GTP和GDP类似物对棉铃虫神经细胞高电压敏感钙通道的调节作用

以Ba²⁺为载流子,采用全细胞膜片钳法,研究了在电极液中分别加入G蛋白稳定 激活剂GTPγS（GTP类似物）和抑制剂GDPβS（GDP类似物）对棉铃虫Helicoverpaarmigera 3龄幼虫神经细胞高电压敏感钙通道的调节作用。Ba²⁺电流记录时间为20 min。对照组 和实验组的Ba²+电流在记录的初期均出现电流的增加现象,随后电流衰减,即“rundown ”。对照组峰电流在第20 min时降为初始值的(72.09±12.80)%。电极内液中加入2 mmol/L GTPγS可缓解电流的衰减现象,在第20 min时,峰电流为初始值的(95.99±7.93)%,明显大 于对照组的峰电流(P<0.01),而且电流 电压（I-V）关系曲线向正电压方向移动。相反 ,电极内液加入2 mmol/L GDPβS则导致峰电流衰减更加严重,第20 min时,峰电流仅为初始水 平的(41.95±9.32)%,显著小于对照组（P<0.01）,但未见电流 电压（I-V）关系曲 线的明显漂移。结果表明,棉铃虫神经细胞钙通道活动受G蛋白激活剂GTPγS和G蛋白抑制剂GDPβS的影响,提示G蛋白活动水平的改变调节钙通道的电流幅值和电压依赖性。     

细胞外Ba2+对大鼠心肌细胞L型钙通道的影响

目的：观察细胞外Ba²⁺对记录大鼠心肌细胞L型钙通道的影响。方法：采用急性酶解分离法获得大鼠的单个心肌细胞,使用全细胞膜片钳技术记录L型钙通道电流。采用Ba²⁺替换台式液中的Ca²⁺和直接向台式液中加入Ba²⁺（0~8 mmol/L）,观察峰值电流15 min内的变化,数据采用5个以上细胞进行重复。结果：（1）台式液中的Ca²⁺被Ba²⁺替换后,L型钙通道电流的失活速率明显减慢（P<0.01）;在台式液中加入少量Ba²⁺（0.2,0.4 mmol/L）时L型钙通道电流的失活速率无明显改变（P>0.05）,加入0.8 mmol/L Ba²⁺时失活速率明显减慢（P<0.05）。（2）与正常台式液比较,在细胞外液中加入Ba²⁺（0.2,0.4 mmol/L）峰值电流衰减减弱,其中10 min和15 min两个时间点衰减差异明显（P<0.01）。（3）在细胞外液中加入Ba²⁺可下移电流电压曲线,改变翻转电位,减弱丹酚酸A对钙电流的抑制强度,使量效关系曲线右移。结论：在细胞外液中加入一定浓度的Ba²⁺,能够减弱全细胞膜片钳技术记录大鼠心室肌细胞L型钙通道时出现的峰值电流衰减,改变通道的电压依赖特性,影响药物量效关系。     

不同浓度葡萄糖对大鼠胰岛β细胞L-型钙通道的影响

采用全细胞膜片钳技术观察不同浓度葡萄糖对新生Wister大鼠胰岛β细胞膜上电压依赖性L-型钙离子通道门控特性的影响,即分别用2.8、5.5、16.7和22.2 mmol/L的葡萄糖刺激单个贴壁胰岛β细胞,以Ba²⁺作为载流子,分析比较葡萄糖对L-型钙通道电流的影响。结果显示：在低糖（2.8 mmol/L）情况下,大鼠胰岛β细胞电压依赖性L-型钙离子通道电流静息膜电位约为－70 mV,钙离子内流不明显,且无明显的时间依赖性关系。在葡萄糖浓度为5.5 mmol/L的条件下,大鼠胰岛β细胞电压依赖性L-型钙离子通道电流在－40 mV激活, +20 mV左右达峰值;高糖（16.7 mmol/L）作用胰岛β细胞后,电压依赖性L-型钙离子通道电流约－40 mV激活,＋10 mV左右达峰值,即峰值电位向负方向移动约10 mV;葡萄糖浓度达22.2 mmol/L时,电活动呈持续性去极化,峰值电位增加不明显,提示葡萄糖降低胰岛β细胞电压依赖性L-型钙通道电流的激活电位阈值,促进其开放,钙电流峰值电位增加,随着高糖作用时间的延长,胰岛β细胞容积变大,细胞膜破坏。提示高浓度葡萄糖在一定范围内可以刺激胰岛素的分泌,但浓度过高则可抑制胰岛素的分泌,通过观察葡萄糖刺激的胰岛β细胞胰岛素第一时相分泌的变化,在一定程度上对高糖毒性作用的可能提供了证据。     

霍乱毒素对三氟氯氰菊酯抗性及敏感棉铃虫神经细胞L型钙通道的调节作用

霍乱毒素（CTX）可激活兴奋性异三聚体G蛋白（Gα_s）的α-亚基和刺激电压门控L-型钙通道,而昆虫的L-型钙通道可能是拟除虫菊酯类杀虫剂的作用靶点。为进一步探讨农业害虫对拟除虫菊酯类杀虫剂产生抗药性的作用机理,我们检测了CTX对三氟氯氰菊酯抗性及敏感棉铃虫Helicoverpa armigera中枢神经细胞电压门控L-型钙通道的调节作用。分别急性分离三氟氯氰菊酯抗性及敏感的3～4龄棉铃虫幼虫胸腹神经节细胞,并在改良的L15培养基（加入或未加入700 ng/mL的CTX）中培养12～16 h。钡离子为载流子,应用全细胞膜片钳技术记录电压门控L_型钙通道电流。结果显示,CTX可使敏感组棉铃虫神经细胞L-型钙通道的峰值电流密度增大36％、峰值电压左移5 mV,但对抗性组棉铃虫神经细胞L-型钙通道无上述作用。并且,CTX对敏感组及抗性组棉铃虫神经细胞L_型钙通道的激活电位、翻转电位、激活曲线和失活曲线等其他一些参数的影响也不明显。在无CTX作用时,所检测到的抗性组与敏感组棉铃虫神经细胞L_型钙通道的上述参数值间差异不显著。结果提示,棉铃虫神经细胞内存在Gs腺苷酸环化酶(AC)-cAMP-蛋白激酶A (PKA)-L-型钙通道信号调节系统;与敏感棉铃虫神经细胞L-型钙通道相比,三氟氯氰菊酯抗性棉铃虫神经细胞L-型钙通道的活性相对不易受到CTX调节,这可能与昆虫对拟除虫菊酯产生抗药性的机理有关。     

前列腺素E_2对四氧嘧啶处理的新生大鼠离体胰岛细胞内环核苷酸含量变化的影响

将分离的新生大鼠胰岛细胞,分别置入四氧嘧啶,PGE_2,PGE_2和四氧嘧啶以及高浓度葡萄糖和四氧嘧啶液进行孵育,用RIA 法测细胞内cAMP和cGMP的含量。结果表明,四氧嘧啶使胰岛细胞内 cAMP含量明显降低 cGMP含量升高cAMP/cGMP 比值比正常鼠低2.79倍,但 PGE_2预处理可以防止由四氧嘧啶引起的cAMP 的降低和 cGMP升高,使胰岛细胞内cAMP/cGMP 比值维持在接近正常水平。高浓度葡萄糖预处理虽不能防止由四氧嘧啶引起的cAMP 降低,但因能使cGMP 和cAMP 值均降低,故cAMP/cGMP 比值仍接近正常水平。单纯 PCE_2可使胰岛细胞内 cAMP含量轻度升高cGMP值略下降,cAMP/cGMP比值比正常值外高1.49倍。上述结果提示PGE_2可能是通过调节胰岛细胞内环核苷酸水平,实现其对胰岛B细胞的细胞保护作用的。     

木黄酮对小鼠胰岛β细胞电压依赖性钙通道表达和电流的影响

目的:研究长期抑制酪氨酸激酶活性对胰岛β细胞中电压依赖性钙通道的影响,探讨酪氨酸激酶在胰岛β细胞中的作用.方法:原代培养小鼠胰岛和胰岛β细胞,经0.1 mmol/L酪氨酸激酶抑制剂木黄酮处理12 h后,运用全细胞电流记录的方法观察电压依赖性钙电流以及动作电位的改变,RT-PCR方法观察电压依赖性钙通道α1亚单位的表达改变.结果:木黄酮处理12 h后,小鼠胰岛β细胞的电压依赖性钙电流明显减小(13.83±1.515pA/pFvs 7.012±1.502 pA/pF,P＜0.01,n=6),动作电位幅度明显减弱(38.50±7.46 mV vs 15.95±4.39 mV,P＜0.01,n=6).木黄酮处理12 h后,小鼠胰岛中电压依赖性钙通道的α1亚单位的表达明显减少,降低为对照组的0.792±0.078(P＜0.01,n=5).结论:木黄酮处理可以抑制小鼠胰岛β细胞中电压依赖性钙通道的表达和电流,提示长期抑制酪氨酸激酶活性在胰岛β细胞功能损害中具有重要作用.     

苦参碱对棉铃虫幼虫神经细胞钠通道的影响

用全细胞膜片钳技术研究了生物碱类植物杀虫剂苦参碱对棉铃虫Helicoverpa armigera幼虫离体培养中枢神经细胞钠离子通道门控过程的影响。结果表明： 苦参碱对棉铃虫幼虫神经细胞所表达的TTX （tetradotoxin, 河豚毒素)敏感钠通道具有浓度依赖性阻滞作用,1,10和100 μmol/L的苦参碱作用5 min后,分别使钠电流峰值较给药前下降（12.49±1.67）%、（18.79±2.16）%和（43.15±8.17）% (n=8, P<0.05)。苦参碱使钠电流的电流 电压关系曲线上移,但并不改变其激活电压、峰电压和电流电压关系曲线的形状。苦参碱对钠通道的阻滞作用可能是其具有某些毒理效应的离子基础。     

棉铃虫幼虫神经细胞的急性分离培养及其电压门控通道的膜片钳研究

探索了棉铃虫Helicoverpa armigera幼虫神经细胞的急性分离与体外培养的条件,并利用全细胞膜片钳技术首次对棉铃虫幼虫急性分离神经细胞的电压门控性钠、钾和钙通道的基本电生理学特性进行了研究。结果表明,棉铃虫幼虫中枢神经细胞在TC-100、L-15和Grace培养基中均可贴壁生长,在DMEM培养基中基本不能存活。在TC-100培养基分别与其它三种培养基按一定比例混合形成的培养液中,TC-100与L-15等量混合培养液更适合于神经细胞的生长。全细胞电压钳条件下,可分别记录到电压门控性钠、钾和钙通道电流。钙电流特征为高电压激活、缓慢失活;钠电流对河豚毒素敏感;钾电流可被细胞外液中的氯化四乙胺和4-氨基吡啶抑制。     

氨基甙类抗生素对家兔离体颈动脉窦压力感受器放电的抑制作用

实验用自制的压力感受器标本灌流－记录一体化装置,研究氨基甙类抗生素,胞外钙离子浓度变化和L－型钙通道拮抗剂对家兔离体颈动脉窦压力感受器活动（CS－BRA）的影响,研究中发现,（1）链霉素（0．24－0．75mmol/L)和庆大霉素（0．43－1．29mmol/L)浓度依赖性地抑制S－BRA,停药后可基本恢复;（2）高钙灌流液（3．3mmol/L)抑制S－BRA,而微量钙灌流液（10^-5mmol/L数量级）兴奋CS－BRA;（3）维拉帕米和地尔硫卓和选择怀阻断L－型钙通道的有效浓度范围内（＜106-7mol/L)对CS－BRA没有显著影响,在更高浓度（＞10^-6mol/L)时,则抑制CSBRA。结果表明。（1）氨基甙类抗生素特异性地抑制CS－BRA,是一种新的研究压力感受器活动的工具药;（2）钙离子不是形成压力感受器发生器电位的主要离子,而且L－型钙通道在CS－BRA中没有显著作用;（3）氨基甙类抗生素对CS－BRA的抑制作用可能不是阻断L－型钙通道实现的。     

三氟氯氰菊酯对棉铃虫神经细胞延迟整流钾电流的抑制作用

用膜片钳技术首次研究了三氟氯氰菊酯对离体培养的棉铃虫中枢神经细胞延迟整流钾通道电流的影响。结果表明,药物作用前有81%和39%的细胞的通道分别在-30 mV 和 -40 mV 激活（n=21）。三氟氯氰菊酯（10^-5 mmol/L）作用15 min后,有63%和38%细胞的通道分别在-40 mV 和 -50 mV 激活（n=8）;作用1 min后电流幅值明显降低,抑制率达到了37.7%（n=19）;加药后激活曲线明显左移且Vh 值变化显著,但k值没有明显变化。实验结果说明,三氟氯氰菊酯作用后,通道更容易激活,但显著抑制电流峰值,导致神经敏感性降低,棉铃虫中枢神经细胞钾通道也是拟除虫菊酯类药物的作用靶标之一。     

Na+和Ca2+对拟南芥根原生质体质膜内向K+通道电流的影响

以拟南芥(Arabidopsis thaliana Columbia)根为材料,利用膜片钳技术测定其根细胞原生质体质膜内向K^ 电流,并对Na^ 对其K^ 电流的影响进行了初步研究,发现Na^2 可明显抑制拟南芥根细胞原生质体的内向K^ 电流,外施Ca^2 可缓解Na^ 对内向K^ 电流的抑制．说明Ca^2 参与了质膜上K^ 通道对K^ ／Na^ 的选择性吸收的调节,从而使植物适应盐胁迫．     

Actin Dynamics Regulates Voltage-Dependent Calcium-Permeable Channels of the Vicia faba Guard Cell Plasma Membrane

Free cytosolic Ca~(2+) ([Ca~(2+)]_(cyt)) is an ubiquitous second messenger in plant cell signaling, and [Ca~(2+)]_(cyt) elevation is associated with Ca~(2+)-permeable channels in the plasma membrane and endomembranes regulated by a wide range of stimuli. However, knowledge regarding Ca~(2+) channels and their regulation remains limited in planta. A type of voltage-dependent Ca~(2+)-permeable channel was identified and characterized for the Vicia faba L. guard cell plasma membrane by using patch-clamp techniques. These channels are permeable to both Ba~(2+) and Ca~(2+), and their activities can be inhibited by micromolar Gd~(3+). The unitary conductance and the reversal potential of the channels depend on the Ca~(2+) or Ba~(2+) gradients across the plasma membrane. The inward whole-cell Ca~(2+) (Ba~(2+)) current, as well as the unitary current amplitude and NP. of the single Ca~(2+) channel, increase along with the membrane hyperpolarization. Pharmacological experiments suggest that actin dynamics may serve as an upstream regulator of this type of calcium channel of the guard cell plasma membrane. Cytochalasin D, an actin polymerization blocker, activated the NP_o of these channels at the single channel level and increased the current amplitude at the whole-cell level. But these channel activations and current increments could be restrained by pretreatment with an F-actin stabilizer, phalloidin. The potential physiological significance of this regulatory mechanism is also discussed.     

Effect of SIN-1 in rat ventricular myocytes: interference with beta-adrenergic stimulation

We have examined the effects of the nitric oxide (NO) donor, 3-morpholino-sydnonimine (SIN-1), on Ca(2+) transients, L-type Ca(2+) current (I(Ca,L)), and cGMP/cAMP content in electrically-stimulated rat ventricular myocytes in the absence and presence of the beta-adrenergic stimulation with isoproterenol. SIN-1 had no effect at low concentrations, but decreased the amplitude of electrically-induced Ca(2+) transients at higher concentrations. SIN-1 attenuated the increase in Ca(2+) transients induced by isoproterenol in a concentration-dependent manner. SIN-1 Also reduced the amplitude of caffeine-induced Ca(2+) transients, and the increase in I(Ca,L) induced by isoproterenol. These effects of SIN-1 were associated with an increased cGMP and a decreased cAMP content in ventricular myocytes in either the absence or presence of isoproterenol. These data suggest that the inhibitory effect of SIN-1 on basal and beta-adrenergic stimulated Ca2+ signal in ventricular myocytes could be due to the depression in the SR function and I(Ca,L), possibly mediated by a cGMP/cAMP-dependent mechanism. Taken together, the present study supports the idea that NO acts as an inhibitory modulator of the cardiac function during pathological conditions associated with an abnormal production of NO such as septic shock.     

A point mutation in the pore region alters gating, Ca(2+) blockage, and permeation of olfactory cyclic nucleotide-gated channels

Upon stimulation by odorants, Ca(2+) and Na(+) enter the cilia of olfactory sensory neurons through channels directly gated by cAMP. Cyclic nucleotide-gated channels have been found in a variety of cells and extensively investigated in the past few years. Glutamate residues at position 363 of the alpha subunit of the bovine retinal rod channel have previously been shown to constitute a cation-binding site important for blockage by external divalent cations and to control single-channel properties. It has therefore been assumed, but not proven, that glutamate residues at the corresponding position of the other cyclic nucleotide-gated channels play a similar role. We studied the corresponding glutamate (E340) of the alpha subunit of the bovine olfactory channel to determine its role in channel gating and in permeation and blockage by Ca(2+) and Mg(2+). E340 was mutated into either an aspartate, glycine, glutamine, or asparagine residue and properties of mutant channels expressed in Xenopus laevis oocytes were measured in excised patches. By single-channel recordings, we demonstrated that the open probabilities in the presence of cGMP or cAMP were decreased by the mutations, with a larger decrease observed on gating by cAMP. Moreover, we observed that the mutant E340N presented two conductance levels. We found that both external Ca(2+) and Mg(2+) powerfully blocked the current in wild-type and E340D mutants, whereas their blockage efficacy was drastically reduced when the glutamate charge was neutralized. The inward current carried by external Ca(2+) relative to Na(+) was larger in the E340G mutant compared with wild-type channels. In conclusion, we have confirmed that the residue at position E340 of the bovine olfactory CNG channel is in the pore region, controls permeation and blockage by external Ca(2+) and Mg(2+), and affects channel gating by cAMP more than by cGMP.     

Prenatal stress on the kinetic properties of Ca2+ and K+ channels in offspring hippocampal CA3 pyramidal neurons

Prenatal stress is known to cause neuronal loss and oxidative damage in the hippocampus of offspring rats. To further understand the mechanisms, the present study was undertaken to investigate the effects of prenatal stress on the kinetic properties of high-voltage-activated (HVA) Ca(2+) and K(+) channels in freshly isolated hippocampal CA3 pyramidal neurons of offspring rats. Pregnant rats in the prenatal stress group were exposed to restraint stress on days 14-20 of pregnancy three times daily for 45 min. The patch clamp technique was employed to record HVA Ca(2+) and K(+) channel currents. Prenatal stress significantly increased HVA Ca(2+) channel disturbance including the maximal average HVA calcium peak current amplitude (-576.52+/-7.03 pA in control group and -702.05+/-6.82 pA in prenatal stress group, p<0.01), the maximal average HVA Ca(2+) current density (-40.89+/-0.31 pA/pF in control group and -49.44+/-0.37 pA/pF in prenatal stress group, p<0.01), and the maximal average integral current of the HVA Ca(2+) channel (106.81+/-4.20 nA ms in control group and 133.49+/-4.59 nA ms in prenatal stress group, p<0.01). The current-voltage relationship and conductance--voltage relationship of HVA Ca(2+) channels and potassium channels in offspring CA3 neurons were not affected by prenatal stress. These data suggest that exposure of animals to stressful experience during pregnancy can exert effects on calcium ion channels of offspring hippocampal neurons and that the calcium channel disturbance may play a role in prenatal stress-induced neuronal loss and oxidative damage in offspring brain.     

cAMP modulates the excitability of immortalized H=hypothalamic (GT1) neurons via a cyclic nucleotide gated channel

GT1 cells are immortalized hypothalamic neurons that show spontaneous bursts of action potentials and oscillations in intracellular calcium concentration [Ca(2+)](i), as well as pulsatile release of GNRH: We investigated the role of cyclic nucleotide gated (CNG) channels in the activity of GT1 neurons using patch clamp and calcium imaging techniques. Excised patches from GT1 cells revealed single channels and macroscopic currents that were activated by either cAMP or cGMP. CNG channels from GT1 cells showed rapid transitions from open to closed states typical of heteromeric CNG channels, were selective for cations, and had an estimated single channel conductance of 60 picosiemens (pS). Ca(2+) inhibited the conductance of macroscopic currents and caused rectification of currents at increasingly positive and negative potentials. The membrane permeant cAMP analog Sp-cAMP-monophosphorothioate (Sp-cAMPS) increased the frequency of spontaneous Ca(2+) oscillations in GT1 cells, whereas the Rp-cAMPS isomer had only a slight stimulatory effect on Ca(2+) signaling. Forskolin, norepinephrine, and dopamine, all of which stimulate cAMP production in GT1 cells, each increased the frequency of Ca(2+) oscillations. The effects of Sp-cAMPS or NE on Ca(2+) signaling did not appear to be mediated by protein kinase A, since treatment with either H9 or Rp-cAMPS did not inhibit the response. The CNG channel inhibitor L-cis-diltiazem inhibited cAMP-activated channels in GT1 cells. Both L-cis-diltiazem and elevated extracellular Ca(2+) reversibly inhibited the stimulatory effects of cAMP-generating ligands or Sp-cAMP on Ca(2+) oscillations. These results indicate that CNG channels play a primary role in mediating the effects of cAMP on excitability in GT1 cells, and thereby may be important in the modulation of GnRH release.     

大鼠背根节神经元后超极化中Ca^2＋激活K^＋电流的蜂毒明肽敏感成分 …

为了明确大鼠背根节(DRG)神经元中存在慢的Ca2+激活K+电流成分,本实验在新鲜分散的DRG神经元胞体上,采用全细胞电压箝技术,给予DRG神经元一定强度的去极化刺激,记录刺激结束后30 ms时的尾电流幅度.结果发现:(1)随着去极化时间从1 ms延长至180 ms时,尾电流幅度由9.3±2.8 pA逐渐增大至64.1±3.4 pA(P＜0.001);(2)当去极化结束后的复极化电位降低时,尾电流幅度先逐渐下降到零,然后改变方向,逆转电位约为-63 mV;(3)细胞外施加500μmol/L Cd2+或细胞内液中施加11 mmol/L EGYA时尾电流明显减小甚至完全消失;(4)尾电流中慢成分的幅度在细胞外给与200 nmol/L蜂毒明肽后,减小了约26.32±3.9%(P＜0.01);(5)细胞外施加10 mmol/L TEA,可明显降低尾电流中的快成分.结果提示,在DRG神经元后超极化中存在Ca2+激活K+电流的蜂毒明肽敏感成分--ⅠAiHP.     

Modulation by internal protons of native cyclic nucleotide-gated channels from retinal rods

Ion channels directly activated by cyclic nucleotides are present in the plasma membrane of retinal rod outer segments. These channels can be modulated by several factors including internal pH (pH(i)). Native cyclic nucleotide-gated channels were studied in excised membrane patches from the outer segment of retinal rods of the salamander. Channels were activated by cGMP or cAMP and currents as a function of voltage and cyclic nucleotide concentrations were measured as pH(i) was varied between 7.6 and 5.0. Increasing internal proton concentrations reduced the current activated by cGMP without modifying the concentration (K(1/2)) of cGMP necessary for half-activation of the maximal current. This effect could be well described as a reduction of single-channel current by protonation of a single acidic residue with a pK(1) of 5.1. When channels were activated by cAMP a more complex phenomenon was observed. K(1/2) for cAMP decreased by increasing internal proton concentration whereas maximal currents activated by cAMP increased by lowering pH(i) from 7.6 to 5.7-5.5 and then decreased from pH(i) 5.5 to 5.0. This behavior was attributed both to a reduction in single-channel current as measured with cGMP and to an increase in channel open probability induced by the binding of three protons to sites with a pK(2) of 6.     

盐胁迫下珠眉海棠与山定子叶片Na+区域化的研究

以盐敏感型山定子实生苗和耐盐型珠眉海棠组培苗为材料,采用灌注离心技术研究了叶片质外体和共质体中Na^＋和Ca^2＋浓度的变化。结果表明：随盐胁迫强度的加强,叶片水势下降;叶片Na^＋含量、质外体和共质体中Na^＋浓度升高,珠眉海棠明显低于山定子;叶片Ca^2＋含量、共质体Ca^2＋浓度随盐胁迫的增加而升高,但珠眉海棠高于山定子,50mmol／L NaCl胁迫对质外体Ca^2＋没有明显影响,100mmol／LNaCl胁迫下增加,珠眉海棠低于山定子;叶片共质体与质外体中Na^＋浓度的比值,珠眉海棠明显高于山定子,说明在盐胁迫下珠眉海棠具有较强的离子区域化能力,离子区域化是珠眉海棠的主要耐盐机制。