蛇床子素对L-和N-型钙通道的影响

目的：比较蛇床子素对不同钙通道亚型的作用差异。方法：首先在tsA201细胞上瞬时转染Cavl．2,Cav1.3,Cav2．2e[37a],和Cav2．2e[37b]通道,然后采用全细胞膜片钳技术,记录tsA201细胞上的钙电流,并观察蛇床子素对各种钙通道亚型的影响。结果：蛇床子素可以浓度依赖性抑制Cav1．2和Cav1.3电流,抑制的半有效浓度分别为162．1μmol·L-1和56．2μmol·L-1．此外,蛇床子素对Cav2．2通道也有一定的抑制作用,在300μmol·L-1。的浓度下,抑制38％的Cav2．2e[37a]电流和61％的Cav2．2e[37b]电流,蛇床子素对钙电流的抑制是快速可逆的。蛇床子素在各个测试电位水平均能抑制上述四种钙通道电流,但不改变电流的激活阂值和最大峰值电流的激活电压。结论：蛇床子素以浓度依赖的方式抑制多种钙通道亚型并表现出不同的亲和力。     

苯丙烯酸对黄瓜幼苗生理特性的影响

采用基质栽培模拟实验,研究了不同浓度苯丙烯酸对黄瓜幼苗生理特性的影响.结果表明,苯丙烯酸对黄瓜幼苗的光合色素、光合速率、蒸腾速率和根系活力产生了抑制作用.当处理浓度为25μmol·L^-1时,对类胡萝卜素产生抑制作用,对叶绿素a、叶绿素b为促进作用;当浓度为50μmol·L^-1时,对光合速率、蒸腾速率和根系活力均产生显著的抑制作用(P<0.05),并随着处理浓度的增加抑制作用增强;当浓度为150μmol·L^-1时,对叶绿素a、叶绿素b产生显著抑制作用(P<0.05);随着处理浓度的增加,对黄瓜上述生理特性的抑制作用增强.低浓度苯丙烯酸(25～50μmol·L^-1)对幼苗根系活力的抑制强度不大,可在处理后期得到恢复;高浓度(100～150μmol·L^-1)处理则表现出显著的抑制作用,随着处理时间延长,抑制作用增强(P<0.05).     

三氯生对峨眉林蛙蝌蚪的毒性效应研究

为了探究水域三氯生(TCS)污染对峨眉林蛙Rana omeimontis蝌蚪生长发育的影响,本研究以峨眉林蛙蝌蚪(G22期)为试验对象,进行了96 h急性毒性实验和21 d慢性毒性实验。急性毒性实验中每隔24 h记录蝌蚪的死亡数;慢性毒性实验分别于饲养0 d、7 d、14 d、21 d测定蝌蚪的体质量、全长、头体长、尾长、吻长和眼间距。结果表明：96 h-LC₅₀为266μg·L^-1、96 h-SC为89μg·L^-1,可知TCS对峨眉林蛙蝌蚪为高毒化合物,强烈影响其生长发育。慢性毒性实验暴露21 d,30μg·L^-1、60μg·L^-1、90μg·L^-1、120μg·L^-1浓度的TCS极显著抑制蝌蚪的体质量增长,60μg·L^-1、90μg·L^-1、120μg·L^-1浓度的TCS极显著抑制蝌蚪的吻长增长。建议减少TCS污水排放,控制水体中TCS浓度。     

芸苔素对铅胁迫下远志种子萌发和幼苗生长生理特征的影响

以远志(Polygala tenuifolia Willd.)为研究对象,采用3 mmol·L^-1醋酸铅溶液模拟铅胁迫,探讨两种处理方式(预浸种、拌种)不同浓度(0～100μmol·L^-1)芸苔素(EBL)对远志种子萌发、幼苗生长、生理生化特性及铅含量的影响。结果表明：(1)远志种子的发芽率、发芽势、发芽指数、活力指数和幼苗的胚芽长度、鲜重、干重在铅胁迫下显著降低,EBL预浸种和拌种处理可有效缓解远志种子和幼苗遭受铅胁迫的伤害且具有剂量效应。(2)在铅胁迫下,远志幼苗游离脯氨酸、可溶性糖和可溶性蛋白含量随EBL浓度的增加而先升后降,它们在0.01μmol·L^-1 EBL拌种处理下分别为0μmol·L^-1 EBL处理的1.99、2.31、1.95倍,且拌种处理影响较浸种处理更为显著。(3)在铅胁迫下,远志幼苗超氧化物歧化酶、抗坏血酸过氧化物酶、过氧化物酶、过氧化氢酶活性均随EBL浓度的增加而先上升后下降,且预浸种和拌种处理的酶活性均在0.01μmol·L^-1 EBL浓度下最强。(...     

不同外源物质对镉砷复合污染胁迫下油菜生理指标和镉砷积累的影响

为缓解重金属对作物的胁迫,降低重金属对作物的毒害作用,通过叶面喷施比较不同外源物质茉莉酸(JA)、褪黑素(MT)、亚精胺(SPD)和2,4-表油菜素内酯(EBL)对Cd、As胁迫下油菜生理指标及吸收积累Cd、As的影响。结果表明:喷施200μmol·L^-1的JA和MT均显著提高油菜叶片的叶绿素SPAD值,而喷施20μmol·L^-1的EBL显著降低油菜叶片的叶绿素SPAD值;与CK处理相比,喷施500μmol·L^-1SPD和20μmol·L^-1EBL分别降低油菜叶片中MDA含量41.0%和52.3%,同时EBL处理提高油菜叶片SOD活性88.2%;喷施200μmol·L^-1MT导致油菜地上部Cd含量比CK处理降低27.8%,而喷施200μmol·L^-1的JA和20μmol·L^-1的EBL却分别提高油菜地上部As含量159.8%和136.8%; JA、MT、SPD和EBL在某种程度上均缓解了Cd、As复合污染对油菜的胁迫,为重金属污染农田...     

Cu2+对铜锈微囊藻生长及叶绿素荧光特性的影响

利用浮游植物荧光仪（Photo-PAM）研究了不同Cu²⁺浓度处理不同时间（12、24、48、72、96 h）对铜锈微囊藻（Microcystis aeruginosa）生长及叶绿素荧光特性的影响。结果表明:在一定时间（96 h）内,低浓度Cu²⁺(1μmol·L^-1)处理的铜锈微囊藻的藻细胞密度以及PSII最大光合效率（Fv/Fm）、PSII实际光合效率（ΦPSII）、光能利用效率（α）与对照相比变化不显著,最大电子传递速率（Pm）显著下降;高浓度Cu²⁺(2、4、6、8μmol·L^-1)处理下,藻细胞密度及Fv/Fm、ΦPSII、α、Pm随着Cu²⁺浓度的加大和胁迫时间的延长均显著降低,浓度为6、8μmol·L^-1 Cu²⁺处理48 h,铜锈微囊藻Fv/Fm、ΦPSII、α、Pm的测得值为0。可见,高浓度Cu²⁺(≥2μmol·L^-1)处理极大地影响铜锈微囊藻的生长及叶绿素荧光特性,胁迫作用显著。     

茉莉酸甲酯对木豆不定根染料木素含量及抗氧化系统影响

为探讨茉莉酸甲酯(Methyl Jasmonate,MJ)对木豆不定根影响,研究了茉莉酸甲酯的不同浓度及处理时间对木豆不定根中染料木素的含量及抗氧化系统的影响。研究结果表明,诱导子的浓度和处理时间对不定根中染料木素的含量有重要影响。在MJ浓度为100μmol·L^-1,处理时间为48 h时,染料木素的含量最高,为1.38 mg·g^-1,是对照组的4.03倍。在此基础上,研究了100μmol·L^-1的MJ处理对木豆不定根抗氧化系统的影响,结果表明,MJ处理的木豆不定根明显发生了氧化胁迫,O_2^-速率、H_2O_2和MDA含量明显升高,同时不定根中SOD和POD活性明显升高,但是抗氧化酶活性的提高并未消除不定根的氧化胁迫,这种氧化胁迫导致了木豆不定根次生代谢产物合成能力提高。     

溴氰菊酯对神经细胞钙通道和 钙库的激活作用

应用膜片钳全细胞记录方式和显微荧光测钙技术,以MN9D神经细胞为材料研究了溴氰菊酯的作用机理。低浓度(10^-9 mol/L～10^-7 mol/L)溴氰菊酯就能使神经细胞Ca²⁺电流显著增加。10^-9 mol/L,1 min时电流增加平均值为20.64%,5 min时为15.48%,表明溴氰菊酯能激活高电位激活钙通道(L型和N型),促使Ca²⁺内流,显微荧光测定细胞内自由钙离子浓度（［Ca²⁺］_I）发现,在含Ca²⁺和无Ca²⁺的胞外液中,溴氰菊酯均能使胞内自由钙离子数量增加,表明它能刺激胞内钙库释放Ca²⁺。［Ca²⁺］_I升高对细胞功能影响很大。     

核糖核酸酶A超家族抗菌活性评价

抗菌肽是机体重要的免疫防御分子，具有广谱杀菌活性。核糖核酸酶A是脊椎动物特异性的分泌型蛋白质，在人基因组中包含8个经典成员(RNase1-8)。它们作为一类重要的抗菌肽，广泛分布于机体需要抵抗外界病原微生物的组织中，除了特有的生物学功能外，均具有一定的抗菌活性。然而，目前各实验室对它们抗菌活性的报道并不一致，有必要开展横向比较分析。为此，我们表达纯化了人核糖核酸酶A超家族8个成员的重组蛋白质，并在同一实验条件下以半致死浓度评估了它们对革兰氏阴性菌(大肠杆菌)和革兰氏阳性菌(金黄色葡萄球菌)的抗菌活性。结果显示，RNase1-8重组蛋白质对大肠杆菌半数致死浓度分别为：0.081,0.046,0.008,0.250,2.028,0.072,0.001μmol·L^-1和1.1416μmol·L^-1;对金黄色葡萄球菌半数致死浓度分别为：3.427,1.856,2.211,5.188,8.274,4.356,2.502μmol·L^-1和9.916μmol·L^-1。该结果提示，RNase1-8对大肠杆菌的抗菌活...     

Myocardin调控心肌H9C2细胞Ca2+通道机制研究

目的：研究同样在维持心脏正常结构和功能过程中发挥着重要作用的转录因子心肌素Myocardin对L型Ca²⁺通道Cav1.2的转录调控作用及分子机制。方法：全细胞膜片钳技术记录心肌细胞膜Ca²⁺电流,慢病毒包装技术制备Myocardin-GFP慢病毒用于感染心肌细胞以过表达Myocardin,Real-time PCR定量检测Cav1.2基因mRNA水平,Western blotting检测Cav1.2蛋白表达水平。PCR介导的定点突变技术得到Ca²⁺通道启动子区特定CarGbox位点突变的突变体。荧光素酶报告系统检测野生型WT和突变体MU启动子活性,以确定Myocardin在Cav1.2基因启动子区的作用位点。结果：全细胞膜片钳技术表明Myocardin激活Cav1.2而增加心肌细胞膜Ca²⁺电流,real-time PCR和Western blotting结果表明,Myocardin激活Cav1.2基因的转录和表达,荧光素酶报告系统检测突变体启动子活性,发现Myocardin激活Cav1.2基因的转录依赖其启动子区的CarGbox。结论：Myocardin通过与Cav1.2基因启动子区CarGbox结合进而激活其转录和表达,促进Ca²⁺通道蛋白装配到心肌细胞膜上,加强Ca²⁺内流,增强膜电流。     

14-3-3τ Promotes Surface Expression of Cav2.2 (α1B) Ca2+ Channels

Surface expression of voltage-gated Ca²⁺ (Cav) channels is important for their function in calcium homeostasis in the physiology of excitable cells, but whether or not and how the α1 pore-forming subunits of Cav channels are trafficked to plasma membrane in the absence of the known Cav auxiliary subunits, β and α2δ, remains mysterious. Here we showed that 14-3-3 proteins promoted functional surface expression of the Cav2.2 α1B channel in transfected tsA-201 cells in the absence of any known Cav auxiliary subunit. Both the surface to total ratio of the expressed α1B protein and the current density of voltage step-evoked Ba²⁺ current were markedly suppressed by the coexpression of a 14-3-3 antagonist construct, pSCM138, but not its inactive control, pSCM174, as determined by immunofluorescence assay and whole cell voltage clamp recording, respectively. By contrast, coexpression with 14-3-3τ significantly enhanced the surface expression and current density of the Cav2.2 α1B channel. Importantly, we found that between the two previously identified 14-3-3 binding regions at the α1B C terminus, only the proximal region (amino acids 1706–1940), closer to the end of the last transmembrane domain, was retained by the endoplasmic reticulum and facilitated by 14-3-3 to traffic to plasma membrane. Additionally, we showed that the 14-3-3/Cav β subunit coregulated the surface expression of Cav2.2 channels in transfected tsA-201 cells and neurons. Altogether, our findings reveal a previously unidentified regulatory function of 14-3-3 proteins in promoting the surface expression of Cav2.2 α1B channels.     

Calreticulin negatively regulates the surface expression of Cav1.3 L-type calcium channel

Background

The neuroendocrine Cav1.3 L-type Ca channels have been recently found in the Human fetal heart and shown to play a vital role in Ca entry from the sarcolemma into the cell and in Ca homeostasis. Calreticulin, a Ca binding endoplasmic reticulum (ER) resident protein, has been recently shown to translocate to the cell surface where its role and function are just emerging. Here, we demonstrated a novel mechanism of Cav1.3 and calreticulin interaction resulting in downregulation of Cav1.3 channel densities in native Human fetal cardiac cells and Human Embryonic Kidney cell lines (tsA201).

Methods and results

Cell surface and cytoplasmic staining of calreticulin was demonstrated first in cultured human fetal cardiomyocytes (HFC), gestational age 18–24 weeks, using confocal microscopy thereby establishing that calreticulin is present at the cell surface in HFC. Co-immunoprecipitation from HFC using anti-Cav1.3 Ca channel antibody, and probing with anti-calreticulin antibody revealed a 46 kDa band corresponding to calreticulin suggesting that Cav1.3 Ca channel and calreticulin co-assemble in a macromolecular complex. Co-expression of Cav1.3 and calreticulin in tsA201 cells resulted in a decrease in surface expression of Cav1.3 Ca channels. These findings were consistent with the electrophysiological studies showing that co-transfection of Cav1.3 Ca channel and calreticulin resulted in 55% reduction of Cav1.3 Ca current densities recorded from tsA201 cells.

Conclusions

The results show the first evidence that calreticulin: (1) is localized outside the ER on the cell surface of HFC; (2) coimmunoprecipitates with Cav1.3 L-type Ca channel; (3) negatively regulates Cav1.3 surface expression thus resulting in decreased Cav1.3 Ca current densities. The data demonstrate a novel mechanism of modulation of Cav1.3 Ca channel by calreticulin, which may be involved in pathological settings such as autoimmune associated congenital heart block where Cav1.3 Ca channels are downregulated.     

Ciliary Neurotrophic Factor-treated Astrocyte Conditioned Medium Regulates the L-type Calcium Channel Activity in Rat Cortical Neurons

Astrocytes are activated by ciliary neurotrophic factor (CNTF) in vivo and in vitro, however, the consequences on the L-type calcium channel (LCC) of neurons are still poorly understood. Therefore, in the present study, whole-cell patch clamp, western-blot and RT-PCR assay were performed to evaluate the effects of CNTF-treated astrocyte conditioned medium (CNTF-ACM) on LCC current (I_Ca-L) and the expression of Cav1.2 and Cav1.3 in Sprague–Dawley rat cortical neurons. The results revealed that CNTF-ACM enhanced the amplitude of Ica-L and the expression of Cav1.3 significantly, but had no effects on Cav1.2 expression. We also found an increase in the concentration of fibroblast growth factor-2 (FGF-2) in CNTF-ACM by ELISA assay. Taken together, these findings indicate that CNTF induces the release of factors, including FGF-2, from astrocytes, thereby potentiating the activity of LCC in cortical neurons. Xiaojing Wang and Honghua Zheng contributed equally.     

Modeling interactions between voltage-gated Ca2+ channels and KCa1.1 channels

High voltage-activated (HVA) Cav channels form complexes with KCa1.1 channels, allowing reliable activation of KCa1.1 current through a nanodomain interaction. We recently found that low voltage-activated Cav3 calcium channels also create KCa1.1-Cav3 complexes. While coimmunoprecipitation studies again supported a nanodomain interaction, the sensitivity to calcium chelating agents was instead consistent with a microdomain interaction. A computational model of the KCa1.1-Cav3 complex suggested that multiple Cav3 channels were necessary to activate KCa1.1 channels, potentially causing the KCa1.1-Cav3 complex to be more susceptible to calcium chelators. Here, we expanded the model and compared it to a KCa1.1-Cav2.2 model to examine the role of Cav channel conductance and kinetics on KCa1.1 activation. As found for direct recordings, the voltage-dependent and kinetic properties of Cav3 channels were reflected in the activation of KCa1.1 current, including transient activation from lower voltages than other KCa1.1-Cav complexes. Substantial activation of KCa1.1 channels required the concerted activity of several Cav3.2 channels. Combined with the effect of EGTA, these results suggest that the Ca²⁺ domains of several KCa1.1-Cav3 complexes need to cooperate to generate sufficient [Ca²⁺]_i, despite the physical association between KCa1.1 and Cav3 channels. By comparison, Cav2.2 channels were twice as effective at activating KCa1.1 channels and a single KCa1.1-Cav2.2 complex would be self-sufficient. However, even though Cav3 channels generate small, transient currents, the regulation of KCa1.1 activity by Cav3 channels is possible if multiple complexes cooperate through microdomain interactions.     

Low Voltage Activation of KCa1.1 Current by Cav3-KCa1.1 Complexes

Calcium-activated potassium channels of the KCa1.1 class are known to regulate repolarization of action potential discharge through a molecular association with high voltage-activated calcium channels. The current study examined the potential for low voltage-activated Cav3 (T-type) calcium channels to interact with KCa1.1 when expressed in tsA-201 cells and in rat medial vestibular neurons (MVN) in vitro. Expression of the channel α-subunits alone in tsA-201 cells was sufficient to enable Cav3 activation of KCa1.1 current. Cav3 calcium influx induced a 50 mV negative shift in KCa1.1 voltage for activation, an interaction that was blocked by Cav3 or KCa1.1 channel blockers, or high internal EGTA. Cav3 and KCa1.1 channels coimmunoprecipitated from lysates of either tsA-201 cells or rat brain, with Cav3 channels associating with the transmembrane S0 segment of the KCa1.1 N-terminus. KCa1.1 channel activation was closely aligned with Cav3 calcium conductance in that KCa1.1 current shared the same low voltage dependence of Cav3 activation, and was blocked by voltage-dependent inactivation of Cav3 channels or by coexpressing a non calcium-conducting Cav3 channel pore mutant. The Cav3-KCa1.1 interaction was found to function highly effectively in a subset of MVN neurons by activating near –50 mV to contribute to spike repolarization and gain of firing. Modelling data indicate that multiple neighboring Cav3-KCa1.1 complexes must act cooperatively to raise calcium to sufficiently high levels to permit KCa1.1 activation. Together the results identify a novel Cav3-KCa1.1 signaling complex where Cav3-mediated calcium entry enables KCa1.1 activation over a wide range of membrane potentials according to the unique voltage profile of Cav3 calcium channels, greatly extending the roles for KCa1.1 potassium channels in controlling membrane excitability.     

Different roles attributed to Cav1 channel subtypes in spontaneous action potential firing and fine tuning of exocytosis in mouse chromaffin cells

This study examines the Cav1 isoforms expressed in mouse chromaffin cells and compares their biophysical properties and roles played in cell excitability and exocytosis. Using immunocytochemical and electrophysiological techniques in mice lacking the Cav1.3α1 subunit (Cav1.3(-/-) ) or the high sensitivity of Cav1.2α1 subunits to dihydropyridines, Cav1.2 and Cav1.3 channels were identified as the only Cav1 channel subtypes expressed in mouse chromaffin cells. Cav1.3 channels were activated at more negative membrane potentials and inactivated more slowly than Cav1.2 channels. Cav1 channels, mainly Cav1.2, control cell excitability by functional coupling to BK channels, revealed by nifedipine blockade of BK channels in wild type (WT) and Cav1.3(-/-) cells (53% and 35%, respectively), and by the identical change in the shape of the spontaneous action potentials elicited by the dihydropyridine in both strains of mice. Cav1.2 channels also play a major role in spontaneous action potential firing, supported by the following evidence: (i) a similar percentage of WT and Cav1.3(-/-) cells fired spontaneous action potentials; (ii) firing frequency did not vary between WT and Cav1.3(-/-) cells; (iii) mostly Cav1.2 channels contributed to the inward current preceding the action potential threshold; and (iv) in the presence of tetrodotoxin, WT or Cav1.3(-/-) cells exhibited spontaneous oscillatory activity, which was fully abolished by nifedipine perfusion. Finally, Cav1.2 and Cav1.3 channels were essential for controlling the exocytotic process at potentials above and below -10 mV, respectively. Our data reveal the key yet differential roles of Cav1.2 and Cav1.3 channels in mediating action potential firing and exocytotic events in the neuroendocrine chromaffin cell.     

L-type calcium channels as drug targets in CNS disorders

L-type calcium channels are present in most electrically excitable cells and are needed for proper brain, muscle, endocrine and sensory function. There is accumulating evidence for their involvement in brain diseases such as Parkinson disease, febrile seizures and neuropsychiatric disorders. Pharmacological inhibition of brain L-type channel isoforms, Cav1.2 and Cav1.3, may therefore be of therapeutic value. Organic calcium channels blockers are clinically used since decades for the treatment of hypertension, cardiac ischemia, and arrhythmias with a well-known and excellent safety profile. This pharmacological benefit is mainly mediated by the inhibition of Cav1.2 channels in the cardiovascular system. Despite their different biophysical properties and physiological functions, both brain channel isoforms are similarly inhibited by existing calcium channel blockers. In this review we will discuss evidence for altered L-type channel activity in human brain pathologies, new therapeutic implications of existing blockers and the rationale and current efforts to develop Cav1.3-selective compounds.     

Decreasing expression of alpha1C calcium L-type channel subunit mRNA in rat ventricular myocytes upon manganese exposure

Manganese is an essential trace element found in many enzymes. As it is the case of many essential trace elements, excessive level of manganese is toxic. It has been proven that excessive manganese could cause heart problems. In order to understand the mechanism of manganese toxicity in the heart, the effects of manganese on isolated rat ventricular myocytes were studied. The L-type calcium channel current was measured by whole-cell patch clamp recording mode. In the electrophysiology experiments, both 50 microM Mn2+ and 100 microM Mn2+ could effectively decrease the channel current amplitude density by 35.7% and 68.2%, respectively. Moreover, Mn2+ shifted the steady-state activation curve toward more positive potential and the steady-state inactivation curve toward more negative potential. Investigation by RT-PCR showed that the mRNA expression of alpha1C/Cav1.2 treated with manganese was decreased depending on its concentration, while the mRNA expression of alpha1D/Cav1.3 was almost unchanged. Fluo-3/AM was utilized for real-time free calcium scanning with laser scanning confocal microscopy (LSCM), and the results showed that Mn2+ could elicit a slow and continuous increase of [Ca2+]i in a concentration-dependent manner. These results have suggested that manganese could interfere with the function of the L-type calcium channel, downregulate the mRNA expression of alpha1C/Cav1.2, and thus causing long-lasting molecular changes of L-type calcium channel which have probably been triggered by overloading of calcium in myocytes.