异丙酚对大鼠海马神经元NMDA受体通道电流的影响

目的 研究异丙酚对大鼠海马神经元N-甲基-D-天冬氨酸（NMDA）受体通道电流的影响。方法 体外培养新生Wistar大鼠海马神经元8—12d，采用全细胞膜片钳技术和压力喷射给药方式，钳制电压为-80mV，记录3、48μg∥ml异丙酚对100μmol／LNMDA诱发NMDA受体通道电流的影响；然后用7-氨基丁酸（GABA）受体拮抗剂荷包牡丹碱100μmol／L阻断GABA．受体，观察3、48μg／ml异丙酚对NMDA受体通道电流的影响。结果 3、48μg／ml异丙酚抑制自发兴奋性突触后电流并直接激动GABA．受体，使Cl．内流，产生外向超极化的GABA电流，从而间接抑制NMDA受体通道电流。用荷包牡丹碱100μmol／L阻断GABA．受体后，3、48μg∥ml异丙酚仍可抑制100μmol／LNMDA诱发的NMDA受体通道电流（P〈0．05）。结论 异丙酚通过激活GABA．受体影响NMDA受体通道电流，对NMDA受体通道也有直接抑制作用。     

瑞芬太尼和芬太尼对大鼠脊髓背角神经元NMDA受体通道电流的影响

目的 探讨瑞芬太尼和芬太尼对大鼠脊髓背角神经元NMDA受体通道电流的影响.方法 采用全细胞膜片钳技术记录NMDA受体通道电流.原代培养的E14SD大鼠脊髓背角神经元(DH细胞)30个,采用随机数字表法,将其分为3组(n=10):瑞芬太尼组(R组)、芬太尼组(F组)、对照组(C组).4 nmol/L瑞芬太尼(R组)、10 μmol/L芬太尼(F组)灌流DH细胞60 min后洗脱.于给药后即刻(T0)、药物作用15 min(T1)、30 min(T2)、45 min(T3)、60 min(T4)、洗脱后15 min(T5)、30 min(T6)时记录NMDA受体通道电流.结果 与C组比较,F组各时点NMDA受体通道峰电流差异无统计学意义,R组T0、T1时NMDA受体通道峰电流差异无统计学意义(P＞0.05),T2-T6时NMDA受体通道峰电流升高(P＜0.01);与T0时比较,R组T3-T6时NMDA受体通道峰电流升高(P＜0.01);与T5时比较,R组T2-T4时、T6时NMDA峰电流下降(P＜0.01).结论 瑞芬太尼可增强大鼠脊髓背角神经元NMDA受体功能,于洗脱后达峰效应,芬太尼无此作用.     

M胆碱能受体激动剂对大鼠脊髓Ⅱ板层甘氨酸能神经元sIPSCs和mIPSCs的影响

目的 探讨不同浓度M胆碱能受体激动剂Oxotremorine-M(Oxo-M)对大鼠脊髓Ⅱ板层甘氨酸能神经元自发抑制性突触后电流(sIPSCs)和微小抑制性突触后电流(mIPSCs)的影响.方法 健康雄性清洁级SD大鼠15只,周龄3～4周,体重160～180 g,取L_(1-5)脊髓节段制备脊髓薄片,采用全细胞膜片钳技术,依次持续灌注终浓度为1、3、5、10 μmol/L Oxo-M,分别记录不同浓度Oxo-M作用前后脊髓Ⅱ板层甘氨酸能神经元sIPSCs和mIPSCs的频率及幅度.结果 3～10 μmol/L Oxo-M可增加脊髓Ⅱ板层甘氨酸能神经元sIPSCs频率,其中3 μmol/L Oxo-M作用最明显,但3～10 ～mol/L Oxo-M对sIPSCs幅度无影响.2 μmol/L甘氨酸受体特异性阻断剂士可宁可完全阻断记录到的sIPSCs,2 μmol/L阿托品可完全阻断Oxo-M增加sIPSCs频率的效应.1～10 μmol/L Oxo-M对mIPSCs的频率及幅度均无影响.结论 Oxo-M可通过激活大鼠脊髓Ⅱ板层甘氨酸能神经元胞体上的M胆碱能受体增加甘氨酸能神经元递质的释放,但不呈剂量依赖性.     

氟哌利多对大鼠海马锥体细胞钠通道电流的影响

目的　研究氟哌利多对大鼠海马锥体细胞钠通道电流的影响。方法　用酶消化法急性分离SD大鼠 (1 0～ 1 4d)海马锥体细胞 ,全细胞膜片钳技术记录氟哌利多对钠通道电流的影响。结果　在钳制电压 (Vh) 80mV、刺激电压 (Vt) 0mV条件下 ,0 3～ 30 0 μmol/L氟哌利多对钠电流抑制率为 1 3 1 2 %～ 82 2 5 % (P <0 0 5 ,n =7) ,IC50 为 2 6 0 1 μmol/L。 30 μmol/L的氟哌利多使电流 电压曲线峰值电流平均降低 4 1 93% (P <0 0 1 ) ,但不影响其形状 ,对激活曲线无明显的影响 (P >0 0 5 )。用药前、后 5 0 %通道激活时的去极化电压 (V1 / 2 )分别为 6 6 89mV和 6 5 35mV ;使稳态失活曲线向超极化方向移动 ,用药前、后 5 0 %的通道灭活时的条件脉冲电压 (V1 / 2 )分别为 5 8 75mV和 6 8 81mV。结论　氟哌利多对海马锥体细胞钠通道电流有明显浓度依赖性的抑制作用 ,其抑制作用主要与优先结合钠通道的失活状态而影响钠通道的失活有关     

甘氨酸对缺氧大鼠心肌细胞的保护作用及机制

目的探讨甘氨酸对缺氧大鼠心肌细胞的保护作用及机制。方法分离培养SD乳鼠心肌细胞,用生化分析仪检测缺氧后6h及甘氨酸处理后心肌细胞培养液中肌酸激酶(CK)、乳酸脱氢酶(LDH)的释放量;用免疫组织化学方法观察常氧及缺氧心肌细胞甘氨酸受体α1亚基(GlyRα1)的表达;激光共聚焦显微镜检测细胞内游离钙及心肌细胞膜电位的变化。结果缺氧后6h心肌细胞培养液中CK、LDH值[(393.8±5.3)、(1564±41)U/L)]均升高,甘氨酸处理后CK、LDH值[(56.3±2.7)、(716±18)U/L]均明显下降(P<0.01).常氧及缺氧心肌细胞GlyRα1均呈阳性表达。常氧心肌细胞钙离子平均荧光强度为27±8,缺氧后6h增加为139±29(P<0.01);而甘氨酸处理后细胞钙离子平均荧光强度为51±11,与缺氧后6h比较明显减少(P<0.01),与常氧下比较却明显增加(P<0.01).常氧心肌细胞膜电位为177±20,缺氧后6h膜电位发生去极化(62±9,P<0·01);而加入甘氨酸后心肌细胞膜电位为123±16,较缺氧后6h时的去极化程度明显减轻(P<0·01).结论甘氨酸对缺氧心肌细胞有保护作用,其可能的机制是甘氨酸与其受体结合后,减轻了心肌细胞膜去极化,从而减少了细胞膜电压依赖性钙通道开放,使钙离子内流减少。     

异丙酚对交感神经元钠通道电流的影响

目的 研究异丙酚对交感神经元他细胞钠通道电流的影响。探讨基金上 因管扩张机制。方法 酶消化法急性分离ＳＤ大鼠（８～１２ｄ）颈上交感神经节细胞,应用全细胞膜片钳技术记录异丙酚对钠通道电流以的影响。结果 在钳制电压（Ｖｈ）～８０ｍＶ,刺激电压（Ｖｔ）０ｍＶ条件下,临床相关浓度的异丙酚（５．６μｍｏｌ／Ｌ）使钠通道电流峰值降２７．６６％（Ｐ〈０．０１）,随浓度增加,抑制作用逐渐增强（ｒ＝０．９８２。Ｐ〈     

DAGO及可乐定对大鼠背根神经节神经元GABAA受体介导膜电流的影响

γ-氨基丁酸(13ABA)是中枢神经系统一种重要的抑制性神经递质，其递质／受体系统在脊髓伤害性信息调制中起着重要的作用。背根神经节(Dorsal Root Ganglion，DRG)是伤害性感受传入的初级感觉神经元，其胞体膜受体通道可反映脊髓突触前膜的功能。本研究拟采用全细胞膜片钳技术研究μ阿片受体激动剂DAGO及α2受体激动剂可乐定对DRG细胞GABA电流的影响，以探讨DAGO及可乐定是否经GABA递质／受体系统产生外周镇痛作用。     

延迟整流性K+通道在肿瘤坏死因子α抑制人肝癌细胞增殖中的作用

目的　研究延迟整流性 Ｋ＋ 通道在肿瘤坏死因子α（ Ｔ Ｎ Ｆα） 抑制人肝癌细胞增殖中的作用。方法　采用膜片钳技术的全细胞记录方式记录人肝癌细胞的跨膜电流,氚标记的胸腺嘧啶核苷掺入技术测定人肝癌细胞的 Ｄ Ｎ Ａ 合成能力。结果　 Ｔ Ｎ Ｆα抑制肝癌细胞增殖的同时,未能使肝癌细胞膜离子通道的跨膜 Ｋ＋ 电流和细胞膜电导降低。 Ｋ＋ 通道阻断剂四乙胺不能进一步增加 Ｔ Ｎ Ｆ 抑制 Ｄ Ｎ Ａ 合成的作用,且无明显量效关系和时间依赖性。结论　 Ｔ Ｎ Ｆα抑制人肝癌细胞增殖不是通过对细胞膜 Ｋ＋通道的干扰来实现。     

新福林增强人心房肌细胞肿胀激活的氯离子电流

目的 通过全细胞膜片钳技术研究α1-肾上腺素受体激动剂新福林对人心房肌细胞肿胀激活的氯离子电流(ICl.swell)的调节作用.方法 术中取冠脉搭桥患者右心耳组织,酶解分离出心房肌细胞.全细胞膜片钳技术记录氯离子电流.等渗、低渗台氏液灌流;分别加入新福林、氯离子电流阻滞剂4,4'-二异硫氰基芪-2,2'-二磺酸(DIDS)、α1受体阻断剂哌唑嗪,观察电流变化.结果 低渗台氏液灌流心房肌细胞,可记录到ICl.swell.用含有100μmol/L新福林的低渗台氏液灌流,ICl.swell电流强度增加.在-90 mV,电流强度由(-1.11±0.55)pA/pF增加到(-1.56±0.69)pA/pF(P＜0.05,n=7);+40mV,由(3.24±2.04)pA/pF增加到(4.64±2.61)pA/pF(P＜0.01,n=7).这种效应为浓度依赖性,加入DIDS或者用等渗灌流液灌流,该效应被逆转.加入哌唑嗪1 μmol/L可以使新福林的量效关系曲线右移.结论 在人心房肌细胞新福林可以增强ICl.swell,这和在其他动物得到的结果不同.该现象是首次发现,表明新福林对ICl.swell的调节具有种属特异性.     

降钙素基因相关肽对大鼠心室肌细胞膜ATP敏感性钾离子通道的影响

目的探讨降钙素基因相关肽(CGRP)与大鼠心室肌细胞膜ATP敏感性钾通道(KATP)之间的关系及其信号转导通路。方法酶解法急性分离SD大鼠心室肌细胞,采用Langendroff灌流装置,分别用含0.1nmol/L CGRP、1nmol/LCGRP8-37(CGRP特异性阻滞剂)、1μmol/L H-89(蛋白激酶A阻滞剂)的细胞外液灌流心室肌细胞,每个细胞采用给药前后自身对照,标准的全细胞膜片钳技术记录KATP电流,待细胞电流稳定后,观察不同药物对KATP电流的影响。结果 0.1nmol/L CGRP对KATP电流有明显的激活作用,电流密度-电压(I-V)曲线明显上移(P0.05)。1nmol/L CGRP8-37和1μmol/L H-89可使CGRP激活的KATP电流明显降低,I-V曲线明显下移(P0.05)。结论心肌细胞上CGRP与其特异性受体结合,通过蛋白激酶A激活KATP,增强KATP电流。     

Alpha4beta2 Neuronal Nicotinic Acetylcholine Receptors in the Central Nervous System Are Inhibited by Isoflurane and Propofol, but alpha7-type Nicotinic Acetylcholine Receptors Are Unaffected

Background: The mechanisms of action of general anesthetics are not completely understood. Many general anesthetics are reported to potentiate gamma-aminobutyric acid (GABAA) and glycine receptors in the central nervous system (CNS) and to inhibit the muscle-type nicotinic acetylcholine receptor (nAChR). The effects of general anesthetics on another family of ligand-gated ion channel in the CNS, the nAChRs, have not been defined.

Methods: Two types of CNS acetylcholine receptor, the alpha4beta2 receptor or the alpha7 homomeric receptor, were expressed heterologously in Xenopus laevis oocytes. Using the standard two-microelectrode voltage-clamp technique, peak acetylcholine-gated current was measured before and after coapplication of isoflurane or propofol.

Results: Coapplication of either isoflurane or propofol with acetylcholine resulted in potent, dose-dependent inhibition of the alpha4beta2 receptor current with median inhibitory concentrations of 85 and 19 micro Meter, respectively. The inhibition of the alpha4beta2 receptor by both isoflurane and propofol appears to be competitive with respect to acetylcholine. The alpha7 receptor current was not effected by either anesthetic.     

The anesthetic potency of propanol and butanol versus propanethiol and butanethiol in alpha1 wild type and alpha1(S267Q) glycine receptors

Although similar in shape and size, and although differing only by substitution of a sulfur atom for an oxygen atom, propanethiol and butanethiol differ markedly from propanol and butanol in their in vivo potency and physical properties. Recent theories of narcosis suggest that anesthetics may act by enhancing the effect of inhibitory agonists, such as glycine, on their receptors. We tested whether propanol, butanol, propanethiol, and butanethiol enhance the effect of glycine on alpha1 glycine receptors expressed in Xenopus laevis oocytes in a manner that reflects the in vivo differences found for potencies. As anticipated, we found an immediate parallel between in vivo (rat minimum alveolar concentration of anesthetic required to eliminate movement in response to a noxious stimulus in 50% of subjects) and in vitro (recombinant receptor) effects. All four compounds enhanced the effect of glycine on wild type receptors, and the extent of enhancement for a given minimum alveolar concentration-multiple was approximately the same for all compounds. We also found that propanethiol, butanethiol, propanol, and butanol did not affect, or minimally affected, the action of glycine in anesthetic resistant mutants in which the amino acid serine at position 267 was replaced by glutamine [alpha1(S267Q)]. Implications: The in vivo potencies of propanethiol, butanethiol, propanol, and butanol correlate with their capacities to enhance the effect of glycine on alpha1 glycine receptors expressed in Xenopus laevis oocytes. These results support the notion that a protein mediates anesthetic action.     

Modulation of Recombination Human gamma-Aminobutyric Acid sub A Receptors by Isoflurane: Influence of the Delta Subunit

Background: The gamma-aminobutyric acid (GABA)A receptor/chloride channel has a broad-spectrum anesthetic sensitivity and is a key regulator of arousal. Each receptor/channel complex is an assembly of five protein subunits. Six subunit classes have been identified, each containing one to six members; many combinations are expressed throughout the brain. Benzodiazepines and intravenous anesthetic agents are clearly subunit dependent, but the literature to date suggests that volatile anesthetics are not. The physiological role of the delta subunit remains enigmatic, and it has not been examined as a determinant of anesthetic sensitivity.

Methods: Combinations of GABAA receptor subunit cDNAs were injected into Xenopus laevis oocytes: alpha1 beta1, alpha1 beta1 gamma2L, alpha1 beta1 delta, and alpha1 beta1 gamma2L delta. Expression of functional ion channels with distinct signalling and pharmacologic properties was demonstrated within 1-4 days by established electrophysiological methods.

Results: Co-expression of the delta subunit produced changes in receptor affinity; current density; and the modulatory efficacy of diazepam, zinc, and lanthanum; it also produced subtle changes in the rate of desensitization in response to GABA. Isoflurane enhanced GABA-induced responses from all combinations: alpha beta delta (> 10-fold) > alpha beta > alpha beta gamma >or= to alpha beta gamma delta ([nearly =] 5-fold). Dose-response plots were bell shaped. Compared with alpha beta gamma receptors (EC50 = 225 micro Meter), both alpha beta delta (EC50 = 372 micro Meter) and alpha beta gamma delta (EC sub 50 = 399 micro Meter) had a reduced affinity for isoflurane. Isoflurane (at a concentration close to the EC50 for each subunit) increased the affinity of GABA for its receptor but depressed the maximal response (alpha beta gamma and alpha beta gamma delta). In contrast, the small currents through alpha beta delta receptors were enhanced, even at saturating agonist concentrations.     

The effects of isoflurane on native and chimeric muscarinic acetylcholine receptors: the role of protein kinase C

By using two electrode voltage clamps, we investigated the effects of isoflurane on m3 and chimeric m1/m3 muscarinic receptors and the role of protein kinase C (PKC) in the effects. Muscarinic receptors were expressed by injection of mRNA into Xenopus oocytes, and Ca(2+)-activated Cl(-) currents were measured after the application of acetyl-beta-methylcholine. We constructed chimeric m1/m3 receptor DNA encoding the third intracellular loop of m1 and the remainder from the m3 receptor. Chimeric and m3 receptors were inhibited by isoflurane, but the m1 receptor was not. PKC activation with phorbol-12-myrisate-13-acetate (50 nM) decreased signaling of both chimeric and m3 receptors significantly. Chelerythrine (20 microM, PKC inhibitor) abolished the effect of isoflurane on chimeric and m3 signaling. Whereas isoflurane inhibition of chimeric and m3 receptors was completely reversible after washout with Tyrode's solution for 3 min, treatment with okadaic acid (500 nM, protein phosphatase inhibitor) rendered the inhibition irreversible. Taken together, our results suggest that isoflurane inhibits m3 and chimeric m1/m3 muscarinic signaling by enhancing PKC activity and that the site of action is located outside of the third intracellular loop. IMPLICATIONS: By use of the Xenopus oocyte expression system, we investigated the effects of isoflurane on muscarinic signaling and the role of protein kinase C in these effects. Our findings suggest that isoflurane inhibits muscarinic receptors through activation of protein kinase C and that the relevant phosphorylation sites are located outside the third intracellular loop.     

Remifentanil directly activates human N-methyl-D-aspartate receptors expressed in Xenopus laevis oocytes

BACKGROUND: Clinical studies suggest that intraoperative administration of the clinical remifentanil formulation Ultiva (GlaxoWellcome GmbH & Co, Bad Oldesloe, Germany) increases postoperative pain and postoperative analgesic requirements, but mechanisms remain unclear. N-methyl-D-aspartate (NMDA) receptors are thought to play a major role in development of postoperative pain and opiate tolerance. The authors hypothesized that Ultiva directly stimulates human NMDA receptors. METHODS: To test this hypothesis, the authors expressed human NR1A/NR2A and NR1A/NR2B NMDA receptors in Xenopus laevis oocytes by injection of messenger RNA prepared in vitro. After protein expression, they used a two-electrode voltage clamp to measure currents induced by NMDA receptor agonists and opioids. RESULTS: Noninjected cells were unresponsive to all compounds tested. Glutamate/glycine (1 nM-1 mM each) or Ultiva (0.01 pM-0.1 mM) stimulated NMDA receptors concentration dependently. NR1A/2A EC50 values were 8.0 microM/12 microM for glutamate/glycine and 3.5 nM for Ultiva, and NR1A/2B EC50 values were 3.9 microM/1.9 microM for glutamate/glycine and 0.82 microM for Ultiva. Glycine in combination with Ultiva showed no additive effect compared with Ultiva alone. Ultiva-induced currents were inhibited by MK-801 (pore blocker) but not by 7-CK (glycine antagonist), D-AP5 (glutamate antagonist), or naloxone. Fentanyl (10 microM) did not stimulate NMDA receptors. CONCLUSION: These data indicate that Ultiva but not fentanyl stimulates NMDA receptors of different subunit combinations (NR1A/2A, NR1A/2B). The mechanism seems to be allosteric activation of the NMDA receptor.     

Modulation of Xenopus laevis Ca-activated Cl currents by protein kinase C and protein phosphatases: implications for studies of anesthetic mechanisms

Ca-activated Cl currents (I(Cl(Ca))) are used frequently as reporters in functional studies of anesthetic effects on G protein-coupled receptors using Xenopus laevis oocytes. However, because anesthetics affect protein kinase C (PKC), they could indirectly affect I(Cl(Ca)) if this current is regulated by phosphorylation. We therefore studied the effect of modulation of either PKC or protein phosphatases PP1alpha and PP2A on I(Cl(Ca)) stimulated either by lysophosphatidate (LPA) signaling or by microinjection of Ca. X. laevis oocytes were studied under voltage clamp. Rat PP1alpha and PP2A were overexpressed in oocytes. PP, inositoltrisphosphate (IP(3)), the PP inhibitor okadaic acid (OA), the PKC inhibitor chelerythrine, or CaCl(2) were directly injected into the oocyte. Responses to agonists (LPA 10(-6) M, IP(3) 10(-4) M, CaCl(2) 0.5 M) were measured at a holding potential of -70 mV in the presence or absence of the PP inhibitors cantharidin or OA. PP1 alpha and PP2A inhibited I(Cl(Ca)) from 7.6 +/- 0.9 microC to 2.5 +/- 0.9 microC and 3.2 +/- 1.4 microC, respectively. PP inhibition enhanced I(Cl(Ca)) in control oocytes and reversed the inhibitory effect in oocytes expressing PP1 alpha or PP2A. PKC inhibition by chelerythrine enhanced both LPA- and CaCl(2)-induced I(Cl(Ca)). Our data indicate that the Xenopus I(Cl(Ca)) is modulated by phosphorylation. This may complicate design and interpretation of studies of G protein-coupled receptors using this model.     

内毒素/脂多糖及烧伤血清对豚鼠肠道平滑肌细胞离子通道的影响

目的观察内毒素/脂多糖(LPS)及烧伤血清对豚鼠结肠带平滑肌细胞钙激活钾通道(KCa)的影响,探讨烧伤后发生胃肠动力障碍的分子电生理机制。方法用急性酶分离法获取单个健康豚鼠结肠带平滑肌细胞,在对称性高钾溶液中采用细胞膜片钳单通道记录技术,分别记录细胞贴附式膜片(电极位于细胞膜外面)和内面向外式膜片(电极位于细胞膜内面)上的电流。引出的电流信号经转换器转换,输入计算机,用离子通道计算机分析系统进行数据处理,并测定以下指标：(1)电流幅值(CA)；(2)开放概率(PO)；(3)开放时间(OT)；(4)关闭时间(CT),以检测其特性并鉴定是否为KCa。确定为KCa后,向浴液中分别加入20、40、60、80、100 mg/L的LPS,观察LPS对两种膜片方式上KCa的影响。在浴液中分别加入正常血清和烧伤血清,观察血清对KCa的影响。结果在对称性高钾溶液中,豚鼠结肠带平滑肌细胞内面向外式膜片上的KCa电导值为(271±7)ps,是高电导的离子通道。随着膜去极化及细胞内Ca~(2+)增加,通道PO增加,该通道可被细胞外低浓度的钾通道阻断剂四乙胺(TEA,1 mmol/L)所阻断,而膜内面较高浓度的TEA(40 mmol/L)对该通道无阻断作用,证实为KCa。当Ca~(2+)为0 mol/L时,向浴液中分别加入20、40、60、80、100 mg/L的LPS,两种膜片方式KCa活性随LPS浓度的增加而增加。40 mg/L以上的LPS对KCa具有明显激活作用,通道PO显著增加(P＜0．05或0．01),用不含LPS的浴液灌流后亦不能回转。两种膜片方式下烧伤血清对KCa有激活作用,而正常血清无此作用。结论LPS和烧伤血清可通过激活肠道平滑肌细胞KCa抑制肠道蠕动。