Protective Effect of Carvedilol on Abnormality of L-type Calcium Current Induced by Oxygen Free Radical in Cardiomyocytes

The protective effect of carvedilol on abnormality of L-type calcium current induced by oxygen free radical in single guinea pig ventricular myocytes was studied. Whole-cell patch clamp technique was used to study the effect of H 2O 2 (0.5 mmol/L) on L-type calcium current in single guinea pig ventricular myocytes and the action of pretreatment with carvedilol (0.5 μmol/L). 0.5 μmol/L carvedilol had no significant effect on I Ca,L and its channel dynamics. In the presence of 0.5 mmol/L H 2O 2, peak current of I Ca,L was reduced significantly (P<0.001), the I-V curve of I Ca,L was shifted upward, steady-state activation curve and steady-state deactivation curve of I Ca,L were shifted left and recovery time of I Ca,L was delayed significantly (P<0.001). 0.5 μmol/L carvedilol significantly alleviated the inhibitory effect of H 2O 2 on I Ca,L as compared with that in H 2O 2 group (P<0.01). In addition, carvedilol reversed the changes of dynamics of I Ca,L induced by H 2O 2. It was concluded that carvedilol could alleviate the abnormality of L-type calcium current induced by oxygen free radical in cardiomyocytes. It shows partly the possible mechanism of the special availability of carvedilol in chronic heart failure.

Gender difference of transmural heterogeneity of calcium current in rabbit ventricle

Objective: To study the distribution of I_ Ca,L in endomyocardium(Endo), mid-myocardium(Mid) and epicardium(Epi) in female and male rabbit ventricle and to elucidate the mechanism of sex difference in drug-associated torsade de pointes. Methods: Whole-cell patch clamp technique was used to record action potential and I_Ca,L. Results: Action potential duration ofMidinfemale rabbit heart waslonger thanthat in male and transmural dispersion of repolarization in female was largerthan thatinmale. The densities of I_ Ca,L in Endo, Mid and Epi of female rabbits were (7.1±0.6), (10.4±0.9) and (9.6±1.1) pA/pF and they were (9.1±0.9), (10.5±1.0) and (9.8±0.9) pA/pF in male respectively. Transmural heterogeneity of I_ Ca,L in female ventricle was more significant than that in male rabbit.Conclusion:Female rabbitspossessmoresignificant transmural heterogeneityof I_Ca,L,whichmaybe responsiblefor largertransmuraldispersion of repolarization and more drug-associated torsade de pointes in female.

The Effect of Extrogenous Phosphocreatine on L-type Calcium Current in Ischemic Guinea Pig Ventricular Myocytes

Objectives Heart failure （HF） is one of the most common outcome for all kinds of heart diseases, the effects of energetic therapy on HF remains controversial, especially to ischemic HF. The aim of this study was to explore the effect of exogenous phosphocreatine with different concentration on L-type calcium（I Cc-L） current in ischemic ventricular myocytes of guinea pig and to investigate its underlying electrophysiological mechanism for the treatment of ischemic HF. Methods Single ventricular myocytes were isolated enzymatically from left ventricle of guinea pig. Peak I Ca-L current were recorded using patch clamp techniques in the whole-cell configuration when myocytes had been superfused with normal Tyrode solution, simple ischemic solution, ischemic solution containing phosphocreatine with different concentration for 10 minutes respectively. Results Peak I Ca-L current density of myocytes superfused with simple simulated ischemic solution was remarkably inhibited by 80.6 ± 5.2% compared with myocytes superfused with normal Tyrode solution（P〈0.05）. Ischemic solution containing phosphocreatine of 5, 10, 20, 30mmol/L inhibited Peak I Ca-L current density by （53.8±6.7）%, （41.8 ± 8.2）%, （38.1±7.4）%, （36.6±9.7）% respectively. There was no statistical significance among phosphocreation of 10, 20, 30 mmol / L. Conclusions Extrogenous phosphocreatine could reverse the inhibition of I Ca-L current under ischemic condition, which could be the ionic basis for the treatment of ischemic heart failure. 0-10 mmol/L phosphocreatine exerted significant dose-effect relationship which no longer existed as concentration more than 10 mmol/L. It is supposed that phosphocreatine increased I Ca-L current by many pathways rather than simple substrate for ATP synthesis.

Sevoflurane postconditioning alleviates action potential duration shortening and L-type calcium current suppression induced by ischemia/reperfusion injury in rat epicardial myocytes

Background It has been proved that sevoflurane postconditioning （SpostC） could protect the heart against myocardial ischemia/reperfusion injury, however, there has been few research focused on the electrophysiological effects of SpostC. The objective of the study was to investigate the effects of SpostC on action potential duration （APD） and L-type calcium current （Ica, L） in isolated cardiomyocytes. Methods Langendorff perfused SD rat hearts were randomly assigned to one of the time control （TC）, ischemia/reperfusion （I/R, 25 minutes of ischemia followed by 30 minutes of reperfusion）, and SpostC （postconditioned with 3% sevoflurane） groups. At the end of reperfusion, epicardial myocytes were dissociated enzymatically for patch clamp studies. Results Sevoflurane directly prolonged APD and decreased peak Ica, L densities in epicardial myocytes of the TC group （P〈0.05）. I/R injury shortened APD and decreased peak Ica, L densities in epicardial myocytes of the I/R group （P〈0.05）. SpostC prolonged APD and increased peak Ica, L densities in epicardial myocytes exposed to I/R injury （P〈0.05）. SpostC decreased intracellular reactive oxygen species （ROS） levels, reduced the incidence of ventricular tachycardia and ventricular fibrillation, and decreased reperfusion arrhythmia scores compared with the I/R group （all P〈0.05）. Conclusions SpostC attenuates APD shortening and Ica, L suppression induced by I/R injury. The regulation of APD and lea, L by SpostC might be related with intracellular ROS modulation, which contributes to the alleviation of reperfusion ventricular arrhvthmia.

L-type calcium current in right ventricular outflow tract myocytes of rabbit heart

The mechanism of idiopathic ventricular tachycardia originating from the right ventricular outflow tract （RVOT） is not clear. Many clinical reports have suggested a mechanism of triggered activity. However, there are few studies investigating this be- cause of the technical difficulties associated with examining this theory. The L-type calcium current （/Ca-L）, an important in- ward current of the action potential （AP）, plays an important role in arrhythmogenesis. The aim of this study was to explore differences in the APs of right ventricular （RV） and RVOT cardiomyocytes, and differences in electrophysiological character- istics of the ICa-L in these myocytes. Rabbit RVOT and RV myocytes were isolated and their AP and Ic,-L were investigated us- ing the patch-clamp technique. RVOT cardiomyocytes had a wider range of AP duration （APD） than RV cardiomyocytes, with some markedly prolonged APDs and markedly shortened APDs. The markedly shortened APDs in RVOT myocytes were abolished by treatment with 4-AP, an inhibitor of the transient outward potassium current, but the markedly prolonged APDs remained, with some myocytes with a long AP plateau not repolarizing to resting potential. In addition, early afterdepolariza- tion （EAD） and second plateau responses were seen in RVOT myocytes but not in RV myocytes. RVOT myocytes had a high- er current density for/Ca-L than RV myocytes （RVOT （13.16±0.87） pA pF-1, RV （8.59±1.97） pA pF-1; P〈0.05）. The ICa-L and the prolonged APD were reduced, and the EAD and second plateau response disappeared, after treatment with nifedipine （10 μmol L^-1）, which blocks the Ica-L. In conclusion, there was a wider range of APDs in RVOT myocytes than in RV myocytes, which is one of the basic factors involved in arrhythmogenesis. The higher current density for ICa-L is one of the factors causing prolongation of the APD in RVOT myocytes. The combination of EAD with prolonged APD may be one of the mechanisms of RVOT-VT generation.

Effect of Sodium Salicylate on Calcium Currents and Exocytosis in Cochlear Inner Hair Cells:Implications for Tinnitus Generation

Sodium salicylate is an anti-inflammatory medication with a side-effect of tinnitus.Here,we used mouse cochlear cultures to explore the effects of salicylate treatment on cochlear inner hair cells(IHCs).We found that IHCs showed significant damage after exposure to a high concentration of salicylate.Whole-cell patch clamp recordings showed that 1–5 mmol/L salicylate did not affect the exocytosis of IHCs,indicating that IHCs are not involved in tinnitus generation by enhancing their neuronal input.Instead,salicylate induced a larger peak amplitude,a more negative half-activation voltage,and a steeper slope factor of Ca^(2+)current.Using noise analysis of Ca^(2+)tail currents and qRT-PCR,we further found that salicylate increased the number of Ca^(2+)channels along with CaV1.3 expression.All these changes could act synergistically to enhance the Ca^(2+)influx into IHCs.Inhibition of intracellular Ca^(2+)overload significantly attenuated IHC death after 10 mmol/L salicylate treatment.These results implicate a cellular mechanism for tinnitus generation in the peripheral auditory system.

Effect of allitridi on calcium current in rat ventricular myocytes

Background Allitridi is an active compound that is extracted from the garlic. It has effects of anti-atherosclerosis, anti-arrhythmias and lowering blood pressure. But the controversy about the effect on cardiac contractility still exists. Methods Whole-cell patch clamp recording technique was used to record ICa,Lin single cell isolated rat ventricular myocytes. The nifedipine- sensitive L-type calcium current was recorded in the rat ventricular myocytes. Results Allitridi decreased the calcium channel current in a dose-dependent and voltage-dependent manner in ventricular myocytes of rats. The current-voltage curve was shifted upwards, on which active potential,peak potential and reverse potential showed no significant changes. The inactivation curve was shifted to more negative potential, but the activation curve and recovery curve were not altered. Allitridi had no effect on frequent-dependency of calcium current. Conclusion These results show that allitridi could concentration-dependently decrease calcium channel current in ventricular myocytes of rats.

Effect of gingerol on colonic motility via inhibition of calcium channel currents in rats

AIM: To investigate the effect of gingerol on colonic motility and the action of L-type calcium channel currents in this process.METHODS: The distal colon was cut along the mesenteric border and cleaned with Ca^(2+)-free physiological saline solution. Muscle strips were removed and placed in Ca^(2+)-free physiological saline solution, which was oxygenated continuously. Longitudinal smooth muscle samples were prepared by cutting along the muscle strips and were then placed in a chamber. Mechanical contractile activities of isolated colonic segments in rats were recorded by a 4-channel physiograph. Colon smooth muscle cells were dissociated by enzymatic digestion. L-type calcium currents were recorded using the conventional whole-cell patch-clamp technique.RESULTS: Gingerol inhibited the spontaneous contraction of colonic longitudinal smooth muscle in a dose-dependent manner with inhibition percentages of 13.3% ± 4.1%, 43.4% ± 3.9%, 78.2% ± 3.6% and 80.5% ± 4.5% at 25 μmol/L, 50 μmol/L, 75 μmol/L and 100 μmol/L, respectively(P < 0.01). Nifedipine, an L-type calcium channel blocker, diminished the inhibition of colonic motility by gingerol. Gingerol inhibited L-type calcium channel currents in colonic longitudinal myocytes of rats. At a 75 μmol/L concentration of gingerol, the percentage of gingerolinduced inhibition was diminished by nifedipine from 77.1% ± 4.2% to 42.6% ± 3.6%(P < 0.01). Gingerol suppressed IBa in a dose-dependent manner, and the inhibition rates were 22.7% ± 2.38%, 35.77% ± 3.14%, 49.78% ± 3.48% and 53.78% ± 4.16% of control at 0 m V, respectively, at concentrations of 25 μmol/L, 50 μmol/L, 75 μmol/L and 100 μmol/L(P < 0.01). The steady-state activation curve was shifted to the right by treatment with gingerol. The value of half activation was-14.23 ± 1.12 m V in the control group and-10.56 ± 1.04 m V in the 75 μmol/L group(P < 0.05) with slope factors, Ks, of 7.16 ± 0.84 and 7.02 ± 0.93(P < 0.05) in the control and 75 μmol/L groups, respectively. However, the steady-state inactivation curve was not changed, with a half-inactivation voltage, 0.5 V, of-27.43 ± 1.26 m V in the control group and-26.56 ± 1.53 m V in the 75 μmol/L gingerol group(P > 0.05), and a slope factor, K, of 13.24 ± 1.62 in the control group and 13.45 ± 1.68(P > 0.05) in the 75 μmol/L gingerol group.CONCLUSION: Gingerol inhibits colonic motility by preventing Ca^(2+) influx through L-type calcium channels.

环状RNA mmu_circ_0005019影响小鼠心肌细胞钙激活钾通道电流及动作电位

目的 探索环状RNA mmu_circ_0005019调控小电导钙激活钾(small-conductance calcium-activated potassium, SK)通道蛋白亚基SK3编码基因Kcnn3的表达,以及对小电导钙激活钾通道电流(IK,Ca)和动作电位时程(action potential duration, APD)的影响。方法 在小鼠HL-1细胞中分别构建mmu_circ_0005019过表达和干扰模型,分为过表达组(n=3)、空质粒组(n=3)、干扰1组(n=3)、干扰2组(n=3)、对照组(n=3),通过RT-qPCR、Western blot分析mmu_circ_0005019调节Kcnn3表达的分子机制,应用膜片钳技术电流钳模式记录全细胞的IK,Ca,并用电压钳模式记录APD。结果 成功构建了环状RNA mmu_circ_0005019过表达和干扰的HL-1细胞模型。过表达组的Kcnn3基因表达与空质粒组相比明显上调(P<0.05);干扰1组和干扰2组的Kcnn3基因表达与对照组相比均明显下调(P<0.05)。电生理发现过表达mmu_circ_0005019增加了HL-1细胞IK,Ca电流密度,APD显著缩短;相反,干扰mmu_circ_0005019减少了IK,Ca电流密度,APD显著延长。结论 mmu_circ_0005019可以上调小鼠HL-1细胞Kcnn3的表达水平,从而改变IK,Ca和APD,提示环状RNA mmu_circ_0005019可能在房颤发生中起到促进作用。

慢性间歇性低压低氧对发育大鼠心肌细胞中的L型钙通道的影响

目的探讨慢性间歇性低压低氧(chronic intermittent hypobaric hypoxia,CIHH)对发育大鼠L型钙电流(L-type calcium current,I_(CaL))的影响。方法新生雄性SD大鼠随机被分为:28 d CIHH干预组(CIHH28)、42 d CIHH干预组(CIHH42)、28 d对照实验组(CON28)和42 d对照实验组(CON42)。其中CIHH干预组分别在模拟3000 m海拔高度低压低氧的条件下(P_(B)=525 mmHg,PO_(2)=108.8 mmHg,5 h/d)(1 mmHg=0.133 kPa)干预28 d和42 d。采用全细胞膜片钳技术记录心室肌细胞中的I_(CaL)。结果CON和CIHH组心肌细胞I_(CaL)的峰值电流和电流-电压(I-V)关系曲线差异无统计学意义(P>0.05)。在模拟缺血-复灌(ischemia-reperfusion,I/R)过程中,I_(CaL)峰值降低,I-V曲线向上偏移(P<0.05),但CIHH组心肌细胞中I_(CaL)的变化明显小于CON心肌细胞(P<0.05)。CIHH组的I_(CaL)稳态激活曲线和稳态失活曲线在I/R前后均无显著性变化(P>0.05)。在I/R过程中,CON28和CON42组的I_(CaL)稳态失活曲线向左偏移(P<0.05),而CIHH28和CIHH42组心肌细胞没有变化(P>0.05)。结论CIHH可减轻缺血再灌注损伤对发育大鼠心室肌细胞I_(CaL)的影响。

Sex Difference in the Repolarization Currents of Rabbit Ventricular Cells

The current difference between male and female rabbit ventricular myocytes was investigated for elucidating the mechanism of longer QT interval and higher incidence of drug-associated torsade de pointes in female rabbits than in male rabbits. Whole cell patch clamp technique was used to record APD, I_to, I_K,tail, I_K1 and I_Ca,L of myocytes from left ventricular apex. There was no difference in the membrane capacitance between male and female rabbit myocytes. APD_90 was longer in female rabbits (560.4±26.5 ms, n=15) than in male ones (489.0±20.7 ms, n=14), P<0.05. In female rabbit myocytes, I_K,tail, I_to, I_K1 and I_Ca,L were 0.71±0.05 pA/pF (n=17), 8.28±1.03 pA/pF (n=18), 24.5±3.6 pA/pF (n=12) and 9.0±2.3 pA/pF (n=15) respectively. In male rabbit myocytes, they were 0.84±0.07 pA/pF (n=18), 8.60±1.20 pA/pF (n=18), 25.9±4.5 pA/pF (n=14) and 9.3±2.6 pA/pF (n=16) respectively. I_K,tail in female rabbits was significantly lower than that of male rabbits (P<0.05), but there was no difference in I_to, I_K1 and I_Ca,L between male rabbits and female rabbits (P>0.05). The lower I_K,tail of female rabbit myocytes may contribute to the longer repolarization and the higher incidence of drug-associated torsade de pointes.

Effects of 2-APB on Store-operated Ca^(2+) Channel Currents of Hepatocytes after Hepatic Ischemia/Reperfusion Injury in Rats

The effects of hepatic ischemia/reperfusion (I/R) injuries on hepatocellular viability and store-operated calcium current (Isoc) in isolated rat hepatocytes and the effects of 2-APB on store-operated calcium current (Isoc) in isolated rat hepatocytes after hepatic ischemia/reperfusion injuries were studied. Hepatic ischemia and reperfusion injury model was established and whole cell patch-clamp techniques were used to investigate the effects of 2-APB on Isoc. The results showed that ischemia/reperfusion injuries could significantly reduce hepatocellular viability and further increase Isoc in hepatocytes and 2-APB (20, 40, 60, 80, 100 μmol/L) produced a concentration-dependent decrease of Isoc with IC 50 value of 64.63±10.56 μmol/L (n=8). It was concluded that ischemia/reperfusion injuries could reduce hepatocellular viability, probably through increased Isoc in hepatocytes and 2-APB had a protective effect on ischemia/reperfusion-induced liver injury, probably though inhibiting Isoc.

直流和脉冲电流对碳酸钙电沉积的影响

电化学沉积碳酸钙是减轻工业水系统中结垢的方法之一。采用脉冲电流和直流电研究CaCO_(3)的电化学沉积过程,以及不同离子对脉冲沉积CaCO_(3)的影响,并通过扫描电子显微镜和X射线衍射对其晶型和晶貌进行表征。实验结果表明,脉冲电流下CaCO_(3)在304SS电极的成核速率快于直流电流,其促进了碳酸钙的熟化过程。Fe^(3+)、PO_(4)^(3-)、SiO_(3)^(2-)和Mg^(2+)的加入加速了CaCO_(3)的结晶,但降低了沉积的致密性和厚度。脉冲电沉积的熟化过程使得结晶倾向于形成粒径更大的层叠形貌方解石,且方解石粒径大于直流电沉积所得。

Inhibitory Mechanism of Carvedilol on L-type Ca^(2+) Current in Rat Ventricular Myocytes

Objectives The effects of car-vedilol on calcium current (ICa) were investigated inisolated adult rat ventricular myocytes. Methods ICawas recorded by using whole-cell patch-clamp recordingtechnique. Results Carvedilol reversibly inhibited ICain a concentration-dependent manner, carvedilol at 0.1,0.3, 1 and 10 μmol/L in the extracellular solution dec-reased peak ICa by 1.52%, 18.04%, 37.34%and72.18%,respectively. The steady-state inactivation curve of ICawas shifted to more negative potentials, while the activ-ation curve was not altered. The recovery from inactiva-tion was shifted to right direction, it could not berecovered completely. In addition, Pretreatment ofventricular myocytes with prazosin and propranololcouldn't block the carvedilol-induced reduction of ICa.Conclusions Carvedilol inhibits I C a i n adult ratventricular myocytes by mechanisms involvingpreferential interaction with the inactivated state ofcalcium channel.

电石炉电极电流智能切换控制系统

当原料、炉况和加料过程发生未知变化时,电石炉电极控制系统的特性发生变化,导致PI控制系统难以满足电石炉的控制目标。将电石炉的动态特性与模糊自适应PI控制、规则切换控制相结合,提出了电石炉电极控制系统智能切换控制方法。设计了可编程逻辑控制器(programmable logic controller,PLC)系统软硬件平台,开发了基于所提控制方法的应用软件并研发了电石炉电极电流控制系统,成功应用于神木电化发展有限公司电石分厂。长期运行结果表明,采用所研制的电石炉电极电流控制系统,实现了生产过程的安全稳定运行,可以将电石炉电极电流控制在工艺规定的目标值范围内,提高了产品质量,降低了生产能耗,提高了企业的经济效益。

Effects of Ca^(2+) channel blockers on store-operated Ca^(2+) channel currents of Kupffer cells after hepatic ischemia/reperfusion injury in rats

AIM： To study the effects of hepatic ischemia/ reperfusion （I/R） injury on store-operated calcium channel （SOC） currents （Isoc） in freshly isolated rat Kupffer cells, and the effects of Ca^2＋ channel blockers, 2-aminoethoxydiphenyl borate （2-APB）, SK＆F96365, econazole and miconazole, on Isoc in isolated rat Kupffer cells after hepatic I/R injury.METHODS： The model of rat hepatic I/R injury was established. Whole-cell patch-clamp techniques were performed to investigate the effects of 2-APB, SK＆F96365, econazole and miconazole on Isoc in isolated rat Kupffer cells after hepatic I/R injury.RESULTS： I/R injury significantly increased Isoc from -80.4±25.2pA to -159.5±34.5pA （^bp 〈 0.01, n = 30）. 2-APB （20, 40, 60, 80, 100 pmol/L）, SK＆F96365 （5, 10, 20, 40, 50 pmol/L）, econazole （0.1, 0.3, 1, 3, 10 μmol/L） and miconazole （0.1, 0.3, 1, 3, 10 μmol/L） inhibited Isoc in a concentration-dependent manner with IC50 of 37.41 μmol/L （n = 8）, 5.89 μmol/L （n = 11）, 0.21 μmol/L （n = 13）, and 0.28 μmol/L （n = 10）. The peak value of Isoc in the I-V relationship was decreased by the blockers in different concentrations, but the reverse potential of Isoc was not transformed. CONCLUSION： SOC is the main channel for the influx of Ca^2＋ during hepatic I/R injuries. Calcium channel blockers, 2-APB, SK＆F96365, econazole and miconazole,have obviously protective effects on I/R injury, probably by inhibiting Isoc in Kupffer cells and preventing the activation of Kupffer cells.

高盐饮食上调跨膜蛋白16A致C57BL/6J小鼠脑动脉重构的机制

目的探讨高盐上调跨膜蛋白16A(transmembrane protein 16A,TMEM16A)致小鼠脑动脉重构的机制。方法40只C57BL/6J小鼠随机分为4组(10只/组,模型制备8周),空白对照组(正常饮水、摄食)、低盐组(2%高盐饲料)、中盐组(4%高盐饲料)和高盐组(8%高盐饲料)。HE染色观察脑动脉形态学变化;血管渗透性实验比较脑组织颜色及吸光度值;免疫荧光检测脑动脉TMEM16A的表达;PCR和Western blot检测脑动脉TMEM16A的mRNA和蛋白表达;离体肌张力检测脑动脉舒缩反应;膜片钳记录脑动脉平滑肌细胞钙激活氯通道(calcium-activated chloride channels,CaCC)电流。结果与空白对照组相比,2%、4%和8%高盐饮食可浓度依赖性引起脑动脉管壁逐渐增厚、管腔狭窄;注射埃文斯兰后,4%和8%高盐组脑组织逐渐变蓝,且吸光度值明显增加;2%、4%和8%高盐组脑动脉对60 mmol·L^(-1) KCl的收缩反应逐渐增强,对10-5 mol·L^(-1)乙酰胆碱的舒张反应逐渐减弱;TMEM16A阻断剂可抑制脑动脉对60 mmol·L^(-1)KCl的收缩反应;高盐组脑动脉TMEM16A不仅荧光表达逐渐增强,且mRNA和蛋白表达逐渐增多,脑动脉平滑肌细胞的CaCC电流逐渐增大。结论高盐可致C57BL/6J小鼠脑动脉重构,其机制与上调TMEM16A表达和/功能有关。

The Effect of Levobunolol Hydrochloride on the Calcium andPotassium Channels in Isolated Ventricular Myocytes of Guinea Pig

The effects of levobunolol hydrochlorid (Bun) on the type L calciumchannel currents (Ica) and delayed rectifier potassium channel currents (Ik) in isolated ventricular myocytes of guinea pig were studied by using patch clamp wholecell recording techniques. The results were showed that: 1) Bun caused a dosedependent decrease in Ica and a dose-dependent increase in Ik of the ventricular myocytes.The threshold concentrations of Bun for Ica and Ik were 10-8 mol/L and10-7 mol/L respectively. The maximum effective concentration of Bun for both Ica and Ik was 3 × 10-5 mol/L, and half-maximal concentration was 3 × 10-6 mol/L;2 ) Ik was blocked by 2× 10-6mol/L tetraethylammonium (TEA). A concentration of 3 × 10-6 mol/L Bun showed a decreasing effect on the Ica as revealed by the current-voltage relationship curve, i. e., Bun caused an elevation of the curve; 3)When Ica was blocked by 2 × 10-6 mol/L Isoptin (Verapamil), at a concentrationof 3 × 106- mol/L Bun showed an increasing effect on Ik and the effect could be blocked by TEA. The above-mentioned results indicated that Bun had an inhibito-ry effect on Ica and a fascilitatory effect on Ik The results suggested that themolecular mechanisms of antihypertensive, heart rate slowing and β-receptorblocking effects of Bun might be due to decrease of Ica and increase of Ik.

O2-芋螺毒素Tx7.29抑制钙通道电流及镇痛活性研究

目的 海洋肉食性软体动物芋螺的毒液是生物活性多肽的一个宝贵来源。这些活性多肽大多是富含二硫键的神经毒素,通常称为芋螺毒素。在本研究中,发现了一个全新的O2超家族芋螺毒素Tx7.29,通过对其进行功能研究,期望发现新的镇痛药候选物。方法 从织锦芋螺毒管c DNA文库中克隆得到Tx7.29的c DNA序列。通过化学合成,制得了Tx7.29的成熟肽,并通过质谱鉴定了其分子质量。通过膜片钳实验和动物实验确定Tx7.29的生物学功能。结果 Tx7.29的c DNA序列编码了一个包含68个氨基酸残基的芋螺毒素前体,由19个残基的信号肽、28个残基的前片段和22个残基的成熟肽组成。圆二色谱分析表明,β转角和反平行片层是Tx7.29二级结构中的主要组分。通过膜片钳实验发现,Tx7.29可以显著抑制大鼠背根神经节细胞的钙通道电流,但对钠电流和钾电流没有明显作用。在小鼠热板疼痛试验中,从0.5到4小时,Tx7.29以剂量依赖性的方式,增加了试验小鼠的热板潜伏时间。Tx7.29对ND7/23细胞无明显细胞毒性。结论 Tx7.29有望成为一种镇痛药物先导化合物,同时它的发现也扩大了O2-芋螺毒素的作用范围。

异丙肾上腺素对NCX1.1基因编码钠钙交换体电流的影响

目的探讨β肾上腺素受体对钠钙交换电流(I_(NCX))的调控及可能的信号转导途径。方法利用免疫磁珠阳性标记表达系统将NCX1.1质粒转染人胚肾293(HEK-293)细胞,选取103个转染阳性的HEK-293细胞,随机分成空白对照组(n=20)、灌流异丙肾上腺素(ISO)组(n=20)、灌流Gi蛋白抑制剂百日咳毒素及ISO组(n=20)、灌流环磷酸腺苷(cAMP)合成激动剂毛喉素(forskolin)及ISO组(n=22),灌流蛋白激酶A(protein kinase A,PKA)抑制剂H89及ISO组(n=21)。运用全细胞膜片钳技术记录各组I_(NCX)的变化。采用SPSS 21.0统计软件进行数据分析。根据数据类型,分别采用t检验或ANOVA方差分析进行组间比较。结果以灌流用药前I_(NCX)为空白对照组,ISO组可使内向I_(NCX)密度从(-6.07±1.53)pA/pF增加至(-7.89±1.61)pA/pF(n=20,P<0.05),平均增加约30%。而预先使用G蛋白-cAMP-PKA通路关键分子激动剂或抑制剂后,ISO对I_(NCX)的效应均发生明显改变。其中,PTX及forskolin可使ISO对I_(NCX)密度增加效应更加显著,电流密度分别增至(-10.02±1.99)pA/pF及(-10.78±1.77)pA/pF,与单纯使用ISO相比,差异有统计学意义(n=20,P<0.01),提示Gi蛋白抑制和环磷酸腺苷激动均可增加ISO的作用。而预先应用H89,ISO增加I_(NCX)的效应几乎消失,电流密度约为(-6.22±1.70)pA/pF,与对照组相比无显著差异(n=20,P>0.05),提示抑制PKA可基本阻断ISO对I_(NCX)的刺激效应。结论β肾上腺素受体激动可增加NCX1.1内向电流,其可能通过G蛋白-cAMP-PKA信号通路参与调节。