Perfect cellular platform for electrophysiology:acutely isolated vagal ganglion neurons from adult canines / 中国药理学与毒理学杂志

OBJECTIVE To establish a platform of electrophysiology using vagal ganglion neurons (VGNs) isolated from adult canines. METHODS The VGNs were enzymatically isolated from adult canines of either gender and cultured under experimental conditions. Action potential (AP), repetitive firing, voltage-gated outward K+ currents (IK) and hyperpolarization-mediated inward currents (Ih) were recorded under current-and voltage-clamp configurations before and after treatment. RESULTS Analysis of AP waveform showed that ① inaddition to traditionally classified myelinated A- and unmyelinated C-types, myelinated Ah-types could also be identified in females rather than in males; ② step current depolarization evoked a stimulus intensity-dependent repetitive discharge, and to reach a similar firing frequency, the lowest stimulus intensity was required for A-types, a similar or slightly higher stimulus intensity was needed for Ah-types, and the highest stimulus intensity was required for C-types;③tetro?dotoxin significantly reduced the rate of depolarization and positively shifted the AP firing threshold of Ah-types, and iberiotoxin dramatically increased the neuroexcitability of Ah-types;④all tested neurons functionally expressed IK and Ih, and the current density for both channels on average was A-types>Ah-types>C-types; ⑤ although the distribution of afferent types of VGNs differed between males and females, the known difference in discharge profiles of A- and C-types isolated from male and female rats was not studied here. CONCLUSION The VGNs can be successfully isolated from adult canines, AP, IK and Ih can be recorded. The tight seal can be held for at least 30 min, which may be enough for pharmacological investigation.

Effects of bone morphogenetic protein 2 and basic fibroblast growth factor 2 on proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cell sheet / 中国组织工程研究

BACKGROUND: The effects and mechanisms of bone morphogenetic protein 2 and basic fibroblast growth factor 2 on the proliferation and osteogenic differentiation of bone mesenchymal stem cells still remain unknown. How to combine the growth factors with tissue-engineered cell patch clamp techniques is of great significance for bone defect repair. OBJECTIVE: To explore the effects of bone morphogenetic protein 2 and basic fibroblast growth factor 2 applied alone or in combination on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cell sheet. METHODS: Bone marrow mesenchymal stem cells from Sprague-Dawley rats were isolated, cultured and identified in vitro to construct cell sheet. Bone morphogenetic protein 2 and basic fibroblast growth factor 2 at different concentrations were individually or jointly used to induce bone marrow mesenchymal stem cell sheet. The cell counting kit-8 assay combined with alkaline phosphatase activity assay was used to determine the optimal concentration of the two factors in promoting cell proliferation and osteogenic differentiation. Osteogenic induction of bone marrow mesenchymal stem cell sheet was assessed by gross and microscopic observations, Vonkossa staining, alizarin red staining, and RT-PCR detection. RESULTS AND CONCLUSION: The single application of bone morphogenetic protein 2 enhanced the alkaline phosphatase activity of the bone marrow mesenchymal stem cell sheet, and the optimal concentration was 100 μg/L (P < 0.001). The single application of basic fibroblast growth factor 2 accelerated the proliferation of bone marrow mesenchymal stem cell sheet, and the optimal concentration was 20 μg/L (P < 0.001). Their combination facilitated the proliferation of the cell sheet, and boosted the alkaline phosphatase activities (P < 0.001). The four groups of cell sheet showed no significant morphological difference, and the osteogenic differentiation of the bone marrow mesenchymal stem cell sheet could all be induced through the osteogenic induction. Calcium nodules were most significant in the combination group (P < 0.001), suggesting that the combination significantly facilitated late osteogenic differentiation, suppressed early osteogenic differentiation of the sheet and showed significant synergistic effect (P < 0.001). In summary, the application of bone morphogenetic protein 2 combined with basic fibroblast growth factor 2 plays a synergistic role in promoting the proliferation of bone marrow mesenchymal stem cell sheet and significantly enhances the osteogenic induction.

Effects of L-borneol on chloride channel and cell volume in human umbilical vein endothelial cells / 中国药理学通报

Aim To study the effects of L-borneol on the chloride channel and cell volume of human umbili-cal vein endothelial cells (HUVECs). Methods Whole-cell patch-clamp technique was used to record chloride currents. The expression of ClC-3 protein was down-regulated by siRNA interference technique. The cell volume was measured by dynamic image analysis. Results 20 nmol·L-1L-borneol significantly activa-ted chloride current in HUVEC (79.59 ± 4.90) pA/pF, which could be inhibited by chloride channel blockers,NPPB and DIDS. The outward current inhib-itory rate of NPPB was (95.57 ± 2.57)%, while that of DIDS was (97.28 ± 6.36)%. The chloride current activated by L-borneol significantly decreased after the silence of ClC-3 (27.03 ± 3.89) pA/pF. Cell volume was markedly reduced by L-borneol (14.38 ± 1.58)%,which was inhibited after NPPB appliance. Conclusion L-borneol can activate ClC-3 chloride channel in HUVECs, which induces Cl- outflow then cell volume decrease.

Change in hippocampal voltage-gated Ⅰ sodium channel of Lithium chloride-Pilocarpine epileptic rat model / 中华实用儿科临床杂志

Objective To research the changes in hippocampal voltage-gated sodium channel of Lithium chloride-Pilocarpine epileptic rat models,including Ⅰ sodium channel α subunit protein (Nav1.1),mRNA of Ⅰ sodium channel alpha subunit protein gene and function of sodium channel.Methods Epileptic rat models of Lithium chloride-Pilocarpine were established.Nav1.1 expression in the hippocampus of experimental rats was detected by immunohistochemical staining method,and the changes in voltage-gated sodium channel function (the current-voltage curves,activation and inactivation curves and the recovery curve) of hippocampus nerve cells were detected by whole cell patch-clamp technique.Results (1) The Lithium chloride-Pilocarpine rat models were successfully reproduced.Three stages of behavior (acute,latent and chronic) of rat models were observed.The blank control group was free of seizure.(2) Immunohistochemistry results:neurons in CA1 and DG regions of hippocampal of epileptic rats were normal,and there was no obvious change in the expression of Nav1.1.In CA3 area,the degeneration and necrosis of neurons were obvious.Staining of Nav1.1 became superficial and even disappeared in these areas,but the normal tissues were enhanced around degenerative and necrotic neurons.Compared with the blank control group,the expression of Nav1.1 in the model group was higher(0.235 ±0.008 vs.O.210 ±0.002),and there was statistically significant difference (t'=-7.426,P ＜ 0.05).(3) The whole-cell patch-clamp technique showed that the sodium current density of the model group increased significantly compared with that of the blank group [(-319.70 ± 28.24) pA/pF vs.(-229.06 ± 26.01) pA/pF,t =8.178,P ＜ 0.05],the threshold value of activation curve decreased (4.15 ± 0.80 vs.4.50 ±0.85,t =11.020,P ＜ 0.05),the threshold value of inactivation curve increased (7.47 ± 0.53 vs.6.24 ±0.31,t =6.940,P ＜ 0.05),and the recovery time after inactivation shortened [(1.36 ± 0.15) ms vs.(1.86 ± 0.21)ms,t =6.712,P ＜ 0.05],and there were all statistically significant differences.Conclusion Repeated seizures can lead to increase Nav1.1 compensatory expression of,and significantly increase sodium channel current density,while the threshold value of activation curve decreases,the threshold value of inactivation curve rises,and the recovery time after inactivation is shortened,which eventually leads to increased neuron excitability and is more likely to cause seizures.

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.

An improved method for isolation of human atrial cardiomyocytes / 重庆医学

Objective To study an improved isolated method of single human atrial myocytes.Methods Enzyme digestion method was used to isolate single myocytes from human atrial and whole-cell patch clamp technique was used to record small conductance calcium activated potassium current.Results This method obtained a large number of atrial myocytes.The total amount of atrial myocytes in SR group was 320±30 while AF group was 230±20 and the difference was statistically significant(P<0.01).In this study,a large number of simple and striated single atrial myocytes were obtained,and a typical small-conductance calcium-activated potassium channel current was recorded on the isolated atrial myocytes.Conclusion The established isolated method is simple,stable and effective.We can acquire a large amount of single atrial myocytes with good quality.

Investigation of effects of monoclonal antibody NCX-3F10against Na+/Ca2+ exchanger on rat cardiac ionic currentsand its suppression on ischemia-reperfusion induced cardiac arrhythmias / 中国药理学通报

Aim To observe the effect of antibody NCX-3F10 on the main ion current of rat ventricular myocytes and its effect on arrhythmias induced by ischemia/reperfusion(I/R).Methods ① The whole-cell patch clamp technique was employed to record the Na+/Ca2+ exchange current(INa/Ca) and other major ion currents in rat ventricular myocytes.② The rat models of arrhythmia induced by ischemia/reperfusion were established by ligating the left coronary artery to in vivo and in vitro.Then the effects of antibody on the arrhythmia were observed.③ The IonOptix ion imaging system was used to observe the effect of antibody on calcium transients in single ventricular myocytes.Results ① The antibody NCX-3F10 dose-dependently inhibited INa/Ca from 5 to 40 mg·L-1.The IC50 for outward and inward currents was 11.15 and 11.69 mg·L-1, and the maximum inhibitory rates were 61% and 62%, respectively.The antibody also had an inhibitory effect on calcium current(ICa-L), and had no significant effect on inward rectifier potassium current(IK1), transient outward potassium current(Ito) and sodium current(INa).② In the isolated rat heart group I/R, 100% rats showed ventricular tachycardia, and 88.89% rats had ventricular fibrillation.After administration of antibody NCX-3F10(10 mg·L-1) 5 min before reperfusion, the incidence of ventricular tachycardia decreased to 44.43%(P<0.05), and the duration of ventricular tachycardia and ventricular fibrillation was also shortened remarkably(P<0.05).③ In the anesthetized rats after administration of antibody NCX-3F10(50 μg·kg-1) 5 min before reperfusion, the incidence and duration of ventricular tachycardia,the incidence and duration of ventricular fibrillation, and total number of ventricular premature beats were significantly decreased(P<0.05).④ From 5 to 40 mg·L-1, NCX-3F10 antibody decreased calcium transient amplitude in rat single ventricular myocytes dose-dependently(P<0.05).Conclusions The NCX-3F10 antibody shows significant arrhythmic effects on ischemia-reperfusion induced arrhythmia in rats both in vitro and in vivo, the underlying mechanism of which is related to NCX and L-type calcium current inhibition and calcium overload reduction by the NCX antibody.

Effects of stress on the rapid component of delayed rectifier potassium current in rat cardiomyocytes / 解放军医学杂志

Objective To observe the effect of stress on the rapid component of delayed rectifier potassium current (IKr) in rat cardiomyocytes. Methods Forty male SD rats were randomly divided into four groups (10 each): control group (Ctrl), exhaustive group (ES), noise group (WN) and composite group (ES+WN). Stress animal models were prepared as follows: Rats in ES group were undergoing exhaustive swimming as the stress factor, in WN group undergoing white noise and in ES+WN group undergoing exhaustive swimming + white noise as the stress factor. Langendorff device was used to reversely perfuse collagenase for isolating the rat's ventricular myocytes. The effect of stress on IKr current and gating mechanism of single ventricular myocyte was recorded by whole-cell patch clamp technique. Results Compared with the Ctrl group, the tail current density of IKr (IKr,tail) of ventricular myocytes increased significantly in ES group and WN group (P<0.01). The IKr,tail current density of the ventricular myocytes in ES+WN group was significantly higher than that in ES group and WN group (P<0.01), and the effect was voltage dependent. Gating mechanism revealed that the half inactivation voltage of IKr,tail (V1/2,inact) can be shifted to the right in ES group, WN group and ES+WN group when compared with the Ctrl group, and the recovery time constant shortened after inactivation (P<0.01). However, the steady-state activation, fast inactivation constant and voltage dependence of IKr,tail were not statistically significant in ES group, WN group and ES+WN group when compared with the Ctrl group. Conclusion Stress increases the IKr current in rat cardiomyocytes, suggesting it be one of the electrophysiological bases of stress-induced arrhythmia.

Acepromazine inhibits hERG potassium ion channels expressed in human embryonic kidney 293 cells

The effects of acepromazine on human ether-à-go-go-related gene (hERG) potassium channels were investigated using whole-cell voltage-clamp technique in human embryonic kidney (HEK293) cells transfected with hERG. The hERG currents were recorded with or without acepromazine, and the steady-state and peak tail currents were analyzed for the evaluating the drug effects. Acepromazine inhibited the hERG currents in a concentration-dependent manner with an IC₅₀ value of 1.5 µM and Hill coefficient of 1.1. Acepromazine blocked hERG currents in a voltage-dependent manner between –40 and +10 mV. Before and after application of acepromazine, the half activation potentials of hERG currents changed to hyperpolarizing direction. Acepromazine blocked both the steady-state hERG currents by depolarizing pulse and the peak tail currents by repolarizing pulse; however, the extent of blocking by acepromazine in the repolarizing pulse was more profound than that in the depolarizing pulse, indicating that acepromazine has a high affinity for the open state of the channels, with a relatively lower affinity for the closed state of hERG channels. A fast application of acepromazine during the tail currents inhibited the open state of hERG channels in a concentration-dependent. The steady-state inactivation of hERG currents shifted to the hyperpolarized direction by acepromazine. These results suggest that acepromazine inhibits the hERG channels probably by an open- and inactivated-channel blocking mechanism. Regarding to the fact that the hERG channels are the potential target of drug-induced long QT syndrome, our results suggest that acepromazine can possibly induce a cardiac arrhythmia through the inhibition of hERG channels.

Effect of Xingnao Jieyu Decoction on the function of Notch/Hes signaling pathway in hippocampus of rats with post-stroke depression / 新乡医学院学报

Objective To investigate the effect of Xingnao Jieyu Decoction on the function of Notch/Hes signaling pathway in hippocampus of rats with post-stroke depression (PSD).Methods A total of 80 healthy male specific pathogen-free Wistar rats were randomly divided into control group,model group,prozac group and Xingnao Jieyu Decoction group;20 rats in each group.The rats in the model group,prozac group and Xingnao Jieyu Decoction group were used to prepare the cerebral ischemia model by middle cerebral artery occlusion method,and then the rats were used to prepare the depressive model by a variety of chronic unpredictable stimuli.The rats in the control group did not have any intervention.After the preparation of PSD model,the rats in the control group and model group were given saline 2.5 mL by intragastric administration,the rats in prozac group were given prozac 2.5 mL (1.8 mg · kg-1) by intragastric administration,the rats in Xingnao Jieyu Decoction group were given Xingnao Jieyu Decoction 2.5 mL (150.0 mg · kg-1) by intragastric administration;one times daily for 28 days.Ten rats in each group were sacrificed immediately on the nineteenth and twenty-eighth day of drug intervention,and the hippocampus tissues were isolated.The expression of Notch and Hes mRNA and protein in hippocampus were detected by real-time quantitative polymerase chain reaction and Western blot method.The open probability,current amplitude,opening time and closing time of Notch signaling pathway were detected by patch clamp technique.Results On the nineteenth and twenty-eighth day of drug intervention,the expression of Notch and Hes protein and mRNA in hippocampus of rats in the model group,prozac group and Xingnao Jieyu Decoction group was significantly higher than that in the control group (P ＜ 0.05);the expression of Notch and Hes protein and mRNA in hippocampus of rats in the prozac group and Xingnao Jieyu Decoction group was significantly higher than that in the model group (P ＜ 0.05);the expressions of Notch and Hes protein and mRNA in hippocampus of rats in the Xingnao Jieyu Decoction group was significantly higher than that in the prozac group (P ＜ 0.05).There was no significant difference in Notch and Hes protein and mRNA expression in hippocampus of rats between the nineteenth and twenty-eighth day of drug intervention (P ＞ 0.05).The expression of Notch and Hes protein and mRNA in hippocampus of rats on the twenty-eighth day was significantly higher than that on the nineteenth day in the model group,prozac group and Xingnao Jieyu Decoction group (P ＜ 0.05).Compared with the control group,the open probability and current amplitude of Notch signaling pathway in hippocampus of rats increased significantly,the opening time prolonged significantly,and the closing time shortened significantly in the model group,prozac group and Xingnao Jieyu Decoction group on the nineteenth and twenty-eighth day of drug intervention (P ＜ 0.05).Compared with the model group,the open probability and current amplitude of Notch signaling pathway in hippocampus of rats increased significantly,the opening time prolonged significantly,and the closing time shortened significantly in the prozac group and Xingnao Jieyu Decoction group on the nineteenth and twenty-eighth day of drug intervention (P ＜ 0.05).Compared with the prozac group,the open probability and current amplitude of Notch signaling pathway in hippocampus of rats increased significantly,the opening time prolonged significantly,and the closing time shortened significantly in the Xingnao Jieyu Decoction group on the nineteenth and twenty-eighth day of drug intervention (P ＜ 0.05).There was no significant difference in the open probability,current amplitude,opening time and closing time of Notch signaling pathway between the nineteenth and twenty-eighth day of drug intervention in the control group(P ＞ 0.05).Compared with the nineteenth day of drug intervention,the open probability and current amplitude of Notch signaling pathway in hippocampus of rats increased significantly,the opening time prolonged significantly,and the closing time shortened significantly on the twenty-eighth day of drug intervention in the model group,prozac group and Xingnao Jieyu Decoction group (P ＜ 0.05).Conclusion Xingnao Jieyu Decoction can mediate the activation of Notch / Hes signaling pathway in hippocanpus of PSD rats,thus effectively promote the recovery of neurological function.Notch/Hes signaling pathway is expected to be an important target for drug action.

Cardiac arrhythmia suppression by I K1 channel agonist in isoproterenol-induced myocardial hypertrophic rats and investigation on its mechanism / 中国药理学通报

Aim To investigate the effect of zacopride ( Zac) on cardiac arrhythmia in isoproterenol ( ISO)-in-duced myocardial hypertrophic rats and the underlying electrophysiological mechanisms .Methods ① Fifty-one rats were randomly divided into control group ( n=17 ) , ISO group ( n=17 ) and ISO +Zac group ( n =17 ) .Rat model with cardiac arrhythmia and hypertro-phy was established by intraperitoneal ISO ( 5 mg?kg -1 ) injection.②ECGs were recorded to observe the effects of Zac on arrhythmia in model rats .③ Whole-cell patch clamp was applied to record inwardly rectifi-er potassium current(IK1), resting membrane potential ( RMP ) and amplicated delayed afterdepolarizations (DADs).Results ① Echocardiographic examination showed that , left ventricular end-diastolic dimension (LVEDD) and left ventricular end-systolic dimension (LVESD) significantly decreased in rats in ISO group compared with control group , whereas left ventricular posterior wall end-diastolic thickness ( LVPWd) and in-terventricular septum end-diastolic thickness ( IVSd ) increased ( P<0.05 ) , suggesting rat model of isoprot-erenol-induced myocardial hypertrophy was successfully established .② ECGs showed that 88.89% of rats in ISO group had ventricular premature beats ( VPBs ) , which significantly decreased to 11.11% after the ap-plication of Zac ( P <0.05 ) .③ Values of RMP de-creased from ( -71.05 ±1.27 ) mV in control group to (-69.38 ±1.21 ) mV in ISO group ( P<0.05 ) . After Zac administration , RMP significantly increased to ( -73.86 ±1.33 ) mV compared with control and ISO group(P<0.05).④DADs and TA incidence sig-nificantly decreased from 88.24% in ISO group to 11.76%in ISO+Zac group ( P<0.05 ) .⑤ Compared with control group , IK1 density was markedly reduced in ISO group, whereas Zac could effectively rescue IK1 suppression to normal level .Conclusions Zac, as a selective IK1 channel agonist , can significantly inhibit cardiac arrhythmia in isoproterenol-induced myocardial hypertrophic rats , which is mainly attributed to in-creased RMP by enhancing IK1 and subsequent suppres-sion of DADs.

The reverse effects of allitridum on sodium current decrease caused by SCN5A-F1473S mutation / 药学学报

This study was designed to test the allitridum (All) activity in correction of sodium current decrease caused by SCN5A-F1473S mutation in HEK293 cells. The result may provide a theoretical basis for screening of new drugs in the treatment of Brugada syndrome. We transferred SCN5A-F1473S channel plasmids into HEK293 cells in a transient transfection. All was administrated acutely and chronically using extracellular irrigation flow and co-culture model. The concentration of All was 30 μmol·L-1. We used whole cell patch clamp technique in voltage clamp mode to record current and gating kinetics. In order to explore the rescue function of All on decreased sodium peak current, we used confocal microscopy and Western blot to detect the expression of channel proteins in the cell membrane. We found a significant increase in sodium peak current of the 30 μmol·L-1 All HEK293 cells (269.8±16.6 pA/pF, PPV1/2,inact returns to -79.5±2.4 mV, PPPSCN5A-F1473S mutation cells. We consider that the main mechanism may be related to the reduced channel inactivation by the drug with an improvement of the migration barrier of the mutational channel.

Protective effects of choline on myocardial ischemic rat heart and its potential mechanisms / 重庆医学

Objective To study the protective effects of choline on myocardial ischemia rat heart and its potential mecha -nisms .Methods Ischemia hypoxia environment was simulated with low value of pH (pH 6 .8) and lack of oxygen .Calcium currents were recorded by whole cell patch under the voltage clamp configuration .The alternations in[Ca2 + ]induced by KCl was detected by laser scanning confocal microscope in ventricular myocytes ,then disccuss the effects of choline on calcium and calcium store in cells . Results The normalized peak currents of ICa-L in ventricular myocytes were larger in pH 6 .8 group than those in pH 7 .4 group , which can be attenuated by choline .The(Ca2 + )i induced by KCl in ventricular myocytes were significantly increased in pH 6 .8 Ty-rode solution and its increasing can be suppressed by choline .4-DAMP can inhibit the suppressing effect of choline .Conclusion The possible mechanism of M 3 receptor involved in the protection of ischemic myocardium by inhibiting myocardial cells in ICa-L ,in-hibiting intracellular calcium overload .

Protein kinase C enhances the swelling-induced chloride current in human atrial myocytes / 华中科技大学学报（医学）（英德文版）

Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.

Influence of new baicalin-metal complexes on Kv1.4 and Cav3.2 ion channels / 中国药理学与毒理学杂志

OBJECTIVE To investigate the effect of new baicalin(BC) metal ions(Co2+,Cu2+, and Ni2+)complexes(BMCs)on ion channels Kv1.4 and Cav3.2. METHODS HEK293 or CHO cells loaded with various ion channels(hERG,Kv1.2,Kv1.3,Kv1.4,Kv1.5,Kv1.6,Kv1.7,Kv1.8,Kir1.1, Kir2.1,KCNQ and Cav3.2)were obtained by stable transfection method. Whole-cell patch-clamp tech?nique was used to record current changes of each ion channel induced by BC and BMC in 10μmoL · L-1. The effect of different concentrations(0.3,1,3,10 and 30μmoL · L-1)of BC-Co and BC-Cu on Kv1.4 and Cav3.2 current was detected by whole-cell patch-clamp technique. RESULTS A model of HEK293 cells or CHO cells that stably expressed various ion channels was obtained. BMCs (BC-Co,BC-Cu and BC-Ni)had some impact on various ion channels,especially on Kv1.4 and Cav3.2. The inhibitory rate induced by BC-Co,BC-Cu and BC-Ni(10 μmol · L-1)was 91%,76% and-10%,respectively,for Kv1.4;and 43%,57%and-14%,respectively,for Cav3.2. IC50 of BC-Co was 1.69 and 0.81μmoL·L-1 for Kv1.4 and Cav3.2. IC50 of BC-Cu was 1.66 and 0.58μmoL · L-1 for Kv1.4 and Cav3.2. CONCLUSION BC-Cu and BC-Co concentration-dependently inhibit Kv1.4 and Cav3.2 ion channels.

Roles of chloride channels in apoptosis induced by adriamycin in nasopharyngeal carcinoma cells / 中国药理学通报

Aim To investigate the roles of chloride channels in the apoptosis and apoptotic volume de-crease (AVD)induced by adriamycin in nasopharyn-geal carcinoma CNE-2Z cells.Methods Apoptotic rates were detected by flow cytometry,and the volume changes were measured by the time-lapse live cell ima-ging technique.The patch clamp technique was used to record whole-cell chloride currents.Results Adria-mycin induced apoptosis of CNE-2Z cells.An early ap-optotic volume decrease was observed in the cell trea-ted with adriamycin.The cell volume was decreased by about 10% in 2 h.Adriamycin activated a chloride current which showed outward rectification.The chlo-ride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB ) could inhibit the adriamycin-in-duced chloride currents,apoptosis and prevent cell shrinkage.Conclusions Our findings suggest that ad-riamycin causes cell apoptosis by activation of chloride channels.Chloride channels may be involved in the apoptosis and apoptotic volume decrease induced by adriamycin in CNE-2Z cells.

Regulatory effect of protein tyrosine phosphatase non-receptor type on the cardiac Kv11.1 (HERG) potas-sium channel / 医学研究生学报

Objective Cardiac HERG potassium channel currents can cause long QT syndrome .The function of the cardiac HERG potassium channel is regulated by tyrosine kinase and phosphatase , and protein tyrosine phosphatase non-receptor type 11 ( PTPN11) negatively regulates the HERG potassium channel .This study aimed to investigate the effect of PTPN 11 on the electrophysio-logical characteristics of the HERG channel . Methods The plasmids of pcDNA3.1-PTPN11-EGFP were constructed by PCR technique and transfected or cotransfected with the pcDNA 3.1-PTPN11-EGFP plasmid into HEK293 cells using Lipofectamine 2000.The patch clamp technique was employed to record the HERG channel currents in the control group ( HEK293 cells transfected with pcDNA3.0-HERG-EGFP), PTPN11 overexpression group (pcDNA3.0-HERG and pcDNA3.1-PTPN6-EGFP cotransfected HEK293 cells), and PTPN11 overexpression with PAO group . Results The pcDNA3.1-PTPN11-EGFP plasmid was successfully constructed .Green fluorescence was observed in the HEK293 cells transfected with pcDNA3.0-HERG-EG-FP or cotransfected with pcDNA3.0-HERG and pcDNA3.1-PTPN11.The maximum densities of pulse and tail currents were significantly decreased in the PTPN11 overexpression group as compared with the control ([31.85 ±5.54] vs [45.92 ±3.18] pApF, P<0.05;[73.82 ±11.31] vs [108.43 ±7.98] pApF, P<0.05) but markedly in-creased in the PTPN11 overexpression with PAO group ([48.08 ±4.32] pApF;[120.06 ±8.02] pApF) (P<0.05).The time con-stant of deactivation was significantly higher in the PTPN 11 overexpression group than in the control ([622.16 ±46.49] vs [440.70 ± 49.49] ms, P<0.05). Conclusion The overexpression of PTPN11 decreases HERG potassium channel currents , which can be re-versed by PAO.This finding provides a theoretical basis for the application of certain regulatory enzymes in the HERG channel as a treat -ment of long QT syndrome .

Preparation and measurement of sodium tanshinone Ⅱ-A sulfonate in mice serum and effects on big conductance calcium-activated potassium channels in porcine coronary artery smooth muscle cells / 重庆医学

Objective To discuss a method combining serum‐pharmacology and electrophysiology technology ,and to research the mechanism of dilating porcine coronary artery of sodium tanshinone Ⅱ‐A sulfonate (DS‐201) .Methods To give mice intragastric administration solution and measure DS‐201 concentration in mice serum ,and apply the serum to single channel patch to research its effect on big conductance calcium‐activated potassium channels(BKca ) in porcine coronary artery smooth muscle cells (PCASMCs) . Results The linear range of concentration containing DS‐201 serum was 0 .73 to 1 .91 μg/mL (r=0 .997 7) ,the recycle rate was 99 .85% -101 .47% ,and the concentration was(7 .32 ± 4 .25)μg/mL ;the result indicates that serum containing DS‐201 has activa‐tion effects on BKCa in PCASMCs ,while there was no statistical significance (P>0 .05) .Conclusion The establishment method of the alcohol extraction of Danshen is useful and reliable .The HPLC method of measuring DS‐201 concentration is precise .Choosing higher quality drugs or raising bioavailability can improve combination of the serum pharmacology and electrophysiological tech‐nique .

Phasic and tonic type A γ‑Aminobutryic acid receptor mediated effect of Withania somnifera on mice hippocampal CA1 pyramidal Neurons.

Background: In Nepali and Indian system of traditional medicine, Withania somnifera (WS) is considered as a rejuvenative medicine to maintain physical and mental health and has also been shown to improve memory consolidation. Objective: In this study, a methanolic extract of WS (mWS) was applied on mice hippocampal CA1 neurons to identify the receptors activated by the WS. Materials and Methods: The whole cell patch clamp recordings were performed on CA1 pyramidal neurons from immature mice (7‑20 postnatal days). The cells were voltage clamped at ‑ 60 mV. Extract of WS root were applied to identify the effect of mWS. Results: The application of mWS (400 ng/μl) induced remarkable inward currents (‑158.1 ± 28.08 pA, n = 26) on the CA1 pyramidal neurons. These inward currents were not only reproducible but also concentration dependent. mWS‑induced inward currents remained persistent in the presence of amino acid receptor blocking cocktail (AARBC) containing blockers for the ionotropic glutamate receptors, glycine receptors and voltage‑gated Na+ channel (Control: ‑ 200.3 ± 55.42 pA, AARBC: ‑ 151.5 ± 40.58 pA, P > 0.05) suggesting that most of the responses by mWS are postsynaptic events. Interestingly, these inward currents were almost completely blocked by broad GABAA receptor antagonist, bicuculline‑ 20 μM (BIC) (BIC: ‑1.46 ± 1.4 pA, P < 0.001), but only partially by synaptic GABAA receptor blocker gabazine (1 μM) (GBZ: ‑18.26 ± 4.70 pA, P < 0.01). Conclusion: These results suggest that WS acts on synaptic/extrasynaptic GABAA receptors and may play an important role in the process of memory and neuroprotection via activation of synaptic and extrasynaptic GABAA receptors.

Effects of taurine-magnesium coordination compound on abnormal calcium current induced by hypoxia-reoxygenation in rat ventricular cardiomyocytes / 中国药理学通报

Aim To investigate the effects of TMCC on abnormal L-type calcium current (ICa,L) in rat ventric-ular cardiomyocytes during hypoxia-reoxygenation to find out the mechanism of antiarrhythmic effect. Methods Whole-cell patch clamp was used to record ICa,L in the ventricular cardiomyocytes during hypoxia-reoxygenation in rat under amiodarone and different concentrations of TMCC. Results In hypoxia-reoxy-genation model, peak ICa,L increased from ( 3. 35 ± 0. 50 ) pA/pF to ( 5. 69 ± 0. 25 ) pA/pF ( n =6 , P 0. 05),(4. 41 ± 0. 22) pA/pF, (3. 82 ± 0. 21)pA/pF(n=6, P<0. 01) by TMCC(100, 200, 400 μmol·L-1 ) and amidodarone 24. 24 μmol·L-1 restored peak ICa,L to(3. 66 ± 0. 27)pA/pF (n=6,P<0. 01 ) . Compared to control group, hypoxia-reoxy-genation turned ICa,L steady-state activation curves to left and inactivation curves to right, which quickened activation and slowed inactivation, TMCC ( 200, 400μmol · L-1 ) and amiodarone could restore the left shift activation curves and right shift inactivation curves. Conclusion TMCC can concentration-de-pendently restore the increase of calcium current due to hypoxia-reoxygenation by promoting inactivation process and inhibiting activation process, and the effect is equal to that of amiodarone. TMCC blocks ICa,L of the ventricular cardiomyocytes, which may be one of its antiarrhythmic mechanisms.