Inhibition of ω-conotoxin-sensitive Ca2+ channel currents by internal Mg2+ in cultured rat cerebellar granule neurones

The effects of changing the intracellular concentrations of either free Mg²⁺ ions ([Mg²⁺]_i) or Mg²⁺-bound adenosine triphosphate ([Mg · ATP]_i) on Ca²⁺ channel currents were assessed in cultured rat cerebellar granule neurones using the whole-cell patch-clamp technique. Raising [Mg²⁺]_i from 0.06 mM to 1.0 mM inhibited Ca²⁺ channel currents by approximately 50%. The action of -conotoxin GVIA (-CgTX), a selective inhibitor of N-type Ca²⁺ channels was also investigated. With increasing [Mg²⁺]_i, the proportion of current irreversibly blocked by -CgTX was reduced, and was negligible (approximately 5 pA of current) in the presence of [Mg²⁺]_i values of 0.5 mM or greater. Block of the -CgTX-sensitive current accounted for the reduction in total current by concentrations of [Mg²⁺]_i to 0.5 mM. Raising [Mg²⁺]_i had no effect on the rate of decay of Ca²⁺ currents, but did produce a negative shift in current activation, possibly due to a non-specific interaction with negative surface charge. Altering [Mg · ATP]_i from 0.3 to 5.0 mM caused a twofold increase in the size of currents without affecting the proportion of current sensitive to -CgTX. [Mg²⁺]_i was also effective in inhibiting the Ca²⁺ channel current following potentiation by increasing [Mg · ATP]_i. These data suggest that -CgTX-sensitive current in these cells is selectively inhibited by internal Mg²⁺ whereas both -CgTX-sensitive and -resistant components of current are potentiated by internal Mg · ATP. The mechanism by which Mg²⁺ inhibits N-type channels is unclear, but may involve an open channel block.     

Effects of acetyl-l-carnitine on the survival of adult rat sensory neurons in primary cultures

Acetyl-L-carnitine produces a significant increase in the survival time-course of adult rat sensory neurons maintained in primary cultures up to 40 days. The analysis of our data suggests that 200 microM acetyl-L-carnitine added to the medium, slows down neuronal decay especially in the first 10 days in vitro, sparing a fraction of cells which would otherwise be lost. Patch-clamp recordings from these neurons show that superfusion with acetyl-L-carnitine (100-1000 microM) does not induce any membrane current. In addition an agonist muscarinic effect particularly concerning high-voltage activated calcium channel modulation appears to be ruled out. In conclusion our data favour the role of acetyl-L-carnitine in the trophism of sensory neurons in adult rats. In agreement with other in vivo experiments our data reinforce the hypothesis that this substance might be involved in reducing neuronal loss observed in nervous system aging.     

A voltage-dependent calcium current in mouse Swiss 3T3 fibroblasts

Patch-clamp experimens in the whole-cell mode have been performed in Swiss 3T3 mouse fibroblasts. Depolarizations from negative holding potential (V _h<–60mV) gave rise to a rapidly activating, fully inactivating, inward current of few tenths of nA in physiological saline at 35°C. The current persisted when external Na⁺ was replaced by impermeant TMA⁺ and disappeared in O Ca²⁺, 1 mM EGTA. The current was reversible blocked by Co²⁺ and it was slightly reduced when external Ca²⁺ was substituted by Ba²⁺. Finally its reversal potential changed with Nernstian slope with increasing external Ca²⁺ concentrations. It is concluded that these cells possess a voltagedependent Ca²⁺ channel.     

茶多酚对豚鼠心肌细胞钙电流及动作电位的影响

 目的 观察茶多酚(tea polyphenols,TP)对豚鼠心肌细胞L型钙通道电流(I_Ca-L )及动作电位的影响。方法 采用常规微电极和全细胞膜片钳技术记录豚鼠心肌细胞I_Ca-L 和动作电位。结果 ①不同浓度的TP均能缩短复极50%和90%水平的动作电位时程 (APD₅₀,APD₉₀)。②不同浓度的TP明显抑制I_Ca-L, 5,10,15 mg·L-1的TP使I_Ca-L 的峰电流密度从(12.84±1.36) pA/pF减少到(10.24±1.47), (8.84±1.39),(6.65±0.56) pA/pF (n=6,P<0.01 )。结论 TP通过抑制心肌细胞钙内流使动作电位位时程缩短。     

慢性孵育β-淀粉样肽(25-35)对培养大鼠海马神经元外向钾电流的影响

目的　研究慢性孵育β-淀粉样肽_(25-35) (β-AP_25-35)对海马神经元上瞬时外向钾电流(I_A)和延迟整流钾电流(I_K)的影响。方法　在培养的大鼠海马神经元上用膜片钳全细胞记录钾通道电流。结果　β-AP_25-35 3μmol·L^-1 孵育细胞24h ,I_K 电流幅度增加(44.3±5.4)% ,电流密度由(30.4±6.4)pA·PF^-1 增加至(43.8±4.7)pA·PF^-1 ;β-AP_25-3510μmol·L^-1 孵育12h ,I_K 电流幅度增加(69.8±4.1) % ,电流密度增加至(51.6±7.9)pA·PF^-1,呈浓度依赖性;β-AP_25-35引起的I_K 增加对TEA 5mmol·L^-1 敏感;β-AP_25-35上调I_K 的作用主要发生在β-AP_25-355用药后48h内。β-AP_25-35对I_A无显著性影响。结论　β-AP_25-35选择性地增加海马神经元上I_K,这一作用可能与β-AP的神经毒性有关     

神经元N受体功能失敏对M受体拮抗剂药理作用的影响

目的研究神经元N受体失敏对宾赛克嗪和阿托品拮抗M受体功能药理作用的影响。方法选用原代培养的大鼠颈上交感神经元为模型,以烟碱和氧化震颤素为工具药,选取拮抗剂对M受体介导的IM-抑制的拮抗作用为指标,采用膜片钳全细胞记录技术进行研究。结果与正常状态相比,在N受体失敏状态下,宾赛克嗪和阿托品对M受体功能的拮抗作用强度均减弱,且宾赛克嗪减弱的幅度小于阿托品。随着N受体逐渐从失敏状态恢复到正常状态,宾赛克嗪和阿托品对M受体功能的拮抗作用强度也呈逐渐恢复的趋势。结论 N受体失敏使M受体拮抗剂对M受体功能的拮抗作用强度减弱。     

TTX-sensitive Na+ channels and Ca2+ channels of the Land N-type underlie the inward current in acutely dispersed coeliac-mesenteric ganglia neurons of adult rats

Inward membrane currents of sympathetic neurons acutely dispersed from coeliac-superior mesenteric ganglia (C-SMG) of adult rats were characterized using the whole-cell variant of the patch-clamp technique. Current-clamp studies indicated that C-SMG neurons retained electrical properties similar to intact ganglia. Voltage-clamp studies designed to isolate Na⁺ currents revealed that tetrodotoxin (TTX, 1 M) completely inhibited the large transient inward current. Half activation potential (V _h) and slope factor (K) were –26.8 mV and 6.1 mV, respectively. Inactivation parameters for V _h and K were –65 mV and 8.2 mV, respectively. Voltage-clamp studies also revealed a high-voltage-activated sustained inward Ca²⁺ current which was blocked by the removal of external Ca²⁺ or the presence of Cd²⁺ (0.1 mM). The dihydropyridine agonist, (+)202–791 (1 M), caused a small increase (20%) in the amplitude of the Ca²⁺ current at more negative potentials and markedly prolonged the tail currents. -Conotoxin GIVA (, CgTX, 15 M) caused a 66% inhibition of the high-voltage-activated Ca²⁺ current amplitude. Norepinephrine (1 M) caused a 49% reduction in the peak Ca²⁺ current. This study is the first demonstration that dispersed C-SMG neurons from adult rats retain electrical characteristics similar to intact ganglia. A TTX-sensitive Na⁺ current as well as a high voltage-activated sustained Ca²⁺ current underlie the inward current in C-SMG neurons. The macroscopic Ca²⁺ current is composed of a small dihydropyridinesensitive (L-type current) and a large -CgTx-sensitive (N-type current) component. Thus, acutely dispersed CSMG neurons are suitable for examining the biophysical properties and modulation of membrane currents of adult prevertebral sympathetic neurons in normal and diseased states.     

笑气对大鼠杏仁中央核神经元自发放电活动的影响

目的　研究吸入麻醉剂笑气 (N2 O)对大鼠杏仁中央核 (Ce)神经元自发放电活动的影响 .方法　应用膜片钳全细胞记录技术在新生 SD大鼠脑薄片上观察了吸入麻醉剂笑气对 Ce自发放电频率的影响 .将新生 SD大鼠迅速断头取全脑 ,把脑置于通入 950 m L· L-1 O2 和 50 m L· L-1 CO2 混合气体的 4℃人工脑脊液 (ACSF)中 ,再用振动切片机制成 30 0～ 40 0μm含 Ce的脑薄片 ,然后用膜片钳全细胞记录技术分别观察不同浓度的笑气对 Ce神经元自发放电频率的影响 .结果　 30 m L· L-1 的笑气对 Ce神经元似有增加放电频率的作用 ,但无统计学差异 .50 0 m L· L-1和 70 0 m L· L-1的笑气可产生明显的抑制作用 (P<0 .0 5) ,冲洗 5min后均能使放电频率逐渐恢复到给药前水平 .结论　笑气对大鼠 Ce内的神经元自发放电活动可产生明显可逆性的抑制作用 ,表明 Ce是笑气在中枢神经系统的重要作用部位     

Matrix metalloproteinase‐9 reversibly affects the time course of NMDA‐induced currents in cultured rat hippocampal neurons

Matrix Metalloproteinase 9 (MMP‐9) has been demonstrated to play a crucial role in maintenance of NMDA receptor‐dependent LTP and in lateral mobility of these receptors. However, the effect of MMP‐9 on NMDA receptor (NMDAR) functional properties is unknown. For this purpose we have investigated the impact of recombinant MMP‐9 on the whole‐cell NMDAR‐mediated current responses in cultured hippocampal neurons. Treatment with MMP‐9 induced a reversible acceleration of desensitization and deactivation kinetics but had no effect on current amplitude. Interestingly, phorbol ester, a PKC activator known to enhance NMDAR lateral mobility, induced kinetic changes of currents similar to those produced by MMP‐9. In conclusion, our results show that MMP‐9 reversibly modulates the NMDAR kinetics and raise a possibility that this modulation could be related to the lateral mobility of these receptors. © 2009 Wiley‐Liss, Inc.     

黄芩甙对大鼠心室肌细胞触发性心律失常的影响及其机制

目的研究黄芩甙对触发性心律失常的影响,探讨黄芩甙抗心律失常的机制。方法酶解法分离大鼠心室肌细胞,全细胞膜片钳技术记录黄芩甙作用前后的L型钙电流(ICa-L)的变化,外科手术得到心室乳头肌,标准玻璃微电极技术记录黄芩甙作用前后跨膜动作电位(TAP)的变化以及哇巴因诱发的延迟后除极(DAD)和触发活动(TA)的影响。结果①在电压钳制下,黄芩甙对ICa-L均有明显抑制作用,随浓度的增加,对ICa-L的抑制作用逐渐增强。10,20和40μmol/L的黄芩甙对ICa-L的最大电流密度抑制作用分别由15.8±1.2pA/pF减小到11.3±0.9,8.2±0.8,4.9±0.6pA/pF(P均<0.05)。黄芩甙对ICa-L的抑制作用具有非常好的量效性,半效抑制浓度为27.7±1.9μmol/L。显著上抬I-V曲线。②20μmol/L黄芩甙明显缩短动作电位时程,抑制哇巴因诱导的DAD和TA。结论黄芩甙能抑制触发性心律失常,这可能与黄芩甙抑制心肌细胞ICa-L内流,减少细胞内Ca2+超载有一定关系。