奥拉西坦对大鼠下丘脑神经元钠钾通道电流的作用

目的 研究在奥拉西坦作用下大鼠下丘脑神经元钠钾通道电流的变化。方法 应用膜片钳技术的全细胞方法记录奥拉西坦作用下大鼠下丘脑神经元钠钾通道电流。结果 在奥拉西坦作用下，下丘脑神经元平均Na^＋电流由（4．130±0．210）hA上升为（5．950±0．411）nA，平均K＋电流由（5．601±0．221）nA增加为（7．550±0．403）nA，而且作用前后比较有统计学差异。结论 在奥拉西坦的作用下，下丘脑神经元Na^＋，K^＋电流均增大，从而增加了神经元细胞动作电位的幅值及传导速度，使动作电位的传导更加容易。     

依卡精对大鼠皮层神经元钠通道的影响

目的:研究依卡精对体外培养大鼠皮层神经元钠电流的影响。方法:采用全细胞膜片钳技术,首先在大鼠皮层神经元诱导出钠电流,然后观察依卡精对钠电流的影响。结果:依卡精可以浓度依赖的方式可逆地抑制皮层神经元钠电流,抑制的半有效浓度为125μM。依卡精不改变钠通道的激活动力学特征。结论:依卡精能够抑制皮层神经元钠通道电流,并呈浓度依赖性。     

Effect of verapamil on tachycardia-induced early cellular electrical remodeling in rabbit atrium

We investigated the effects of a 7-day verapamil pretreatment (VPT, 7.5 mg/kg bodyweight subcutaneously every 12 h) on ionic currents and molecular mechanisms underlying tachycardia-induced early electrical remodeling after 24-h rapid atrial pacing (RAP, 600 bpm) in rabbit atrium. Animals were divided into four groups (n = 6 each group): control (not paced, no verapamil), paced only, verapamil only and verapamil and paced, respectively. VPT doubled ICa,L [7.0 ± 0.7 pA/pF (control) vs 14.2 ± 0.6 pA/pF (verapamil only)]. RAP reduced ICa,L by 48% to 3.6 ± 0.7 pA/pF (paced only). RAP did not affect ICa,L in verapamil-treated animals and averaged 15.3 ± 0.2 pA/pF (paced and verapamil). RAP resulted in a significant decrease of the expression of the α1c subunit (−24.7%) and the β2A subunit (−13.3%), respectively. VPT led to a similar alteration of subunit expression as RAP [“control” vs “verapamil only”, decrease of α1c subunit (−25.4%), but no significant change in β2A subunit expression]. However, after VPT, further diminishment of α1c and β2A subunit expression after rapid atrial pacing was absent. (“verapamil” vs “verapamil and paced”, n = 6 both groups). RAP decreased Ito [−45%, 51.5 ± 3.9 pA/pF (control) vs 26.8 ± 1.5 pA/pF (paced only)] and was not influenceable by VPT. IK1 was neither affected by RAP nor verapamil pretreatment. Downregulation of α1c and β2A subunit expression and the resulting decay of ICa,L current densities were prevented by verapamil. However, these effects are abolished by multiple other adverse effects of verapamil on atrial electrophysiology.     

The Venom of the Spider Selenocosmia Jiafu Contains Various Neurotoxins Acting on Voltage-Gated Ion Channels in Rat Dorsal Root Ganglion Neurons

Selenocosmia jiafu is a medium-sized theraphosid spider and an attractive source of venom, because it can be bred in captivity and it produces large amounts of venom. We performed reversed-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analyses and showed that S. jiafu venom contains hundreds of peptides with a predominant mass of 3000–4500 Da. Patch clamp analyses indicated that the venom could inhibit voltage-gated Na⁺, K⁺ and Ca²⁺ channels in rat dorsal root ganglion (DRG) neurons. The venom exhibited inhibitory effects on tetrodotoxin-resistant (TTX-R) Na⁺ currents and T-type Ca²⁺ currents, suggesting the presence of antagonists to both channel types and providing a valuable tool for the investigation of these channels and for drug development. Intra-abdominal injection of the venom had severe toxic effects on cockroaches and caused death at higher concentrations. The LD₅₀ was 84.24 μg/g of body weight in the cockroach. However, no visible symptoms or behavioral changes were detected after intraperitoneal injection of the venom into mice even at doses up to 10 mg/kg body weight. Our results provide a basis for further case-by-case investigations of peptide toxins from this venom.     

钩藤碱和异钩藤碱对~(45)Ca转运的影响

在离体家兔主动脉条.钩藤碱和异钩藤碱明显减少KCl(77.0 mmol·L~(-1))溶液所致45Ca内流量、不影响去甲肾上腺素所引起的45Ca内流和外溢。结果表明.钩藤碱和异钩藤碱对电位依赖性钙通道具有阻滞作用。     

体外培育牛黄抑制大鼠三叉神经节细胞电压依赖性钙通道电流参与镇痛机制

目的:研究体外培育牛黄(CBS)对大鼠三叉神经节(TRG)细胞电压依赖性钙通道电流(ICa)的影响,探讨牛黄镇痛作用的电生理机制。方法:在培养大鼠TRG细胞上采用全细胞膜片钳记录ICa。结果:CBS能够剂量依赖性地抑制TRG细胞电压依赖性钙通道电流,0.2,2,20μg.ml-1CBS可使钙电流幅值分别减少(30.3±4.7)%、(41.9±3.6)%和(56.7±6.8)%(n=6,P<0.01)。结论:CBS对钙电流的阻滞作用可能是其镇痛作用机制之一。     

钩藤碱对大鼠苯丙胺条件性位置偏爱效应的影响

目的:观察钩藤碱对大鼠苯丙胺条件性位置偏爱效应的影响及钩藤碱的奖赏效应或厌恶效应.方法:连续给予苯丙胺(2 mg/kg,sc,每日1次)4 d,引起大鼠产生显著的条件性位置偏爱效应,建立苯丙胺位置偏爱模型.钩藤碱低、中、高剂量(10、20、60 mg/kg) 苯丙胺组在训练阶段第2天sc苯丙胺12 h后分别按剂量ip给予钩藤碱,氯胺酮 苯丙胺组在训练阶段第3天sc苯丙胺之前15 min给予氯胺酮(15 mg/kg,ip),均连续3 d.钩藤碱 生理盐水组连续给予钩藤碱(60 mg/kg,ip,每日1次)4 d.结果:钩藤碱各剂量组和阳性药氯胺酮组大鼠在伴药箱的逗留时间均明显缩短,钩藤碱三剂量组随剂量的增加其作用相应增强.钩藤碱 生理盐水组大鼠不形成条件性位置偏爱.结论:预先给予钩藤碱能在一定程度上消除苯丙胺诱导的条件性位置偏爱效应,而钩藤碱本身未显示精神依赖性潜力.     

Inhibition of voltage-gated calcium currents in type II vestibular hair cells by cinnarizine

Cinnarizine is pharmaceutically used in conditions with vestibular vertigo such as Menieres disease. It is thought to act on extra-vestibular targets. We hypothesized that cinnarizine, as a blocker of L-type Ca²⁺ channels, may directly target vestibular hair cells where Ca²⁺ currents are important for the mechano-electrical transduction and transmitter release. Our aim was to clarify whether cinnarizine affected voltage-dependent Ca²⁺ currents in vestibular type II hair cells. Such cells were isolated from inner ears of guinea pigs by enzymatic and mechanical dissection from the gelatinous otolithic membrane and studied with the patch-clamp technique in conventional whole-cell mode. Ca²⁺ currents were elicited by depolarizing pulses in a solution containing 1.8 mM Ca²⁺ and 40 mM Ba²⁺. These currents resembled L-type currents (I_Ca,L) with respect to their voltage-dependence and their inhibition by nifedipine and Cd²⁺ but did not show time-dependent inactivation. The currents were inhibited by cinnarizine in a concentration-dependent and reversible manner. The IC₅₀ was 1.5 M. A block exceeding 80% was achieved with 10 µM. The onset of current block was faster with higher concentrations but the reversibility after wash-out was less, suggesting accumulation in the membrane. We conclude that these direct actions of cinnarizine on hair cells should be considered as molecular mechanisms contributing to therapeutic effects of cinnarizine in vertigo.The first two authors contributed equally to the work     

川芎嗪对大鼠大脑皮层神经细胞的体外存活及其钙超载的影响

目的:研究川芎嗪对体外培养的大鼠大脑皮层神经细胞的保护作用及其钙超载的影响。方法:取出生1d内的乳鼠大脑皮层制成细胞悬液,接种于经多聚赖氨酸包被的培养板。于培养6d后,加入不同浓度的川芎嗪,继续培养1d,采用MTT比色法测量其存活细胞的吸收值,同时行NSE检查。采用激光扫描共聚焦显微镜(LSCM)技术检测药物对谷氨酸(500μmol·L-1)、KCl(50mmol·L-1)诱导培养6d的大脑皮层神经细胞内钙超载的影响,计算荧光强度变化率。结果:NSE检查结果表明,培养6d的存活细胞大部分均为神经元。MTT比色检查结果表明,川芎嗪浓度为1.6～200.0μg·mL-1时,存活细胞数均明显增加(P<0.05)。LSCM测定结果表明,100μg·mL-1川芎嗪能明显降低荧光强度变化率(P<0.01)。结论:川芎嗪能促进体外培养的大脑皮层神经细胞存活,且可抑制谷氨酸、KCl诱导的神经细胞内钙超载,这可能是其治疗缺血性脑血管病的作用机制。     

维司力农结构改造物FPA对豚鼠心肌细胞钙电流的影响

目的用膜片钳全细胞记录法观察FPA对分离的单个豚鼠心室肌细胞L型钙电流(Ica-L)的影响。方法采用急性酶分离法(胶原酶 白蛋白)分离单个豚鼠心室肌细胞,在生物倒置显微镜下对豚鼠心室肌细胞进行膜片钳全细胞记录。实验采用空白试验组,加药组(FPA组),二甲亚砜组进行对造,在实验中FPA以三种浓度给药(0.75,1.0和1.25 mmol.L-1)。实验中保持电位为-40 mV,刺激频率为0.1 Hz,刺激时间200 ms,以10 mV为一阶跃逐级去极化到 60 mV,通道电流经膜片钳负反馈放大器放大,再用pClamp 7.0软件的数据采集系统采样和最后分析。结果FPA增加Ica-L。0.75 mmol.L-1FPA使Ica-L最大峰值电流从(4.1181±1.404)pA/pF增至(4.907±1.208)pA/pF(n=6,P均<0.05),增加率为19.16%;1.0 mmol.L-1FPA使Ica-L最大峰值电流从(4.519±1.106)pA/pF增至(7.483±2.154)pA/pF(n=6,P均<0.05),增加率为65.58%;1.25 mmol.L-1FPA使Ica-L最大峰值电流从(3.288±1.038)pA/pF增至(8.345±0.172)pA/pF(n=4,P均<0.05),增加率为153.80%。FPA使Ica-L的电流-电压曲线下移,但不改变其激活、峰值和反转电位,亦不改变细胞的静息电位。结论FPA对Ica-L的促进作用为其强心效应的离子基础之一。     

急性缺氧犬氰化钠中毒后心脏功能的变化

目的研究氰化钠（NaCN）中毒对急性缺氧犬心脏功能的影响。方法将18只四川杂种家犬随机均分为常氧对照组和缺氧实验组。动物麻醉后行气管和左心室插管,常氧对照组吸入空气,缺氧实验组吸入氧-氮混合气体。术后1 h背部皮下注射NaCN3.6 mg/kg,记录中毒前即刻（0 min）及中毒后10、20、30、50 min各时间点心率、左心室收缩压峰值（LVSP）、左心室收缩压最大上升速率（＋dp/dtmax）,取静脉血测定乳酸脱氢酶（LDH）、肌酸激酶（CK）及肌酸激酶同工酶（CK-MB）的含量。结果常氧对照组NaCN中毒后,心率减慢、＋dp/dtmax下降,LVSP和3种心肌酶含量升高。缺氧实验组上述指标变化趋势同常氧对照组,但变化时间提前、变化率增加。结论急性缺氧加重氰化物中毒对心脏功能的影响。     

甘草次酸对大鼠心室肌细胞L型钙离子电流的影响

目的观察甘草次酸（glycyrrhetic acid ，Gta）对大鼠单一心室肌细胞L型钙离子电流（ICa L）的影响。方法以Langendoff匀速灌流体外大鼠心脏，Ⅰ型胶原酶酶解分离单一大鼠心室肌细胞， 将心室肌细胞悬液放入容积为0.2～0.3 mL的浴槽内，间隔相同时间分别向浴槽内加入0.1，1.0，10.0 μmol·L^-1 Gta，应用全细胞膜片钳技术记录不同浓度Gta对单一大鼠心室肌细胞ICa-L的影响；保持电位为－30 mV、指令电位为－40～＋60 mV、步阶脉冲为10 mV、时程为300 ms、刺激频率为0.5 Hz的条件下，引出钙离子电流，取指令电位与相对应的钙离子通道电流作用结果制作不同浓度Gta下L型钙离子通道的电流-电压(I-V)关系曲线。结果Gta浓度分别为0.1，1.0， 10.0 μmo1·L^-1 时可剂量依赖性地降低ICa-L，分别使ICa-L从加入Gta前的（2.30±0.29） nA降至(1.96±0.34) ，(1.37±0.24) ，（0.66±0.20） nA（与加入Gta前比较，均P＜0.01）；Gta 0.1，1.0， 10.0 μmo1·L^-1 也可抑制L型钙离子I-V曲线，使I V曲线上移，但峰值电流不变。结论Gta通过阻滞L型钙通道抑制L型钙离子内流，这种机制可能与Gta抗心律失常作用有关。     

Methanandamide activation of a novel current in mouse trigeminal ganglion sensory neurons in vitro

Anandamide is an endogenous agonist for cannabinoid receptors and produces analgesia by acting at these receptors in several sites in the brain and peripheral nervous system. Anandamide is also an agonist at the TRPV1 receptor, a protein that serves as an important integrator of noxious stimuli in sensory neurons. Although anandamide actions at CB1 and TRPV1 receptors can explain many of its effects on sensory neurons, some apparently CB1- and TRPV1-independent effects of anandamide have been reported. To explore possible mechanisms underlying these effects we examined the actions of the stable anandamide analog methanandamide on the membrane properties of trigeminal ganglion neurons from mice with TRPV1 deleted. We found that methanandamide and anandamide activate a novel current in a subpopulation of small trigeminal ganglion neurons. Methanandamide activated the current (EC(50) 2 microM) more potently than it activates TRPV1 under the same conditions. The methanandamide-activated current reverses at 0 mV and does not inactivate at positive potentials but declines rapidly at negative membrane potentials. Activation of the current is not mediated via cannabinoid receptors and does not appear to involve G proteins. The phytocannabinoid Delta(9)-tetrahydrocannabinol, the endocannabinoid-related molecules N-arachidonoyl dopamine and N-arachidonoyl glycine and the non-specific TRPV channel activator 2-aminoethoxydiphenyl borate do not mimic the effects of methanandamide. The molecular identity of the current remains to be established, but we have identified a potential new effector for endocannabinoids in sensory neurons, and activation of this current may underlie some of the previously reported CB1 and TRPV1-independent effects of these compounds.     

Effects of cinnarizine on calcium and pressure-dependent potassium currents in guinea pig vestibular hair cells

In vestibular hair cells, K⁺ currents induced by rises in hydrostatic pressure have recently been demonstrated. These currents are inhibited by charybdotoxin, a blocker of Ca²⁺-dependent K⁺ channels. On the other hand, cinnarizine is a blocker of voltage-gated Ca²⁺ currents in hair cells and is used as a drug in conditions with vestibular vertigo. Our aim was to test in patch-clamp experiments (conventional whole-cell mode) whether cinnarizine, by reducing Ca²⁺ influx, inhibited Ca²⁺ and pressure-sensitive K⁺ currents in vestibular type-II hair cells of guinea pigs. A quantitatively similar inhibition of K⁺ currents was evoked by extracellular Ca²⁺ removal, cinnarizine (0.5 M), and the L-type Ca²⁺ channel blocker nifedipine (3 M). Cinnarizine abrogated increases of K⁺ currents induced by increases in the hydrostatic pressure (from 0.2 to 0.5 cm H₂O). At a higher concentration (1 M), cinnarizine elicited K⁺ current inhibitions larger than those elicited by Ca²⁺ removal. Moreover, it reduced K⁺ currents in the absence of Ca²⁺, in contrast to nifedipine. However, charybdotoxin abolished these effects of cinnarizine. We thus conclude that cinnarizine inhibits, by two mechanisms, pressure-induced currents that are sensitive to charybdotoxin and Ca²⁺. It reduces Ca²⁺ influx and exerts a Ca²⁺-independent inhibition, with a lower IC₅₀ than that required for Ca²⁺ channel blockade. These two actions may importantly contribute to its therapeutic effects.P. Düwel and T. Haasler contributed equally to this work.     

Nitric Oxide Modulation of GABAergic Synaptic Transmission in Mechanically Isolated Rat Auditory Cortical Neurons

The auditory cortex (A1) encodes the acquired significance of sound for the perception and interpretation of sound. Nitric oxide (NO) is a gas molecule with free radical properties that functions as a transmitter molecule and can alter neural activity without direct synaptic connections. We used whole-cell recordings under voltage clamp to investigate the effect of NO on spontaneous GABAergic synaptic transmission in mechanically isolated rat auditory cortical neurons preserving functional presynaptic nerve terminals. GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) in the A1 were completely blocked by bicuculline. The NO donor, S-nitroso-N-acetylpenicillamine (SNAP), reduced the GABAergic sIPSC frequency without affecting the mean current amplitude. The SNAP-induced inhibition of sIPSC frequency was mimicked by 8-bromoguanosine cyclic 3'',5''-monophosphate, a membrane permeable cyclic-GMP analogue, and blocked by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a specific NO scavenger. Blockade of presynaptic K⁺ channels by 4-aminopyridine, a K⁺ channel blocker, increased the frequencies of GABAergic sIPSCs, but did not affect the inhibitory effects of SNAP. However, blocking of presynaptic Ca²⁺ channels by Cd²⁺, a general voltage-dependent Ca²⁺ channel blocker, decreased the frequencies of GABAergic sIPSCs, and blocked SNAP-induced reduction of sIPSC frequency. These findings suggest that NO inhibits spontaneous GABA release by activation of cGMP-dependent signaling and inhibition of presynaptic Ca²⁺ channels in the presynaptic nerve terminals of A1 neurons.     

Lowering Intracellular Calcium Concentration May Reduce the Cytotoxicity of Triamcinolone on Human Retinal Pigment Epithelial (ARPE19) Cells

1. The present study investigated the use of drugs that affect calcium (Ca^{2 +}) levels and thus reduction of triamcinolone (TA)‐induced cytotoxicity on human retinal epithelial (ARPE19) cells. 2. Four groups were compared: ARPE19 cells alone, cells exposed to TA (0.1 mg/mL), cells that have been pretreated with one of the testing agents for 30 min before the addition of TA, and cells that have only been treated with one of the testing agents. Pinacidil (PIN) and its analogue, P1060, were used to test the effect of potassium (K⁺) channel opening on TA‐induced toxicity. Verapamil (VP) and diltiazem (DZ) were used to test their Ca^{2 +} channel blocking effect. The cell viability under different settings was assessed using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Ca^{2 +}‐imaging was used to determine the changes in intracellular Ca^{2 +} levels [(Ca^{2 +})_i] upon different treatments. 3. Both PIN and P1060 reduced TA‐induced toxicity. Verapamil and DZ increased the viability of cells treated with TA significantly, suggesting that the excessive influx of Ca^{2 +} was one of the main contributory factors to the TA‐induced toxicity. 4. The results suggest that the prevention of Ca^{2 +} entry may be effective in the reduction of cell necrosis in the presence of TA.     

Effects of potassium channel openers on single potassium channels in mouse skeletal muscle

Summary The patch-clamp technique was used to study the effects of the potassium channel openers cromakalim, pinacidil, RP 49356 and diazoxide on single potassium channels in mouse skeletal muscle.In excised patches in the inside-out configuration, one type of potassium channel, the ATP-sensitive potassium channel, could be activated by internally applied RP 49356 even in the absence of internal ATP. At a concentration of 0.4 and 0.8 mmol/l, RP 49356 increased the open-probability of the channels by a factor of 2.7 and 17.4 respectively. The stimulating effect of cromakalim (0.2–0.8 mmol/l) and pinacidil (0.4 mmol/l) depended on the presence of ATP (0.1 mmol/l) at the cytoplasmic side of the patch membrane. The two drugs were able to restore the open-probability of the channels blocked by internal ATP (0.1 mmol/l) to 50–90% of its value in ATP-free solution. No channel reactivation could be observed at a higher ATP concentration (1 mmol/l). Diazoxide (0.4 mmol/l) had almost no effect. None of these channel openers could stimulate the other prominent type of potassium channel in skeletal muscle, the large-conductance Ca²⁺-activated potassium channel.The results show that cromakalim, pinacidil and RP 49356 are specific openers of ATP-sensitive potassium channels in skeletal muscle. It is suggested that the drugs displace the channel blocker ATP and that RP 49356 in addition recruits inactive channels. Send offprint requests to B. Neumcke at the above address     

克拉霉素对糖氧剥离大鼠皮质神经细胞的保护作用及其抑制凋亡的机制研究

目的：研究克拉霉素对大鼠皮质神经细胞糖氧剥离（OGD）后的保护作用及其与抑制凋亡相关的作用机制。方法：提取培养原代大鼠皮质神经细胞,建立皮质神经细胞糖氧剥离模型,OGD前予以克拉霉素（CAM）预处理;通过存活/凋亡检测试剂盒于荧光显微镜下观察各组细胞凋亡情况;LDH试剂盒测定各组培养基LDH含量定量评估细胞凋亡情况;蛋白免疫印迹法测定各组细胞Caspase-3,Bax,Bcl-2的表达情况。结果：OGD处理后皮质神经细胞凋亡及乳酸释放含量较正常组明显增加,差异具有显著统计学意义（76.51±14.92% VS 15.37±3.91%,p＜0.01）,100μM CAM预处理可显著减少OGD造成的细胞凋亡及LDH释放（21.64±7.16% VS 76.51±14.92%,p＜0.01）;OGD处理后皮质神经细胞Caspase-3,Bax/Bcl-2表达显著升高,与Control组相比,均具有显著统计学意义（p＜0.01）,而CAM预处理有效降低了Caspase-3,Bax/Bcl-2表达。结论：克拉霉素对糖氧剥离大鼠皮质神经细胞具有保护作用,其作用可能与促进了Caspase-3,Bax上调,Bcl-2蛋白下调的凋亡相关通路有关。