social defeatstress was established.
Results:(1) Incubation with NE (10μM) or5-HT (30μM) for17minincreased expression of both the total and the membrane PI4KIIIβ in the midbrain slices of rat but not in mice.(2) Incubation with NE increasedexpression of membrane PKC and blockage of PKC by Bis-1inhibited the NEand5-HT induced-increase of PI4KIIIβ expression.(3) PI4KIIIβ interactedwith PKCs in rat midbrain, which was enhanced by NE or5-HT. On the otherhand, the interaction between PI4K and PKC was down-regulated in theprefrontal cortex of mice.(4) Incubation with5-HT increased synaptosomalPIP2in the midbrain of rats, but decreased synaptosomal PIP2in the prefrontalcortex of mice.(5) Expression of PI4KIIIβ was down-regulated in themidbrain and prefrontal cortex of the model MDD mice.
Conclusion: NE and5-HT increased the expression of PI4K and PIP5Kin the midbrain of rats, increased the interaction of PI4K and PKC in both themidbrain and the prefrontal cortex of rats, and increased the PIP2level in themidbrain of rats. On the other hand, none of the above effects of NE and5-HTdescribed on rats were found in C57mice, but reduced the interaction betweenPI4K and PKC and the PIP2level in the prefrontal cortex of the mice. In socialdefeat depression model of mice, the expression of PI4K in both the midbrainand the prefrontal cortex were reduced compared with the control mice. Theseresults indicate the monoamine transmitters such as NE and5-HT are able tomodulate the metabolism of PIP2and such modulation may be related with thedevelopment of major depressive disorder.
PIP2是磷脂酰肌醇（PI）在肌醇环4位和5位磷酸化的产物。一方面，在细胞信号转导中它可以被激活的磷脂酶C（PLC）水解生成二脂酰甘油（DAG）和肌醇1,4,5-三磷酸（IP3）；也可以被磷脂酰肌醇-3-激酶（PI3K）磷酸化成为磷脂酰肌醇-3,4,5-三磷酸（PIP3），后者是激活PI3K/Akt细胞生存通路的关键信号分子。另一方面，磷脂酰肌醇-4-激酶（PI4K）可以将细胞中的PI磷酸化成为磷脂酰肌醇-4-磷酸（PI4P），后者则会被细胞膜上的磷脂酰肌醇-4-磷酸-5-激酶（PIP5K）磷酸化从而完成PIP2的合成。
除酪氨酸激酶通路外，PLC的激活一般是通过Gq蛋白耦联受体（GqPCR）介导的。儿茶酚胺类化合物、血管紧张素II等激素能够激活细胞膜上的GqPCR。激活的Gq蛋白的α亚基与βγ亚基分离，GTP水解释放的能量将PLC激活，后者完成PIP2的水解。在较短时间内PIP2通过水解局部的再合成机制来维持自身含量水平的稳定，在加大刺激强度并延长刺激时间的情况下细胞中PIP2的水平则可能发生改变。与短时刺激造成的局部影响不同，在增大作用浓度或延长刺激时间的情况下，G蛋白耦联受体激动剂能够增加成年大鼠心肌细胞或脑组织中PIP2的含量。许多重要的体内活性物质都是G蛋白耦联受体激动剂，这些活性物质往往在疾病状态下发生改变，因此在疾病的发生过程中相应细胞中PIP2的代谢状态也可能发生改变。所以，研究在这些情况下PIP2的代谢特征对于探究疾病的发生机制以及寻找可能的治疗方法十分有意义。本研究首先针对去甲肾上腺素和血管紧张素II对心肌PIP2代谢的影响及机制，以及这些作用与心肌肥厚、心脏功能之间的关系进行了研究；此外还研究了去甲肾上腺素和5-羟色胺对脑PIP2相关代谢机制的影响及其与抑郁症的相关性。研究结果表明，在激动剂刺激的过程中PIP2合成酶PI4K与PIP5K的活性发生变化是PIP2含量改变的原因；而且，这种PIP2含量的改变可能是细胞在激动剂刺激下为维持自身功能正常而做出的一种适应性改变。具体的研究内容如下：
第一部分去甲肾上腺素和血管紧张素II对大鼠心肌细胞膜PIP2以及心肌功能的调节
目的：早在上个世纪研究者们就发现了Gq蛋白耦联受体激动剂对细胞膜磷脂酰肌醇代谢的调节作用，然而对其中具体的调节机制和生理意义却仍不清楚。本部分实验对Gq蛋白耦联受体激动剂去甲肾上腺素（NE）、血管紧张素II（Ang II）调节大鼠心肌细胞膜PIP2代谢的分子机制进行深入研究，并初步探讨了PIP2代谢改变对心肌细胞功能的影响及其与心肌肥厚发展不同阶段的相关性。
方法：分离大鼠心室肌细胞，使用薄层色谱（TLC）和脂-蛋白交叠分析（Lipid-protein overlay assay, LPOA）的方法来分析NE、Ang II对心肌细胞PIP2含量的影响；以Gq/PLC通路相关信号分子特异性抑制剂—U73122（抑制PLC活性）和bisindolylmaleimide-1（Bis-1）（抑制PKC活性）预处理细胞，以PIP2合成酶抑制剂wortmannin、PIK93以及腺苷（adenosine）预处理细胞，再按照前面所述的方法分析NE、Ang II刺激后心肌细胞中PIP2的含量变化；利用免疫共沉淀的方法检测激动剂刺激后PIP2合成酶PI4K与蛋白激酶C（PKC）之间的相互作用；使用TLC的实验方法测定NE、Ang II刺激后心肌细胞PIP2水平变化的时程曲线，20分钟到72小时之间取4-5个时间点进行研究；按照PIP2变化时程曲线的时间点，使用细胞动缘探测系统检测NE、Ang II刺激后心肌细胞收缩功能的变化；建立大鼠游泳致心肌肥厚以及异丙肾上腺素（isoprenaline）注射致心肌肥厚的实验模型，使用LPOA的实验方法测定在生理性心肌肥厚以及病理性心肌肥厚的不同条件下大鼠心室肌中PIP2总含量。
结果：（1）原代心肌细胞培养24小时后，再以NE（5μM）刺激2小时，或以Ang II（3μM）刺激24小时，经测定发现刺激后心肌细胞中PIP2的含量明显增加，使用α受体抑制剂—prazosin（3μM）或AT1受体抑制剂—losartan（3μM）处理细胞能够拮抗NE或Ang II对PIP2含量的增加作用；（2）以U73122（0.8μM）、U73343（U73122的同形无效物）、Bis-1（2μM）以及wortmannin（10μM）预处理细胞20-30分钟，再以NE（5μM，2h）、Ang II（3μM，24h）刺激细胞，经测定发现U73122、Bis-1和wortmannin均能够抑制NE或Ang II对心肌细胞PIP2以及PIP含量的增加作用；（3）以III型PI4K的抑制剂—PIK93（250nM）或II型PI4K的抑制剂—adenosine（2μM）提前30分钟处理细胞，再给予NE（5μM，2h）、Ang II（3μM，24h）孵育，经测定发现只有PIK93对NE或Ang II引起的PIP2含量增加具有明显的抑制作用并且显著降低了细胞中PIP的水平。单独给予细胞PIK93或adenosine，PIK93对心肌细胞中PIP2以及PIP的含量均无明显影响，但adenosine则显著下调了细胞中PIP2以及PIP的含量水平；（4）NE（5μM）、Ang I（I3μM）以及PKC激动剂佛波酯（PMA，0.5μM）均能够增加PI4KIIIβ与PKCα之间、PI4KIIIβ与PKCβ之间的相互作用，而在心肌细胞中PI4KIIIα与PKCs之间并无明显的相互作用；（5）当刺激心肌细胞24小时以后，NE或Ang II对PIP2含量的增加作用随刺激时间的延长而逐渐降低，甚至可能逆转为降低作用。与此同时，对应时间点上心肌细胞的收缩功能也出现与PIP2含量类似的变化；（6）使用LPOA的方法测定两种心肌肥厚动物模型心脏中PIP2的含量，相比正常动物，游泳致心肌肥厚大鼠心脏中PIP2的含量水平并无较大改变，而Iso注射组大鼠心脏中PIP2的含量却显著减少。
结论：NE和Ang II激动Gq蛋白耦联受体，并通过受体激动后的下游信号通路水解PIP2，同时激活PIP2的再合成。PKC与PI4KIIIβ之间的相互作用是连接PIP2水解与再合成通路的分子基础。通过对心肌细胞收缩功能以及两种不同心肌肥厚模型的研究我们发现，在应激刺激下PIP2含量的增加可能与心肌肥厚初期心脏的代偿功能有关。
第二部分PIP5Kγ在G蛋白耦联受体调节大鼠心肌细胞膜PIP2中的作用
目的：了解磷脂酰肌醇-4-磷酸-5-激酶（PIP5K）在去甲肾上腺素（NE）、血管紧张素II（Ang II）及异丙肾上腺素（Iso）等G蛋白耦联受体激动剂在调节心肌细胞PIP2代谢中的作用及细胞信号机制。
方法：利用western blot的方法检测PIP5Kγ的表达水平；使用TLC的实验方法测定心肌细胞中PIP2的含量水平，观察NE、Ang II及Iso对心肌细胞PIP2含量的影响；使用药理学手段了解上述激动剂影响心肌细胞中PIP5Kγ的表达以及PIP2的含量的信号机制；使用蛋白合成酶抑制剂放线菌酮（cycloheximide）处理细胞，通过测定激动剂刺激后心肌细胞PIP2含量的改变，并以此来分析激酶表达与PIP2合成之间的联系；利用免疫共沉淀的方法检测激动剂刺激后PIP2合成酶PIP5Kγ与其水解酶PLC之间的相互作用。
结果：（1）NE（5μM）、Ang II（3μM）、PMA（0.5μM）以及β受体激动剂isoprenaline（0.5μM）均能够增加心肌细胞中PIP5Kγ的表达量，但NE与Iso的增加作用要显著大于Ang II与PMA的作用；（2）使用propranolol（3μM）抑制β受体或使用H89（30μM）抑制Gs蛋白耦联受体信号通路下游效应分子PKA，均能够抑制激动剂对PIP5Kγ表达水平的上调作用，但prazosin和Bis-1却对此无效；（3）β受体拮抗剂propranolol预处理细胞30分钟能够抑制NE对PIP2的增加作用。与NE类似，Iso也能够增加心肌细胞中PIP2的含量，但对PIP的含量却无明显影响；（4）Cycloheximide不仅能够抑制NE引起的PIP5Kγ表达量的增加，也能够显著降低心肌细胞中PIP2的含量并抑制NE的作用；（5）NE（5μM）、Ang II（3μM），以及Iso（0.5μM）均能够增强PIP2合成酶PIP5Kγ与水解酶PLCγ之间的相互作用。
结论：NE、Iso激动Gs蛋白耦联受体，并通过提高PIP5Kγ的表达水平来增加细胞中PIP2的再合成。由于能够同时激活Gq/PLC/PKC与Gs/cAMP/PKA两条通路，NE引起的PIP2含量增加要明显快于Ang II的作用。
第三部分去甲肾上腺素和5-羟色胺对鼠脑PIP2代谢的调节以及与抑郁症的关系
目的：PIP2是突触囊泡释放以及回收过程中不可缺少的关键物质，它与突触可塑性之间存在关系，同时也可能影响突触间递质的释放。因此研究脑磷脂酰肌醇特别是PIP2的代谢对于了解疾病状态下如抑郁症的发病机制以及寻找新的治疗手段具有重要意义。去甲肾上腺素（NE）及5-羟色胺（5-HT）是重要的与抑郁症相关的单胺类神经递质。在本研究中我们将研究NE和5-HT对大鼠和小鼠脑PIP2代谢的影响，通过测定给药后PIP2合成酶PI4K表达水平以及突触中PIP2含量的变化，初步探究中脑PIP2代谢改变与抑郁症之间的联系。
方法：利用western blot的方法检测NE、5-HT孵育后鼠脑内PIP2合成酶—PI4K的表达水平；提取膜蛋白，观察NE、5-HT孵育后PKC在细胞膜中的表达情况；利用免疫共沉淀的方法检测NE、5-HT孵育后PI4K与PKC之间的相互作用是否发生变化；分离突触小体，利用LPOA的方法（ELISA试剂盒）测定其中PIP2的含量，并观察NE、5-HT孵育后PIP2水平是否发生改变；建立小鼠的慢性社交失败抑郁症模型，初步研究模拟精神疾病状态下鼠脑内PI4K的表达状况。
结果：（1）给予NE（10μM）、5-HT（30μM）17分钟后，大鼠中脑切片中总PI4KIIIβ以及膜PI4KIIIβ的表达水平均出现明显增高；（2）通过使用PKC阻断剂Bis-1（1.5μM）预处理中脑切片我们发现，NE、5-HT对PI4KIIIβ表达量的增加作用依赖于PKC的激活；（3）NE、5-HT能够增强大鼠中脑切片中PI4KIIIβ与PKCα以及PI4KIIIβ与PKCβ之间的相互作用并增加其中脑突触小体中PIP2的含量；（4）虽然对小鼠中脑区域PI4K和PKC在的相互作用并无明显影响，但NE、5-HT能够显著降低PI4K和PKC在小鼠前额叶皮层中的相互作用以及该区域突触小体中PIP2的水平；（5）社交失败模型小鼠脑内的PI4KIIIβ表达水平下调。
结论：NE和5-HT能够促进大鼠中脑PI4K、PIP5K的表达，增强大鼠中脑和前额皮层PI4K与PKC间的相互作用，升高大鼠中脑PIP2水平。相对于大鼠，NE和5-HT对小鼠有不同的作用：只降低前额皮层PI4K与PKC间的相互作用和PIP2水平，而没有其他上述对大鼠的作用。在小鼠社交失败抑郁模型中，小鼠中脑和前额皮层中PI4K的表达相对于对照小鼠均有降低。这些初步结果表明，与抑郁症相关的中枢单胺类神经递质可以影响以PIP2为代表的磷脂酰肌醇代谢通路，并可能以此影响抑郁症的发生。
Phosphatidylinositol-4,5-bisphosphate (PIP2), which is a majorphosphoinositide of the plasma membrane, comprises about1%of plasmamembrane phospholipids. PIP2is enriched in the inner leaflet of the plasmamembrane, where it serves as a precursor of the second messengers, a ‘lipidanchor’ that attaches the cytoskeleton to the plasma membrane, and stabilizesor activates many membrane proteins, such as ion channels and transporters.Thus, changes of membrane PIP2metabolism may have roles in pathologicalconditions.
One of the well established cellular events involved in PIP2dynamics ismediated by the activation of the Gqprotein-coupled receptor (GqPCR): thestimulation of GqPCR activates phospholipase C (PLC), which in turn,hydrolyzes PIP2into inositol1,4,5-trisphosphate and diacylglycerol.Meanwhile, both PI(4,5)P2generation by phosphatidylinositol4-phosphate5-kinases (PIP5K) and the subsequent generation of PI(3,4,5)P3by thephosphoinositide3-kinases are dependent on an initial phosphorylation ofphosphatidylinositol (PI) on the D4position by one of the four mammalian PI4-kinase enzymes synthesising phosphatidylinositol4-phosphate (PI4P). AndPIP3is also an important signaling molecule that can stimulate theAkt-mediated cellular survival pathways.
Catecholamines and angiotensin II are well established GqPCR agonists.After ligand binding, a conformational shift in the G protein complex occursresulting in activation of a number of effectors such as PLC, which hydrolyzesmembrane PIP2. Generally, depletion of local PIP2is followed by thephosphoinositide reproduction. The recruitment of PIP2induced by GqPCRactivation is essential for the maintaince of local ‘PIP2pool’ and stabilization of global PIP2. However, prolonged stimulation, such as treatment withGqPCR agonists, may change the total phosphoinositides level incardiomyocytes or brain. It is well established that many GqPCR agonists areinvolved in the pathophysiological processes of diseases and as such theseagonists-mediated modulation of membrane PIP2metabolism may well beinvolved in these dieases. In this study, we studied the modulation andmechanism of phosphoinositides turnover of cardiac mocytes understimulation of norepinephrine (NE) and angiotensin II (Ang II) at both thecellular and whole animal levels, and related the changes of membrane PIP2with the development of cardiac hypertrophy and cardiac function. Also westudied the modulation of brain membrane PIP2by NE and5-HT and relatedthese modulations with the development of major depressive disorder (MDD).We described an increase of PIP2levels in cardiomyocytes and cerebral slicesunder stimulation of NE, Ang II and5-HT. We also present a novelmechanism for these increases to be the result of the enhanced activity ofPI4KIIIβ, mediated by an up-regulated interaction between PI4KIIIβ and PKC;furthermore, an increased activity of phosphatidylinositol4-phosphate5-kinase γ was involved in NE-induced increase of cardiac PIP2. These GPCRagonists mediated modulation of phosphoinositides turnover was related to thecardiac hypertrophy and major depression.
Part1Molecular mechanisms for norepinephrine and angiotensinII-induced increase in PIP2and modulation of cardiac function
Objective: The seemly paradoxical GqPCR agonist-stimulatedphosphoinositide production has long been known, but the underlyingmechanism and its physiological significance are not known. In this study, westudied cardiac phosphoinositide levels in both cells and whole animals underthe stimulation of norepinephrine (NE), angiotensin II (Ang II), and otherphysiologically relevant interventions.
Methods:(1) The thin layer chromatography (TLC) and lipid-proteinoverlay assay (LPOA) were used to measure the change of PIP2in the primarycultured cardiomyocytes after treated with NE and Ang II.(2) The effects of NE and Ang II on PIP2of cardiomyocytes were studied in the presence ofPLC inhibitors (U73122), PKC inhibitors (Bis-1), or three kinds of PI4Kinhibitors (wortmannin, PIK93, and adenosine).(3) Immunoprecipitation wasused to detect the interaction between PI4K and PKC.(4) PIP2level incardiomyocytes was measured after treated with NE for the time of20min,1h,2h,24h and48h, and with Ang II for the time of2h,24h,48h and72h.(5) The systolic function of the cultured cardiomyocytes was assessed bymeasuring sarcomere shortening induced by an electrical stimulation, whichrepresents the amplitude of cardiomyocyte contraction.(6) Lipid-proteinoverlay assay was used to measure the changes of total PIP2from rat heartswith hypertrophy induced either with isopreternol or with swimming excise.
Results:(1) After incubation with NE (5μM,2h) or Ang II (3μM,24h),the total abundance of PIP2in cardiomyocytes, measured using TLC, wasfound to be significantly increased. We also used a lipid-protein overlay assay,which confirmed the TLC results. Specific antagonists of the α-adrenergicreceptor prazosin, or Ang II receptor losartan (3μM) abolished the effects ofNE and Ang II, respectively.(2) The PLC inhibitor U73122(0.8μM; inactiveanalog U73343,0.8μM, was used as a control), the PKC inhibitor Bis-1(2μM), and the PI4K inhibitor wortmannin (10μM) were used to assess the rolesof these molecules. Pretreatment of cardiomyocytes with U73122, Bis-1, andwortmannin prevented the NE-or Ang II-induced increase in PIP and PIP2.(3)Pretreatment of cardiomyocytes with PIK93(250nM) prevented the NE-orAng II-induced increase in PIP and PIP2. Adenosine (2μM,2h), but notPIK93(250nM,2h), reduced basal PIP2levels in primary culturedcardiomyocytes.(4) Co-immunoprecipitation results showed that both PKCisoforms of α and βII were able to interact with PI4KIIIβ, but not withPI4KIIIα.(5) When cardiomyocytes were incubated with NE (5μM) or Ang II(3μM) for up to72h, both NE and Ang II induced a gradual increase in PIP2levels within the first24h, but afterward, the effects of NE and Ang II beganto recede, and PIP2levels fell back to control or even lower levels. Furtherly,the agonists affected the contraction of cardiomyocytes with a pattern and time course similar to their effect on PIP2levels.(6) The PIP2levels in the rathearts of Iso-induced hypertrophy were significantly reduced compared withthe time matched controls; PIP2levels in the rat hearts of swimming-inducedhypertrophy were increased compared with control hearts, although theincrease did not reach statistical significance.
Conclusion: Stimulation of rat cardiomyocytes with NE and Ang IIincreased both cellular PIP and PIP2levels. The increased PIP and PIP2induced by NE and Ang II was the result of an enhanced activity of PI4KIIIβ,mediated by an up-regulated interaction between PI4KIIIβ and PKC. Thisenhanced PIP2turnover is correlated with maintained cardiac systolic functionin compensative cardiac hypertrophy.
Part2The role of PIP5Kγ plays in GPCR agonists-mediated regulation ofmembrane PIP2in rat cardiomyocytes
Objective: To investigate the role of PIP5Kγ plays in NE-, Ang II-andisoprenaline (Iso)-induced regulation of PIP2turnover in rat cardiac myocytesand the related signaling mechanism.
Methods:(1) Western blot was used to detect the expression of PIP5Kγ,a key enzyme of PIP2production, in the cardiomyocytes.(2) Pharmacologicaltools were used to dissect the signaling mechanism for receptoragonists-induced modulation of PIP5Kγ expression and PIP2level.(3)Cycloheximide (CHX), a protein synthesis inhibitor, was used to relate theturnover of PIP5Kγ expression with the increased PIP2level.(4)Immunoprecipitation method was used to detect the interaction betweenPIP5Kγ and PLC.
Results:(1) In rat cardiomyocytes, PIP5Kγ was found to be up-regulatedafter stimulation with NE (5μM,2h), Iso (0.5μM,2h), Ang II (3μM,2h),and PMA (0.5μM,20min). However, the effect of NE and Iso on PIP5Kγwas significantly greater than that of Ang II or PMA.(2) The enhancedexpression of PIP5Kγ by NE was abolished by propranolol (a β receptorblocker) and H89(a PKA blocker), but not by prazosin and Bis-1.(3)Propranolol abolished the NE-induced increase in PIP2, and Iso induced an increase in PIP2levels.(4) In the presence of CHX, the NE-induced increasein total PIP2and PIP5Kγ expression in cardiomyocytes was inhibited.(5) Theinteraction between PIP5Kγ and PLCγ was increased by NE (5μM), Ang II (3μM) and Iso (0.5μM).
Conclusion: NE and Iso, through activation of GsPCR and PKA pathwayincreased the expression and activity of PIP5Kγ which enhanced synthesis ofPIP2. This effect of NE, combined with its effect of enhanced activity of PI4Kdescribed in the first part may explain a faster and stronger modulation ofcellular PIP2by NE than Ang II.
Part3The modulation of cerebral PIP2turnover by norepinephrine and5-hydroxytryptamine and its possible role in major depressivedisorder
Objective: Phosphoinositide lipids regulate key cellular functionsincluding endocytosis, signaling and secretion. In neuronal systems,well-studied areas include the roles of PIP2in synaptic vesicle recycling.Recent evidence suggests that PI4K and PIP2have important neuronalfunctions and that dysfunction of PI4K may have neuropathologicalconsequences. NE and5-HT are important monoamines involved in thedevelopment of major depressive disorder (MDD). In this study, we studiedthe effects and the mechanism of NE and5-hydroxytryptamine (5-HT) oncerebral PIP2turnover, and investigated consequence of these effects on thedevelopment of major depressive disorder.
Methods:(1) Western blot was used to measure total PI4KIIIβ in rat andmice brain slices after incubation with NE or5-HT.(2) Ultracentrifugationwas performed to extract membrane protein.(3) Immunoprecipitation wasused to detect the interaction between PI4K and PKC.(4) The component ofsynaptosome was extracted, and the synaptosomal PIP2was measured byLPOA (ELISA) method.(5) Major depression model of mice social defeatstress was established.
Results:(1) Incubation with NE (10μM) or5-HT (30μM) for17minincreased expression of both the total and the membrane PI4KIIIβ in the midbrain slices of rat but not in mice.(2) Incubation with NE increasedexpression of membrane PKC and blockage of PKC by Bis-1inhibited the NEand5-HT induced-increase of PI4KIIIβ expression.(3) PI4KIIIβ interactedwith PKCs in rat midbrain, which was enhanced by NE or5-HT. On the otherhand, the interaction between PI4K and PKC was down-regulated in theprefrontal cortex of mice.(4) Incubation with5-HT increased synaptosomalPIP2in the midbrain of rats, but decreased synaptosomal PIP2in the prefrontalcortex of mice.(5) Expression of PI4KIIIβ was down-regulated in themidbrain and prefrontal cortex of the model MDD mice.
Conclusion: NE and5-HT increased the expression of PI4K and PIP5Kin the midbrain of rats, increased the interaction of PI4K and PKC in both themidbrain and the prefrontal cortex of rats, and increased the PIP2level in themidbrain of rats. On the other hand, none of the above effects of NE and5-HTdescribed on rats were found in C57mice, but reduced the interaction betweenPI4K and PKC and the PIP2level in the prefrontal cortex of the mice. In socialdefeat depression model of mice, the expression of PI4K in both the midbrainand the prefrontal cortex were reduced compared with the control mice. Theseresults indicate the monoamine transmitters such as NE and5-HT are able tomodulate the metabolism of PIP2and such modulation may be related with thedevelopment of major depressive disorder.
引文
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