干扰素-α联合GM－CSF诱导慢性髓性白血病细胞向树突状细胞分化

目的：研究干扰素-α（IFN-α）对慢性髓性白血病（CML）骨髓单个核细胞来源的树突状细胞（DCs）发育的影响。 方法： 12例初发慢性期CML患者的骨髓单个核细胞，分别与含如下细胞因子,RPMI-1640培养液共育：rhGM-CSF 1×106U/L联合rhIFN-α 2×106U/L（IFN-α组）、rhGM-CSF 1×10⁶U/L联合rhIL-4 5×10⁵U/L（IL-4组）、单用rhGM-CSF 1×10⁶U/L和单用rhIFN-α 2×106U/L，培养7 d；于第8-10 d，部分孔加入rhTNF-α 5×10⁴U/L。形态学（Wright染色、倒置显微镜）、免疫学（CD80、CD86、CD83、CD1a、HLA-DR）检测；磷脂酰丝氨酸（PS）转位检测 DCs凋亡情况；荧光原位杂交（FISH）对1例CML进行细胞遗传学分析；混合淋巴细胞反应（MLR）检测刺激同种异体T淋巴细胞增殖的能力。 结果： CML骨髓单个核细胞经上述细胞因子诱导7 d后，IFN-α组和IL-4组均呈现树突状细胞的典型形态；免疫学鉴定，IFN-α组DCs CD80、CD86、CD83、HLA-DR的表达显著高于IL-4组（P<0.05），经5×104U/L rhTNF-α作用后，两组DCs CD80、CD86、CD83、HLA-DR进一步上调，其中IFN-α组DCs CD80、CD86、CD83、HLA-DR的表达显著高于IL-4组（P<0.05）；经FISH证实来源于白血病细胞；两组DCs均具有刺激同种异体T淋巴细胞增殖的能力，IFN-α组刺激淋巴细胞增殖的能力明显高于IL-4组(P<0.05)。 结论： IFN-α可促进CML骨髓单个核细胞来源的树突状细胞的分化、活化。这可能是IFN-α在CML中的治疗机制之一。     

急性白血病患者bcrp、mdr-1、mrp-1和lrp耐药基因表达的临床意义

目的：定量检测急性白血病(AL) 患者乳腺癌耐药相关蛋白基因（bcrp）、多药耐药基因（mdr-1）、多药耐药相关蛋白基因（mrp-1）及肺耐药相关蛋白基因（lrp）表达及其与临床的关系。方法：应用实时荧光定量逆转录-聚合酶链反应RT-PCR （real time fluorescent quantitative RT-PCR，FQ RT-PCR）检测105例AL 患者bcrp、mdr-1、mrp-1、lrp等基因的拷贝数,分析其与临床预后的关系。结果： AL组bcrp基因拷贝数（2.8×10³)±(8.4×10³）明显高于正常对照组（7.6×10²)±(2.3×10³）,P<0.05；mdr-1基因拷贝数（1.3×10⁵)±(2.2×10⁵）及lrp基因拷贝数（8.3×10⁵)±(1.0×10⁶）显著高于正常对照组（P<0.01）。复发组AL患者的mdr-1拷贝数（3.7×10⁴)±(1.1×10⁵）明显高于初治组患者（1.1×10⁴)±(3.6×10⁴）,P<0.05。在AL耐药组mdr-1拷贝数（2.1×10⁴)±(9.1×10⁴）明显高于敏感组（1.0×10⁴)±(3.8×10⁴）,P<0.05）。经直线相关分析显示mdr-1和mrp-1，mdr-1和lrp，mrp-1和lrp之间呈显著正相关，经等级相关分析显示mdr-1和lrp的表达与耐药性密切相关。结论：急性白血病多药耐药的出现与bcrp、mdr-1、mrp-1、lrp中一项或几项过度表达有关，mdr-1较其它基因更具有预测临床预后的意义。     

锌对镉诱导人淋巴细胞姊妹染色单体交换的影响

目的：探讨镉与锌的致突变性以及锌对镉致突变性的影响。 方法： 采用正常人外周血淋巴细胞体外培养的方法，观察镉、锌以及镉和锌的混合物诱导细胞姊妹染色单体交换（SCE）频率增加的情况。 结果： 镉在1×10-8mol/L－1×10-6mol/L浓度范围内具有诱发人外周血淋巴细胞SCE频率增加的作用，而锌在1×10-6mol/L－1×10-4mol/L浓度范围内不具有诱发人外周血淋巴细胞SCE频率增加的作用。锌可拮抗镉诱导SCE频率的增加，并且在1×10-6mol/L－1×10-4mol/L浓度范围内随着锌浓度的增加，其抑制作用也增强，并有明显的剂量-效应关系。 结论： 锌在体外培养的细胞中对镉的致突变性具有一定程度的抑制作用。     

粉防己碱(Tet)对MDCK细胞钙激活钾通道(KCa)的影响

目的MDCK细胞钙激活钾通道(KCa)的鉴定及探讨Tet对该通道的影响.方法内面向外式(inside-out)膜片钳技术.结果 (1)在对称性高钾溶液([K+].[K+]i=140mmolL1/140mmolL-1)中,主要记录到电导为50.475±0.600pS(n=5)的外向通道电流,随电压增加而增加.(2)浴液中钾离子浓度由140mmolL-1改变为45mmolL-1,在0mV记录到内向电流,从+30mv开始记录到记录到外向电流,电导为50.275±0.294pS(n=5).(3)在对称性高钾溶液、膜电位+60mV时.向浴液中加入不同浓度的Ca2+,从10-7molL-1开始,通道活动浓度依赖性增加.(4)对称性高钾溶液、膜电位为+60mV,向浴液中加入Tet,通道活动在1.5×10-5molL-1时最大,在6.0×10-5molL-1时明显受到抑制,几乎为0,洗脱后,部分恢复.结论(1)在MDCK细胞上记录到中电导钙激活钾通道(IKCa),该通道具有电压依赖性和胞内游离Ca2+浓度依赖性,无整流特性.(2)Tet在7.5×10-6molL-1时对MDCK细胞Ka没有显著影响;在1.5×10-5molL-1促进KCa开放,增加钾离子外流;在6.0×10-5molL-1时抑制MDCK细胞Kca开放,减少钾离子外流.     

PPARs激动剂上调内皮细胞表达eNOS并增加NO生成

目的： 观察PPARα激动剂非诺贝特及PPARγ激动剂赛格列酮对人脐静脉内皮细胞（HUVECs）血管紧张素Ⅱ（AngⅡ）抑制NO生成的作用。 方法： 体外培养HUVECs，用1×10-7-1×10-4mol/L赛格列酮和10-5、10-4mol/L非诺贝特预处理HUVECs 24 h，再与10-6mol/L AngⅡ 共同孵育12 h，通过RT-PCR和Western blotting分别检测eNOS mRNA和蛋白表达水平；通过Griees反应测定NO2－/NO3－浓度。 结果： 与对照组相比，10-7mol/L AngⅡ刺激HUVEC 12 h下调eNOS mRNA（0.38±0.19 vs 0.13±0.18，P<0.01）和蛋白（35.90±3.18 vs 6.95±2.19，P<0.01）表达，减少NO生成（50.21 μmol/L vs 21.33 μmol/L，P<0.01）。用10-7、10-6、10-5、10-4 mol/L赛格列酮预处理24 h，上调eNOS mRNA表达（分别为0.36±0.03、0.36±0.14、0.37±0.16、0.43±0.06，与AngⅡ组比较，均P<0.01）和蛋白表达（分别为11.60±3.31、11.78±5.45、13.93±2.46、22.93±3.17，与AngⅡ组相比，均P<0.01），增加细胞培养液NO2-/NO3-浓度。非诺贝特也上调eNOS mRNA和蛋白表达，增加细胞培养液NO2-/NO3-浓度（P<0.01）。 结论： AngⅡ减少eNOS表达，从而减少NO生成。赛格列酮和非诺贝特预处理24 h，可拮抗AngⅡ对HUVECs eNOS mRNA和蛋白表达的抑制作用，增加NO的释放。     

用SYBR-GreenⅠ实时荧光PER结合融解曲线分析法诊断α-地中海贫血

目的建立自动化、高通量、准确快速检测缺失型α-地中海贫血基因型的技术。方法应用SYBR-Greenl进行两个实时荧光聚合酶链反应（real-time fluorescence polymerase chain reaction with SYBR-Green 1，SYBR-PCR），检测左缺（-α^4.2）、右缺（-α^3.7）等位基因，同时进行融解曲线（dissociation calve，DC）和Tm（melting temperature）值分析。PCR产物重组到pCR2．1，重组子梯度稀释作为模板检测灵敏度，确定两种等位基因型（-α^4.2，-α^3.7）的检测下限，并对110份DNA样品进行检测。结果检测-α^4.2和-α^3.7的PCR产物长度分别为1．65kb、1．9kb，Tm分别为（81．5±0．5）℃、（82．5±0．5）℃，检测下限分别为9×10^2个拷贝、4．3×10^2个拷贝。该检测技术的灵敏度较常规PCR结合琼脂糖凝胶电泳法高10倍。结论SYBR-Greenl实时荧光PCR结合融解曲线分析及Tm分析可以灵敏、准确地检测-α^4.2、-α^3.7、αα（包括α^Tα）及--^SEA4种等位基因，从而为各种缺失型α-地中海贫血做出基因诊断。该技术具有自动化程度高，不需荧光标记探针，成本低，易质控，防污染，高通量等优点，适于临床推广应用。     

焦亚硫酸钠对大鼠海马CA1区神经元钠电流的影响

目的：探讨SO2 及其体内衍生物（亚硫酸盐和亚硫酸氢盐）对中枢神经元钠通道的影响。 方法: 采用全细胞膜片钳技术研究了焦亚硫酸钠（SMB）对大鼠海马CA1区神经元钠电流的影响及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)及谷胱甘肽过氧化物酶(GPx)相应的保护作用。 结果： ① 焦亚硫酸钠可剂量依赖性地增大全细胞钠电流，剂量为2 μmol/L和20 μmol/L时，钠电流分别增大（22.36±3.28）% 和（65.05±5.75）%（n=10）。② 10　μmol/L的焦亚硫酸钠不影响钠电流的激活过程，却非常显著地影响其失活过程，使失活曲线显著右移，作用前后的半数失活电压分别为（-82.38±0.54）mV和（-69.39±0.41）mV (n=10, P<0.01), 但失活曲线的斜率因子未见改变。③ SOD(1×106 U/L)、CAT(2×106 U/L) 及GPx (1×104 U/L) 均可使SMB（10 μmol/L）增大的钠电流部分恢复。 结论： SMB增大钠电流并抑制其失活过程，从而影响神经细胞的兴奋性，这一效应可能与硫中心或氧中心自由基的损伤作用有关。     

金雀异黄酮对冈田酸诱导大鼠血小板Tau蛋白过度磷酸化的保护作用及机制

目的探讨金雀异黄酮（GS）对冈田酸（OA）诱导大鼠血小板Tau蛋白过度磷酸化的保护作用及其机制。方法通过MqT法观察不同浓度的OA对大鼠血小板存活的影响。应用蛋白免疫印迹方法,检测OA处理后大鼠血小板Tau蛋白磷酸化Serl99和Thr231位点、Tau-1（非磷酸化蛋白）、Tau-5（总Tau蛋白）、糖原合成激酶3β（GSK-3β）和抑制性磷酸化GSK．3βSer9位点的表达情况：并观察GS对OA诱导大鼠血小板上述指标变化的影响。结果MTT检测结果表明,不同浓度的OA对血小板均有损伤作用,其中80nmol／LOA对大鼠血小板损伤程度较为合适。Westernblot检测结果表明,80nmol／LOA处理12h后,Tau蛋白Serl99位点的磷酸化水平增高（P〈0．05）,Thr231位点的磷酸化水平显著增高（P〈0．01）;Tau-1的磷酸化水平显著降低（P〈0．01）;Tau-5无明显变化;GSK．3B的表达无变化而抑制性磷酸化GSK．3βSer9位点的表达减少（P〈0．05）。用0．5μmol／LGS预处理后可减轻OA所诱导的Tau蛋白过度磷酸化,同时抑制性磷酸化GSK-3βSer9位点的表达增加（P〈0．05）。结论OA可诱导大鼠血小板Tau蛋白Serl99和Thr231位点的磷酸化水平增高,该作用可能通过激活GSK-3β实现。GS可能通过抑制血小板GSK．3B的活性,最终达到减轻OA所诱导的大鼠血小板Tau蛋白过度磷酸化的作用。     

Baclofen对机械分离的大鼠海马锥体细胞GABA-激活电流的抑制作用

在机械分离的海马锥体细胞上,应用全细胞膜片钳技术。证明大多数细胞(88.5％,46／52)对GABA敏感。10-5-10-3mol／L的GABA引起一剂量依赖性、有明显去敏感作用的内向电流。预加3×10-5mol／L baclofen(GABAB受体的特异性激动剂)30 s后再加GABA,84.8％(39／46)的细胞GABA-激活电流被抑制,其中仅有一个细胞(2.2％,1／46)GABA-激活电流幅值增强,13％(6／46)的细胞GABA-激活电流幅值无变化。预加baclofen后,GABA-激活电流量-效曲线明显下移。预加baclofen前后IGABA量效曲线的Kd值非常接近(1.0×10-4 vsl.4×10-4 mol／L)。经saclofen预处理可消除baclofen对GABA-激活电流的抑制。这与我室以往在外周神经元上的研究结果一致。本文结果不仅证明了GABAA和GABAB受体在海马锥体细胞上的共存,而且也证明了GABAB受体激活后对GABAA受体功能抑制这一现象无论在外周或中枢神经系统均具有普遍性。     

抗利尿激素单克隆抗体的制备及初步应用

作者应用B淋巴细胞杂交瘤技术,成功地建立了分泌抗抗利尿激素(ADH)的McAb细胞株,所获的3株细胞分泌的McAb分别为IgG1,IgG2a,IgG2b。其诱生腹水滴度可达1×10~(-5)～5×10~(-5),灵敏度为2.5ng/ml。抗体亲和力K值在(3～4)×10~(-9)L/M之间,受温度影响明显。McAb与11种神经肽进行竞争抑制试验,均无交叉反应。静脉注射McAb可使新西兰兔尿量明显增加。用免疫组化的方法对大鼠下丘脑神经细胞进     

大鼠骶髓后连合核神经元兴奋性氨基酸诱导的反应

应用制霉菌素穿孔全细胞电压钳技术，研究急性分离的大鼠骶髓后连合核（ＳＤＣＮ）神经元对谷氨酸受体激动剂的反应。钳制电压为－４０ｍＶ的条件下，Ｌ－谷氨酸（Ｇｌｕ），Ｎ－甲基－Ｄ－门冬氨酸（ＮＭＤＡ），使君子酸（ＱＡ），α－氨基－３－羟基－５－甲基异倾阶－４－丙酸（ＡＭＰＡ）和红藻氨酸（ＫＡ）均诱导产生内向电流。随着激动剂浓度的增高，这些电流的量效关系曲线呈典型的Ｓ型。ＥＣ５０值分别是Ｇｌｕ３．３×１０－５Ｍ，ＮＭＤＡ９．０×１０－ＳＭ，ＱＡ６．４×１０７Ｍ，ＡＭＰＡ１．３×１０－４及ＫＡｇ．６×ｕ１０－５Ｍ。Ｎｉｌｌ系数分别是Ｇｌｕ０．７４，ＮＭＤＡ０．８３，ＱＡ１．３，ＡＭＰＡ１．１和ＫＡ１．３。代谢型谷氨酸受体激动剂ｔＡＣＰＤ（１０－３Ｍ）未能诱导出电流．Ｃｙｃｌｏｔｈｉａｚｉｄｅ显著增强ＫＡ和ＡＭＰＡ诱导的反应。相反，伴刀豆球蛋白Ａ（ＣｏｎＡ）对ＫＡ和ＡＭＰＡ反应作用甚微，提示ＳＤＣＮ神经元主要表达ＡＭＰＡ型非ＮＭＤＡ受体。     

Potentiation, activation and blockade of GABAA receptors by etomidate in the rat sacral dorsal commissural neurons

Etomidate (ET), an imidazole general anesthetic, has been medically widely used. Recent evidence suggests that the inhibitory neurotransmitter GABA receptor may be one of the important molecular target(s) of general anesthetics. Up to date, little attention has been directed toward the sacral dorsal commissural nucleus (SDCN), which serves as a relay of sensory information from the pelvic viscera in the spinal cord. Therefore, the effect of ET on GABA(A) receptor function in neurons acutely dissociated from the SDCN was investigated using the nystatin-perforated patch-recording configuration under voltage-clamp conditions. At a holding potential of -40 mV, ET (above 10 microM) induced an inward ET-activated current (I(ET)) with the EC(50) value of 33 +/- 3 microM, which was reversibly blocked by bicuculline and picrotoxin. The reversal potential of I(ET) was close to the Cl(-) equilibrium potential. ET also displayed a biphasic modulatory effect on GABA responses. At lower concentrations (0.1-100 microM), ET reversibly potentiated GABA (1 microM)-activated Cl(-) currents in a bell-shaped manner, with the maximal facilitative effect at 10 microM, whereas at concentrations >100 microM, the peak of the ET-induced current was suppressed in the absence or presence of GABA (1 microM). These results suggest that in SDCN, in addition to the potentiation of GABA(A) receptor-mediated responses at low concentrations and the direct activation of GABA(A) receptors at moderate concentrations as expected, ET produced a fast blocking action at high concentrations. The general anesthetic-induced effects in SDCN, at least the potentiation of GABA responses, may significantly contribute to anesthesia of pelvic viscera during the general anesthesia.     

Excitatory amino acid response in isolated spiral ganglion cells of guinea pig cochlea.

1. The physiological and pharmacological properties of excitatory amino acid (EAA)-induced responses were investigated in acutely isolated spiral ganglion cells of guinea pig by a conventional patch-clamp technique combined with a rapid drug application (Y-tube) method. 2. L-glutamate (Glu) and its agonists, quisqualate (QA) and kainate (KA), induced inward currents in a concentration-dependent manner at a holding potential (VH) of -70 mV. The values of half-maximal concentration (EC50) were 4.0 x 10(-4) M for Glu, 2.3 x 10(-5) M for QA, and 1.4 x 10(-4) for KA. The Hill coefficients were 0.96, 1.00, and 1.56 for Glu, QA, and KA, respectively. However, one of Glu agonists, N-methyl-D-aspartate (NMDA), and another excitatory amino acid, L-aspartate (Asp), did not induce any responses even in Mg2(+)-free external solution containing 10(-6) M glycine (Gly). 3. The current-voltage (I-V) relationships for the Glu-, QA-, and KA-induced responses were linear, and these reversal potentials were near 5 mV. 4. Kynurenic acid (Kyn), 6,7-dichloro-3-hydroxy-2-quinoxalinecarboxylic acid (diCl-HQC), and quinoxalinediones such as 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitro-quinoxaline-2,3-dione (DNQX) suppressed the Glu-, QA-, and KA-induced currents in a concentration-dependent manner. The inhibitory potency was in the order of DNQX = CNQX greater than diCl-HQC greater than Kyn. 5. CNQX antagonized the Glu-, QA-, and KA-induced currents without affecting the maximum responses showing no voltage-dependency, indicating the competitive inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Excitatory amino acid response in isolated nucleus tractus solitarii neurons of the rat

The excitatory amino-acid-induced currents in nucleus tractus solitarii neurons freshly isolated from rats were investigated in a whole-cell recording mode using a conventional patch-clamp technique. At a holding potential of -70 mV, L-glutamate (Glu), N-methyl-D-aspartate (NMDA) with 10(-9) M glycine, kainate (KA), quisqualate (QA) and L-aspartate (Asp) evoked inward currents. The currents increased in a sigmoidal fashion with increasing agonists concentration. The half-maximum concentration (EC50) values were 5 x 10(-5) M for Glu, 10(-6) M for QA, 10(-4) M for KA, 6 x 10(-5) M for NMDA and 5 x 10(-5) M for Asp. The Hill coefficients of the Glu-, QA-, KA-, NMDA- and Asp-induced responses were 1.0, 1.3, 1.1, 1.3 and 1.1, respectively. The Glu-, QA-, NMDA- and Asp-induced currents consisted of a transient initial peak and a successive steady-state component showing no desensitization. These currents had the same reversal potential near +5 mV. In the current-voltage (I-V) relationships for the Glu-, NMDA- and Asp-induced currents, slight outward rectifications were observed in Mg2(+)-free external solution at membrane potentials negative to 0 mV. In the presence of extracellular Mg2+, the currents induced by Glu, NMDA and Asp were suppressed at negative membrane potentials, but the suppression was less for the Glu response. The I-V relationships for QA- and KA-induced responses were almost linear at a membrane potential between -90 and +50 mV with or without the presence of Mg2+.     

Morphological evidence for GABA/glycine-cocontaining terminals in synaptic contact with neurokinin-1 receptor-expressing neurons in the sacral dorsal commissural nucleus of the rat

Previous studies have shown that neurons in the sacral dorsal commissural nucleus (SDCN) express neurokinin-1 receptor (NK1R) and can be modulated by the co-release of GABA and glycine (Gly) from single presynaptic terminal. These results raise the possibility that GABA/Gly-cocontaining terminals might make synaptic contacts with NK1R-expressing neurons in the SDCN. In order to provide morphological evidence for this hypothesis, the triple-immunohistochemical studies were performed in the SDCN. Triple-immunofluorescence histochemical study showed that some axon terminals in close association with NK1R-immunopositive (NK1R-ip) neurons in the SDCN were immunopositive for both glutamic acid decarboxylase (GAD) and glycine transporter 2 (GlyT2). In electron microscopic dual- and triple-immunohistochemistry for GAD/GlyT2, GAD/NK1R, GlyT2/NK1R, or GAD/GlyT2/NK1R also revealed dually labeled (GAD/GlyT2-ip) synaptic terminals upon SDCN neurons, as well as GAD- and/or GlyT2-ip axon terminals in synaptic contact with NK1R-ip SDCN neurons. These results suggested that some synaptic terminals upon NK1R-expressing SDCN neurons co-released both GABA and Gly.     

Activity-dependent disinhibition. II. Effects of extracellular potassium, furosemide, and membrane potential on ECl- in hippocampal CA3 neurons

1. Single-electrode voltage-clamp recordings were made from CA3 pyramidal cells in organotypic hippocampal slice cultures for measurement of membrane currents underlying both the gamma-aminobutyric acid (GABA)-mediated, Cl- -dependent inhibitory postsynaptic potential (IPSC), evoked in response to stimulation of the mossy fiber pathway, and responses to iontophoretically applied GABA. Their reversal potentials are presumed to equal the equilibrium potential for Cl- (37). Mechanisms underlying activity-dependent increases in the intracellular concentration of Cl- ([Cl-]i) were investigated by describing active and passive pathways for Cl- influx and efflux. 2. During 99-s applications of GABA, driving force declined by 51% due to increases in [Cl-]i; thus passive Cl- influx through GABA-activated pathways can significantly affect [Cl-]i. 3. Decreasing the extracellular K+ concentration ([K+]o) from 5.8 to 1 mM caused a rapid hyperpolarizing shift in the mean IPSC reversal potential (EIPSC) from -67.6 to -81.9 mV, even when membrane potential (Vm) was maintained constant and depolarized with respect to EIPSC. 4. Decreasing [K+]o from 5.8 to 1 mM caused a rapid hyperpolarizing shift in the mean GABA reversal potential (EGABA) from -64.7 to -81.1 mV, even when Vm was maintained constant and depolarized with respect to EGABA. Reducing the extracellular Cl- concentration from 153 to 89 mM, while maintaining [K+]o constant at 1 mM, shifted the mean EGABA from -81.1 to -66.2 mV, an amount close to that predicted by the Nernst equation for Cl-. We conclude that reducing [K+]o caused a hyperpolarizing shift in EGABA and EIPSC by decreasing [Cl-]i. 5. The shift of EIPSC and EGABA upon alteration of [K+]o did not result from contamination of the responses by additional K+-mediated components because it was unaffected by block of K+ channels with intracellular Cs+. 6. Reducing the extracellular Na+ concentration from 141 to 70 mM had no effect on EGABA. 7. Furosemide, bath-applied at 5 X 10(-4) M while holding Vm depolarized with respect to EIPSC, caused a rapid, reversible decrease in IPSC driving force averaging 69%, consistent with the presence of a furosemide-sensitive outward Cl- -transport system. 8. Reducing [K+]o from 5.8 to 1 mM in the presence of 5 X 10(-4) M furosemide produced a smaller shift of EIPSC from -61.0 to -71.2 mV, however, after washout of furosemide from [K+]o = 1 mM saline, EIPSC shifted further to -89.8 mV.(ABSTRACT TRUNCATED AT 400 WORDS)     

gamma-Aminobutyric acid-induced response in rat dissociated paratracheal ganglion cells.

1. The pharmacologic properties of gamma-aminobutyric acid (GABA)-induced Cl- current (ICl) were studied in the paratracheal ganglion cells freshly dissociated from 7- to 10-day-old rat trachea in a whole-cell recording mode by the use of a conventional patch-clamp technique. 2. GABA- and muscimol-induced currents increased sigmoidally in a concentration-dependent manner, and both currents reversed at approximately -3 mV, which was close to the Cl- equilibrium potential (ECl). 3. Strychnine (STR) at low concentration and bicuculline (BIC) inhibited GABA response competitively, whereas STR at the higher concentrations, benzylpenicillin (PCG), or picrotoxin (PTX) inhibited noncompetitively. Inhibition of GABA response by PCG but not other antagonists was voltage dependent, indicating that PCG acts as a Cl- channel blocker. 4. The concentration-response curve of pentobarbital sodium (PB)-induced ICl was bell shaped. At concentrations higher than 10(-3) M, both the peak and plateau currents decreased, and a transient "hump" current appeared immediately after washing out PB. In the presence of PB, the concentration-response curve of GABA shifted toward left without changing the maximum response. 5. Although diazepam (DZP) at concentration used did not induce a response, it potentiated the GABA response in a concentration-dependent manner between 10(-8) and 10(-6) M. DZP also caused a parallel shift toward left in the concentration-response curve of GABA. 6. PB or DZP further enhanced the GABA response in the presence of the other agent. 7. It is concluded that the properties of GABAA receptors in the paratracheal ganglion cells are essentially similar to those reported in other preparations.     

Excitatory amino acid responses in relay neurons of the rat lateral geniculate nucleus

Responses to glutamate receptor agonists were recorded from identified relay neurons in the dorsal lateral geniculate nucleus of the rat, using the nystatin-perforated patch-clamp technique. Rapid application of glutamate, N-methyl-D-aspartate, (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate induced inward currents at a holding potential of -44 mV. The responses to low concentrations of each agonist were composed only of steady-state currents, but the responses to high concentrations were additionally composed of a rapid transient peak component except in the kainate-induced current. The currents induced by 10(-3)M N-methyl-D-aspartate in the external solution containing 0 mM Mg2+ and 10(-6)M glycine were reduced in amplitude when the external solution contained 1 mM Mg2+, and were abolished when the solution contained no glycine. The currents induced by a neurotransmitter candidate at retinogeniculate synapses, N-acetyl-aspartyl-glutamate, were markedly reduced in amplitude when the solution contained 1 mM Mg2+ or 10(-4)M DL-2-amino-5-phosphonovaleric acid. The current abolished in the Mg2+-containing, glycine-free solution (N-methyl-D-aspartate component) and the current remaining in the same solution (non-N-methyl-D-aspartate component) of the N-acetyl-aspartyl-glutamate response were both increased in a concentration-dependent manner, as the N-acetyl-aspartyl-glutamate concentration was increased. The current-voltage relationship of the currents induced by N-methyl-D-aspartate and N-acetyl-aspartyl-glutamate was characterized by Mg2+-dependent block at hyperpolarized potentials. The inward currents induced by 3 x 10(-4)M AMPA and 3 x 10(-4)M glutamate were markedly potentiated by 10(-4)M cyclothiazide, but the currents induced by 3 x 10(-4)M kainate and 10(-3)M N-acetyl-aspartyl-glutamate (non-N-methyl-D-aspartate component) were little affected. The currents induced by any agonist were not affected by 3 x 10(-4)g/ml concanavalin A. The current induced by 10(-4)M kainate was markedly suppressed by pretreatment with 10(-4)M AMPA or 10(-4)M glutamate, but only weakly by 10(-3)M N-acetyl-aspartylglutamate. The Ca2+ permeability (PCa/PCs) of the N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors was 9.57 and 0.16, respectively. These results suggest that dorsal lateral geniculate nucleus relay neurons of the rat possessed both Ca2+-permeable N-methyl-D-aspartate receptors and less permeable non-N-methyl-D-aspartate (presumably AMPA) receptors, and that N-acetyl-aspartyl-glutamate mainly acts at N-methyl-D-aspartate receptors with a weak kainate-like action on non-N-methyl-D-aspartate receptors.     

Effects of two volatile anesthetics and a volatile convulsant on the excitatory and inhibitory amino acid responses in dissociated CNS neurons of the rat.

1. Effects of two volatile anesthetics [halothane (Hal) and enflurane (Enf)] and a volatile convulsant [hexafluorodiethyl ether (HFE)] on amino acid-induced membrane currents in neurons dissociated from the nucleus tractus solitarius of the rat were examined. The dissociated neurons were voltage clamped in the whole-cell mode of the patch-clamp technique. All drugs were applied with a microperfusion system, termed the "Y-tube" method. 2. The glutamate (Glu)-induced excitatory response was slightly reduced by both the anesthetics. The responses to three agonists at Glu receptor were depressed by Hal (10(-3) M) in the rank order of quisqualate greater than N-methyl-D-aspartate greater than kainate. HFE slightly increased the Glu response at a high concentration of 2 x 10(-3) M. 3. The gamma-aminobutyric acid (GABA)-induced chloride current (ICl) was enhanced by both anesthetics. The dissociation constant (Kd) for the enhancement was 2.3 x 10(-4) M for Hal and 2.1 x 10(-4) M for Enf, and the Hill coefficient was 1.6 for Hal and 1.5 for Enf. HFE depressed the GABA response with a Kd of 8.7 x 10(-5) M and a Hill coefficient of 0.84. 4. Hal (10(-3) M) and Enf (10(-3) M) decreased the Kd of the GABA concentration-response curve from 3.5 x 10(-6) to 10(-6) and 1.9 x 10(-6) M, respectively, without changing the maximum response or the Hill coefficient (1.5). In the presence of HFE (10(-4) M), the Kd was increased to 1.4 x 10(-5) M and the Hill coefficient was slightly changed to 1.2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Response of plasma membrane to applied hydrostatic pressure in chondrocytes and fibroblasts

Effects of applied hydrostatic pressure on transmembrane potentials were investigated in sheep articular chondrocytes and human skin fibroblasts in non-confluent monolayer cultures. Resting potentials in chondrocytes (about –12 mv) and in fibroblasts (about –15 mV) were increased and decreased respectively by over 40% after pressure was applied cyclically (0.33 Hz, 120 mm Hg, 20 minutes). Continuous pressure (120 mm Hg, 20 minutes) caused deplorization in both cell types. Low frequency pressure application (< 0.08 Hz) caused depolarization in chondrocytes and hyperpolarization in fibroblasts. Quinidine (2 × 10^?5M) blocked and verapamil (10^?5M) reduced hyperpolarization responses, suggesting involvement of Ca²⁺-dependent K⁺channels. A23187 (1.9 × 10^?6M) caused hyperpolarization in chondrocytes, augmented further by subsequent pressure application (0.33 Hz). Tetrodotoxin (10^?6M) blocked depolarization responses indicating that these were due to Na⁺influx. Blockade of histamine H1 receptors by chlorpheniramine maleate (5.1 × 10^?6M), H2 receptors by cimetidine (7.9 × 10^?6M) and β-adrenoreceptors by sotolol (1.3 × 10^?4M) had no effect on hydrostatic pressure-induced hyperpolarization in chondrocytes. Cytochalasin B (2 × 10^?5M and at 4 × 10^?6M) abolished pressure-induced hyperpolarization in chondrocytes; in contrast, applied cyclical hydrostatic pressure to cytochalasin-treated fibroblasts caused hyper-polarization, suggesting that cytoskeletal changes were involved.