羟基红花黄色素A对肝纤维化大鼠血清学指标及脂质过氧化的影响

目的观察羟基红花黄色素A(HYSA)对四氯化碳(CCl4)诱导的肝纤维化(HF)大鼠血清学指标及脂质过氧化的影响。方法清洁级雄性SD大鼠60只,体重(180±30)g,随机分为A、B、C、D、E组,A组(正常对照组)、B组(HF模型组)、C组(红花组)、D组(HYSA组)、E(秋水仙碱组),除A组外,其余各组采用CCl4灌胃,诱导大鼠肝纤维化模型;造模第2周起,腹腔注射给予相应药物,8w后处死大鼠取血清及肝组织标本,光景下观察肝组织结构变化及肝纤维化程度,放射免疫技术检测测定血清中ALT、AST、ALB含量,分光光度计检测肝组织匀浆中SOD和MDA的含量。结果与B组比较,C、D、E组大鼠血清中ALT、AST活性显著降低,ALB含量显著升高,肝细胞变性、坏死明显减少,肝组织纤维化程度明显减轻,肝组织MDA含量显著降低,SOD活性显著升高;与C组比较,D组的肝纤维化程度显著减轻,两者比较有统计学意义(P<0.05)。结论 HYSA作为红花抗肝纤维化的主要有效作用成分,能够保护肝组织,减轻肝细胞水肿程度,减轻其纤维化程度,并能促进损伤肝组织的修复,且其抗肝纤维化作用机制可能与其抗脂质过氧化作用有关。     

羟基红花黄色素A抑制人肝癌细胞系Huh7发生上皮-间质转化

目的 研究羟基红花黄色素A(HSYA)对人肝癌细胞系Huh7迁移及侵袭的影响,并通过体内外实验探究其是否影响肝癌细胞上皮-间质转化(EMT).方法 体外培养Huh7细胞,分为对照组、HSYA 80和160μmol/L实验组,分别处理24 h,用transwell小室法和细胞划痕实验检测细胞侵袭和迁移能力;Western...     

羟基红花黄色素A对缺血/再灌注大鼠肺线粒体通透性转换功能的影响

目的: 研究羟基红花黄色素A(HSYA)对缺血/再灌注(I/R)损伤肺组织细胞的保护作用,并探讨其对肺I/R损伤时线粒体通透性转换(MPT)功能的影响。方法: 开胸阻断左肺门45 min,松开阻断带形成再灌注,复制在体肺I/R模型。健康SD大鼠50只,随机分为5组,每组10只:假手术对照组(对照组) 、缺血/再灌注1 h组(I/R 1 h组)、缺血/再灌注3 h组(I/R 3 h组)、HSYA干预+I/R 1 h组(SI 1 h组)和HSYA干预+I/R 3 h组(SI 3 h组)。SI 1 h组和SI 3 h组分别在缺血前20 min和再灌注即刻静注HSYA (2.0 mg/kg),其余步骤分别同I/R 1 h组和I/R 3 h组,对照组、I/R 1 h组和I/R 3 h组分别注射同等体积生理盐水。观察各组肺湿干重比(W/D)、肺泡损伤数定量评价指标(IQA)、肺组织细胞凋亡情况、胞浆内和线粒体基质内细胞色素C (CytC)、凋亡诱导因子(AIF)的水平以及MPT功能。结果: 与对照组相比,其余各组肺组织细胞凋亡指数(AI)、W/D和IQA均增高,CytC和AIF胞浆含量增加而线粒体内含量减少;SI各组与相应的I/R组比,AI、W/D和IQA均显著降低,但仍高于对照组;SI组CytC和AIF较相应的I/R组胞浆含量减少而线粒体内含量增加;线粒体在540 nm处的吸光度值SI 1 h较IR 1 h、SI 3 h较IR 3 h增高,但仍低于对照组。结论: 羟基红花黄色素A在一定程度上抑制I/R肺组织细胞凋亡,发挥细胞保护作用,该作用可能与其维持MPT功能、减少CytC和AIF由线粒体向胞浆内释放有关。     

CD4~+T淋巴细胞Kv1.3通道在大鼠动脉粥样硬化中的作用

目的:探讨大鼠动脉粥样硬化(AS)模型中CD4+T淋巴细胞电压门控钾通道(Kv)Kv1.3的表达、功能及其在AS中的作用。方法:采用高脂饮食法建立大鼠动脉粥样硬化模型,流式细胞术分析淋巴细胞的比例,采用免疫磁珠法分离CD4+T淋巴细胞,研究脾组织CD4+T淋巴细胞Kv1.3mRNA表达、细胞内钙离子浓度及细胞因子分泌的变化。结果:(1)AS组脾组织CD4+T淋巴细胞占总T淋巴细胞比例较对照组明显升高(74.93%±2.15%vs67.80%±2.54%,P0.05)。(2)经刀豆蛋白A(ConA)刺激,AS组T淋巴细胞增殖程度明显高于对照组(1.1321±0.1750vs0.7971±0.0955,P0.05)。(3)AS组脾组织CD4+T淋巴细胞在ConA刺激状态下胞内钙离子浓度明显高于对照组(H=82,P0.05)。(4)AS组脾组织CD4+T淋巴细胞在刺激48h后较刺激24h后细胞因子(IL-2,TNF-α)分泌显著增加。(5)AS组脾组织CD4+T淋巴细胞Kv1.3mRNA表达明显高于对照组(3.670±1.579vs1)。结论:AS组脾组织CD4+T淋巴细胞比例高于对照组,CD4+T淋巴细胞Kv1.3mRNA表达增多,提示高表达Kv1.3的CD4+T淋巴细胞可能在AS的发生发展中发挥重要的作用。     

羟基红花黄色素A抑制H22小鼠肝癌移植瘤血管生成及减少MMP-3的表达

目的研究红花组分羟基红花黄色素A(HSYA)对H22小鼠肝癌移植瘤组织新血管生成的抑制作用及对基质金属蛋白酶-3(MMP-3)的影响。方法建立H22小鼠肝癌移植瘤模型,第2天将小鼠随机分为对照组、索拉非尼组及HSYA组(1.125和2.25 mg/kg),HE观察肝癌移植瘤组织病理;用免疫组织化学法检测肿瘤组织中CD34的表达,计数微血管密度(MVD);免疫组化和Western blot分别检测瘤组织中MMP-3的表达。结果与对照组相比,HSYA组(1.125和2.25 mg/kg)的MVD显著降低(P0.01),MMP-3蛋白在移植瘤组织中的表达明显减少(P0.01),以HSYA组(2.25 mg/kg)效果最为显著,但不及索拉非尼组。结论 HSYA在一定浓度范围内抑制H22小鼠肝癌移植瘤血管生成,其作用可能与降低基质金属蛋白酶-3的蛋白表达有关。     

羟基红花黄色素A干预脑缺血再灌注损伤后星形胶质细胞脂质运载蛋白2的表达

背景：缺血性脑卒中严重威胁人类健康,缺血缺氧后星形胶质细胞大量表达脂质运载蛋白2加重脑损伤,但其具体机制并不清楚。羟基红花黄色素A具有抗缺血、抗氧化、抗血栓及抗炎等作用,其是否影响脑缺血缺氧后星形胶质细胞表达脂质运载蛋白2,目前尚不清楚。目的：探究羟基红花黄色素A对脑缺血再灌注损伤后星形胶质细胞中脂质运载蛋白2表达的影响及机制。方法：(1)将30只成年SD大鼠随机分成3组：假手术组、大脑中动脉闭塞再灌注组、羟基红花黄色素A组,后2组建立大脑中动脉闭塞再灌注模型,羟基红花黄色素A组在再灌注后以12 mg/kg的剂量腹腔注射羟基红花黄色素A。采用Longa评分法评估神经功能缺损程度,采用TTC染色法测定脑梗死体积,Western blot和免疫荧光检测JAK2/STAT3通路及脂质运载蛋白2的表达,ELISA法检测白细胞介素1β、白细胞介素6、肿瘤坏死因子α水平。(2)将星形胶质细胞分为4组：正常组、糖氧剥夺复糖氧组、羟基红花黄色素A组、AG490组,后3组建立糖氧剥夺复糖氧模型,在糖氧剥夺期间分别用75μmol/L羟基红花黄色素A、10μmol/L酪氨酸磷酸化抑制剂AG490处理星形胶质...     

羟基红花黄色素A对四氯化碳致大鼠肝纤维化的干预作用

目的:探讨羟基红花黄色素A(HYSA)对四氯化碳(CCl4)诱导的大鼠肝纤维化(HF)形成的影响及其机制.方法:清洁级雄性SD大鼠随机分为模型组、红花注射液组、HYSA组和秋水仙碱组,腹腔注射给药,8周后取材,放射免疫技术检测血清纤维化指标透明质酸(HA)、层黏蛋白(LN)、Ⅲ型前胶原(PC-Ⅲ)、Ⅳ型胶原(CⅣ),Masson三色染色观察胶原纤维面积比变化,Hoechest 33342染色荧光显微镜观察肝细胞凋亡情况,免疫组织化学法检测肝组织基质金属蛋白酶组织抑制因子(TIMP-1)的表达情况.结果:与生理盐水组比较,红花注射液组、HYSA组和秋水仙碱组大鼠血清HA、LN、PCⅢ、CⅣ的含量降低,大鼠肝组织胶原纤维增生程度减轻,细胞变性、坏死减少,TIMP-1表达减少.结论:HYSA具有抗大鼠肝纤维化的作用,其机制可能与减少TIMP-1的表达有关.     

H2O2对大鼠肺动脉平滑肌细胞Kv通道的作用

 目的:观察H₂O₂在常氧时对大鼠肺动脉平滑肌细胞（PASMCs）的Kv的影响, 探讨H₂O₂对肺动脉平滑肌细胞的Kv通道的作用。 方法:用酶解法急性分离单个PASMCs，以全细胞膜片钳技术记录PASMCs膜上的电压依赖性钾通道 (voltage-gated potassium channel, Kv) 电流。 结果:常氧下 H₂O₂可显著增加Kv电流，电流-电压关系曲线左上移；而且Kv电流呈浓度依赖性增加。 结论:常氧下H₂O₂ 可使Kv通道开放。     

5-羟基癸酸盐对慢性低氧大鼠肺动脉Kv1.5通道表达的影响

目的: 以大鼠为研究对象,研究线粒体ATP敏感性钾通道(mitoK_ATP)的抑制剂5-羟基癸酸盐(5-HD)对慢性低氧肺动脉高压大鼠的影响及其潜在机制。方法: 24只SD雄性大鼠随机分成对照组、低氧组、低氧+5-羟基癸酸盐干预组,每组8只。将低氧组和5-HD干预组大鼠放入常压低氧舱内 以建立低氧肺动脉高压模型。 4周后测定平均肺动脉压(mPAP)及右心室与左心室及室间隔的重量比 ,并采用RT-PCR及Western blotting技术,分析各组肺动脉Kv1.5 mRNA及蛋白表达。结果: (1) 慢性低氧组大鼠的mPAP及RV/(LV+S)显著高于正常对照组(P<0.05),5-HD干预组mPAP及RV/(LV+S)显著低于低氧组,均P<0.05。(2) 低氧组Kv1.5通道mRNA及蛋白表达显著低于正常组,5-HD组Kv1.5通道表达显著高于低氧组, 均P<0.05。结论: mitoK_ATP通道的抑制剂5-HD通过降低mPAP及RV/(LV+S),在慢性低氧肺动脉高压中起保护作用。mitoK_ATP通道的抑制及Kv1.5通道表达的上调可能与该保护作用有关。     

羟基红花黄色素A对阿尔茨海默病的干预作用

<正>阿尔茨海默病(AD)是最常见的神经退行性疾病,其临床特征主要表现为进行性记忆丧失及认知功能下降。AD的两大病理学特征为脑中细胞内过度磷酸化的tau蛋白聚集形成神经原纤维缠结(NFTs)和细胞外β样淀粉蛋白肽(A)形成的老年斑(SP)。研究显示,tau蛋白的过度磷酸化由蛋白激酶及磷酸酯酶调节失衡引起,其中糖原合酶激酶-3(GSK-3)是最关键的激酶。大鼠侧脑室共注射Wortmannin(WT,PI3K的特异性抑制剂)及GF-109203X(GFX,PKC的特异性抑制剂)可导致GSK-3过度激活,进而引起大鼠tau蛋白过度磷酸化及空间记忆障碍。     

Recording, analysis, and function of dendritic voltage-gated channels

Ever since the publication of the Hamill et al. [Hamill et al., Pflügers Arch, 391:85–100, 1981] paper and the following increase in popularity of acute brain slice preparations, there has been a large increase in the volume of publications investigating voltage-gated channels in the central nervous system using the patch-clamp technique. In the preceding decade, investigations of voltage-gated channels have moved out of the somatic region into dendrites providing much needed information about dendritic voltage-gated channels. In this study, we review some aspects related to the investigation of voltage-gated ion channels in dendrites: recording, analysis, and function.     

Effect of acidosis on Ca2+-activated K+ channels in cultured porcine coronary artery smooth muscle cells

 Although acidosis induces vasodilation, the vascular responses mediated by large-conductance Ca²⁺-activated K⁺ (K_Ca) channels have not been investigated in coronary artery smooth muscle cells. We therefore investigated the response of porcine coronary arteries and smooth muscle cells to acidosis, as well as the role of K_Ca channels in the regulation of muscular tone. Acidosis (pH 7.3–6.8), produced by adding HCl to the extravascular solution, elicited concentration-dependent relaxation of precontracted, endothelium-denuded arterial rings. Glibenclamide (20 μM) significantly inhibited the vasodilatory response to acidosis (pH 7.3-6.8). Charybdotoxin (100 nM) was effective only at pH 6.9–6.8. When we exposed porcine coronary artery smooth muscle cells to a low-pH solution, K_Ca channel activity in cell-attached patches increased. However, pretreatment of these cells with 10 or 30 μM O, O′-bis(2-aminophenyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl)ester (BAPTA-AM), a Ca²⁺ chelator for which the cell membrane is permeable, abolished the H⁺-mediated activation of K_Ca channels in cell-attached patches. Under these circumstances H⁺ actually inhibited K_Ca channel activity. When inside-out patches were exposed to a [Ca²⁺] of 10^–6 M [adjusted with ethyleneglycolbis(β-aminoethylester)-N,N,N′,N′-tetraacetic acid (EGTA) at pH 7.3], K_Ca channels were activated by H⁺ concentration dependently. However, when these patches were exposed to a [Ca²⁺] of 10^–6 M adjusted with BAPTA at pH 7.3, H⁺ inhibited K_Ca channel activity. Extracellular acidosis had no significant direct effect on K_Ca channels, suggesting that extracellular H⁺ exerts its effects after transport into the cell, and that K_Ca channels are regulated by intracellular H⁺ and by cytosolic free Ca²⁺ modulated by acute acidosis. These results indicate that the modulation of K_Ca channel kinetics by acidosis plays an important role in the determination of membrane potential and, hence, coronary arterial tone. Received: 20 January 1998 / Received after revision: 9 April 1998 / Accepted: 22 April 1998     

pH regulation of voltage-dependent K+ channels in canine pulmonary arterial smooth muscle cells

 Voltage-dependent K⁺ currents (K_v) may play a role in hypoxic pulmonary vaso constriction. The effects of changes in extracellular pH (pH_o) and intracellular pH (pH_i) on K_v currents in smooth muscle cells isolated from canine pulmonary artery were studied using the amphotericin B perforated-patch technique for whole-cell recording. Under these conditions, cellular mechanisms for pH_i regulation remain intact, and the effects of pH_o were examined by directly changing the pH of external solutions and changes in pH_i were produced by external application of weak extracellular acids and bases and the cation/H⁺ ionophore, nigericin. Ca²⁺-free external solutions were used to isolate whole-cell K_v currents from contaminating Ca²⁺-activated K⁺ currents. Extracellular acidification (pH_o = 6.4–7.0) reduced K_v currents, produced a positive voltage shift in steady-state activation and reduced maximum K_v conductance (-g _K). Extracellular alkalinization (pH_o = 8.0–8.4) increased K_v currents, produced a small negative voltage shift in steady-state activation, and increased -g _K. Intracellular acidification produced by exposure of cells to external sodium butyrate (20 mM) or nigericin (5 μg/ml) increased K_v currents, produced a negative voltage shift in steady-state activation, and increased -g _K. Intracellular alkalinization produced by exposure of cells to external trimethylamine (20 mM) reduced K_v currents, produced a small positive voltage shift in steady-state activation and reduced -g _K. These results suggest that the effects of pH_o and pH_i on K_v currents are distinctly different, but are consistent with reported effects of pH_o and pH_i on hypoxic pulmonary vasoconstriction, suggesting that such modulation may be mediated in part by pH-induced alterations in K_v channel activity. Received: 1 November 1996 / Received after revision: 19 December 1996 / Accepted: 3 January 1997     

酸中毒致大鼠离体冠状动脉收缩的机制

目的:探讨细胞外酸中毒(pH_(ex)6.8)致大鼠离体冠状动脉(RCA)收缩的机制。方法:采用离体微血管张力法,通过观察Na~+-H~+交换体1(NHE-1)选择性抑制剂cariporide(HOE-642)或Na~+-HCO_3~-共同转运体(NBC)抑制剂S0859预孵对pH_(ex)6.8所致RCA收缩的影响,探讨酸碱转运体在酸中毒收缩RCA中的作用;通过观察氯通道抑制剂NPPB和尼氟酸(NFA)预孵及细胞外去除氯离子对pH_(ex)6.8所致RCA收缩的影响,探讨氯离子通道在酸中毒收缩RCA中的作用。结果:pH_(ex)6.8引起RCA静息张力升高,最大张力为(3.90±0.95)mN。30μmol/L HOE-642和100μmol/L S0859均抑制pH_(ex)6.8引起的RCA收缩(P0.01)。NPPB和NFA均呈浓度依赖性地抑制pH_(ex)6.8引起的RCA收缩和KCl(60 mmol/L)引起的收缩。100μmol/L的NPPB和NFA均抑制U46619(1μmol/L)引起的RCA收缩(P0.01)。等摩尔NaBr代替细胞外液中NaCl后,几乎完全抑制pH_(ex)6.8引起的RCA收缩(P0.01),但是对KCl(60 mmol/L)或U46619(1μmol/L)引起的RCA收缩无显著影响。结论:细胞外液酸化所致的RCA收缩与激活NHE-1和NBC及促进氯离子跨细胞膜运动有关。     

Adaptative modifications of right coronary myocytes voltage-gated K+ currents in rat with hypoxic pulmonary hypertension

Chronic hypoxia (CH)-induced pulmonary hypertension (PHT) is well known to alter K+ channels in pulmonary myocytes. PHT induces right ventricle hypertrophy that increases oxygen demand; however, coronary blood flow and K+ channel adaptations of coronary myocytes during PHT remain unknown. We determined whether CH and PHT altered K+ currents and coronary reactivity and what impact they might have on right myocardial perfusion. Right ventricle perfusion, as attested by microspheres, was redistributed toward hypertrophied right ventricle [RV/LV (%)=0.59+/-0.07% in CH rats vs. 0.29+/-0.03 in control rats, P<0.05]. Whole-cell patch clamping showed a reduction of global outward current in hypoxic right coronary artery myocytes (H-RCA), whereas hypoxic left coronary artery myocytes exhibited an increase. K+ channel blockers revealed that a 4-aminopyridine (4AP)-sensitive current (Kv current) was decreased in H-RCA (14.3+/-1.1 vs. 23.4+/-2.5 pA/pF at 60 mV in control RCA, P<0.05) and increased in hypoxic left coronary artery myocytes (H-LCA; 26.4+/-3.8 vs. 11.8+/-1.6 pA/pF at 60 mV in control LCA, P<0.05). Constriction to 4AP was decreased in H-RCA when compared to normoxic control and increased in H-LCA when compared to LCA. Finally, we observed that the expression of Kv1.2 and Kv1.5 were lower in H-RCA than that in H-LCA. This study reveals that CH differentially regulates Kv channels in coronary myocytes. Hypoxia decreases Kv currents and therefore reduces vasoreactivity that contributes to an adaptative response leading to right hypertrophied ventricle perfusion enhancement at rest.     

槲皮素改善高糖损伤大鼠冠脉肌原性反应的机制研究

目的:通过大鼠冠状动脉(coronary artery,CA)张力测定和全细胞膜片钳记录CA血管平滑肌细胞(vascular smooth muscle cells,VSMC)的电压门控性钾通道(voltage-gated K~+channel,Kv)电流,研究槲皮素改善高糖损伤大鼠CA肌原性反应的机制。方法:从正常SD大鼠急性分离得到CA环,然后分成6组:(1)正常对照组;(2)高糖组;(3)高糖+低剂量(3μmol/L)槲皮素组;(4)高糖+中剂量(10μmol/L)槲皮素组;(5)高糖+高剂量(30μmol/L)槲皮素组;(6)高糖+蛋白激酶C(protein kinase C,PKC)抑制剂C6303+高剂量槲皮素组。利用血管张力测定,检测CA环对血管收缩剂(60 mmol/L KCl和0.1 mmol/L U46619)的反应,并将CA环用60 mmol/L KCl预收缩后,用乙酰胆碱(acetylcholine,ACh;10~(-9)~10~(-5)mol/L)进行舒张,计算ACh引起CA环张力下降幅度与预先收缩的百分比。急性分离得到大鼠CA的VSMC,然后记录Kv电流。结果:与正常対照组相比,高糖组CA环对60mmol/L KCl和0.1 mmol/L U46619的反应明显升高;中、高剂量槲皮素的干预可以使高糖所致CA环对血管收缩剂的反应降低,孵育PKC特异性抑制剂C6303可减弱槲皮素的作用。与正常对照组相比,高糖组大鼠离体CA环对ACh的舒张幅度明显减弱。槲皮素的干预可以改善高糖孵育的大鼠离体CA环对ACh的舒张反应,C6303可以减弱槲皮素的作用。高糖孵育明显抑制CA VSMC的Kv电流,槲皮素干预可以减弱高糖对CA VSMC Kv电流的影响,孵育C6303可减弱槲皮素的作用。结论:槲皮素对高糖损伤大鼠CA肌原性反应具有保护作用,该作用与增大VSMC的Kv电流和激活PKC有关。     

Extracellular Mg2+ blocks endothelin-1-induced contraction through the inhibition of non-selective cation channels in coronary smooth muscle

This study investigated the effects of changing the extracellular [Mg²⁺] ([Mg²⁺]_o) on endothelin-1 (ET-1)-induced contraction of rabbit coronary artery smooth muscle and the involvement of non-selective cation (NSC) channels in this response. Increased [Mg²⁺]_o shifted the concentration/contraction relationship curve of ET-1 to the right. In whole-cell patch clamp recordings, ET-1 (10^–7 M) induced a long-lasting inwards current (94.7±7.2 pA) that was inhibited by 8 mM [Mg²⁺]_o (45.3±4.4%) and NSC channel blockers (10^–3 M streptomycin and 10^–3 M La³⁺), but not by the voltage-dependent Ca²⁺ channel blocker nicardipine. The current/voltage (I/V) curve was linear. Furthermore, in pressurized arteries, the ET-1-induced contraction was also inhibited by La³⁺ and streptomycin, but not by nicardipine. U-73122, a selective phospholipase C (PLC) inhibitor and staurosporine and GF 109203X, which block protein kinase C (PKC), reduced ET-1-activated NSC currents by 54.2±5.1%, 60.3±5.5% and 48.5±2.9%, respectively. The inwards current was increased by 1-oleoyl-2-acetyl-sn-glycerol (OAG) and phorbol 12,13-dibutyrate (PDBu), which activate PKC selectively. Like transient receptor potential channel (TRPC3) currents, ET-1-activated NSC currents had a linear I/V relationship, were blocked by flufenamate and activated by a diacylglycerol analogue. These results suggest that [Mg²⁺]_o blocks ET-1-induced contraction of coronary arteries by inhibiting NSC channels. Activation of PLC and PKC might be involved in activation of NSC channels.     

Biophysical properties of Ca2+- and Mg-ATP-activated K+ channels in pulmonary arterial smooth muscle cells isolated from the rat

A novel class of Ca²⁺-activated K⁺ channel, also activated by Mg-ATP, exists in the main pulmonary artery of the rat. In view of the sensitivity of these K_Ca,ATP channels to such charged intermediates it is possible that they may be involved in regulating cellular responses to hypoxia. However, their electrophysiological profile is at present unknown. We have therefore characterised the sensitivity of K_Ca,ATP channels to voltage, intracellular Ca²⁺ ([Ca²⁺]_i) and Mg-ATP. They have a conductance of 245 pS in symmetrical K⁺ and are approximately 20 times more selective for K⁺ ions than Na⁺ ions, with a K⁺ permeability (P _K) of 4.6×10^–13cm s^–1. Ca²⁺ ions applied to the intracellular membrane surface of K_Ca,ATP channels causes a marked enhancement of their activity. This activation is probably the result of simultaneous binding of at least two Ca²⁺ ions, determined using Hill analysis, to the channel or some closely associated protein. This results in a shift of the voltage activation threshold to more hyperpolarized membrane potentials. The activation of K_Ca,ATP channels by Mg-ATP has an EC₅₀ of approximately 50 M. Although the EC₅₀ is unaffected by [Ca²⁺]_i, channel activation by Mg-ATP is enhanced by increasing [Ca²⁺]_i. One possible interpretation of these data is that Mg-ATP increases the sensitivity of K_Ca,ATP channels to Ca²⁺. It is therefore possible that under hypoxic conditions, where lower levels of Mg-ATP may be encountered, the sensitivity of K_Ca,ATP channels to Ca²⁺ and therefore voltage is reduced. This would tend to induce a depolarising influence, which would favour the influx of Ca²⁺ through voltage-activated Ca²⁺ channels, ultimately leading to increased vascular tone.     

A common local anesthetic receptor for benzocaine and etidocaine in voltage-gated μ1 Na+ channels

 According to Hille’s modulated receptor hypothesis, benzocaine shares a common receptor with all other local anesthetics (LAs) in the voltage-gated Na⁺ channel. We tested this single receptor hypothesis using mutant muscle Na⁺ channels of μ1-I1575A, F1579A, and N1584A transiently expressed in Hek-293t cells. Both benzocaine and etidocaine are more effective at blocking μ1-N1584A current than the wild-type current, while they are less potent at blocking μ1-F1579A current. Such concurrent changes of both benzocaine and etidocaine potency towards F1579A and N1584A mutants suggest that they share a common LA receptor. Consistent with results found in studies of native Na⁺ channels, permanently charged QX-314 at 1 mM is not effective at blocking wild-type, F1579A, and N1584A current via external application. In contrast, QX-314 is relatively potent at blocking I1575A current when applied externally. This increased potency of external QX-314 against the μ1-I1575A mutant has been reported previously in a study of the brain counterpart. Mutant I1575A also appears to be highly sensitive to the external divalent cation Cd²⁺, probably because of the presence of cysteine residues near the μ1-I1575 position in the IV-S6 segment. To our surprise, neutral benzocaine becomes more effective at blocking μ1-I1575A current than the wild-type current, whereas the opposite is found for etidocaine. We hypothesize that an increase in accessibility of external QX-314 to the μ1-I1575A mutant is accompanied by a reduction of binding towards the charged amine component. Received: 27 May 1997 / Received after revision: 29 August 1997 / Accepted: 5 September 1997     

Localization of N-type Ca2+ channels in the rat spinal cord following chronic constrictive nerve injury

Previous studies have shown that spinal L-type, N-type, and P-type Ca²⁺-channel blockers are effective in modulating pain behavior caused nerve injury. In the present work, using the loose ligation of the sciatic nerve model, we characterized the time course of the appearance of tactile and cold allodynia and the corresponding spinal expression of the N-type Ca²⁺ channel α_1B-subunit after nerve ligation. Within 1 week after ligation, the majority of rats developed a unilateral sensitivity to mechanical stimulation (von Frey filaments), as well as sensitivity to cold, which persisted for 30 days. Immunocytochemical analysis of the spinal cord in sham-operated animals for the α_1B-subunit showed a smooth, moderate staining pattern in the superficial laminae I–II, as well as in ventral α-motoneurons. In nerve-ligated animals, an intense, dot-like immunoreactivity in the ipsilateral dorsal horn was observed from 5–20 days after nerve ligation. The most prominent α_1B-subunit upregulation was found in the outer as well as the inner part of lamina II (II_o, II_i), extending from the medial toward the lateral region of the L4 and L5 spinal segments. The behavioral changes which developed after chronic constriction injury directly correlated with the α_1B-subunit upregulation in the corresponding spinal cord segments. These data suggest that upregulation of the spinal α_1B-subunit may play an important role in the initiation and maintenance of pain state after peripheral nerve injury. Electronic Publication