正常人与囊性纤维化病人汗腺细胞膜单离子通道对Forskolin的反应

本文报道用片膜钳技术研究正常人及囊性纤维化病人(CF)的汗腺上皮细胞膜单个离子通道活动的特性,并比较分析了它们对Forskolin的反应。在400多个膜片上找出了至少七种或七个状态的离子通道的特性。观察到正常人和CF的部分汗腺上皮细胞(包括腺细胞与管细胞)膜离子通道对Forskolin有兴奋反应。发现腺细胞的兴奋反应率在正常人明显高于CF病人,差异非常显著。而管细胞的兴奋反应率,两组问无明显差异。以上结果提示,用Forskolin可协助鉴定CF的缺损,但宜选用腺细胞,不宜选用管细胞。

离子通道门控动力学的随机建模

片膜钳技术为我们了解离子通道的特性奠定了实验基础,而根据片膜钳实验记录建立离子通道的门控数学模型是定量地刻画通道的门控行为和深刻理解其运行机制的必经途径。本文在简述用片膜钳技术记录离子通道电信号的科学背景之后,介绍了离子通道门控动力学随机建模的进展,并着重评论这一过程中出现的基本问题和富有特色的研究方法。

离子通道门控动力学的随机建模(二)

片膜钳技术为我们了解离子通道的特性奠定了实验基础,而根据片膜钳实验记录建立离子通道的门控数学模型,是定量地刻划通道的门控行为,深刻理解其运行机制的必经途径。本文在简述用片膜钳技术记录离子通道电信号的科学背景之后,介绍离子通道门控动力学随机建模的进展,并着重评论这一过程中出现的基本问题和富有特色的研究方法。

The Effect of Benzyltetrahydropalmatine (BTHP) on Action Potentials and the Two Components of Delayed Rectifying Potassium Currents in Guinea Pig Ventricular Myocytes

The effects of BTHP on Ca 2+ independent action potential and the two components of delayed rectifier potassium currents were studied in guinea pig single ventricular myocytes by using whole cell patch clamp technique. BTHP 30 μmol·L -1 significantly prolonged APD 90 from 143±16 ms to 184±21 ms ( P 【0.01, n=5) without affecting either the RP or APA, and the APD prolonging effects of BTHP were independent of extracellular Ca 2+ . BTHP inhibited both I kr (IC 50 =7 9 μmol·L -1 ) and I ks (IC 50 =22 4 μmol·L -1 ) in a concentration dependent fashion. The results demon strated that BTHP had no obvious selectivity for I kr and I ks .

Cardiac Electrophysiological Differences Between Kunming and C57BL6/J Mice

Objective To investigate the alterations of cardiac electrophysiological properties and substantial mechanism and find the stable arrhythmia mouse model in Kunming （KM） and C57BL6/J （C57） mice. Methods Electrocardiogram recordings were used to analyze the QT interval in vivo, and mono- phasic action potential of right and left ventricular epicardium was recorded to elicit changes of action potential duration （APD） in conventional and programmed electrical stimulation （PES）. Transient outward potassium current （Its,） was recorded via whole-cell patch-clamp technique in single right and left epicardial myocytes. Results QT interval was prolonged in KM mice relative to C57 mice （62.51±4.47 ms vs. 52.59±4.85 ms, P〈0.05）. The APD at 50% repolarization of the left ventricular epicardium （18.60±0.91 ms vs. 12.90±0.35 ms）, and APDs at 50% （17.31±6.05 ms vs. 12.00±3.24 ms） and 70% repolarization （36.13±5.32 ms vs. 2 1.95±8.06 ms） of the right ventricular epicardium in KM mice were significantly pro- longed compared with C57 mice, respectively （all P〈0.05）. KM mice were more sensitive to PES-induced ventricular tachycardia （25%, 3 of 12 hearts）, and especially to Burst-induced ventricular tachycardia （50%, 6 of 12 hearts） compared with C57 mice, which were 20% （2 of 10 hearts） and 30% （3 of 10 hearts） respec- tively. It,, densities both in the left and right ventricular epicardial myocytes from KM mice were significantly decreased compared with C57 mice, respectively （all P〈0.01）. Conclusion Our data showed that KM mice with tile prolonged QT interval and APD are ruiner- abilities to ventricular arrhythmia, which are attributed to lower Ito densities in ventricular myocytes ob- tained from KM mice than that from C57 mice.

Characteristics of Transient Outward Potassium Channel Exposed to 3 mT Static Magnetic Field

Acutely isolated mouse hippocampal CA3 pyramidal neurons were exposed to 3 mT static magnetic field,and the characteristics of transient outward K+ channel were studied using the whole-cell patch-clamp technique.The experiment revealed that the amplitude of transient outward potassium channel current was reduced.The maximum activated current densities of control group and exposure group were 163.62±20.68 pA/pF and 98.74±16.57 pA/pF(n=12,P<0.01) respectively.The static magnetic field exposure affected the activation and inactivation process of transient outward potassium channel current.Due to the magnetic field exposure,the half-activation voltage of the activation curves changed from 5.59±1.96 mV to 27.87±7.24 mV(n=12,P<0.05) ,and the slope factor changed from 19.43±2.11 mV to 25.87±4.22 mV(n=12,P<0.05) .The half-inactivation voltage of the inactivation curves also changed from-56.09±0.89 mV to-57.16±1.10 mV(n=12,P>0.05) and the slope factor of the inactivation curves from 8.69±0.80 mV to 10.87±1.02 mV(n=12,P<0.05) .The results show that the static magnetic field can change the characteristics of transient outward K+ channel,and affect the physiological functions of neurons.

Upregulated voltage-gated potassium channel Kvl.3 on CD4＋CD28null T lymphocytes from patients with acute coronary syndrome

Objective The purpose of our study is to observe the voltage-gated potassium channel Kvl.3 expressed on CD4＋CD28~ T cells from the peripheral blood of acute coronary syndrome （ACS） patients by the patch clamp technique. Methods Kvl.3 potassium channels expression from 17 patients with ACS and 11 healthy age-match controls was detected in single cell（CD4＋CD28null T cells and CD4＋CD28＋T cells） by fluorescence mieroscopy and patch clamp. Results The percentage of CD4＋CD28mullT cells was higher in the ACS patients [（6.97±2.05）%] than that in the controls [（1.38±0.84）%, P〈0.05]. The concentration of hsCRP was directly correlated with the number of the CD4~CD28nul~ T cells in the ACS patients （r=0.52, P〈0,05）. The conductance （6.89±1.17ns vs 3.36±0.66ns）, dens （1.95±0.80 μm2 vs 1.13±0.57 μm2） and numbers （574.5±97.6 n/cell vs. 280.3±55.3 n/cell） of the Kv1.3 channels on the CIM＋CD28null T cells were significantly higher than those on the CD4＋CD28＋ T cells （all P〈0.01） in ACS patients, but were similar on CD4＋CD28＋T betweenACS patients and controls. Conclusion The CD4＋CD28nullT cells and the numbers of Kvl.3 channels on the CD4＋CD28nullT cells from patients with ACS are significantly upregulated and might contribute to the pathogenesis of ACS （d Geriatr Cardio12010; 7：40-46）.

Effects of hypoxia on sodium current in rat cardiomyocytes

The aim of this study was to investigate the effect of hypoxia on the sodium current of rat cardiomyocytes in order to explore ion channel mechanism of cardiomyocyte hypoxia.The rat cardiomyocytes were isolated by acute enzymatic hydrolysis.A group of untreated cells were used to record sodium currents using whole-cell patch-clamp technique,another group was subjected to hypoxia and record sodium currents using same technique.The results showed that the morphological trajectory of sodium hypoxia was not changed compared with that of normal cells.The I-V curve of hypoxic cells was significantly higher than that of normal cells,and the peak current of INa was 15.68%higher than that of normal cells(P<0.0001).Activation potential of normal and hypoxia cells was about-40mV,the maximum peak current corresponds to the stimulation voltage of-25mV.The above results suggest that rat cardiomyocytes sodium current increases in the case of hypoxia.

Sodium channels in the apical membrane of human nasal epithelial cells

Objective To study the electrophysiological properties of sodium channels in the apical membrane of human nasal epithelial cells Method Nasal epithelial cells of human inferior turbinate from patients with obstructive sleep apnea syndrome were cultured in serum free medium on collagen gel coated membranes at an air liquid interface and studied by a patch clamp technique Results In cell attached patches, a typical single channel current with a conductance of 21 09?pS and reversal potential of -50 96 were recorded The permeability ratio P Na /P K was more than 5 80 In the presence of 10 4 mmol/L amiloride in the pipette, the incidence of sodium channels decreased from 26 67% to 5 13% This revealed that a population of channels were inhibited by amiloride at a dose of 10 4 mmol/L Ca 2+ at dose of 10 3 mmol/L did not influence the incidence of sodium channels There was no obvious association between voltage and the open probability of the channels Conclusions Our results indicate that most Na + channels in cell attached patches of human nasal epithelial cells are amiloride sensitive and Na + selective Only a few channels are amiloride insensitive The channels were not activated by extracellular Ca 2+ and the open probability followed a voltage independent manner

Effects of SKF-96365, a TRPC inhibitor, on melittin-induced inward current and intracellular Ca^2+ rise in primary sensory cells

Objective Melittin （MEL） is a major component of bee venom and can produce both persistent spontaneous nociception and pain hypersensitivity when injected subcutaneously in the periphery. The present study aimed to examine the roles of transient receptor potential canonical （TRPC） channels in mediation of MEL-indueed activation of primary nociceptive cells. Methods Whole-cell patch-clamp and laser scanning confocal calcium detection were used to evalu- ate the effects of SKF-96365, a TRPC inhibitor, applied on the acutely isolated dorsal root ganglion （DRG） cells of rat, on MEL-induced increase in intracellular calcium concentration （[Ca2＋]i） and inward current. Results Under voltage- clamp mode, 43.9% （40/91） DRG cells were evoked to give rise to the inward current by 2 pmol/L MEL, which could be significantly suppressed by 3 doses of SKF-96365 （1, 5 and 10μmol/L） in a dose-dependent manner. Of the other 210 cells, 67.6% responded to MEL with an intracellular Ca2＋ rise, as revealed by confocal calcium imaging. Of these MEL- sensitive cells, 46.5% （66/142） were suppressed by the highest dose of SKF-96365. Conclusion MEL-induced activation of small to medium-sized DRG cells can be suppressed by SKF-96365, suggesting the involvement of TRPC channels in the mediation of MEL-induced activation of primary nociceptive cells.

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

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

Astragaloside IV Regulates Expression of ATP-sensitive Potassium Channel Subunits after Ischemia-reperfusion in Rat Ventricular Cardiomyocytes

Objective: Astragaloside IV (AsIV) is the major effective component extracted from the Chinese herb Astragalus membranaceus, which has been widely used to treat cardiovascular disease. Recent studies have shown that AsIV can potentially protect the heart from myocardial ischemic injury, but the mechanisms of action are unknown. ATP-sensitive potassium (KATP) channels are activated during ischemia and exert a compensatory protective effect on cardiomyocytes. We therefore examined the effects of AsIV on KATP channel currents and channel expression in isolated rat ventricular cardiomyocytes after ischemia-reperfusion injury. Methods: Forty Wistar rats were divided into five groups: control group, ischemia-reperfusion (IP) group, IP + glibenclamide group, IP + pinacidil group and IP + AsIV group. The ischemia-reperfusion injury model was established in enzymatically isolated ventricular cardiomyocytes by perfusion with calcium-free Tyrode solution for 10 min, arrest for 30 min, and reperfusion for 45 min. The different drugs were applied for 10-15 min, and the KATP channel current (IKATP) was recorded with voltage-clamp mode by whole-cell patch-clamp technique. Protein and mRNA expression of the KATP channel subunits Kir6.1, Kir6.2, SUR2A and SUR2B was quantified using western blotting and real-time PCR. Results: The KATP current in IP group was significantly greater than that in control group (211.45±33.67 vs 83.51±23.67 pA; P<0.01). Glibenclamide (10 μmol/L) blocked KATP currents, whereas both AsIV (1 mg/L) and the known channel opener pinacidil (50 μmol/L) significantly increased IKATP (P<0.05). Consistent with this, AsIV significantly up-regulated protein and mRNA expression of Kir6.1, Kir6.2, SUR2A, SUR2B (P<0.01 vs IP group). Conclusion: The protective effects of AsIV in ischemia-reperfusion injury may be related to the up-regulation of several KATP channel subunits and facilitation of KATP currents.

Increased intracellular calcium concentration causes electrical turbulence in guinea pig ventricular myocytes

Dysregulation of intracellular Ca2+ homeostasis is associated with various pathological conditions and arrhythmogenesis of the heart.The objective of this study was to investigate the effects of an acute increase in intracellular Ca2+ concentration ([Ca2+] i) on the electrophysiology of ventricular myocytes by mimicking intracellular Ca 2+ overload.The [Ca2+] i was clamped to either a controlled (65-100 nmol L-1) or increased (1 μmol L-1) level.The transmembrane action potentials and ionic currents were recorded using whole-cell patch clamp techniques.We found that the acute increase in [Ca2+] i shortened the action potential duration,reduced the action potential amplitude,maximum depolarization velocity and resting membrane potential,caused delayed after-depolarizations (DADs),and triggered activity--compared with these parameters in the control.The increased [Ca2+] i augmented late I Na in a time-dependent manner,reduced ICaL and IK1,and increased IKr but not IKs.The results of this study can be used to explain calcium overload-induced ventricular arrhythmias.

Inhibition of P2X_7 Receptor by Extracts of Chinese Medicine

Objective： To investigate the influence of Acorus gramineus （Soland）, a crude extract, SCP01, and a purified component, SCP02, and of Rosmarinus officinalis L., X0728 on human mast cells （HMC-1 Cell Line）. Methods： Current-voltage of P2X7 receptors on human mast cell membrane activated by ATP was recorded by the whole-cell patch clamp technique. Results： The current at -100 mV mediated by P2X7 was inhibited by （27.6 ± 2.0） % in the presence of 40 μg/mL SCP01 and by （29.5 ± 2.2） % in the presence of 40 μg/mL SCP02, which was identified as α-asarone. 42 μg/mL of the commercially available α-asarone inhibited the P2X7-mediated current by （52.2 ± 2.0） %. In contrast to SCP01 and SCP02, 40 μg/mL X0728 provoked stimulation of the current by （28.6 ± 2.8） %. All effects were voltage- independent. Conclusion： The inhibition of P2X7 by α-asarone will inhibit intracellular calcium increase and this may account for the inhibition of reported excitotoxic cell death. The pharmacological function of P2X7 stimulation by X0728 needs further investigation.