Radioiodine therapy for castration-resistant prostate cancer following prostate-specific membrane antigen promoter-mediated transfer of the human sodium iodide symporter

Radioiodine therapy, the most effective form of systemic radiotherapy available, is currently useful only for thyroid cancer because of the thyroid-specific expression of the human sodium iodide symporter （hNIS）. Here, we explore the efficacy of a novel form of gene therapy using prostate-specific membrane antigen （PSMA） promoter-mediated hNIS gene transfer followed by radioiodine administration for the treatment of castration-resistant prostate cancer （CRPC）. The androgen-dependent C33 LNCaP cell line and the androgen-independent C81 LNCaP cell line were transfected by adenovirus. PSMA promoter-hNIS （Ad.PSMApro-hNIS） or adenovirus.cytomegalovirus-hNIS containing the cytomegalovirus promoter （Ad.CMM-hNIS） or a control virus. The iodide uptake was measured in vitro. The in vivo iodide uptake by C81 cell xenografts in nude mice injected with an adenovirus carrying the hNIS gene linked to PSMA and the corresponding tumor volume fluctuation were assessed. Iodide accumulation was shown in different LNCaP cell lines after Ad.PSMApro-hNIS and Ad.CMV-hNIS infection, but not in different LNCaP cell lines after adenovirus.cytomegalovirus （Ad.CMV） infection. At each time point, higher iodide uptake was shown in the C81 cells infected with Ad.PSMApro-hNIS than in the C33 cells （P 〈 0.05）. An in vivo animal model showed a significant difference in 1311 radioiodine uptake in the tumors infected with Ad.PSMApro-hNIS, Ad.CMV-hNIS and control virus （P 〈 0.05） and a maximum reduction of tumor volume in mice infected with Ad.PSMApro-hNIS. These results show prostate-specific expression of the hNIS gene delivered by the PSMA promoter and effective radioiodine therapy of CRPC by the PSMA promoter-driven hNIS transfection.

Mechanism of intervention of medicine combined with mild moxibustion on hypothyroidism rat model

Objective:To explore the intervention effect of medicine combined with mild moxibustion on immune factor and Na/I symporter(NIS)in hypothyroidism rat model.Methods:The model was successfully made by intragastric administration of propylthiouracil(PTU)solution medicine and medicine combined with mild moxibustion groups were given levothyroxine sodium suspension 60μg/kg body weight by gavage,once a day.In the medicine combined with mild moxibustion group,mild moxibustion was applied in“Dazhui”,“Mingmen”,“Pishu”,“Shenshu”,and 10 minutes per point,once a day,and one day off every six days;four weeks in a row.The model group and medicine was fixed in the same way as the medicine combined with mild moxibustion group.The blank group received no treatment.The contents of thyrotropin-releasing hormone(TSH),tatalthyroxine(TT4),thyroid peroxidase antibody(TPOAb),thyroglobulin antibodies(TGAb),Interleukin-4(IL-4)and Interleukin-23(IL-23)in serum were determined by Enzyme Linked Immunosorbent Assay(ELISA).The content of NIS and the expression level of NISmRNA in thyroid tissues of each group were detected by immunohistochemistry and real-time polymerase chain reaction(R-T PCR).Results:Compared with the blank group,the contents of TSH,TPOAb,TGAb and IL-23 in the serum of rats in the model group were increased,the contents of IL-4 and TT4 were decreased,and the contents of NIS and NISmRNA in thyroid tissue were decreased,with statistical significance(P<0.01).Compared with model group,the contents of TSH,TPOAb,TGAb and IL-23 in serum of medicine group and medicine combined with mild moxibustion group were decreased,while the contents of IL-4 and TT4 were increased;the NIS content and NISmRNA expression in thyroid tissues were increased,and the differences were statistically significant(P<0.01).Compared with the medicine group,NISmRNA expression in thyroid tissues of medicine combined with mild moxibustion groups was increased,and the difference was statistically significant(P<0.05).Conclusion:Medicine combined with mild moxibustion can decrease the contents of TPOAb,TGAb and IL-23,and increase the content of IL-4,increasing the content and expression of NIS to interfere with the hypothyroidism rat model.

皮状丝孢酵母同步利用葡萄糖/木糖的糖转运动力学

皮状丝孢酵母(Trichosporon cutaneum)能够同步利用葡萄糖和木糖生产油脂。以2-脱氧葡萄糖(2-DOG)为底物,考察皮状丝孢酵母糖跨膜运输的转运动力学。结果表明:2-DOG转运符合米氏方程,表观米氏常数K m为0.19 mmol/L,最大转运速率V max为14.1 nmol/(min·mg)。葡萄糖和木糖均竞争性抑制2-DOG转运,葡萄糖表观抑制常数K i远低于木糖,表明存在一个共用转运体系,且该转运体系对葡萄糖亲和力更高。大量木糖与2-DOG同时转运到胞内,进一步说明木糖与葡萄糖共运输。代谢抑制剂和pH对糖转运有明显影响,说明质子/底物同向运输系统是该酵母的主要糖转运系统。

通讯膜计算系统研究综述

膜计算是自然计算的一个分支,文章主要研究从活细胞的结构和功能中或从组织和器官等细胞群协作中抽象的计算模型.根据膜结构不同,膜计算系统可以分为细胞型膜系统(树状结构)和组织型膜系统或脉冲神经膜系统(任意图结构).细胞间的通讯是膜系统中的一个重要特征,文章讨论的通讯规则是指同向/异向规则,对细胞型和组织型通讯膜系统的研究现状进行概述,从计算能力和计算复杂性方面介绍这2类通讯膜系统的研究进展.最后给出通讯膜系统中存在的一些问题.

Baculovirus vector-mediated transfer of NIS gene into colon tumor cells for radionuclide therapy

AIM:To investigate the feasibility of radionuclide therapy of colon tumor cells by baculovirus vector-mediated transfer of the sodium/iodide symporter(NIS) gene.METHODS:A recombinant baculovirus plasmid carrying the NIS gene was constructed,and the viruses(BacNIS) were prepared using the Bac-to-Bac system.The infection efficiency in the colon cancer cell line SW1116 of a green fluorescent protein(GFP) expressing baculovirus(Bac-GFP) at different multiplicities of infection(MOI) with various concentrations of sodium butyrate was determined by flow cytometry.An in vitro cytotoxicity assay was also conducted after infection of SW1116 cells with Bac-NIS.Iodine uptake of Bac-NIS infected SW1116 cells and inhibition of this uptake by sodium perchlorate was examined,and the effect of Bac-NISmediated 131 I in killing tumor cells was evaluated by cell colony formation tests.RESULTS:Infection and transgene expression in SW1116with Bac-GFP were significantly enhanced by sodium butyrate,as up to 72% of SW1116 cells were infected with the virus at MOI of 400 and sodium butyrate at 0.5 mmol/L.No obvious cytotoxicity was observed under these conditions.Infection of SW1116 with Bac-NIS allowed uptake of 131 I in these tumor cells,which could be inhibited by sodium perchlorate.The viability of SW1116 cells infected with Bac-NIS was significantly lower than with Bac-GFP,suggesting that NIS gene-mediated 131 I uptake could specifically kill tumor cells.CONCLUSION:Baculovirus vector-mediated NIS gene therapy is a potential approach for treatment of colon cancer.

钠碘同向转运体上游增强子序列的特异性结合蛋白文库的构建及初步筛选

目的:构建酵母单杂交文库,筛选出与钠碘同向转运体上游增强子(NUE)特异性结合的蛋白质分子,进一步研究其与NUE相互作用的分子机制。方法:应用酵母单杂交系统,以NUE为酵母单杂交的"诱饵",对甲状腺癌K1细胞的c DNA文库进行初步筛选,采用DNA测序技术及生物信息学技术对筛选的克隆进行进一步功能分析。结果:对文库中的阳性克隆测序,获得53条可读序列,Blast X比对分析及基因功能注释发现其中11条具有DNA结合功能,包括Rho GTP酶激活蛋白35(ARHGAP35)、锌指蛋白394(ZNF394)、上游刺激因子2(USF2)、SOX9等。结论:应用酵母单杂交技术初步筛选出与甲状腺癌K1细胞NUE相互作用的候选蛋白,并对这些蛋白质进行了初步的功能分析。

钠⁃钾⁃氯协同转运蛋白1相关神经系统疾病研究进展

钠⁃钾⁃氯协同转运蛋白1(NKCC1)是阳离子⁃氯化物协同转运蛋白之一,为介导钠、钾、氯离子转运的膜蛋白,对保持氯离子动态平衡至关重要。病理状态下NKCC1表达或活性发生变化,进而调节氯离子细胞内浓度和神经元兴奋性。本文拟对NKCC1表达及活性调节方式,以及NKCC1在多种神经系统疾病发病机制、治疗中的作用进行综述,以为后续相关研究提供理论线索。

BRAF V600E/TERT promoter mutations and NIS/TSHR expression in differentiated thyroid carcinomaand their clinical significance

Objective Telomerase reverse transcriptase(TERT) promoter mutations have recently been described in thyroid carcinoma.The purpose of this study was to investigate the clinical significance of(v-raf murine sarcoma viral oncogene homolog B1) BRAF V600 E and TERT promoter mutations in differentiated thyroid carcinoma(DTC).The relationship between the two mutations and NIS/TSHR expression was also analyzed.Methods We have detected BRAF V600 E and TERT promoter mutations by direct sequencing and NIS/TSHR expression by immunohistochemistry in 229 cases of DTC,52 cases of benign nodular goiter,and 31 cases of normal thyroid tissue.Results The BRAF V600 E mutation was detected in 142(62.0%) of 229 cases of DTC [141 cases of papillary thyroid carcinoma(PTC) and 1 case of follicular thyroid carcinoma(FTC)].TERT promoter mutations were detected in 18(7.9%) of 229 cases of DTC(14 cases of PTC and 4 cases of FTC),including the mutations C228T(0.9%) and C250T(7.0%),which were mutually exclusive.Moreover,11(61.1%) cases also harbored the BRAF V600 E mutation,which was not associated with gender,age,tumor size,lymph node metastasis,and recurrence risk stratification(P >0.05).The rate of TERT promoter mutation was higher in males,age ≥45,and in the middle/high-risk group(P <0.05),and the rate of simultaneous BRAF V600 E and TERT promoter mutations were higher in the middle/high-risk group(P <0.05).In addition,NIS positive rate in the concurrent BRAF V600 E and TERT promoter mutation group(45.5 %) was lower than in other groups(that is,the DTC group with BRAF V600 E or TERT promoter mutations(55.1%),the DTC group with no BRAF V600 E or TERT promoter mutation(57.5%),the nodules and normal group(75.9%);| r | = 0.171,P = 0.002).Conclusion TERT promoter mutations were lower in patients with DTC,with the C250 T mutation being the most common.The detection of BRAF V600 E mutation combined with TERT promoter mutations was instructive for the prognosis assessment and treatment of DTC.

Monitoring adenoviral based gene delivery in rat glioma by molecular imaging

AIM:To determine whether endothelial progenitor cells(EPCs)can be used as delivery vehicle for adenoviral vectors and imaging probes for gene therapy in glioblastoma.METHODS:To use cord blood derived EPCs as delivery vehicle for adenoviral vectors and imaging probes for glioma gene therapy,a rat model of human glioma was made by implanting U251 cells orthotopically.EPCs were transfected with an adenovirus(AD5/carrying hNIS gene)and labeled with iron oxide and inoculated them directly into the tumor 14 d following implantation of U251 cells.Magnetic resonance imaging(MRI)was used to in vivo track the migration of EPCs in the tumor.The expression of gene products was determined by in vivo Tc-99m single photon emission computed tomography(SPECT).The findings were validated with immunohistochemistry(IHC).RESULTS:EPCs were successfully transfected with the adenoviral vectors carrying hNIS which was proved by significantly(P<0.05)higher uptake of Tc-99m in transfected cells.Viability of EPCs following transfection and iron labeling was not altered.In vivo imaging showed the presence of iron positive cells and the expression of transgene(hNIS)product on MRI and SPECT,respectively,all over the tumors following administration of transfected and iron labeled EPCs in the tumors.IHC confirmed the distribution of EPC around the tumor away from the injection site and also showed transgene expression in the tumor.The results indicated the EPCs’ability to deliver adenoviral vectors into the glioma upon intratumor injection.CONCLUSION:EPCs can be used as vehicle to deliver adenoviral vector to glioma and also act as imaging probe at the same time.

Primary lung cancer with radioiodine avidity: A thyroid cancer cohort study

BACKGROUND A proportion of lung cancers show sodium/iodide symporter(NIS)expression.Lung cancers with NIS expression may uptake radioiodine(RAI)and show RAIavid lesions on RAI scan for differentiated thyroid cancer(DTC)surveillance.AIM To investigate the possibility of RAI uptake by lung cancer in a cohort with thyroid cancer.METHODS RAI-avid lung cancers were analyzed using a prospectively maintained database of patients with thyroid cancer who were registered at a medical center between December 1,1976 and May 28,2018.NIS expression in lung cancer was assessed using immunohistochemical staining.RESULTS Of the 5000 patients with thyroid cancer from the studied dataset,4602 had DTC.During follow-up,33 patients developed primary lung cancer.Of these patients,nine received an iodine-131(131I)scan within 1 year before the diagnosis of lung cancer.One of these nine lung cancers was RAI-avid.NIS expression was evaluated,and three of the eight available lung cancers revealed NIS expression.The proportions of lung cancer cells with NIS expression were 60%,15%,and 10%.The RAI-avid lung cancer had the highest level of expression(60%).The RAI-avid lung cancer had a spiculated border upon single-photon emission computed tomography/computed tomography,which led to an accurate diagnosis.CONCLUSION A proportion of lung cancer demonstrates NIS expression and is RAI-avid.Clinicians should be aware of this possibility in the interpretation of RAI scintigraphy.

带膜分裂和促进剂的通讯膜系统求解QSAT问题

膜计算是自然计算的一个分支,膜计算中所研究的模型均称为膜系统,而细胞间通讯是膜系统的一个重要特征。带膜分裂的通讯膜系统是一种分布式并行计算模型,可以在多项式时间内解决计算困难问题。文中将促进剂引入带膜分裂的类细胞型通讯膜系统,提出了膜系统的一种变型——带膜分裂和促进剂的通讯膜系统,其中,一个促进剂可以同时控制多条规则,而促进剂本身不参与该条规则的进化。文中研究了带膜分裂和促进剂的通讯膜系统的计算效率,证明该类膜系统在使用同向规则长度为2,每条规则中促进剂的个数最多为1时,可以在多项式时间内求解PSPACE完全问题(QSAT问题)的统一解。

带通道状态通讯膜系统的研究综述

膜计算是一种生物启发式计算,其来源于对活细胞组织或器官结构及其功能的抽象模拟.细胞间最多建立一条链接,这条链接又称为突触,突触上有与其相关的通道状态.因此,根据此生物现象,提出了一种新型的计算模型:带有通道状态的通讯膜系统.这类膜系统是一类分布式并行计算模型,系统中细胞间的通讯主要依赖于细胞膜通道上同向/异向转运规则的使用,其中每个通道上的规则以一种串行的方式执行,且通道上的状态用来控制细胞间或细胞与环境间的通讯.文章根据带通道状态的同向/异向转运规则对细胞型通讯膜系统、组织型通讯膜系统,带有细胞分裂和通道状态的细胞型识别通讯膜系统及带有细胞分裂和通道状态的组织型识别通讯膜系统的概念分别进行了介绍,并说明了这些系统的计算能力以及计算复杂性.最后进行了总结并给出了未来展望.

Molecular rotaxane shuttle-relay accelerates K^(+)/Cl^(-) symport across a lipid membrane

Synthetic molecules that can mediate the coupled transport of Cl^(-) with K^(+) and/or Na+across the lipid bilayers have aroused great interest due to their potential as a novel therapeutic strategy by disrupting cellular ion homeostasis.Based on the structural advantages of molecular rotaxanes,we herein show that two rotaxane-based transporters[2]R and[3]R induce coupled K^(+)/Cl^(-) channel transport by introducing Cl^(-) recognition sites in the thread and K^(+) binding group in the wheel,respectively.The welldesigned molecular structures allow the insertion of unimolecular rotaxanes into the lipid bilayer,thus achieving effective ion transport by means of thermodynamically controlled movement and driven by the difference in ion concentration inside and outside the vesicles.In addition,the use of a three-component rotaxane can accelerate ion transport through a cooperative shuttlerelay mechanism in which two wheels move along the thread in the lipid membrane,thereby enabling[3]R to have higher ion transport capacity.This work represents a major advance in the use of rotaxane molecules to accomplish more complex and effective tasks.

Adenovirus-mediated and tumor-specific transgene expression of the sodium-iodide symporter from the human telomerase reverse transcriptase promoter enhances killing of lung cancer cell line in vitro

Background The sodium-iodide symporter （NIS） protein can mediate the active radioiodine uptake.The human telomerase reverse transcriptase （hTERT） promoter is known to be selectively reactivated in majority of tumors and hence could be used for tumor targeting.We constructed a recombinant adenovirus containing the human sodium iodide symporter （hNIS） gene directed by the hTERT promoter, characterized the ability of infected cells in uptaking iodide, and explored the therapeutic efficacy of 131I in a lung cancer cell line in vitro.Methods The hTERT promoter was amplified by PCR from DNA isolated from log-phase HepG2 cells, subcloned into lineralized FL＊-hNIS/pcDNA3, and then the hTERT-hNIS sequence was subcloned into the shuttle plasmid pAdTrack.The recombinant adenovirus Ad-hTERT-hNIS was constructed by AdEasy system.A positive control adenovirusAd-CMV-hNIS and a negative control adenovirus Ad-CMV were created similarly.A549 cells were transduced with recombinant adenoviruses.125I uptake studies and sodium perchlorate suppression studies were used to confirm hNIS expression and function.Toxic effects of 131I on tumor cells were studied by in vitro clonogenic assay.Results We first successfully constructed an adenovirus mediated transgene expression system of the hNIS under the control of hTERT promoter.When infected with recombinant adenovirus constructs expressing hNIS directed by hTERTand CMV-promoters （Ad-hTERT-hNIS and Ad-CMV-hNIS, respectively）, the lung cancer cell line A549 had increased ability to uptake radioiodide up to 23- and 30- fold compared to the control parental cells, respectively.The radioiodide uptake ability of both the Ad-CMV-hNIS and Ad-hTERT-hNIS transduced cell lines were repressed 11-fold by sodium perchlorate （NaCIO4）.The subsequent in vitro clonogenic assay of the infected A549 cell line was further repressed to 23% （Ad-CMV-hNIS） and 30% （Ad-hTERT-hNIS） of the control group after receiving radioiodide for 7 hours （P 〈0.001）.Conclusion Our preliminary study indicates that an adenovirus mediated transgene expression system of the hNIS under the control of hTERT promoter has the potential to become an effective wide-spectrum yet highly specific anti-cancer strategy.

Capsaicin restores sodium iodine symportermediated radioiodine uptake through bypassing canonical TSH–TSHR pathway in anaplastic thyroid carcinoma cells

Anaplastic thyroid cancer(ATC)is a rare but highly lethal disease.ATCs are resistant to standard therapies and are extremely difficult to manage.The stepwise cell dedifferentiation results in the impairment of the iodine-metabolizing machinery and the infeasibility of radioiodine treatment in ATC.Hence,reinducing iodine-metabolizing gene expression to restore radioiodine avidity is considered as a promising strategy to fight against ATC.In the present study,capsaicin(CAP),a natural potent transient receptor potential vanilloid type 1(TRPV1)agonist,was discovered to reinduce ATC cell differentiation and to increase the expression of thyroid transcription factors(TTFs including TTF-1,TTF-2,and PAX8)and iodine-metabolizing proteins,including thyroidstimulating hormone receptor(TSHR),thyroid peroxidase,and sodium iodine symporter(NIS),in two ATC cell lines,8505C and FRO.Strikingly,CAP treatment promoted NIS glycosylation and its membrane trafficking,resulting in a significant enhancement of radioiodine uptake of ATC cells in vitro.Mechanistically,CAP-activated TRPV1 channel and subsequently triggered Ca2þinflux,cyclic adenosine monophosphate(cAMP)generation,and cAMP-responsive element-binding protein(CREB)signal activation.Next,CREB recognized and bound to the promoter of SLC5A5 to facilitate its transcription.Moreover,the TRPV1 antagonist CPZ,the calcium chelator BAPTA,and the PKA inhibitor H-89 effectively alleviated the redifferentiation exerted by CAP,demonstrating that CAP might improve radioiodine avidity through the activation of the TRPV1–Ca2þ/cAMP/PKA/CREB signaling pathway.In addition,our study indicated that CAP might trigger a novel cascade to redifferentiate ATC cells and provide unprecedented opportunities for radioiodine therapy in ATC,bypassing canonical TSH–TSHR pathway.

The effects of thyroid-stimulating hormone,estradiol and prolactin on sodium/iodide symporter mRNA expression in mouse lactating mammary gland cells under different iodine levels

Objective The present study investigated the sodium/iodide symporter mRNA expression in mouse lactating mammary gland cells under different iodine levels and the effects of thyroid-stimulating hormone(TSH),estradiol(E2)and prolactin(PRL)on NIS mRNA expression in mouse lactating mammary gland cells.

Crown ether-thiourea conjugates as ion transporters

Na^(+),Cl^(‒)and K^(+)are the most abundant electrolytes present in biological fluids that are essential to the regulation of pH homeostasis,membrane potential and cell volume in living organisms.Herein,we report synthetic crown ether-thiourea conjugates as a cation/anion symporter,which can transport both Na+and Cl^(–)across lipid bilayers with relatively high transport activity.Surprisingly,the ion transport activities were diminished when high concentrations of K+ions were present outside the vesicles.This unusual behavior resulted from the strong affinity of the transporters for K^(+)ions,which led to predominant partitioning of the transporters as the K^(+)complexes in the aqueous phase preventing the transporter incorporation into the membrane.Synthetic membrane transporters with Na^(+),Cl^(‒)and K^(+)transport capabilities may have potential biological and medicinal applications.

NES1/KLK10 and hNIS gene therapy enhanced iodine-131 internal radiation in PC3 proliferation inhibition

NES1 gene is thought to be a tumor-suppressor gene.Our previous study found that overexpression of NES1 gene in PC3 cell line could slow down the tumor proliferation rate,associated with a mild decrease in BCL-2 expression.The BCL-2 decrease could increase the sensitivity of radiotherapy to tumors.Thus,we supposed to have an“enhanced firepower”effect by combining overexpressed NES1 gene therapy and 131I radiation therapy uptake by overexpressed hNIS protein.We found a weak endogenous expression of hNIS protein in PC3 cells and demonstrated that the low expression of hNIS protein in PC3 cells might be the reason for the low iodine uptake.By overexpressing hNIS in PC3,the radioactive iodine uptake ability was significantly increased.Results of in vitro and in vivo tumor proliferation experiments and 18F-fluorothymidine(18F-FLT)micro-positron emission tomography/computed tomography(micro-PET/CT)imaging showed that the combined NES1 gene therapy and 131I radiation therapy mediated by overexpressed hNIS protein had the best tumor proliferative inhibition effect.Immunohistochemistry showed an obvious decrease of Ki-67 expression and the lowest BCL-2 expression.These data suggest that via inhibition of BCL-2 expression,overexpressed NES1 might enhance the effect of radiation therapy of 131I uptake in hNIS overexpressed PC3 cells.

钠钾氯共转运体在γ-氨基丁酸引起的肠神经元兴奋中的作用

γ-氨基丁酸(γ-aminobutyric acid, GABA)是成年个体中枢神经系统(central nervous system, CNS)的一种抑制性神经递质,但对未成熟CNS神经元有兴奋作用。出生后GABA对CNS神经元的作用由去极化逐渐转为超极化,这种转变主要是由于大脑Na^+-K^+-2Cl^-共运体1 (Na^+-K^+-2Cl^-symporter 1, NKCC1)表达逐渐减少和K^+-Cl^-共运体2 (K^+-Cl^-cotransporter 2, KCC2)表达增加。与CNS神经元不同,肠神经系统(enteric nervous system, ENS)中未成熟和成体神经元都可被GABA去极化。GABA激发ENS神经元兴奋的分子机制尚不清楚,但与ENS神经元内的高Cl^-浓度有关。本研究目的是检验一个假设,即ENS神经元的细胞内高Cl^-浓度是由NKCCs活动维持的。结果显示,NKCC2免疫反应性(immunoreactivity, IR)在出生后第1天(P1)大鼠结肠ENS中出现,随后持续升高,在P14达到稳定的高水平,并在成年后保持在这一水平。NKCC1的IR出现在P14大鼠的ENS中,并维持到成年。在任何发育阶段的大鼠ENS中都检测不到KCC2的IR。在ENS神经元中,NKCC1和NKCC2均与GABAA受体共同表达。外源性GABA (1 mmol/L)可引起ENS神经元膜去极化。GABA诱导的去极化逆转电位约为-16 m V。NKCC抑制剂布美他尼(50μmol/L)或速尿(300μmol/L)可显著抑制GABA诱导的去极化。布美他尼(50μmol/L)使GABA诱导的去极化逆转电位向超极化方向移动。无论是KCC抑制剂DIOA (20μmol/L)还是Cl^-/HCO3-交换抑制剂DIDS (200μmol/L)对GABA诱发的去极化都没有明显影响。以上结果提示,ENS表达NKCC1和NKCC2,但是不表达KCC2。NKCC1和NKCC2在GABA引起的肠神经元兴奋中起重要作用。