Influence of CO_2 pneumoperitoneum on intracellular pH and signal transduction in cancer cells

Object: The authors studied the influence of CO2 pneumoperitoneum on intracellular pH and signal transduction arising from cancer cell multiplication in laparoscopic tumor operation. Method: They set up a simulation of pneumoperitoneum under different CO2 pressure, and then measured the variation of intracellular pH (pHi) at different time and the activity of protein kinase C (PKC) and protein phosphatase 2a (PP2a) at the end of the pneumoperitoneum. After 1 week, the concentration of cancer cells in the culture medium was calculated. Result: When the pressure of CO2 pneumoperitoneum was 0, 10, 20, 30 mmHg respectively, the average pHi was 7.273, 7.075, 6.783, 6.693 at the end of the pneumoperitoneum; PKC activity was 159.4, 168.5,178.0, 181.6 nmol/(g.min) and PP2a was 4158.3, 4066.9, 3984.0, 3878.5 nmol/(g.min) respectively. After 1 week, the cancer cells concentration was 2.15×105, 2.03×105, 2.20×105, 2.18×105 L-1. Conclusion: CO2 pneumoperitoneum could promote acidosis in cancer cells, inducing the activation of protein kinase C and deactivation of protein phosphatase 2a, but it could not accelerate the mitosis rate of the cancer cells.

Functional and molecular mechanism of intracellular pH regulation in human inducible pluripotent stem cells

AIM To establish a functional and molecular model of the intracellular pH(pH_i) regulatory mechanism in human induced pluripotent stem cells(hiPSCs).METHODS hiP SCs(HPS0077) were kindly provided by Dr. Dai from the Tri-Service General Hospital(IRB No. B-106-09). Changes in the pH_i were detected either by microspectrofluorimetry or by a multimode reader with a pH-sensitive fluorescent probe, BCECF, and the fluorescent ratio was calibrated by the high K^+/nigericin method. NH_4Cl and Na-acetate prepulse techniques were used to induce rapid intracellular acidosis and alkalization, respectively. The buffering power(β) was calculated from the ΔpH_i induced by perfusing different concentrations of(NH_4)_2SO_4. Western blot techniques and immunocytochemistry staining were used to detect the protein expression of pH_i regulators and pluripotency markers.RESULTS In this study, our results indicated that(1) the steadystate pH_i value was found to be 7.5 ± 0.01(n = 20) and 7.68 ± 0.01(n =20) in HEPES and 5% CO_2/HCO_3^- buffered systems, respectively, which were much greater than that in normal adult cells(7.2);(2) in a CO_2/HCO_3^--buffered system, the values of total intracellular buffering power(β) can be described by the following equation: β_(tot) = 107.79(pH_i)~2-1522.2(pH_i) + 5396.9(correlation coefficient R^2 = 0.85), in the estimated pH_i range of 7.1- 8.0;(3) the Na^+/H^+ exchanger(NHE) and the Na^+/HCO_3^- cotransporter(NBC) were found to be functionally activated for acid extrusion for pHi values less than 7.5 and 7.68, respectively;(4) V-ATPase and some other unknown Na^+-independent acid extruder(s) could only be functionally detected for pHi values less than 7.1;(5) the Cl^-/OH^- exchanger(CHE) and the Cl^- /HCO_3 anion exchanger(AE) were found to be responsible for the weakening of intracellular proton loading;(6) besides the CHE and the AE, a Cl^--independent acid loading mechanism was functionally identified; and(7) in hiPSCs, a strong positive correlation was observed between the loss of pluripotency and the weakening of the intracellular acid extrusion mechanism, which included a decrease in the steady-state pH i value and diminished the functional activity and protein expression of the NHE and the NBC.CONCLUSION For the first time, we established a functional and molecular model of a pHi regulatory mechanism and demonstrated its strong positive correlation with hiPSC pluripotency.

Dynamic Measurement of Intracellular pH Based on Bioluminescent Bacteria

Intracellular pH(pHi)is a fundamental indicator of cellular physiological state,regulating cellular state and function,and has important research values.Although various probes for measuring intracellular pH were available,it is challenging to reflect pHi in real-time and reversible manners.Herein,we developed a whole-cell bioluminescent(BL)probe based on wild type BL bacteria,photobacterium phosphoreum(P.phosphoreum),to determine and image pHi.The dependence of BL intensity of P.phosphoreum on pH values of culture solutions was established.It was found that BL intensity could respond to the change of pH values rapidly and reversibly.We further revealed that P.phosphoreum maintained pH homeostasis in the extracellular pH(pHe)within the range of 5.0–7.0,while intracellular pH homeostasis was destroyed at the alkaline pHe.This method opens up the enormous potential of BL bacteria as an alternative to fluorescence for monitoring and imaging pHi.

LipR functions as an intracellular pH regulator in Bacillus thuringiensis under glucose conditions

Intracellular pH critically affects various biological processes,and an appropriate cytoplasmic pH is essential for ensuring bacterial growth.Glucose is the preferred carbon source for most heterotrophs;however,excess glucose often causes the accumulation of acidic metabolites,lowering the intracellular pH and inhibiting bacterial growth.Bacillus thuringiensis can effectively cope with glucose-induced stress;unfortunately,little is known about the regulators involved in this process.Here,we document that the target of the dual-function sRNA YhfH,the lipR gene,encodes a LacI-family transcription factor LipR as an intracellular pH regulator when B.thuringiensis BMB171 is suddenly exposed to glucose.Under glucose conditions,lipR deletion leads to early growth arrest by causing a rapid decrease in intracellular pH(~5.4).Then,the direct targets and a binding motif(GAWAWCRWTWTCAT)of LipR were identified based on the electrophoretic mobility shift assay,the DNase-I footprinting assay,and RNA sequencing,and the gapN gene encoding a key enzyme in glycolysis was directly inhibited by LipR.Furthermore,Ni^(2+)is considered a possible effector for LipR.In addition to YhfH,the lipR expression was coregulated by itself,CcpA,and AbrB.Our study reveals that LipR plays a balancing role between glucose metabolism and intracellular pH in B.thuringiensis subjected to glucose stress.

pH-sensitive polymeric micelles triggered drug release for extracellular and intracellular drug targeting delivery

Most of the conventional chemotherapeutic agents used for cancer chemotherapy suffer from multidrug resistance of tumor cells and poor antitumor efficacy.Based on physiological differences between the normal tissue and the tumor tissue,one effective approach to improve the efficacy of cancer chemotherapy is to develop pH-sensitive polymeric micellar delivery systems.The copolymers with reversible protonationedeprotonation core units or acid-liable bonds between the therapeutic agents and the micelle-forming copolymers can be used to form pH-sensitive polymeric micelles for extracellular and intracellular drug smart release.These systems can be triggered to release drug in response to the slightly acidic extracellular fluids of tumor tissue after accumulation in tumor tissues via the enhanced permeability and retention effect,or they can be triggered to release drug in endosomes or lysosomes by pH-controlled micelle hydrolysis or dissociation after uptake by cells via the endocytic pathway.The pH-sensitive micelles have been proved the specific tumor cell targeting,enhanced cellular internalization,rapid drug release,and multidrug resistance reversal.The multifunctional polymeric micelles combining extracellular pH-sensitivity with receptor-mediated active targeting strategies are of great interest for enhanced tumor targeting.The micelles with receptor-mediated and intracellular pH targeting functions are internalized via receptor-mediated endocytosis followed by endosomal-pH triggered drug release inside the cells,which reverses multidrug resistance.The pH sensitivity strategy of the polymeric micelles facilitates the specific drug delivery with reduced systemic side effects and improved chemotherapeutical efficacy,and is a novel promising platform for tumor-targeting drug delivery.

Monitoring intracellular pH fluctuation with an excited-state intramolecular proton transfer-based ratiometric fluorescent sensor

Intracellular pH is a key parameter related to various biological and pathological processes.In this study,a ratiometric pH fluorescent sensor ABTT was developed harnessing the amino-type excited-state intramolecular proton transfer(ESIPT) process.Relying on whether the ESIPT proceeds normally or not,ABTT exhibited the yellow fluorescence in acidic media,or cyan fluorescence in basic condition.According to the variation,ABTT behaved as a promising sensor which possessed fast and reversible response to pH change without interference from the biological substances,and exported a steady ratiometric signal(I_(478)/I_(546)).Moreover,due to the ESIPT effect,large Stokes shift and high quantum yield were also exhibited in ABTT.Furthermore,ABTT was applied for monitoring the pH changes in living cells and visualizing the pH fluctuations under oxidative stress successfully.These results elucidated great potential of ABTT in understanding pH-dependent physiological and pathological processes.

AgInS_(2)/ZnS quantum dots for noninvasive cervical cancer screening with intracellular pH sensing using fluorescence lifetime imaging microscopy

Intracellular pH plays a critical role in biological functions,and abnormal pH values are related to various diseases.Here,we report on an intracellular pH sensor AgInS_(2)(AIS)/ZnS quantum dots(QDs)that show long fluorescence lifetimes of hundreds of nanoseconds and low toxicity.Fluorescence lifetime imaging microscopy(FLIM)combined with AIS/ZnS QDs is used for the imaging of live cells in different pH buffers and different cell lines.The FLIM images of AIS/ZnS QDs in live cells demonstrate different intracellular pH values in different regions,such as in lysosomes or cytoplasm.This method can also distinguish cancer cells from normal cells,and the fluorescence lifetime difference of the AIS/ZnS QDs between the two types of cells is 100±7 ns.Most importantly,the exfoliated cervical cells from 20 patients are investigated using FLIM combined with AIS/ZnS QDs.The lifetime difference value between the normal and cervical cancer(CC)groups is 115±9 ns,and the difference between the normal and the precancerous lesion group is 64±9 ns.For the first time,the noninvasive method has been used for cervical cancer screening,and it has shown great improvement in sensitivity compared with a clinical conventional cytology examination.

SciFinder-guided rational design of fluorescent carbon dots for ratiometric monitoring intracellular pH fluctuations under heat shock

Intracellular pH plays a significant role in various biological processes, including cell proliferation,apoptosis, metabolism, enzyme activity and homeostasis. In this work, a novel design strategy for the preparation of pH responsive carbon dots(CDs-pH) for ratiometric intracellular imaging was reported. By using SciFinder database, fluorescent CDs-pH with the required p Kavalue of 6.84 were rationally designed, which is vital important for precise sensing of intracellular pH. As a result, the synthesized CDspH demonstrated robust ability to test pH fluctuations within the physiological range of 5.4-7.4. The CDspH was further utilized for fluorescent ratiometric imaging of pH in living HeLa cells, effectively avoided the influence of autofluorescence from native cellular species. Moreover, real-time monitoring of intracellular pH fluctuation under heat shock was successfully realized. This SciFinder-guided design strategy is simple and flexible, which has a great potential to be used for the development of other types of CDs for various applications.

灵芝多糖对小鼠T细胞胞浆游离Ca^(2+)浓度和胞内pH的影响

目的通过考察灵芝多糖（ＧＬＰ）对免疫细胞信号转导过程的影响，探讨ＧＬＰ免疫调节作用机制。方法采用激光扫描共聚焦显微镜（ＬＳＣＭ）技术，动态监测ＧＬＰ均一体组分ＧＬＢ７对小鼠Ｔ细胞胞浆游离Ｃａ２＋浓度（［Ｃａ２＋）和胞内 ｐＨ（［ｐＨ］ｉ）的影响。结果 ＧＬＢ７（２０ｍｇ·Ｌ－１）引起小鼠 Ｔ细胞中［Ｃａ２＋］；和［ｐＨ］；明显升高，１ｍｉｎ即分别升高至３３４．７０％±１６，４％（ｎ＝３）、１７１．６％ ± １０．４％（ｎ＝３），之后一直分别维持在该平台期，至 １０ ｍｉｎ仍维持高峰水平。加入ＥＣＴＡ和 ｖｅｒａｐａｍｉｌ处理后， １０ ｍｉｎ ＧＬＢ７ 引起 Ｔ细胞［Ｃａ２＋］；和［ｐＨ］；分别升高为 ２０２．４％± １３．６％（ｎ＝３）。１４０．２％±７．８％（ｎ＝３），与正常对照组比较差异有显著性（Ｐ＜０．０1）。以 ＥＧＴＡ和 ｖｅｒａｐａｍｉｌ处理后，再分别以 ｈｅｐ－ｅｒｉｎ和 ｐrocaine处理细胞 １０ ｍｉｎ，之后以 ＧＬＢ７刺激Ｔ细胞，１０ｍｉｎ［Ｃａ２＋］ｉ值分别为１５１．５％±９．４％（ｎ＝３）、１４３．２％± ８．１ ％（ ｎ＝ ３），与 ＥＧＴＡ和 ｖｅｒａｐａｍｉｌ处理组相比差异有显著性（ Ｐ＜ ０．０１）。

热带假丝酵母细胞内pH的测定及其与生长代谢活性的关系

应用荧光探针 5(6)_双醋酸羧基荧光素 (Carboxyfluoresceindiacetate)测定了产长链二元酸热带假丝酵母(Candidatropicalis)细胞内pH (pHi)值 ,确定了该探针载入C .tropicalis细胞的适宜条件。用摇瓶培养C .tropicalis细胞 ,考察了细胞外pH和生长碳源对pHi的影响 ,实验结果表明 :细胞外pH对pHi略有影响 ,而生长碳源对pHi的影响略为明显。利用 5L发酵罐进一步研究了细胞生长代谢活性与pHi的关系 ,结果表明 :细胞比生长速率、CO2 比生产速率和葡萄糖比消耗速率与pHi变化密切相关 ,pHi的增加伴随着细胞生长活力的增加 ,反之亦然。在pH 6 0条件下用葡萄糖和醋酸钠共作碳源培养C .tropicalis细胞时 ,测得的pHi值维持在 5 72～ 6

虎杖苷对休克大鼠微血管平滑肌细胞内钙、pH和膜电位的影响

目的探讨虎杖苷（ＰＤ）改善休克动物微循环的作用机制。方法股动脉放血复制休克模型，取肠系膜微动脉用链霉蛋白酶Ｅ消化以分离单个血管平滑肌细胞（ＶＳＭＣ），再用不同荧光染料分别标记细胞，在激光共聚焦显微镜上测定细胞内钙（［Ｃａ２＋］ｉ）、ｐＨ和膜电位的变化。结果ＰＤ（０４ｍｍｏｌ·Ｌ－１）使休克大鼠ＶＳＭＣ［Ｃａ２＋］ｉ浓度在１０ｍｉｎ内显著下降至加药前的７８４％±５６％、ｐＨ下降至原来的７２８％±８２％；而正常对照组ＶＳＭＣ［Ｃａ２＋］ｉ升高１７５％±６３％、ｐＨ上升３４％±１０１％。当ＰＤ加入前１０ｍｉｎ用钙通道阻断剂维拉帕米（５０μｍｏｌ·Ｌ－１）预处理后，则动物不论休克与否，其［Ｃａ２＋］ｉ、ｐＨ都显著下降。另外ＰＤ使正常及休克大鼠ＶＳＭＣ膜电位负值下降，出现去极化反应。加入ＥＧＴＡ和维拉帕米预处理不能阻断ＰＤ作用，但加入钠通道阻断剂河豚毒素（１μｍｏｌ·Ｌ－１）则可完全阻断ＰＤ的去极化作用。结论ＰＤ对细胞内钙、ｐＨ有双向调节作用，正常情况下ＰＤ增加细胞内游离钙及升高ｐＨ以提高血管张力，休克时ＰＤ降低细胞内钙浓度及降低细胞内ｐＨ以降低血管张力，使血管扩张。此外ＰＤ还可能通过促进细胞外钠离子?

“植物细胞内pH调控系统”是适应环境逆境的一个耐性机制?

本研究把“植物细胞内 pH调控系统”与植物抗逆性的关系作为研究对象 ,并探讨了参与植物细胞内pH调控相关的基因在碳酸盐 (Na2 CO3,NaHCO3)等逆境下的表达特性 ,其结果表明 :NADP苹果酸酶(NADP MalicEnzyme,NADP ME)基因、磷酸烯醇式丙酮酸羧化酶 (PhosphoenolpyruvateCarboxylase,PEP Case)基因 ,还有细胞膜H+ ATPase (plasmamembraneH+ ATPase ,P H+ ATPase)基因、液泡膜H+ AT Pase (vacuolarmembranceH+ ATPase ,V H+ ATPase)基因、液泡膜H+ PPase (vacuolarmembranceH+ PPase ,V H+ Ppase)基因等的表达与NaCl逆境、碳酸盐逆境、外界环境 pH逆境有应答关系 ,显示了这些基因在逆境下的表达有被强化的趋势。通过对逆境胁迫后植物根活体切片进行的pH变化趋势的观察 ,结果表明 :植物根细胞在组织学水平上 pH有明显的变化。在逆境下水稻根的组织学水平的观察 ,暗示了根组织有被酸性化的趋势 ,从而我们推测在逆境胁迫下细胞内 pH发生了改变 ,不正常的细胞内 pH会抑制细胞的生长发育 ,使植物受害。由此 ,我们提出细胞内 pH调控系统是植物在长期的进化过程中获得的抗逆机制。在逆境下强化“细胞内pH调控系统”的调控能力能够提高植物的抗性。

姜半夏乙醇提取物对人胃腺癌SGC7901细胞内pH值的影响

目的:观察姜半夏乙醇提取物对人胃腺癌SGC7901细胞内pH值的影响。方法:不同浓度姜半夏乙醇提取物(终浓度分别为1、0.5、0.25、0.125mg/mL)处理SGC7901细胞后,通过甲基噻唑基四唑(3-(4,5-di methylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide,MTT)法检测细胞增殖状况,使用2,7-双(2-羧基乙基)-5(6)-羧基荧光素四(丙酰甲酯)(2,7-bis-(2-carboxyethyl)-5-carboxyfluorescein-acetoxymethyl,BCECF-AM)荧光探针检测细胞内pH值,pH计测定胞外培养基的pH值;采用荧光定量聚合酶链反应技术检测药物干预细胞72h后细胞钠氢交换蛋白1(Na+/H+exchangerisoform1,NHE1)和空泡质子转运ATP酶(vacuolar-H+-ATPase,V-ATPase)基因的表达水平。结果:不同浓度姜半夏乙醇提取物均能不同程度地抑制人胃癌SGC7901细胞的增殖,降低细胞内pH值,提高细胞外培养基pH值。0.5mg/mL姜半夏乙醇提取物可下调SGC7901细胞V-ATPase和NHE1mRNA的表达。结论:姜半夏乙醇提取物具有降低SGC7901细胞内pH值的作用,其机制可能与抑制细胞V-ATPase和NHE1mRNA表达有关。

肿瘤细胞内pH值改变与肿瘤多药耐药的关系

细胞内pH值(pHi)增高是许多耐药肿瘤的共同特点,其结果引起肿瘤细胞对化疗药物的敏感性下降,降低药物疗效。通过对pHi的调节,可纠正细胞内碱化,逆转肿瘤耐药。Na+-H+交换蛋白(Na+/H+exchanger,NHE)抑制剂可通过抑制NHE的活性调节pHi,为肿瘤的治疗提供了新的思路和手段。

共聚焦镜观察凋亡巨噬细胞内pH的变化

用透射电镜观察巨噬细胞的形态学改变，结果显示，地塞米松处理８小时后，大部分巨噬细胞发生凋亡特征变化：胞突缩短、减少，胞膜完整。胞体皱缩，胞质密度增加，其中出现大量空泡。胞核染色质边聚、浓缩。另外用激光扫描共聚焦显微镜（ＡＣＡＳ５７０）和ｐＨ荧光探针ＳＮＡＲＦ┐１／ＡＭ实时检测地塞米松处理巨噬细胞胞浆ｐＨ的动态变化。加入地塞米松，多数巨噬细胞胞浆马上发生快速和短期的碱化。随后，胞浆ｐＨ缓慢降低，胞浆酸化。结果表明，胞浆酸化是细胞凋亡发展的必然过程，胞浆碱化则很可能与细胞凋亡的发生相关，也可能与细胞种类。

利用三通道实时荧光成像方法研究单个活细胞凋亡与胞浆pH值变化的关系

利用双波长分光器(Dual-View)和自制滤光片滑块,在基于像增强型电荷耦合器件(ICCD)的实时/快速荧光成像系统基础上,建立了一种基于单个ICCD的三通道实时荧光成像方法,同时发展了相应的图像校准处理方法用于消除光谱串扰的影响,并将该方法应用于单个活细胞的研究。利用Annexin V-FITC和SNARF-1两种荧光探针进行双标记,在单细胞水平上对亚硝基谷胱甘肽(GSNO)诱导小鼠胸腺细胞凋亡与其胞浆pH值变化的关系进行实时研究。结果揭示了GSNO诱导的细胞凋亡与细胞发生自发凋亡时胞浆pH值有不同的变化规律,为这种三通道实时荧光成像方法在生物医学领域的应用展示了广阔的前景。

细胞内pH对胆碱能受体介导的Kir3.1/3.4电流的调节

目的 研究细胞内pH对胆碱能受体介导的Kir3 1/3 4电流的调节作用。方法 应用AzideNa、KHCO3 和通过灌流直接降低细胞内 pH ,用双电极电压钳和膜片钳方法观察在蛙卵细胞中表达的Kir3 1/ 3 4钾离子通道电流的变化和M受体激活对Kir3 1/ 3 4电流调节的变化。结果 细胞内 pH降低能抑制Kir3 1/ 3 4的电流 ;Kir3 1/ 3 4对细胞内pH的敏感性介于另外两种Kir通道Kir2 1和Kir2 3之间 ,即这三种通道对细胞内 pH的敏感性依次为Kir2 3>Kir3 1/ 3 4 >Kir2 1;细胞内pH降低能够减弱M1受体激活对Kir3 1/ 3 4的抑制作用 ,加强M2 受体激活Kir3 1/ 3 4电流后的去敏作用。结论 在维持细胞静息电位方面起重要作用的Kir3 1/ 3 4通道在细胞内pH降低的情况下基础电流和M受体激活调节电流均发生了变化 ,这些变化在缺血缺氧引起的细胞 (如心肌细胞 )

甘草水溶物诱导MGC-803细胞凋亡中胞内Ca^(2+)、pH与线粒体△ψ_m的变化

Changes of intracellular Ca 2+ ,pH value and mitochondria membrane potential(△Ψ m) in the apoptosis of MGC 803 cells induced by water soluble constituents of Glycyrrhiza uralensis Fisch(WSCG) were investigated.MGC 803 cells were incubated with 0.5,0.75,1.0 and 1.5 g/L WSCG for 1,4,7,11,15,19 and 23 hours respectively.The percentage of apoptosis and intracellular Ca 2+ content increased in a dose and time dependent manner,except that the intracellular Ca 2+ content of 1.5 g/L WSCG treated cells started to decrease after 11 hours.The cells were alkalized after various treatments,except that 1.5 g/L WSCG treated cells turned to acidify after 11 hours.It was found that the mitochondria △Ψ m of all treated cells decreased drastically at the first hour,then kept decreasing slowly until the 23rd hour.The results indicated that intracellular Ca 2+ ,pH and mitochondria △Ψ m all played pivotal roles in the apoptosis of MGC 803 cells induced by WSCG.

氢离子选择性微电极细胞内pH测量系统的研制

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