Effect of microparticles derived from bone marrow mesenchymal stem cells on cardiomyocyte hypertrophy and its mechanism / 中国生物制品学杂志

@#Objective To investigate the effect of microparticles(MPs)derived from bone marrow mesenchymal stem cells(BMSCs) on myocardial hypertrophy and its mechanism.Methods The osteogenic differentiation and adipogenic differentiation of mesenchymal stem cells(MSCs) were induced. After isolation and purification,the morphological characteristics were observed by transmission electron microscope,and the MPs surface antigen was identified by flow cytometry. Myocardial hypertrophy model was induced by using isoprenaline(ISO)in rats,which were measured for the cardiac structure and function by echocardiography,and then detected for various indexes of the heart and isolated left ventricle. Single ventricular myocytes of rats were acutely isolated and divided into control group(Control group),cardiomyocyte hypertrophy group(ISO group),MPs group(MPs group),and MPs supernatant group(Supernatant group). The mRNA expressions of atrial natriuretic peptide(ANP)and B-type natriuretic peptide(BNP)were detected by qRTPCR. The expression levels of calmodulin-dependent protein kinaseⅡ（CaMKⅡ）and phosphorylated calmodulin-dependent protein kinaseⅡ（p-CaMKⅡ）were detected by ELISA. The L-type calcium current(LCa-L)in single ventricular myocyte of various groups was recorded by whole-cell patch clamp.Results The bone nodules of MSCs osteogenic differentiation turned red after alizarin red staining,and lipid droplets of adipogenic differentiation turned red after oil red O staining;Under transmission electron microscope,MPs membrane had a complete structure,a clear outline and a diameter of about200 nm;The positive rates of CD29 and CD90 on the surface of MPs were(98. 24 ± 0. 82)% and(97. 69 ± 1. 83)%,respectively. Compared with Control group,the left ventricular end diastolic dimension(LVEDD)reduced signifi-cantly(t =5. 065,P < 0. 05),while the interventricular septum end-diastolic dimension(IVSd),left ventricular posterior wall dimension(LVPWd),heart weight to body weight ratio(HW/BW),and heart weight to tibial length ratio(HW/Tibia)significantly increased in ISO group(t = 4. 013,2. 368,4. 392,5. 043 and 6. 120,respectively,each P < 0. 05),indicating that the hypertrophic model was successfully established. The expression levels of ANP and BNP mRNA in hypertrophic cardiomyocytes of rats in ISO group were significantly higher than those in Control group(t = 25. 120 and18. 261,respectively,each P < 0. 01);While the expression levels of ANP and BNP mRNA in MPs group significantly reduced after incubation with 48 μg/mL MPs for 48 h compared with ISO group(t = 12. 110 and 3. 526,respectively,each P < 0. 05);The expression levels of CaMK Ⅱand p-CaMKⅡ in ISO group were significantly higher than those in Control group(t = 3. 278 and 4. 181,respectively,each P < 0. 05),while the expression of p-CaMK Ⅱ in MPs group decreased significantly(t = 5. 420,P < 0. 05);The calcium current density in ISO group was significantly higher than that in Control group(t = 15. 261,P < 0. 01),while that in MPs group was significantly lower than that in ISO group(t =6. 216,P < 0. 05).Conclusion MSC-MPs can significantly inhibit ISO-induced cardiomyocyte hypertrophy in rats,which is related to its down-regulation of cardiomyocyte CaMKⅡ and inhibition of L-type calcium channel.

Potentiation of PIEZO2 mechanically-activated currents in sensory neurons mediates vincristine-induced mechanical hypersensitivity

Vincristine, a widely used chemotherapeutic agent for treating different cancer, often induces severe peripheral neuropathic pain. A common symptom of vincristine-induced peripheral neuropathic pain is mechanical allodynia and hyperalgesia. However, mechanisms underlying vincristine-induced mechanical allodynia and hyperalgesia are not well understood. In the present study, we show with behavioral assessment in rats that vincristine induces mechanical allodynia and hyperalgesia in a PIEZO2 channel-dependent manner since gene knockdown or pharmacological inhibition of PIEZO2 channels alleviates vincristine-induced mechanical hypersensitivity. Electrophysiological results show that vincristine potentiates PIEZO2 rapidly adapting (RA) mechanically-activated (MA) currents in rat dorsal root ganglion (DRG) neurons. We have found that vincristine-induced potentiation of PIEZO2 MA currents is due to the enhancement of static plasma membrane tension (SPMT) of these cells following vincristine treatment. Reducing SPMT of DRG neurons by cytochalasin D (CD), a disruptor of the actin filament, abolishes vincristine-induced potentiation of PIEZO2 MA currents, and suppresses vincristine-induced mechanical hypersensitivity in rats. Collectively, enhancing SPMT and subsequently potentiating PIEZO2 MA currents in primary afferent neurons may be an underlying mechanism responsible for vincristine-induced mechanical allodynia and hyperalgesia in rats. Targeting to inhibit PIEZO2 channels may be an effective analgesic method to attenuate vincristine-induced mechanical hypersensitivity.

Isolation, identification and electrophysiological activity of BmK M2 from Buthus martensii Karsch venom / 中国药科大学学报

@#This study aimed to isolate and identify novel toxin peptides targeting voltage-gated sodium channels (VGSGs) from the venom of the Buthus martensii Karsch (BmK) scorpion. Using G50-gel filtration, HPLC, peptide fingerprinting and amino acid sequencing, a novel sodium channel modulator, BmK M2, was identified from BMK scorpion. BmK M2 is a relatively abundant long chain polypeptide toxin in BmK scorpion venom with a molecular weight of 7 235.59, consisting of 64 amino acids and 4 pairs of disulfide bonds.Sequence alignment showed that the amino acid sequence of BmK M2 had high sequence and structural similarity to that of the discovered sodium channel toxins of BmK M1, BmK M3 and BmK M9, etc.BmK M2 is a potential new sodium channel modulator.Electrophysiological results revealed that BmK M2 can significantly enhance the activation, delay the steady-state inactivation and closed-state inactivation of Nav1.7, but has no activity on Nav1.8.BmK M2 can be used as a novel peptide probe for the study of the structure and function of Nav1.7 and the development of drugs targeting Nav1.7.

Effect of Sodium Salicylate on Calcium Currents and Exocytosis in Cochlear Inner Hair Cells: Implications for Tinnitus Generation / 神经科学通报·英文版

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 Ca2+ current. Using noise analysis of Ca2+ tail currents and qRT-PCR, we further found that salicylate increased the number of Ca2+ channels along with CaV1.3 expression. All these changes could act synergistically to enhance the Ca2+ influx into IHCs. Inhibition of intracellular Ca2+ 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.

Study of electrophysiological mechanism of dopamine inhibiting insulin secretion by Kv channels / 中国药理学通报

Aim To study the electrophysiological mechanism of dopamine inhibiting insulin secretion hv voltage-dependent potassium ( Kv) channels.Methods Islets and (3 cells were isolated from male SD rats.D,-like receptor agonist ( SKP38393), D2-like receptor agonist (Quinpirole) and antagonist (Epiclopride) were used according to the experiment.Insulin secretion was detected by insulin radioimmunoassay.Whole-cell j J patch-clamp technique was applied to detect Kv channel currents and action potential duration of p cells.Di- BAC4(3) staining was used to observe membrane potential.Results SKF38393 did not affect insulin secretion and the Kv channel currents.Quinpirole signifi cantly inhibited insulin secretion and increased Kv channel currents.Dopamine significantly inhibited insulin secretion, increased Kv channel currents and shortened action potential duration of p cells, which could be reversed by epiclopride.In addition, dopamine de-creased membrane potential of INS-1 cells.Conclusions Dopamine inhibits insulin secretion by acting on D2-like receptors, resulting in actived Kv channels, shortened action potential duration and decreased cell membrane potential.

The insulinotropic effects and its relevant mechanism of telmisartan in rats / 中国药理学通报

Aim To investigate the insulinotropic effect of telmisartan anrl the underlying electrophysiological mechanism.Methods Islets and cells were isolated from Wistar rats.Islets were incubated with drugs un¬der different conditions, then supernatant liquid was collected for insulin secretion.Intracellular Ca" + ( Ca'+ j) levels of (3-cells were measured by calcium imaging technology.Patch-clamp technology was ap¬plied to detect effects on voltage-gated potassium chan¬nel ( Kv ) , and voltage-gated calcium channel ( VGCC ).Results Not affecting insulin secretion un¬der low glucose condition, telmisartan dose-dependent- ly stimulated insulin secretion under high glucose con¬ dition, and stimulation was enhanced with increasing glucose concentration.Acute increases of Ca' + concentration were elicited by telmisartan under high glucose condition.Telmisartan decreased current den¬sity of Kv channel, and increased VGCC current densi¬ty.Conclusions Telmisartan enhanced Ca~+ ; lev¬els of p-cells through its action on Kv channel and VGCC, thereby amplifying glucose-stimulated insulin secretion.

Effects of daidzein on Inain rat ventricular myocytes / 中国药理学通报

Aim To study the effects of daidzein on sodium channel current ( /Na) in ventricular myocytes of rats and the mechanism of its antiarrhythmia. Methods The effect of daidzein on the viability of ventricular myocytes was delected by MTT assay; single ventricular myocytes from rats were isolated by single enzymatic hydrolysis; the changes of /N, and its dynamic characteristics in rat ventricular myocytes before and after administration of daidzein were observed, recorded and analyzed by cell patch clamp technique. Results MTT experiments showed that the ICjo of daidzein was 30 to 100 jjimol • L"1 ,so the concentration of 0. 3 - 10 jimol • L"1 was chosen for the subsequent experiments. When daidzein was given 0. 3,1,3,10 pjnol • L"',the /Nb amplitude of ventricular myocytes in rats showed a concentration-dependent inhibition. The concentration of daidzein 0. 3 imol • L"1 also had certain effect on the time course of /Nt. The /Nl, peak decreased gradually over time. The 1,3,10 jimol • L"1 daidzein raised the I-U curve obviously. Under the same condition, the activation curve moved to the direction of depolarization. The steady-state inactivation curve shifted toward hyperpolarization, and the t value of the recovery curve was prolonged after inactivated state. Conclusions Daidzein significantly inhibited the Na∗ channel of ventricular myocardium in rats, which may l)e one of its mechanisms of anti-arrhythmia.

Nitric oxide modulates ATP-evoked currents in mouse Leydig cells

Testosterone synthesis within Leydig cells is a calcium-dependent process. Intracellular calcium levels are regulated by different processes including ATP-activated P2X purinergic receptors, T-type Ca2+ channels modulated by the luteinizing hormone, and intracellular calcium storages recruited by a calcium-induced calcium release mechanism. On the other hand, nitric oxide (NO) is reported to have an inhibitory role in testosterone production. Based on these observations, we investigated the interaction between the purinergic and nitrergic systems in Leydig cells of adult mice. For this purpose, we recorded ATP-evoked currents in isolated Leydig cells using the whole cell patch clamp technique after treatment with L-NAME (300 μM and 1 mM), L-arginine (10, 100, 300, and 500 μM), ODQ (300 μM), and 8-Br-cGMP (100 μM). Our results show that NO produced by Leydig cells in basal conditions is insufficient to change the ATP-evoked currents and that extra NO provided by adding 300 μM L-arginine positively modulates the current through a mechanism involving the NO/cGMP signaling pathway. Thus, we report an interaction between the nitrergic and purinergic systems in Leydig cells and suggest that Ca2+ entry via the purinergic receptors can be regulated by NO.

Inhibitory effect of monoclonal antibody NCX-2D2 on isoproterenol-induced arrhythmias in adult rat hearts / 中国药理学通报

Aim To investigate the effects of mono-clonal antibody NCX-2D2 on isoproterenol-induced ar-rhythmias in rat hearts, and to explore the electrophys-iological mechanism. Methods Using isoproterenol to establish in vitro and in vivo arrhythmic rat models to observe the effect of NCX-2D2 antibody on ventricular arrhythmias in rats. The whole-cell patch clamp tech-nique was used to investigate the effects of NCX-2D2 antibody on INa/Ca, ICa-Lat voltage-clamp mode and on DADs at current-clamp mode in single rat ventricular myocytes. Results 10 mg·L-1NCX-2D2 antibody significantly inhibited cardiac arrhythmias induced by ISO in vitro ( P<0.01) . 80 μg·kg-1NCX-2D2 anti-body markedly inhibit the occurrence of arrhythmias in ISO-induced anesthetized rats in vivo ( P <0.01 ) . 5 mg·L-1NCX-2D2 antibody partially inhibited the in-crease of INa/Ca(P<0.01) and the increase of ICa-L(P<0.01 ) , and could effectively inhibit ISO-induced DADs in rat ventricular myocytes ( P <0.05 ) . Con-clusions The sodium-calcium exchanger monoclonal antibody NCX-2D2 significantly inhibits isoproterenol-induced ventricular arrhythmias in rats. The mecha-nism against ventricular arrhythmias is mainly due to its inhibition of cardiomyocyte sodium-calcium exchanger and L-type calcium channel and marked suppression of DADs in rat ventricular myocytes.

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.

Role of Pre-Synaptic NMDA Receptors in the Modulation of Inhibitory Synaptic Transmission in Sensory-Motor and Visual Cortical Pyramidal Neurons in Brain Slices of Young Epileptic Mice

Background: Previous studies from animal models have shown that pre-synapticNMDA receptors (preNMDARs) are present in the cortex, but the role of inhibition mediated bypreNMDARs during epileptogenesis remains unclear. In this study, we wanted to observe thechanges in GABAergic inhibition through preNMDARs in sensory-motor and visual corticalpyramidal neurons after pilocarpine-induced status epilepticus.Methods: Using a pilocarpine-induced epileptic mouse model, sensory-motor and visualcortical slices were prepared, and the whole-cell patch clamp technique was used to recordspontaneous inhibitory post-synaptic currents (sIPSCs).Results: The primary finding was that the mean amplitude of sIPSC from the sensorymotorcortex increased significantly in epileptic mice when the recording pipette contained MK-801 compared to control mice, whereas the mean sIPSC frequency was not significantly different,indicating that post-synaptic mechanisms are involved. However, there was no significant presynapticinhibition through preNMDARs in the acute brain slices from pilocarpine-inducedepileptic mice.Conclusion: In the acute case of epilepsy, a compensatory mechanism of post-synapticinhibition, possibly from ambient GABA, was observed through changes in the amplitude withoutsignificant changes in the frequency of sIPSC compared to control mice. The role of preNMDARmediatedinhibition in epileptogenesis during the chronic condition or in the juvenile stagewarrants further investigation.

Alizarin induced the function of beta subunits of BKCa channel relaxed interlobar renal artery and reduced SHR systolic pressure / 实用医学杂志

Objective To investigate the effect of alizarin on BKCa channel beta subunit and electrophysiological characteristics in interlobar renal artery of SHR.Methods Tail artery pressure measurement instrument,pressure myograph system,whole cell patch clamp technique and Western blot were used to measure the change of systolic pressure of SHR,renal interlobar arterial diameter,single vascular smooth muscle cell electrophysiological characteristics and expression of the BKCa channel beta subunit before and after alizarin intervention,respectively.Results (1) SHR systolic pressure was decreased after alizarin intervention (P ＜ 0.01).(2) ibTX inhibited alizarin-mediated renal interlobar arterial relaxation (P ＜ 0.01).(3) Alizarin enhanced BKCa channel-mediated outward currents (P ＜ 0.05).(4) Alizarin up-regulated BKCa channel beta subunits after alizarin intervention (P ＜ 0.01).Conclusion Alizarin reduced SHR systolic pressure and relaxed interlobar renal artery by enhancement BKCa currents via mediation of the function of beta subunits.

Relation between esophageal visceral hyperesthesia and expression of acid-sensing ion channel 1 in rats with reflux esophagitis / 中华消化杂志

Objective To explore the role of the visceral afferent nerve hyperesthesia and acid-sensing ion channel 1 (ASIC1) in rats with reflux esophagitis (RE).Methods Sixty male Sprague-Dawley rats were selected and animal model was established.Rats were divided into control group (n=20) and RE group (n=40).The esophageal mocosa biopsy were routinely performed in two groups.The esophageal specific DRG neurons were identified by 1,1'dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate tracing method and the whole-cell patch clamp assay was performed.The expression of ASIC1 in esophageal mucosa and thoracic spine cord three to five segments at protein level and mRNA level were detected by Western blotting and quantitative real time-polymerase chain reaction (qPCR).Two independent samples t test was performed for statistical analysis.Results The body weight of RE group was significantly lower than that of control group ((179.41±-16.38) g vs (290.75 ±-22.20) g),and the difference was statistically significant (t=17.090,P＜ 0.01).Esophageal basal cell hyperplasia,papillary elongation,vascular dialation and congestion,inflammatory cells infiltration were found in RE group rats.The results of whole-cell patchclamp showed depolarization of the resting potential of esophageal-specific DRG neurons of RE group was more significant than that of control group (-(46.20 ± 1.92) mV vs-(51.60 ± 1.52) mV),and the difference was statistically significant (t=4.930,P＜0.01).The threshold current of RE group was much lower than that of control group ((18.00±13.04) pAvs (80.00±12.25) pA),and the difference was statistically significant (t=7.750,P＜0.01).When stimulated with two to three times the threshold current,the frequency of action potential of RE group significantly increased (5.80 ±1.48 vs 3.00 ±1.58,10.60±2.30 vs 5.20±1.92),and the differences were statistically significant (t=2.890 and 4.030,both P＜0.01).The results of Western blotting indicated that the expression of ASIC1 in esophageal mucosa of RE group was significatly lower than that of control group (0.614±0.120 vs 0.976±0.283),and the difference was statistically significant (t =2.885,P＜ 0.05),while there was no statistically significant difference in the expression of ASIC1 in DRG between RE group and control group (0.804 ± 0.182 vs 1.032±0.316;t=1.528,P＞0.05).The results of qPCR showed that the expression of ASIC1 mRNA in esophageal mucosa of RE group was lower than that of control group (0.694 ± 0.118 vs 1.036 ±0.137),and the difference was statistically significant (t=4.642,P＜0.01).However there was no statistically significant difference in ASIC1 at mRNA level between RE group and control group (1.002± 0.074 vs 0.985±0.120;t=0.294,P＞0.05).Conclusion The sensitivity of esophageal visceral afferent nerve of rats in RE group increases and ASIC1 may negatively regulate the formation of esophageal visceral hypersensitivity.

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.

Inhibitory effect of zacopride on ouabain-induced arrhythmias in adult rats / 中国病理生理杂志

AIM: To investigate the effect of zacopride, an inward rectifier potassium channel agonist, on ouabain-induced arrhythmias in adult rats, and to explore the underlying electrophysiological mechanism.METHODS: Using ouabain to establish in vitro and in vivo arrhythmic rat models, the effects of zacopride on ouabain-induced arrhythmias were observed.The technique of whole-cell patch clamp was used to observe the effects of zacopride on inward rectifier potassium current (IK1), resting membrane potential (RMP) and delayed afterdepolarizations (DADs) in single rat ventricular myocyte.RESULTS: Zacopride at 1 μmol/L significantly reduced total number of premature ventricular beats, and the duration and incidence of ventricular tachycardia and ventricular fibrillation induced by ouabain in rat hearts in vitro (P<0.05).In anesthetized rats, zacopride at 15 μg/kg significantly reduced total number of premature ventricular beats, and the duration and incidence of ventricular tachycardia and ventricular fibrillation induced by ouabain (P<0.05).IK1 was significantly inhibited by ouabain (P<0.05), which was partially and even completely reversed by zacopride at 0.1~10 μmol/L.RMP value was significantly reduced by ouabain (P<0.05), and then increased to different levels after treatment with zacopride (0.1~10 μmol/L).Zacopride at 1 μmol/L showed its maximal effect and RMP was restored to normal level.Moreover, zacopride at 1 μmol/L markedly suppressed ouabain-induced DADs in single rat ventricular myocyte.The incidence of DADs decreased from 91.67% to 12.50% after zacopride was applied (P<0.05), and this effect was abolished by 1 μmol/L BaCl2.CONCLUSION: Inward rectifier potassium channel agonist zacopride significantly inhibits ouabain-induced ventricular arrhythmias in adult rats.The mechanism is related to increased RMP level and inhibition of DADs by activation of IK1 channel.

Relation between esophageal visceral hyperesthesia and expression of acid-sensing ion channel 1 in rats with reflux esophagitis / 中华消化杂志

Objective To explore the role of the visceral afferent nerve hyperesthesia and acid-sensing ion channel 1 (ASIC1) in rats with reflux esophagitis (RE).Methods Sixty male Sprague-Dawley rats were selected and animal model was established.Rats were divided into control group (n=20) and RE group (n=40).The esophageal mocosa biopsy were routinely performed in two groups.The esophageal specific DRG neurons were identified by 1,1'dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate tracing method and the whole-cell patch clamp assay was performed.The expression of ASIC1 in esophageal mucosa and thoracic spine cord three to five segments at protein level and mRNA level were detected by Western blotting and quantitative real time-polymerase chain reaction (qPCR).Two independent samples t test was performed for statistical analysis.Results The body weight of RE group was significantly lower than that of control group ((179.41±-16.38) g vs (290.75 ±-22.20) g),and the difference was statistically significant (t=17.090,P＜ 0.01).Esophageal basal cell hyperplasia,papillary elongation,vascular dialation and congestion,inflammatory cells infiltration were found in RE group rats.The results of whole-cell patchclamp showed depolarization of the resting potential of esophageal-specific DRG neurons of RE group was more significant than that of control group (-(46.20 ± 1.92) mV vs-(51.60 ± 1.52) mV),and the difference was statistically significant (t=4.930,P＜0.01).The threshold current of RE group was much lower than that of control group ((18.00±13.04) pAvs (80.00±12.25) pA),and the difference was statistically significant (t=7.750,P＜0.01).When stimulated with two to three times the threshold current,the frequency of action potential of RE group significantly increased (5.80 ±1.48 vs 3.00 ±1.58,10.60±2.30 vs 5.20±1.92),and the differences were statistically significant (t=2.890 and 4.030,both P＜0.01).The results of Western blotting indicated that the expression of ASIC1 in esophageal mucosa of RE group was significatly lower than that of control group (0.614±0.120 vs 0.976±0.283),and the difference was statistically significant (t =2.885,P＜ 0.05),while there was no statistically significant difference in the expression of ASIC1 in DRG between RE group and control group (0.804 ± 0.182 vs 1.032±0.316;t=1.528,P＞0.05).The results of qPCR showed that the expression of ASIC1 mRNA in esophageal mucosa of RE group was lower than that of control group (0.694 ± 0.118 vs 1.036 ±0.137),and the difference was statistically significant (t=4.642,P＜0.01).However there was no statistically significant difference in ASIC1 at mRNA level between RE group and control group (1.002± 0.074 vs 0.985±0.120;t=0.294,P＞0.05).Conclusion The sensitivity of esophageal visceral afferent nerve of rats in RE group increases and ASIC1 may negatively regulate the formation of esophageal visceral hypersensitivity.

Effects of honokiol on TTX-S sodium current in mouse dorsal root ganglion neurons / 中国药科大学学报

@#To elucidate possible mechanisms and targets of honokiol(Hnk)for hte treatment of pain, the effects of Hnk on tetrodoxin-senstive(TTX-S)sodium current(INa)were studied in acutely dissociated mouse dorsal root ganglion(DRG)neurons with whole-cell patch clamp technique. Hnk inhibited TTX-S INa currents in a concentration-dependent manner. At 30 μmol/L, Hnk obviously shifted the steady state activation of TTX-S INa toward more positive potentials by 10. 2 mV and prolonged the time course of recovery of sodium current, yet with no significant difference on recovery from inactivation of TTX-S sodium channel.

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.

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.

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.