七氟醚对大鼠背根神经节电压门控性离子通道的影响

目的 观察七氟醚对大鼠背根神经节(DRG)细胞电压门控性Na~ 、K~ 、Ca~(2 )离子通道的影响。方法 取SD大鼠胸段和上腰段的DRG,在32～34℃下,用胶原酶、胰蛋白酶孵育60～90 min后获取急性分离的DRG细胞。以-70mV为钳制电压,间隔10mV逐步递增测试电压至0mV,膜片钳全细胞记录DRG细胞的Na~ 、K~ 、Ca~(2 )电流。纯氧驱动七氟醚溶解于培养液,观察不同浓度七氟醚对细胞膜Na~ 、K~ 、Ca~(2 )电流的影响,并记录细胞外液中七氟醚浓度。结果 与用药前比较,七氟醚在0.4、0.9、1.8mmol/L时DRG细胞的Na~ 、K~ 电流差异无显著性(P>0.05),而七氟醚在0.4mmol/L时Ca~(2 )电流明显受抑制(P<0.05)。结论 七氟醚在脊髓水平的抗伤害感受作用可能与其抑制DRG细胞Ca~(2 )电流的作用有关。     

A description of Ca2+ channels in human detrusor smooth muscle

OBJECTIVE: To characterize the Ca2+ channels in human detrusor smooth muscle and to investigate their contribution to spontaneous electrical activity. MATERIALS AND METHODS: Isolated human detrusor smooth muscle myocytes were used to measure ionic currents under voltage-clamp or membrane potential under current-clamp. Membrane potential oscillations were analysed in terms of oscillation frequency and amplitude using fast Fourier transforms. RESULTS: Under voltage-clamp an inward current dependent on extracellular Ca2+ was recorded using Cs+-filled patch electrodes. The current could be separated into two components on the basis of their sensitivity to Ni2+, verapamil or nicardipine, and their dependence on holding and clamp potential. A Ni2+-sensitive component activated over a relatively negative range of potentials (-60 to -20 mV) comprised about a third of the total current and was designated a T-type Ca2+ current. A verapamil/nicardipine-sensitive component, activated at more positive potentials, was designated an l-type Ca2+ current. Using K+-based filling solutions spontaneous transient outward currents were recorded that had the characteristics of current flow through BK channels. Membrane potential oscillations, under current-clamp increased in frequency but not amplitude as the mean membrane potential was made less negative. The voltage-dependence of oscillation frequency was similar to that of the l-type, but not T-type, Ca2+ current activation curve. Furthermore oscillation frequency was slowed by verapamil but not Ni2+. CONCLUSION: The study showed, for the first time, the presence of both T- and L-type Ca2+ channels in human detrusor smooth muscle; we propose a role for these channels in spontaneous activity. The results suggest that the L-type Ca2+ current can control membrane potential oscillation frequency. The significance of this finding for spontaneous contractions is discussed.     

电压门控钠离子通道β3亚基在小鼠神经病理性痛中的作用

目的 评价电压门控钠离子通道β3亚基(Scn3b)在小鼠神经病理性痛中的作用.方法 采用转基因手段,将目的基因Scn3b嵌入pBROAD-mcs载体中,成为pBROAD-rosa-Scn3b质粒,孕育原代小鼠.将原代小鼠和C57/B6小鼠进行配种,得到转基因小鼠.通过DNA、RT-PCR和Western blot实验鉴定过表达成功后,进行后续实验.随机选择Scn3b过表达转基因小鼠(Scn3b组)和同窝阴性对照小鼠(Con组)各10只,2月龄,雌雄不拘,体重25 ～ 30 g,制备神经病理性痛模型.分别于术前、术后3、5、7和14 d时,测定机械痛阈.结果 2组间不同时间点机械痛阈比较差异无统计学意义(P＞0.05).结论 Scn3b不参与小鼠神经病理性痛的形成和维持.     

罗比卡因对豚鼠心室肌细胞L型钙通道的影响

目的观察罗比卡因对豚鼠心室肌细胞L型钙通道的影响,探讨其心肌负性肌力作用的机制。方法以急性酶解法获得豚鼠单个心室肌细胞,用全细胞膜片钳技术记录L型钙电流(ICa-L)。结果罗比卡因浓度依赖性阻滞豚鼠心室肌细胞ICa-L,其半最大抑制浓度(IC50)为(212±38)μmol/L,Hill常数为1.15±0.18。罗比卡因100μmol/L使钙电流峰值从(-4.6±1.7)pA/pF减小至(-2.9±0.9)pA/pF(P<0.01),抑制率为(37±3)%;使电流密度-电压曲线上移,但不改变激活电压、最大激活电压和曲线的形状;激活曲线也没有明显改变;使稳态失活曲线向左移,半失活电压由给药前(-11.0±1.0)mV降为给药后(-15.0±0.6)mV(P<0.05);使ICa-L稳态失活后再恢复的时间常数(τ)明显延长,由给药前(260±17)ms增加至给药后(346±32)ms(P<0.05)。结论罗比卡因作用于L型钙通道的失活态,从而抑制了心室肌细胞ICa-L,是其产生负性肌力作用的原因之一。     

不同浓度褪黑激素对新生大鼠海马锥体神经元电压门控性Ca2+通道的影响

目的 探讨不同浓度褪黑激素对新生大鼠海马锥体神经元电压门控性ca2通道的影响.方法 原代培养新生Wistar大鼠(出生时问<12 h)海马锥体神经元7～12 d.配制褪黑激素溶液,终浓度依次为1 nmol/L、10 nmol/L、100 nmol/L、1 μmol/L、10μmol/L、100μmol/L和1 mmol/L.选择胞体清晰、光晕良好、轴突明显的锥体神经元,采用膜片钳全细胞记录模式观察Ca2+通道的基本电生理特点,记录不同浓度褪黑激素作用下Ca2+电流,分析Ca+2通道动力学特性,并计算Ca2+电流变化率.结果 不同浓度褪黑激素均可快速、可逆性地增强Ca2+电流,10 nmol/L和100 nmol/L褪黑激素使Ca2+电流激活曲线向超极化方向移动,其余浓度褪黑激素对Ca2+电流激活曲线的动力学特性无影响.与100 mol/L褪黑激素比较,其余浓度褪黑激素作用后Ca2+电流变化率降低(P<0.05或0.01);与Iμmol/L褪黑激素比较,10 μmol/L、100μmol/L和1 mmol/L褪黑激素作用后Ca2+电流变化率降低(P<0.05).结论 l nmol/L～1 mmol/L褪黑激素均可增强体外培养的新生大鼠海马锥体神经元Ca2+电流,100 nmol/L褪黑激素作用最强.     

大电导钙激活钾通道与心血管保护

背景 大电导钙激活钾通道(large conductance Ca2+-activated K+ channels,BKCa)可能是各类心脏疾病的重要治疗靶点. 目的 主要阐述如何通过调节心肌及血管平滑肌的BKCa通道而发挥保护心肌作用及其相关机制. 内容 在心肌细胞的线粒体上,BKCa通道能够有效地调节线粒体的活性氧、Ca2+和呼吸作用.在血管平滑肌细胞,BKCa通道能调节血管紧张度,促进血管扩张. 趋向 BKCa通道在心肌缺血/再灌注(ischemia/reperfusion,I/R)损伤中有着重要的作用,包括改善心肌功能和减少梗死面积.     

罗哌卡因对豚鼠心室肌细胞膜离子通道的影响

目的 研究罗哌卡因对豚鼠心室肌细胞膜离子通道的影响，探讨其心脏毒性作用的发生机理。方法 用酶解法获得单个的左心室肌细胞，用标准的全细胞膜片钳技术记录用药前后的钠流（ＩＮａ）、Ｌ－型钙流（ＩＣａ－ｌ）、延迟整流性钾流（ＩＫ）和内向整流性钾流（ＩＫ１）的变化。结果 罗哌卡因对ＩＮａ及ＩＣａ－Ｌ都有抑制作用，且呈浓度依赖性。５０ｕｍｏｌ．Ｌ＾－１罗哌卡因可使ＩＮａ、ＩＣａ－Ｌ分别下降３６．８％、７．６８     

酸敏感离子通道参与伤害性感受的研究

背景 组织酸化是炎症、缺血/缺氧、骨质破坏等多种疼痛条件下的共同病理特征.酸敏感离子通道(acid-sensingion channels,ASICs)是一类兴奋性阳离子通道,表达在神经系统,可直接被细胞外质子激活,介导组织酸化所致的伤害性感受. 目的 以ASICs为疼痛治疗靶标,将为疼痛治疗提供一条新途径. 内容 综述ASICs参与组织酸化所致伤害性感受的相关研究. 趋向 近年来,研究发现ASICs在介导组织酸化所致伤害性感受过程中发挥重要作用,以ASICs为靶点,将为开发新型镇痛药物和疼痛治疗提供新思路.     

利多卡因对大鼠海马神经元L-型Ca2+通道电流的影响

目的 观察不同浓度利多卡因对成年大鼠海马CA1区锥体细胞L-型Ca2 通道电流的影响,以探讨其对中枢神经的作用.方法 采用酶加机械分离的方法,急性分离成年大鼠海马CA1区锥体细胞.运用膜片钳细胞贴附模式技术,记录0、2、4、8、16、32 μg/ml的利多卡因对L-型Ca2 通道电流幅度和电导的影响.结果 不同浓度的利多卡因不影响单通道的单一电流幅度,也不影响L型Ca2 通道的单通道电导.低浓度利多卡因(2、4 μg/ml)对L-型Ca2 通道的整体平均电流无明显影响;浓度为8、16、32 μg/ml时,整体平均电流分别为(0.80±0.09)、(0.67±0.07)、(0.37±0.05)pA(与对照浓度比较,P＜0.05).结论 利多卡因影响成年大鼠海马CA1区锥体细胞L-型Ca2 通道电流,随着浓度的增加表现为先兴奋后抑制.     

离子通道在骨关节炎发病机制中的作用研究进展

由于离子通道参与许多细胞过程,作用于离子通道的药物长期以来被用于治疗许多疾病,特别是那些影响组织细胞电兴奋的疾病。随着离子通道病理学机制的不断研究,离子通道已被确定为潜在的新药靶点,许多针对离子通道的基础药物的治疗价值得到了证实。对药物-离子通道相互作用的实验研究表明,离子通道突变可以增加或减少对药物的亲和力,改变其潜在的治疗效果。与可能影响药物药效的通道基因多态性的发现一样,这些发现强调了离子通道研究的必要性,以便识别对通道亚型或突变体有更具体作用的药物,以提高疗效和减少不良反应。随着对离子通道遗传学、结构和功能的更深入理解,以及对新一级和二级离子通道疾病的识别,用于骨关节炎(osteoarthritis, OA)的离子通道药物的数量将大幅增加。笔者就离子通道在OA发病机制中的作用进行了综述,重点介绍了电压门控钠通道Nav1.7、嘌呤类受体P2X3、瞬时感受器电位受体TRPV1、氯离子(Cl^-)通道。从而进一步指导药物研发,以期为OA的诊疗和机制研究提供参考依据。     

Diabetes-induced alterations in latissimus dorsi muscle properties impair effectiveness of dynamic cardiomyoplasty in rats

Short-term diabetes was induced in male Wistar rats with streptozotocin injection. The effects of diabetes on latissimus dorsi (LD) muscle contractile and biochemical properties and acute cardiomyoplasty (CDM) were assessed and compared with data from 16 control rats. Isometric force, contractile properties, and fatigue were measured in electrically stimulated muscles (0.3 ms, 1-256 Hz), and Na+K+ and Ca2+ATPase activities were quantified in muscle membrane preparations. Systolic arterial pressure and aortic blood flow were recorded at rest and during LD muscle stimulation. Compared with control muscle, diabetic muscle showed smaller maximum specific tetanic tension and lower rates of rise and fall in force. Diabetic LD muscle also showed lower muscle enzyme activities. Twitch tension and fatigue did not differ between groups. Smaller increases in aortic flow and systolic pressure after CDM were found in diabetic rats compared to controls. The marked decrease in CDM effectiveness in diabetic rats likely reflected the alterations in muscle properties associated with diabetes.     

Receptor‐induced phasic activity of newborn mouse bladders is inhibited by protein kinase C and involves T‐type Ca2+ channels

OBJECTIVE

To investigate the mechanisms involved in the phasic contractile activity after muscarinic receptor activation of newborn urinary bladders and to compare neonatal and adult bladders.

MATERIALS AND METHODS

Detrusor muscle strips were isolated from newborn mice (aged 0–2 days) and compared with preparations from adult mice (aged 10–12 weeks). The effects of an activator (phorbol 12,13‐dibutyrate, PDBu) and an inhibitor (GF109203X) of protein kinase C (PKC) on contractions were investigated. T‐type Ca²⁺ channels were blocked with NiCl₂.

RESULTS

The newborn urinary bladders responded with prominent phasic contractile activity in response to carbachol (1 µm ). GF109203X (3 µm ) reduced carbachol‐induced force by ≈60% in the newborn, compared with 30% in the adult. PDBu (1 µm ) enhanced the muscarinic receptor‐mediated contraction in adult bladder muscle, whereas it completely abolished the responses in the newborn. There was no inhibition after activation with depolarization (high‐K⁺) or purinergic agonists (ATP, α,β‐methylene ATP). NiCl₂ (>30 µm ) inhibited the peak responses to carbachol in the newborn and at 300 µm it completely abolished the phasic contractile response. The responses of the adult bladder muscle were only marginally affected by NiCl₂.

CONCLUSIONS

Muscarinic receptor stimulation recruits the PKC signalling pathway in both the adult and neonatal urinary bladder. Potent PKC activation is inhibitory on carbachol‐induced activation in the newborn and stimulatory in the adult bladder. Furthermore, muscarinic receptor stimulation activates T‐type Ca²⁺ channels in the newborn, but not the adult bladder.     

蛋白酪氨酸磷酸酶-3通过诱导肿瘤性巨噬细胞钙离子依赖性钾离子通道表达增强LoVo细胞侵袭性

目的 观察结肠癌肿瘤微环境中肿瘤细胞与巨噬细胞(M2)相互作用及对肿瘤细胞功能学的改变.方法 将转入蛋白酪氨酸磷酸酶-3(PRL-3)的LoVo细胞和M2细胞模拟肿瘤微环境进行共培养,通过Western blot检测M2细胞钙离子依赖性钾离子通道(KCNN4)蛋白表达,并检测LoVo细胞侵袭性的改变.结果 Western blot检测示PRL-3能通过共培养后诱导M2细胞KCNN4蛋白表达升高,而未作共培养的M2细胞KCNN4蛋白表达未升高.此外,经过共培养后LoVo细胞侵袭性升高(3367±135比1442±89,P＜0.05),而在阻断KCNN4蛋白表达后,LoVo细胞侵袭性降低(1388 ±87比2893±163,P＜0.05).结论 PRL-3在结肠癌肿瘤微环境中通过提高KCNN4表达从而增强LoVo细胞的侵袭性.     

不同浓度右美托咪定对大鼠肠系膜动脉平滑肌细胞BKCa通道的影响

目的 评价不同浓度右美托咪定对大鼠肠系膜动脉平滑细胞大电导钙激活钾通道(BKCa)的影响.方法 SD大鼠,雌雄不拘,体重180～220 g,经两步法酶消化分离肠系膜动脉平滑肌细胞.选取10个肠系膜动脉平滑肌细胞,采用单通道内面向外式膜片钳技术进行记录,钳制电压为40 mV,游离钙离子浓度为10-7 mol/L,采用浓度累积法加入右美托咪定,分别于0(基础值)、10-9、10-8、10-7、10-6、10-5 mol/L右美托咪定时记录BKCa开放概率(NPo)、电流幅度(Am)、平均开放时间(To)和平均关闭时间(Tc).结果 与基础值比较,10-7、10-6、10-5 mol/L右美托咪定时NPo升高,且呈浓度依赖性,10-9、10-8、10-7、10-6、10-5 mol/L右美托咪定时Tc缩短(P＜0.05或0.01)；与10-9mol/L右美托咪定比较,10-8mol/L右美托咪定时Tc缩短(P＜0.05),不同浓度右美托咪定时Am和To 差异无统计学意义(P＞0.05).结论 10-7、10-6、10-5 mol/L右美托咪定可呈浓度依赖性地激活大鼠肠系膜动脉平滑肌细胞BKCa通道,是其降压作用的机制之一.     

Voltage-Gated calcium channels and nonvoltage-gated calcium uptake pathways in the rat incisor odontoblast plasma membrane

Odontoblasts participate actively in the transport and accumulation of Ca²⁺ ions to the mineralization front during dentinogenesis. These cells are known to carry membrane-bound ATP-driven pumps and Na⁺/Ca²⁺ antiports for Ca²⁺ extrusion, but little is known about Ca²⁺ influx mechanisms into these cells. It has been shown that the administration of Ca²⁺ channel blockers in vivo strongly impairs Ca²⁺ uptake in the mineral phase during dentinogenesis in the rat; the present in vitro study is aimed at further elucidating odontoblast Ca²⁺ uptake mechanisms. Dissected rat incisor odontoblasts exhibited a pronounced fluorescence when incubated with a fluorescently-labeled (STBodipy) dihydropyridine, which is specific for voltage-gated Ca²⁺ channels of the L-type, and this binding was competitively abolished by nifedipine. As assayed by fluorescence spectrometry, odontoblast Ca²⁺ uptake was enhanced by the agonistic dihydropyridine BAYK-8644 (5 μM) as well as by plasma membrane depolarization in a high K⁺ (120 mM) medium. The Ca²⁺ uptake after depolarization was impaired by nifedipine (5 μM). When treated with the Ca²⁺-ATPase inhibitor cyclopiazonic acid (CPA; 10 μM), a nonvoltage-gated uptake of ⁴⁵Ca²⁺ was identified. This uptake was not influenced by nifedipine (20 μM) but was impaired by lanthanum ions (200 μM). A nonvoltage-gated uptake of Mn²⁺ into CPA-treated cells could be traced using the fura-2 quenching technique. This CPA-induced Ca²⁺ flux was not caused by an alteration of the plasma membrane potential, as assayed with di-8-ANEPPS. The results demonstrate that Ca²⁺ flux into dentinogenically active odontoblasts occurs through voltage-gated Ca²⁺ channels of the L-type and by nonvoltage-gated, agonist-sensitive Ca²⁺ uptake pathways. Received: 6 November 1995 / Accepted: 21 February 1996     

Role of ions and ion channels in capacitation and acrosome reaction of spermatozoa

Capacitation and acrosome reaction are important prerequisites of the fertilization process. Capacitation is a highlycomplex phenomenon occurring in the female genital tract, rendering the spermatozoa capable of binding and fusionwith the oocyte. During capacitation various biochemical and biophysical changes occur in the spermatozoa and thespermatozoal membranes. Ions and ion channels also play important roles in governing the process of capacitation bychanging the fluxes of different ions which in turn controls various characteristics of capacitated spermatozoa. Alongwith the mobilization of ions the generation of free radicals and efflux of cholesterol also plays an impo~.nt role in thecapacitation state of the spermatozoa. The generation of free radical and efflux of cholesterol change the mechano-dynamic properties of the membrane by oxidation of the polyunsaturated lipids and by generating the cholesterol freepatches. The process of capacitation renders the spermatozoa responsive to the inducers of the acrosome reaction. Theglycoprotein zona pellucida 3 (ZP3) of the egg coat zona pellucida is the potent physiological stimulator of the acro-some reaction; progesterone, a major component of the follicular fluid, is also an inducer of the acrosome reaction.The inducers of the acrosome reaction cause the activation of the various ion-channels leading to high influxes of calci-um, sodium and bicarbonate. The efflux of cholesterol during the process of capacitation alters the permeability of themembrane to the ions and generate areas which are prone to fusion and ve.siculation process during the acrosome reactioa. this review focuses mainly on effects of the ion and ion-channels, free radicals, and membrane fluidity changesduring the process of capacitation and acrosome reaction.     

The influence of changes in extracellular and intracellular sodium concentration on detrusor contractility

OBJECTIVE: To determine the role of Na+-Ca2+ exchange in the regulation of isolated detrusor smooth muscle contractility. MATERIALS AND METHODS: Isolated guinea-pig detrusor strips were used to record isometric tension generated by; (a) electrical-field stimulation to elicit nerve-mediated responses; and (b) adding carbachol or superfusing with a high-K+ solution. The [Na+] gradient between extracellular and intracellular compartments was altered by: (i) reducing superfusate [Na+] in stages from 140.2 to 10.2 mm; (ii) addition of the cardiac glycoside strophanthidin (200 microm). RESULTS Reducing extracellular [Na+] reversibly reduced the magnitude of nerve-mediated contractions but increased the resting tension and magnitude of carbachol-induced contracture. The mean (sd) [Na+] required for a half-maximum effect on attenuating contractions, at 85.9 (6.2) mm, and developing contracture, at 59.1 (14.3) mm, were significantly different. The time constants of changes to nerve-mediated contractions and carbachol contracture were also significantly different, at 147 (5) vs 1207 (386) s, respectively. These differences suggest that separate mechanisms influence nerve-mediated contraction and contracture in low-Na+ solutions. Exposure to the cardiac glycoside strophanthidin produced a similar effect to low-Na+ solutions for carbachol contracture. Low-Na+ solutions had no significant effect on contractures induced by high extracellular [K+]. CONCLUSION Reducing the transmembrane [Na+] difference increases intracellular [Ca2+]. This increase is largely accommodated in intracellular stores, that can be released by exogenous carbachol. The results are consistent with the presence of a functional Na+-Ca2+ exchanger in the surface membrane. The lack of effect of low-Na+ solutions on contractures evoked by membrane depolarization is consistent with this conclusion. The reduction of the nerve-mediated contraction by low-Na+ solution might result from blockade of the nerve action potential.     

L-type calcium current of isolated rat cardiac myocytes in experimental uraemia.

BACKGROUND: End-stage renal failure is associated with a low-output cardiomyopathy, left ventricular hypertrophy and increased QTc dispersion. Cardiac dysfunction is prevalent in patients at the beginning of dialysis and is an important predictor of mortality. Ca(2+) influx through voltage-gated L-type Ca(2+) channels plays a key role in the excitation-contraction coupling of cardiac myocytes. The purpose of this study was to examine the effect of subtotal nephrectomy (SNx) in the rat on both cardiac L-type Ca(2+) currents and action potential duration. METHODS: Wistar rats underwent two-stage SNx; control rats (C) underwent bilateral renal decapsulation. Animals were sacrificed after 8 weeks, and ventricular myocytes were isolated. SNx rats showed a 2-fold increase in plasma urea and creatinine compared with C rats. Whole-cell patch clamp techniques were used to examine L-type Ca(2+) channel currents in isolated cardiac myocytes at 37 degrees C. In separate experiments, the epicardial monophasic action potentials of isolated perfused whole hearts from C and SNx rats were recorded. RESULTS: The amplitude and current-voltage relationships of the L-type Ca(2+) current were not significantly different in myocytes from C (n=11) and SNx (n=8) rats. However, the rate of inactivation of the Ca(2+) current was increased by approximately 15-25% (P<0. 05) in myocytes from SNx rats. The action potential duration (APD(33)) at the apex of the left ventricle was approximately 20% shorter (P<0.01) in hearts from SNx rats as compared with controls. CONCLUSIONS: Renal failure is associated with rapid inactivation of cardiac ventricular myocyte L-type Ca(2+) currents, which may reduce Ca(2+) influx and contribute to shortening of the action potential duration.     

严重烧伤后早期大鼠肝脏细胞内外钠离子分布和化学状态的变化

目的观察大鼠严重烧伤后肝脏细胞内、外Na 在分布和化学状态上的改变,为烧伤后早期液体复苏方案的选择提供理论指导。方法选取成年雄性SD大鼠19只,随机分为对照组(12只)、烧伤组(7只),采用钠-23磁共振(23NaNMR)波谱技术和位移试剂,测定两组大鼠肝脏细胞内、外Na 的纵向弛豫时间(T1)和横向弛豫时间(T2)的变化。结果输注位移试剂后,烧伤组大鼠肝脏细胞外Na 浓度降低17%,其快T2所占百分比较对照组有所增加(P<0.01),提示细胞外可与Na 结合或可影响Na 的位点增加;细胞内Na 浓度升高了59%,但其弛豫行为却未发生变化。结论烧伤后早期细胞外的Na 可因向细胞内流失或受其周围大分子可逆性结合位点的影响,使瞬时可发挥渗透粒子作用的Na 相对不足,提示烧伤后第1个24h选择适量高钠溶液复苏较合理。