Suxiao Jiuxin Pills Prevent Ventricular Fibrillation from Inhibiting L-type Calcium Currents CaV1.2 in vivo and in vitro / 中国结合医学杂志

OBJECTIVE@#To investigate whether Suxiao Jiuxin Pills (SJP), a Chinese herbal remedy, is an anti-ventricular fibrillation (VF) agent.@*METHODS@#VF was induced by isoproterenolol (ISO) intraperitoneal injection followed by electrical pacing in mice and rabbits. The effects of SJP on the L-type calcium channel current (CaV1.2), voltage-dependent sodium channel current (INa), rapid and slow delayed rectifier potassium channel current (IKr and IKs, respectively) were studied by whole-cell patch-clamp method. Computer simulation was implemented to incorporate the experimental data of SJP effects on the CaV1.2 current into the action potential (AP) and pseudo-electrocardiography (pseudo-ECG) models.@*RESULTS@#SJP prevented VF induction and reduced VF durations significantly in mice and rabbits. Patch-clamp experiments revealed that SJP decreased the peak amplitude of the CaV1.2 current with a half maximal concentration (IC50) value of 16.9 mg/L (SJP-30 mg/L, -32.8 ± 6.1 pA; Verapamil, -16.2 ±1.8 pA; vs. control, -234.5 ±16.7 pA, P<0.01, respectively). The steady-state activation curve, inactivation curve, and the recovery from inactivation of the CaV1.2 current were not shifted significantly. Specifically, SJP did not altered INa, IKr, and IKs currents significantly (SJP vs. control, P>0.05). Computer simulation showed that SJP-reduced CaV1.2 current shortened the AP duration, transiting VF into sinus rhythm in pseudo-ECG.@*CONCLUSION@#SJP reduced VF via inhibiting the CaV1.2 current with in vivo, in vitro, and in silico studies, which provide experimental basis for SJP anti-VF clinical application.

The regulation and gating mechanism of Cav1.2 current by KCNE1 subunit / 中华急诊医学杂志

Objective To explore the regulation and mechanism of Cav1.2 current by KCNE1. Methods Transient transfection was used to transfer Cav1.2 channel plasmids separately or together with KCNE1 plasmids into HEK293 cells. The experiment was divided into 2 groups (15 cells in each group):Cav1.2 group, Cav1.2+KCNE1 group.The whole-cell patch clamp technique was used to record Cav1.2 current and gating dynamics. Results After co-transfection of KCNE1 with Cav1.2, Cav1.2 current decreased significantly. At 0 mV, peak current density of Cav1.2 was reduced from (-14.8±2.5) pA/pF to (-7.5±1.6) pA/pF (n=15, P<0.01). Based on the gate control mechanism, it is found that the regulation of Cav1.2 current by KCNE1 mainly makes the steady-state inactivation curve of the channel shifted to a more negative direction, thus accelerating the inactivation. Meanwhile, the recovery process of the channel after inactivation is slowed down and the recovery time constant was prolonged. Conclusions The KCNE1 subunit can reduce the current density of Cav1.2 by changing the channel inactivation and recovery process.

The effects of low intensity pulsed ultrasound on the expression of calcium transportation associated proteins during dentin-pulp complex injury and repair in rats / 实用口腔医学杂志

Objective: To investigate low intensity pulsed ultrasound (LIPUS) on the expression of L-type calcium ion channels(cav1. 2) and Na +-Ca2 + exchangers(NCX1) during dentin-pulp complex injury and repair in rats. Methods: Cavity preparation was made on the upper right first molar of 40 male adult SD rats,20 of them and the upper left first molar of the other 20 were randomly chosen for LIPUS irradiation(frequency: 1. 5 MHz,200 μs pulses,pulse repetition frequency: 1 KHz,ISATA 30 mW/cm2,20 min /d),so the animals were randomly allocated into 4 groups(n = 10): Control group,LIPUS group,cavity preparation group and cavity preparation + LIPUS group. At 1,3,7,14 d post-irradiation the rats were sacrificed respectively for HE stain and immunohistochemical analysis. Results: Reparative dentin formation was observed at 14 days after cavity preparation and LIPUS irradiation,the expression of Cav1. 2(L-type) and NCX1 in this group were increased significantly at day 1 and day 3. Compared with the control group, the expression of Cav1. 2 in LIPUS group increased at day 1 post-irradiation. Conclusion: LIPUS may enhance tertiary dentin formation and up-regulate the expression of Cav1. 2 and NCX1 at the early period of dentin injury.

Effects of Daurisoline on L-Type Calcium Channel CaV1.2 Expressed in HEK293 Cells / 中国药师

Objective:To investigate the effects of daurinsoline (DS) on L-type calcium channel Cav1.2 expressed in HEK293 cells.Methods:Cav1.2 was transferred into HEK293 cells using Lipofectamine 2000, and the effects of DS on Cav1.2 currents (ICav1.2) were analyzed by whole-cell patch clamp techniques. Results:DS at 1,3 and 10 μmol·L-1could inhibit the ICav1.2in HEK293 cells in a dose-dependent manner. The inhibitory rate was(14.68 ± 4.02) %,(32.37 ± 6.63) % and(59.63 ± 5.23) %,respectively. The inhibitory rate of DS at 3 μmol·L-1was 40 % of that of 3 μmol·L-1isradipine(a L-type calcium channel blocker). Conclusion:DS can inhibit the ICav1.2in HEK293 cells in a dose-dependent manner and the inhibition of DS is weaker than that of isradipine.

Construction of and Protein Preparation from a Recombinant Plasmid Containing the Distal Fragment of the Distal C-terminus of the Cav1.2 Channel / 中国医科大学学报

Objective To construct a recombinant plasmid vector containing the distal fragment of the distal C-terminus (dDCT) of the Cav 1.2 channel,and express,extract,and purify dDCT protein and characterize its biological activity.Methods dDCT cDNA was ligated into the pGEX-6p-1 vector to create a recombinant plasmid that was subsequently transformed into Escherichia coli BL21 competent cells.Expression of GST-dDCT fusion protein from this plasmid was induced with isopropy-β-D-thiogalactoside,and the resulting protein was purified using glutathione-sepharose 4B beads.The biological activity of dDCT was analyzed by GST pull-down assay.Results The recombinant plasmid was verified by restriction enzyme digestion and sequencing.The concentration and purity of the dDCT protein,which was extracted by ultrasonication,were high enough to detect dDCT activity.The binding of dDCT to CT1 was determined to be concentration-dependent.Conclusion The recombinant dDCT plasmid was successfully constructed,providing the fundamental basis for future studies on mechanisms of Cav 1.2 channel autoregulation.

Effect of root saponins of Panax notoginseng on different voltage-dependent calcium and potassium ion channels / 中国药理学与毒理学杂志

OBJECTIVE To investigate the effect of root saponins of Panax notoginseng(RPNS) on different voltage-dependent calcium and potassium ion channels. METHODS By using the two-elec?trode voltage clamp (TEVC), the effect of RPNS 0.01, 0.06, 0.1, 0.6, 1 and 4 g · L- 1 was investigated on Cav1.2,and the effect of RPNS 1 g · L-1 was evaluated on Cav2.1,Cav2.2,Cav3.1, KCNH2,KCNQ1,KCNQ1/KCNE1 and BK channel. All the ion channels examined were expressed in Xenopus laevis oocytes. RESULTS TEVC suggested that the effect of RPNS on Cav1.2 exhibited the concentration-response relationship and its EC50 was 0.048 g · L-1. Compared with cell control,TEVC also showed that RPNS 1 g·L-1 had obviously inhibitory effect on Cav1.2,Cav2.2 and Cav3.1,and the inhibitory rate of RPNS 1 g · L-1 on the peak current of Cav1.2,Cav2.2 and Cav3.1 was(57.1 ± 8.6)%, (17.2 ± 0.7)% and(50.2 ± 7.7)%(P<0.01),respectively. RPNS 1 g · L-1 had obviously activated effect on BK channel,and the activated rate of RPNS 1 g·L-1 on the peak current of BK channel was(37.9± 2.7)%(P<0.01). RPNS 1 g·L-1 showed no significant effect on Cav2.1,KCNH2,KCNQ1 and KCNQ1/KCNE1. CONCLUSION RPNS may effectively inhibit Cav1.2 and Cav3.1,activate BK channel,but have little effect on Cav2.1,Cav2.2,KCNH2,KCNQ1 and KCNQ1/KCNE1.

Effect of tetrahydropalmatine on expression of Cav1. 2 in dorsal root ganglion neurons in mice with sciatic nerve chronic constriction injury / 中国药理学通报

Aim To investigate the analgesic effect of tetrahydropalmatine on Cav1 . 2 expression in the dorsal root ganglion ( DRG) of mice with sciatic nerve chronic constriction injury ( CCI ) -induced neuropathic pain. Methods Forty male C57 BL/6 mice were randomly divided into 5 groups ( n =5 ): sham group ( group S) , CCI group ( group C ) and L-THP group ( group L) . Steady mice models of neuropathic pain were es-tablished by inducing CCI of sciatic nerve. According to development of neuropathic pain in mice, L group was divided into induction period, induction with ma-intenance period and long-term low-dose group. The mice were intraperitoneally administered with 45 mg · kg-1 tetrahydropalmatine in induction ( day 0~5 ) , in-duction with maintenance ( day 0~5 , 14~19 ) period of neuropathic pain state. From the instant after opera-tion, 15 mg · kg-1 tetrahydropalmatine was injected into the long-term low-dose group once per day for 19 days. Then, the behavior changes of mice were moni-tored. Moreover, the threshold of mechanical and ther-mal stimuli was tested. In addition, the expression of Cav1 . 2 protein was detected by Western blot and im-munohistochemical staining. Results The lowest ex-pression of Cav1 . 2 was observed in group C and the highest expression level of Cav1 . 2 was found in group S. Cav1. 2 expression was significantly up-regulated in induction period group, induction with maintenance period group and long-term low-dose group ( P0. 05 ) in long-term low-dose group. Conclusions High dose of tet-rahydropalmatine in induction period group, induction with maintenance period group and low-dose among the whole experiment process obviously relieves the neuro-pathic pain induced by nerve injury. The analgesic effect of tetrahydropalmatine on neuropathic pain may be due to the increased expression of Cav1 . 2 protein in DRG neurons.