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.

Polydatin attenuated food allergy via store-operated calcium channels in mast cell

AIM: To investigate the effect of polydatin (PD), a resveratrol glucoside, on mast cell degranulation and antiallergic activity. METHODS: After the rats were orally sensitized with ovalbumin (OVA) for 48 d and underwent PD treatment for 4 d, all the rats were stimulated by 100 mg/mL OVA for24 h and then sacrificed for the following experiments. The small intestines from all the groups were prepared for morphology examination by hematoxylin and eosin staining. We also used a smooth muscle organ bath to evaluate the motility of the small intestines. The OVA-specific immunoglobulin E (IgE) production and interleu-kin-4 (IL-4) levels in serum or supernatant of intestinal mucosa homogenates were analyzed by enzyme-linked immunosorbent assay (ELISA). Using toluidine blue stain, the activation and degranulation of isolated rat peritoneal mast cells (RPMCs) were analyzed. Release of histamine from RPMCs was measured by ELISA, and regulation of PD on intracellular Ca 2+ mobilization was investigated by probing intracellular Ca 2+ with fluo-4 fluo-rescent dye, with the signal recorded and analyzed. RESULTS: We found that intragastric treatment with PD significantly reduced loss of mucosal barrier integrity in the small intestine. However, OVA-sensitization caused significant hyperactivity in the small intestine of allergic rats, which was attenuated by PD administration by 42% (1.26 ± 0.13 g vs OVA 2.18 ± 0.21 g, P < 0.01). PD therapy also inhibited IgE production (3.95 ± 0.53 ng/mL vs OVA 4.53 ± 0.52 ng/mL, P < 0.05) by suppressing the secretion of Th2-type cytokine, IL-4, by 34% (38.58 ± 4.41 pg/mLvs OVA 58.15 ± 6.24 pg/mL, P < 0.01). The ratio of degranulated mast cells, as indicated by vehicles (at least five) around the cells, dramatically increased in the OVA group by 5.5 fold (63.50% ± 15.51% vs phosphate-buffered saline 11.15% ± 8.26%, P < 0.001) and fell by 65% after PD treatment (21.95% ± 4.37% vs OVA 63.50% ± 15.51%, P < 0.001). PD mediated attenuation of mast cell degranulation was further confirmed by decreased histamine levels in both serum (5.98 ± 0.17 vs OVA 6.67 ± 0.12, P < 0.05) and intestinal mucosa homogenates (5.83 ± 0.91 vs OVA 7.35 ± 0.97, P < 0.05). Furthermore, we demonstrated that administration with PD significantly decreased mast cell degranulation due to reduced Ca 2+ influx through store-operated calcium channels (SOCs) (2.35 ± 0.39vs OVA 3.51 ± 0.38,P < 0.01).CONCLUSION: Taken together, our data indicate that PD stabilizes mast cells by suppressing intracellular Ca 2+ mobilization, mainly through inhibiting Ca 2+ entry via SOCs, thus exerting a protective role against OVA-sensitized food allergy.

Ca^(2+)-induced myelin pathology precedes axonal spheroid formation and is mediated in part by store-operated Ca^(2+)entry after spinal cord injury

The formation of axonal spheroid is a common feature following spinal cord injury.To further understand the source of Ca^(2+)that mediates axonal spheroid formation,we used our previously characterized ex vivo mouse spinal cord model that allows precise perturbation of extracellular Ca^(2+).We performed twophoton excitation imaging of spinal cords isolated from Thy1YFP+transgenic mice and applied the lipophilic dye,Nile red,to record dynamic changes in dorsal column axons and their myelin sheaths respectively.We selectively released Ca^(2+)from internal stores using the Ca^(2+)ionophore ionomycin in the presence or absence of external Ca^(2+).We reported that ionomycin dose-dependently induces pathological changes in myelin and pronounced axonal spheroid formation in the presence of normal 2 m M Ca^(2+)artificial cerebrospinal fluid.In contrast,removal of external Ca^(2+)significantly decreased ionomycin-induced myelin and axonal spheroid formation at 2 hours but not at 1 hour after treatment.Using mice that express a neuron-specific Ca^(2+)indicator in spinal cord axons,we confirmed that ionomycin induced significant increases in intra-axonal Ca^(2+),but not in the absence of external Ca^(2+).Periaxonal swelling and the resultant disruption in the axo-myelinic interface often precedes and is negatively correlated with axonal spheroid formation.Pretreatment with YM58483(500 n M),a well-established blocker of store-operated Ca^(2+)entry,significantly decreased myelin injury and axonal spheroid formation.Collectively,these data reveal that ionomycin-induced depletion of internal Ca^(2+)stores and subsequent external Ca^(2+)entry through store-operated Ca^(2+)entry contributes to pathological changes in myelin and axonal spheroid formation,providing new targets to protect central myelinated fibers.

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

瞄准：学习肝的 ischemia/reperfusion ( I/R )的效果操作店的钙隧道( SOC )上的损害水流(我( SOC ))在刚孤立的老鼠 Kupffer ， Ca (2+)的房间，和效果隧道堵住 ers ， 2-aminoethoxydiphenyl 硼酸盐( 2-APB )， SK 和 F96365 ， econazole 和 miconazole ，在上我( SOC )在在肝的 I/R 损害以后的孤立的老鼠 Kupffer 房间。方法：老鼠的模型肝的 I/R 损害被建立。整个房间的斑夹钳技术被执行调查 2-APB， SK 和 F96365 的效果， econazole 和 miconazole 在上我(SOC ) 在孤立的老鼠 Kupffer 房间在以后肝我 /R 损害。结果：I/R 损害显著地增加了我(SOC ) 从 -80.4 +/- 25.2pA 到 -159.5 +/- 34.5pA ((b) P 【 0.01， n = 30 ) 。 2-APB ( 20 ， 40 ， 60 ， 80 ， 100 micromol/L )， SK 和 F96365 ( 5 ， 10 ， 20 ， 40 ， 50 micromol/L )， econazole ( 0.1 ， 0.3 ， 1 ， 3 ， 10 micromol/L )并且 miconazole ( 0.1 ， 0.3 ， 1 ， 3 ， 10 micromol/L )禁止我( SOC )以有 37.41 micromol/L 的 IC50 的一种集中依赖者方式( n = 8 )， 5.89 micromol/L ( n = 11 )， 0.21 micromol/L ( n = 13 )，并且 0.28 micromol/L ( n = 10 )。山峰价值(SOC ) 我被块 ers 在不同集中，而是反向的潜力在 I-V 关系中减少(SOC ) 我没被转变。结论：SOC 在肝的 I/R 损害期间是为 Ca (2+) 的流入的主要隧道。钙隧道块 ers， 2-APB， SK 和 F96365， econazole 和 miconazole，在 I/R 损害上有显然保护的效果，可能由禁止我(SOC ) 在 Kupffer 房间并且阻止 Kupffer 房间的激活。

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.

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.

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.

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.

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。

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。

Hepatitis B Virus X Protein Upregulates Intracellular Calcium Signaling by Binding C-terminal of Orai1 Protein

Hepatitis B virus X（HBx） protein plays a pivotal role in the development of hepatitis B virus（HBV）-associated hepatocellular carcinoma.Although regulation of cytosolic calcium is essential for HBV replication and is mediated by HBx protein,the mechanism of HBx protein regulating intracellular calcium level remains poorly understood.The present study examined whether HBx protein elevated the intracellular calcium through interacting with storeoperated calcium entry（SOCE） components,Orai1 and stromal interaction molecule 1,and then identified the targets of HBx protein,with an attempt to understand the mechanism of HBx protein upsetting intracellular calcium homeostasis.By employing co-immunoprecipitation and GST-pull-down assay,we found that Orai1 protein interacted with HBx protein,and the C-terminus of Orai1 was implicated in the interaction.Confocal microscopy also revealed that HBx protein could co-localize with full-length Orai1 protein in HEK293 cells.Moreover,live cell calcium imaging exhibited that HBx protein elevated intracellular calcium,possibly by binding to SOCE components.Our results suggest that HBx protein binds to STIM1-Orai1 complexes to positively regulate the activity of plasma membrane store-operated calcium channels.

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.

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.

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.

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。

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。

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。

Store-operated calcium entry in neuroglia

Neuroglial cells are homeostatic neural cells. Generally, they are electrically non-excitable and their activation is associated with the generation of complex intracellular Ca^2＋ signals that define the ＂Ca^2＋ excitability＂ of glia. In mammalian glial cells the major source of Ca^2＋ for this excitability is the lumen of the endoplasmic reticulum （ER）, which is ultimately （re）filled from the extracellular space. This occurs via store-operated Ca^2＋ entry （SOCE） which is supported by a specific signaling system connecting the ER with plasmalemmal Ca^2＋ entry. Here, emptying of the ER Ca^2＋ store is necessary and sufficient for the activation of SOCE, and without Ca^2＋ influx via SOCE the ER store cannot be refilled. The molecular arrangements underlying SOCE are relatively complex and include plasmalemmal channels, ER Ca^2＋ sensors, such as stromal interaction molecule, and possibly ER Ca^2＋ pumps （of the SERCA type）. There are at least two sets of plasmalemmal channels mediating SOCE, the Ca2＊-release activated channels, Orai, and transient receptor potential （TRP） channels. The molecular identity of neuroglial SOCE has not been yet identified unequivocally. However, it seems that Orai is predominantly expressed in microglia, whereas astrocytes and oligodendrocytes rely more on TRP channels to produce SOCE. In physiological conditions the SOCE pathway is instrumental for the sustained phase of the Ca^2＋ signal observed following stimulation of metabotropic receptors on glial cells.

哇巴因和乌头碱诱发豚鼠和大鼠心律失常的离子作用靶点

目的 观察畦巴因和乌头碱对豚鼠和大鼠心肌单细胞离子通道的作用,确定两药诱发心律失常时离子靶点和最佳靶点,建立细胞水平的心律失常模型。方法 用全细胞膜片钳技术记录哇巴因和乌头碱对酶解法分离的豚鼠和大鼠心肌细胞离子通道的作用。结果 5;μmol·L^(-1)哇巴因使豚鼠心肌细胞APD延长、I_(Ca-L)增加、I_k减少、I_(k1)减少;1μmol·L^(-1)乌头碱使大鼠心肌细胞APD延长、I_(Ca-L)增加、I_(to)减少、I_(k1)增加。结论 哇巴因和乌头碱诱发心律失常的离子靶点有APD,I_(Ca-L),I_k,I_(to)和I_(k1)而最佳靶点应为APD,I_(Ca-L),I_k和I_(to)。在单细胞水平分别应用哇巴因和乌头碱诱发豚鼠和大鼠心律失常.具有稳定性高、条件可控、重复性好等优点,可用于药物筛选和机制研究。

麻黄碱、β-细辛醚和去甲乌药碱对大鼠心肌细胞钙离子浓度和细胞膜钙通道的影响

目的初步筛查麻黄细辛附子汤的活性单体麻黄碱、β-细辛醚、去甲乌药碱对大鼠心肌细胞钙离子的作用,探讨3种活性单体对心肌细胞功能的影响。方法用酶解法急性分离大鼠心室肌细胞,采用Fluo-3/AM荧光探针测定麻黄碱、β-细辛醚、去甲乌药碱对大鼠心室肌细胞内钙离子浓度的影响。利用电生理片钳技术的全细胞电压钳方法测定心肌细胞内高电压依赖性钙离子通道钙电流(IC)a的变化。结果 (1)麻黄碱使心肌细胞内钙离子浓度升高,而细胞外液钙离子浓度为零时则无此现象。同时,麻黄碱使心肌细胞ICa峰电流密度显著增大;(2)β-细辛醚和去甲乌药碱分别使心肌细胞内钙离子浓度降低,ICa峰电流密度也呈轻度减弱趋势。结论麻黄碱能够增强ICa峰电流密度,提高细胞内钙离子浓度,因而具有兴奋心肌细胞的作用,此作用与高电压门控性钙通道开放有关;β-细辛醚和去甲乌药碱可通过降低细胞内钙离子浓度和轻微阻断钙离子内流而起到保护心肌细胞作用,避免钙超载的损害。麻黄碱、β-细辛醚和去甲乌药碱3者对心肌细胞内钙浓度和功能的影响符合中药方剂学的相反相成、补偿控制配伍理论。