【作者】 宋丹；

【导师】 彭亮；

【作者基本信息】 中国医科大学 ， 药理学， 2009， 博士

【摘要】 前言锂盐治疗双相情感障碍最主要的机制是“肌醇消耗”假说。最初认为低浓度锂盐通过对肌醇单磷酸酶（inositol monophosphatase,IMPase）和肌醇多磷酸-1-磷酸酶（inositol polyphosphate 1-phosphatase,IPPase）的非竞争性抑制作用,使参与磷酯酶C（phospholipase C,PLC）信号传导的肌醇再生减少。此后研究发现锂盐介导肌醇消耗的另一个机制:锂盐慢性作用可抑制星形胶质细胞对肌醇的摄取。由于脑内只有内皮细胞可以合成肌醇,神经元或胶质细胞从细胞外摄取肌醇以维持细胞内肌醇水平就显得尤为重要。“肌醇消耗”假说不能完全解释锂盐对双相情感障碍躁狂相和抑郁相均有效的现象。一些研究发现,在肌醇水平较高时锂盐才可抑制星形胶质细胞瘤细胞对肌醇的摄取;而肌醇浓度低时,锂盐的慢性处理可引起细胞对肌醇的摄取增加。这可能与星形胶质细胞摄取肌醇的过程中有两个肌醇摄取载体参与有关:高亲合力的Na⁺-依赖性肌醇转运体（Na⁺-dependent inositol transporter,SMIT）和低亲合力的H⁺-依赖性肌醇转运体（H⁺-dependent inositol transporter,HMIT）。SMIT在pH较高时活性增加,而HMIT则在pH增加时活性降低。因此,如果锂盐处理可以使星形胶质细胞碱化,低亲合力的HMIT介导的肌醇摄取将会减少,而高亲合力的SMIT介导的肌醇摄取将会增多。这将可以解释锂盐能够治疗和预防双相情感障碍患者的躁狂发作和抑郁发作。同时经磁共振检查（magnetic resonancespectroscopy,MRS）研究发现双相情感障碍患者脑内pH降低,也进一步证实了这一猜测。在本研究中,我们将观察锂盐对细胞pH_i的影响,采用原代培养的小鼠星形胶质细胞观察:（1）治疗相似浓度Li⁺（0.5 mM、1.0 mM、2.0 mM）急性或慢性处理对星形胶质细胞稳态pH_i的影响;（2）Li⁺急性或慢性处理是否会影响星形胶质细胞NHE的活性;（3）Li⁺是否会调节NHE1 mRNA表达和蛋白的水平。（4）Li⁺持续作用后细胞内Li⁺浓度的大小。通过对治疗相似浓度锂盐急慢性处理后星形胶质细胞内pH_i的变化及机制的研究,为进一步了解锂盐治疗双相情感障碍的作用机制提供依据。方法首先进行小鼠大脑皮质星形胶质细胞的原代培养。用dBcAMP处理细胞1周后,采用Li₂CO₃或LiCl急性或慢性处理（两者浓度相当于Li⁺浓度达0.5 mM、1.0mM、2.0 mM）。处理后用原子分光光度剂进行细胞内Li⁺浓度测定。用5μMBCECF-AM进行荧光负载,以进行星形胶质细胞稳态pH_i的检测。采用NH₄Cl-预刺激技术对同样处理后的星形胶质细胞进行酸负载,以观察NHE活性的变化。Li⁺慢性处理后,进行PT-PCR和Western blot,观察NHE1 mRNA表达和蛋白水平的变化。采用one-way ANOVA方法进行多组间结果比较。P＜0.05表示具有统计学意义。结果1、细胞内Li⁺浓度Li⁺处理过程中,细胞内Li⁺的浓度可迅速增高。在Li⁺处理0.5小时、1小时或3天后细胞内Li⁺升高的幅度及细胞内与细胞外Li⁺浓度的最大比值（0.5 mM Li⁺处理3天时最大比值为0.4）差别不大。而1.0 mM及2.0 mM Li⁺处理3周后,细胞内的Li⁺浓度显著升高。虽然此时细胞内的Li⁺浓度仍低于细胞外,但细胞内与细胞外的Li⁺浓度比值可达0.50。2、稳态pH_i原代培养的星形胶质细胞在培养3-4周时平均pH_i在7.0-7.20之间,而培养6-7周时平均pH_i约为7.30,总的变化范围为6.8-7.4之间。1.0 mM Li₂CO₃（2.0 mMLi⁺）急性作用0.5-2小时对星形胶质细胞pH_i无影响。而锂盐慢性作用3天、1周、2周、3周或4周后发现,在不同时间段,2.0 mM Li⁺可使星形胶质细胞的pH_i升高0.20个pH单位（P＜0.05）,在2周和3周时1.0 mM Li⁺可使星形胶质细胞的pH_i升高0.10个pH单位（P＜0.05）。0.5 mM Li⁺对pH_i没有明显影响。用LiCl替代Li₂CO₃时也能获得相似的结果,表明是Li⁺而不是相伴的阴离子在起作用。3、锂盐急性或慢性处理对NHE活性的影响一般以NH₄Cl预刺激后pH_i恢复过程中的△pH_i/△t代表NHE活性。2.0 mM Li⁺急性处理星形胶质细胞可使NHE活性升高。用0.5 mM、1.0 mM或2.0 mM Li⁺持续作用细胞30分钟,可使NHE活性不同程度地增加（P＜0.05）,增加幅度达50%或以上。同样,使用LiCl也可获得相似的结果。Li⁺慢性作用细胞2周,在检测NHE活性时仍维持细胞内外Li⁺浓度梯度时,0.5 mM Li⁺不能增加NHE活性,甚至有时低于对照组。而1.0 mM或2.0 mM Li⁺慢性作用后可显著增加△pH_i/△t,增加幅度与急性处理时增加的幅度相同（P＜0.05）。但在检测NHE活性前去除细胞内外Li⁺浓度梯度,NHE活性呈下降趋势。1.0 mM或2.0 mM Li⁺慢性处理后可使NHE活性分别降低10%和20%,具有统计学差异（P＜0.05）。4、NHE1 mRNA的表达采用0.5 mM、1.0 mM、2.0 mM Li⁺慢性处理星形胶质细胞1天、3天、1周、2周、3周或4周后,检测NHE1 mRNA表达。不同浓度的Li⁺处理1天后NHE1mRNA表达无变化。0.5 mM Li⁺处理不同时间段后NHE1 mRNA表达也无变化。但是1.0 mM或2.0 mM Li⁺慢性处理3天至4周时间后,NHE1 mRNA表达较对照组下降30%左右。5、NHE1蛋白水平无论采用LiCl还是Li₂CO₃,0.5 mM、1.0 mM或2.0 mM Li⁺慢性处理细胞3周对NHE1蛋白水平无影响。讨论在本研究中,我们使用的Li⁺浓度在0.5.2.0 mM之间,包含了临床治疗时Li⁺产生有效治疗效应的血浆浓度范围。在实验中,我们发现临床治疗相似浓度的锂盐慢性处理星形胶质细胞后,细胞内的Li⁺浓度快速升高,但仍低于细胞外,这与Soares等发现使用锂盐治疗的患者脑内Li⁺浓度为0.23-0.55 mM,低于血浆Li⁺浓度（0.7 mM）的研究结果是一致的。细胞内Li⁺的积聚可能与胞膜上的Na⁺/H⁺交换体和Na⁺,K⁺-ATPas的作用有关,Li⁺可使Na⁺/H⁺交换体对Na⁺的摄取减少30%,也可抑制Na⁺,K⁺-ATPase对K⁺的摄取,通过这些转运机制可将Li⁺转运至细胞内。细胞对Li⁺的摄取还需要Na⁺,K⁺-ATPase介导的Li⁺外排机制以保持Li⁺的平衡,这说明Li⁺与Na⁺,k⁺-ATPase细胞外的K⁺活性位点及细胞内的Na⁺活性位点均有亲合力。在无CO₂/HCO₃^-存在的条件下,我们观察到星形胶质细胞稳态pH_i范围与Bevensee等人检测的哺乳动物星形胶质细胞的稳态pH_i相似。同时,我们也发现锂盐慢性作用能够引起星形胶质细胞碱化。这一现象的机制可能与Li⁺对NHE的活性调节有关。Li⁺与NHE的细胞外位点有较高的亲和力,可与细胞内的H⁺发生交换作用,通过内向的Li⁺浓度梯度刺激H⁺外流。这与我们所发现的1.0 mM和2.0 mM Li⁺引起细胞内碱化相一致。我们还发现无论Li⁺的急性作用还是慢性作用,在维持细胞内外的Li⁺浓度差时均可观察到NHE活性的增高。我们观察到治疗相似浓度的锂盐持续作用后,星形胶质细胞内外Li⁺浓度的比值为0.4-0.5。只要有细胞内外的浓度差存在,NHE的活性则增强。在星形胶质细胞中NHE1是主要的NHE异构体,在应用临床治疗相似浓度的Li⁺慢性作用后可引起星形胶质细胞NHE1 mRNA表达下调。我们发现Li⁺慢性处理后在Li⁺存在的条件下（维持细胞内外的Li⁺浓度差）检测NHE活性,NHE活性增加,但增加的幅度较Li⁺急性作用后引起的NHE活性增加的幅度小。而Li⁺慢性处理后在无Li⁺存在的条件下（去除细胞内外的Li⁺浓度差）,NHE活性是下降的。这可能与Li⁺慢性作用导致NHE1 mRNA表达下调有关。说明Li⁺慢性作用引起的NHE转录抑制对Li⁺刺激NHE活性增强起到负性调节作用。在研究中,我们发现应用治疗相似浓度的锂盐慢性处理星形胶质细胞后,细胞内的NHE1蛋白水平并没有随着NHE1 mRNA表达下调而发生变化。在应用心衰动物模型的研究中也有类似发现。我们发现虽然Li⁺急性作用即可引起NHE活性增高,但Li⁺对细胞稳态pH_i的影响却存在时间延迟。而在临床治疗过程中发现,Li⁺治疗效应的时间延迟与细胞内肌醇含量变化有关。只有肌醇摄取改变后导致细胞内肌醇含量发生变化时,Li⁺的治疗效应才会出现。Li⁺诱导的星形胶质细胞碱化可以调节细胞上肌醇转运体的活性:SMIT活性升高,细胞对肌醇的摄取增加;HMIT活性降低,细胞对肌醇的摄取减少。这可能是Li⁺进行细胞内肌醇含量的双相调节维持细胞内肌醇含量恒定的机制,也有助于解释锂盐即可治疗双相情感障碍躁狂相又可治疗抑郁相的现象。此外,卡马西平和丙戊酸钠的慢性处理也能够引起原代培养的星形胶质细胞出现细胞内碱化。但作用机制与Li⁺不同（与Na/HCO₃共转运体有关）。说明引起细胞内碱化可能与情绪稳定剂治疗双相情感障碍的作用机制有关。细胞内碱化除了可调节肌醇转运体的活性,还可对星形胶质细胞产生其它的作用。细胞内碱化可使cPLA2活性增强,同样应用锂盐、卡马西平或丙戊酸钠慢性处理星形胶质细胞也可使该酶的活性增强,这可能与情绪稳定剂作用机制有关。另外,细胞内碱化导致的D-丝氨酸生成增多,促进谷氨酸对NMDA受体的激动作用。星形胶质细胞的碱化还可激活K⁺通道引起K⁺外流,而抑制Na⁺依赖性的谷氨酸摄取,进而影响谷氨酸能信号传导。细胞的外部-核苷酸酶可以使ATP降解为AMP和焦磷酸,增加pH可刺激该酶活性,由于其为细胞外酶,当NHE活性增高引起细胞外酸化时它的活性会有所降低。细胞外酸化也可使NMDA受体活性降低,可部分补偿了由细胞内碱化导致的D-丝氨酸生成增多及谷氨酸摄取抑制所产生的效应。星形胶质细胞pH_i增加所引起的这些效应是否对神经元也能产生相似的效应还不能确定。然而,由于神经元内的Li⁺浓度高于星形胶质细胞,不会出现依靠细胞内/外的浓度梯度驱使Li⁺向细胞内运动与H⁺进行交换的情况,神经元的pH_i也就不会有所变化。刺激NHE1会引起Na⁺的内流,使细胞内的离子浓度发生变化并可引起细胞肿胀。在一些研究中发现,Li⁺处理后的大鼠脑组织水含量增加,而且临床应用Li⁺进行治疗时可见大脑假瘤等副作用的出现,这些现象均可能与Li⁺刺激NHE1引起细胞水肿有关。因此,Li⁺介导的星形胶质细胞NHE1活性增强不仅与其治疗相关,也与其产生的一些副作用有关。结论治疗相似浓度的锂盐慢性处理可使星形胶质细胞碱化,其机制可能是Li⁺作用于NHE细胞外结合位点,借助向内的浓度梯度与细胞内H⁺发生交换而产生细胞内碱化。而星形胶质细胞内pH_i的升高可能使细胞在高肌醇水平时对肌醇的摄取降低,在低肌醇水平时对肌醇的摄取增加。同时丙戊酸钠和卡马西平慢性处理也可以使星形胶质细胞pH_i升高,这也进一步支持了Li⁺这一效应的假说。此外,激活NHE1还可引起细胞对Na⁺及Cl^-的摄取并导致渗透性水肿,这可能与锂盐治疗时产生的副作用有关。更多还原

【Abstract】 IntroductionThe most widely accepted theory for the mechanism of the therapeutic effect of the lithium ion（Li⁺） in bipolar disorder is the inositol depletion hypothesis.This hypothesis was proposed on the basis of an uncompetitive inhibition of the regeneration of myo-inositol,needed for continued phospholipase C（PLC） signaling,from inositol monophosphate by low concentrations of Li⁺.Later an additional mechanism for Li⁺-mediated myo-inositol depletion was reported,i.e.,an inhibition of the cellular uptake of myo-inositol observed in astrocytes treated chronically with Li⁺.Cellular uptake is important,because the only cells within the brain that synthesize myo-inositol （from glucose） are endothelial cells.These findings do not explain why Li+ treatment is effective against both manic and depressive phases,but additional studies of myo-inositol uptake showed that uptake inhibition in Li⁺-treated astrocytoma cells only occurred at high concentrations of rnyo-inositol,whereas the uptake at a low concentration of myo-inositol was enhanced following Li⁺ treatment.As a possible explanation for this result it was pointed out that myo-inositol uptake in astrocytes occurs by the aid of two inositol uptake carriers,the high-affinity Na⁺-dependent inositol transporter（SMIT） and the lower affinity H⁺-dependent inositol transporter（HMIT）,where SMIT shows an increased activity at higher pH,whereas HMIT shows a reduced activity at higher pH.Accordingly myo-inositol uptake by the low-affinity HMIT would be reduced and that by the high-affiny SMIT increased,if Li⁺ treatment caused an alkalinization of the astrocytes. That this scenario may be relevant for the mechanism of action by Li⁺ treatment and/or prophylaxis of both mania and depression in bipolar patients is supported by magnetic resonance spectroscopy（MRS） studies,which have shown a decrease of pH in the brain of bipolar patients,including drug-free patients and led to the conclusion that cerebral pH may be decreased in bipolar disorder.In the present study we have therefore used primary cultures of mouse astrocytes, which functionally react to chronic treatment with Li⁺,to investigateⅰ) whether therapeutically relevant concentrations of Li⁺（0.5,1.0 and 2.0 mM） have acute and/or chronic effect on steady-state intracellular pH（pH_i）;ⅱ) whether Li⁺ has acute and/or chronic effects on the Na⁺/H⁺ exchanger（NHE）,the most important bicarbonate/CO₂-independent acid-extruding transporter in mammalian cells;ⅲ) whether Li⁺ regulates mRNA and/or protein expression of NHE1,a major NHE isoform in cultured astrocytes;andⅳ) the magnitude of the intracellular Li⁺ concentrations during continued Li⁺ treatment.Experimental MethodsThe Primary astrocytes were dissociated from the neophallia of the cerebral hemispheres.After treated with dBcAMP for 1 week,astrocytes were acute or chronic treated with lithium carbonate or lithium chloride,which both provide Li⁺ concentration of 2.0,1.0,or 0.5 mM.After treatment,the astroyctes were loaded with 5μM fluorescent pH-sensitive indicator,BCECF-AM.Then the steady state pHi was measured.The activity of NHE1 was determined by acute NH₄Cl-prepulse technique. After chronic treatment,the cells were collected for RT-PCR and Western-Blot to detect mRNA and protein expression of NHE1.The results are analysised with one-way ANOVA by Fisher’s LSD multiple comparison test for unequal replications.The level of significance was set at P＜0.05.Results1.Intracellular concentration of Li⁺.The intracellular concentration of Li⁺ increased rapidly during Li⁺ treatment with little,if any difference between cells treated for 0.5 hr,1 hr,or 3 days and a maximum ratio between intracellular and extracellular Li⁺ of 0.40（after treatment with 0.5 mM Li⁺ for 3 days）.After treatment for 3 weeks there was a doubling of the intracellular Li⁺ concentration at 1 and 2.0 mM extracellular Li⁺,although the intracellular concentration of Li⁺ remained well below its extracellular concentration,creating an extracellular/intracellular Li⁺ gradient of at most 0.50.2.pH_i. In astrocytes that had not been treated with Li⁺ the average pH_i was between 7.0 and 7.20 in 3-4-week-old cultures and around 7.30 in 6-7-week-old cultures,with values between 6.8 and 7.4 in individual cultures.Acute treatment with 1.0 mM lithium carbonate（2.0 mM Li⁺） for between 0.5 and 2 h caused no statistically significant change in pH_i,but chronic treatment with 2.0 mM Li⁺ for 3 days or 1,2,3 and 4 weeks increased it by～0.20 at all time points（P＜0.05）;similar chronic treatment with a Li⁺ concentration of 1.0 mM caused an increase of about 0.10,that was statistically significant at 2 and 3 weeks（P⁰.05）.A Li⁺ concentration of 0.5 mM did not significantly change pHi.Similar results were obtained after treatment with lithium chloride for 2 weeks,indicating that the effect was evoked by the lithium ion not the accompanying anion.3.NHE Activity.In untreated cells 2.0 mM Li⁺ exerted an acute effect on NHE activity,measured as△pH_i/△t during recovery after NH₄Cl challenge.The activity after 30 min of continued exposure to 0.5,1 or 2.0 mM Li⁺ was significantly（P＞0.05） increased by almost 50,slightly above 50 and almost 100%.Again,similar effects were observed with lithium chloride.The effect on△pH_i/△t of chronic treatment with 0.5 mM Li⁺ during 2 weeks was not statistically significant and appeared smaller than that in cells that had not been chronically treated,even when Li⁺ was present during the measurement of NHE activity.However,after treatment with 1 or 2.0 mM Li⁺ there was a statistically significant（P＜0.05） increase of almost the same magnitude as during acute treatment. When Li⁺ was withdrawn immediately before the measurement of NHE activity,there was a tendency towards a decrease of NHE activity in cells treated with Li⁺ for two weeks.After treatment with a Li⁺ concentration of 0.5 mM there was little,if any effect but after treatment with 1 or 2.0 mM Li⁺,the activity of NHE was reduced by～10 and～20%,respectively,a statistically significant effect（P＜0.05）.4.mRNA expression of NHE.mRNA Expression of NHE1 was examined after 1 or 3 days,or 1,2,3 or 4 weeks of treatment with 0.5,1 or 2.0 mM Li+.It was unchanged after 1 day,regardless of the concentration of Li⁺ and at all times studied after exposure to 0.5 mM Li⁺.However it was significantly decreased to～30%（P＜0.05） of control after treatment with 1 or 2.0 mM Li⁺ for between 3 days and 4 weeks.5.Protein expression of NHE1Regardless whether the cultures were treated with LiCl or lithium carbonate, treatment with 0.5,1,or 2.0 mM Li⁺ for 3 weeks had no significant effect on protein expression of NHE1.DiscussionBy studying the range 0.5-2.0 mM lithium,the pharmacologically relevant plasma concentration range has been included.The observation that the intracellular Li⁺ concentration is lower than its extracellular concentration is consistent with the observation of Li⁺ brain concentrations in the human brain in vivo between 0.23 and 0.55 mM at a plasma Li⁺ concentration of 0.7 mM.Accumulation of Li⁺ is likely to occur both via the Na⁺/H⁺ exchanger⁺,in agreement with the observation that Li⁺ inhibits Na⁺ uptake by this transporter by 30%at 1.0 mM Li⁺,and the Na⁺,K⁺-ATPase, consistent with an inhibition of K⁺ uptake.The uptake must be balanced by extrusion of Li⁺,also mediated by the Na⁺,K⁺-ATPase,which is in agreementt with the observation that Li⁺ has affinity for both the extracellular K⁺-activated site and the intracellular Na⁺-activated site of the Na⁺,K⁺-ATPase.The observed values for pHi in control cultures are similar to literature data for mammalian astrocytes in the absence of bicarbonate/CO₂.The intracellular alkalinization by Li⁺ is consistent with the conclusion in a review by Aronson that inward gradients of Li⁺ stimulate H⁺ efflux by an exchange of external Li⁺ for internal H⁺ by a Li⁺-mediated stimulation at the extracellular site of NHE.The inward gradients of Li⁺ also improve the activity of NHE in astrocytes with acute or chronic treatment with Li⁺.The down-regulation of NHE 1 mRNA in response to continuous stimulation by Li⁺ is in agreement with NHE1 being the predominant NHE isoform in astrocytes.The tendency towards a smaller Li⁺-mediated stimulation of NHE activity in cells during chronic treatment with Li⁺ than in acutely treated cells,and the reduction in NHE activity immediately after withdrawal of Li⁺ after chronic treatment are consistent with a down-regulation of NHE in response to continued stimulation.The mechanism of this homeostatic regulation is unknown but a similar response occurs in the case of K⁺ uptake,the rate of which becomes normalized（i.e.,upregulated） after chronic treatment with Li⁺.There is precedence from other systems,e.g.,experimentally induced heart failure that down-regulation of mRNA does not necessarily imply that expression of the corresponding protein is also down-regulated,even after a considerable length of time.Although NHE responded acutely to extracellular Li⁺,the change in intracellular pH required chronic treatment,especially at the lower Li⁺ concentrations.Moreover,in the clinical setting,there will be another lag phase until changes in uptake of myo-inositol lead to changes in myo-inositol content,all of which may contribute to the considerable length of time required for Li⁺ treatment to become effective.That the intracellular alkalinization in response to Li⁺ treatment may be relevant for its efficacy in treatment of bipolar disorder is suggested by a preliminary finding that chronic treatment with therapeutically relevant concentrations of carbamazepine or valproate also leads to an increased pHi in cultured astrocytes,albeit by a different mechanism （stimulation of Na/HCO₃ cotransporter）.Besides increasing myo-inositol uptake by SMIT and decreasing myo-inositol uptake by HMIT an increased pHi has additional effects on astrocytes.It may be of special interest that cPLA₂ is stimulated by elevated pHi,since chronic treatment with Li⁺,carbamazepine or valproic acid has been found to increase this enzyme in cultured astrocytes and with the exception of valproic acid also in the brain in vivo.Another reaction that is stimulated by increased phi is the astrocyte-specific racemation of L-serine,generating D-serine,an important agonist at the glycine-binding site of the NMDA receptor.An ecto-nucleotidase,degrading ATP to AMP and pyrophosphate is also stimulated by increased pH,at least in C6 glioma cells,but since this is an extracellular enzyme its activity is decreased by enhanced NHE activity.An increased extracellular acidity also decreases NMDA receptor activity.To what extent these other effects of increased pHi in astrocytes might affect astrocytic activity or whole-brain function is unknown,as is their possible correlation with bipolar disorder.The influx of Na⁺ by stimulation of NHE1 might affect intracellular ion content and cell swelling.Several studies show that stimulation of NHE1 might lead to uptake of water,which could explain the slight increase in brain water in Li⁺-treated rats and the occurrence of pseudotumor cerebri as a side effect of Li⁺ treatment in humans.Thus Li⁺-mediated stimulation of astrocytic NHE1 exchanger might be associated with,not only therapeutic effects of Li⁺,but also some of its side effects. ConclusionChronic treatment with a pharmacologically relevant Li⁺ concentration increases pHi in astrocytes,the mechanism is that inward gradients of Li⁺ sitmulate H⁺ efflux by an exchange of external Li⁺ for internal H⁺ by a Li⁺-mediated stimulation at the extracellular site of NHE.The astrocytic alkalinization may create conditions for decreased uptake of high myo-inositol concentrations and increased uptake of low concentrations.The pharmacological relevance of this effect is supported by literature data suggesting brain acidosis that increased that carbamazepine and valproate also increase pH_i in astrocytes.Stimulation of NHE1-stimulated sodium ion uptake might also trigger uptake of chloride ions and osmotically obliged water.更多还原