乙酰胆碱对犬右室三层心肌细胞L型钙电流不均一性的影响

目的测定犬右室三层心肌细胞上的L型钙电流(ICa,L),并研究其对自主神经递质乙酰胆碱的反应。方法经酶解法分离获得犬右室三层心肌细胞,应用全细胞膜片钳技术,记录并比较三层心肌细胞的ICa,L,以及应用2μmol/L乙酰胆碱前后电流-电压曲线的差异。结果ICa,L的峰值电流密度外膜下大于M细胞,而M细胞又大于内膜下心肌细胞,分别为-4.896±1.907pA/pF(n=31),-3.406±0.904pA/pF(n=37),-2.788±0.756pA/pF(n=33)(P<0.05)。使用乙酰胆碱后,右室外膜下及M细胞的峰值电流密度减小[-4.921±1.023pA/pF vs -3.462±0.997pA/pF(n=12);-3.803±1.115pA/pF vs -2.959±0.883pA/pF(n=13),P均<0.05]。心内膜下心肌细胞用药前后无差异(P>0.05)。结论ICa,L在犬右室三层心肌细胞存在不均一性,乙酰胆碱可以减小心外膜下、M细胞的ICa,L,对内膜下心肌细胞的ICa,L无影响。     

正常大鼠心室肌细胞动作电位和瞬时外向钾离子流的特点

目的研究正常Spraque-Dawley大鼠外层、中层和内层心室肌细胞动作电位(AP)和瞬时外向钾离子流(Ito)的特点。方法采用酶消化法获得大鼠外层、中层和内层心室肌细胞,以全细胞膜片钳技术记录心室肌细胞AP和Ito。结果成功记录到大鼠心室肌细胞外、中和内层心肌细胞AP和Ito。外层至内层心室肌细胞动作电位时程(APD)逐渐延长(P<0.05)。在+70mV刺激时外层至内层心室肌细胞Ito电流密度逐渐减小,分别为59.50±15.99,29.15±5.53和12.29±3.62pA/pF(P<0.05)。三层心室肌细胞曲线半激活电压、半失活电压及失活后恢复时间均无差异(P>0.05)。结论大鼠三层心室肌细胞AP形态和Ito大小存在分层差别。     

Differences in the effect of metabolic inhibition on action potentials and calcium currents in endocardial and epicardial cells

BACKGROUND. Ischemia-induced electrophysiological changes are more prominent in epicardial cells than in endocardial cells. Epicardial action potentials shorten more than endocardial action potentials during ischemia. Since the L-type Ca2+ current plays an important role in the maintenance of action potential duration, we hypothesized that the Ca2+ current is affected more in epicardial cells than in endocardial cells during ischemia. METHODS AND RESULTS. To test this hypothesis, we examined the effect of metabolic inhibition, a major component of ischemia, on action potentials and the Ca2+ current in single cells isolated from the endocardial and epicardial layers of the feline left ventricle. The membrane voltage and current were measured by using the whole-cell mode of the patch-clamp technique. During control periods, action potentials recorded from epicardial myocytes had lower amplitude, a prominent notch between phases 1 and 2, and shorter action potential duration compared with those recorded from endocardial myocytes. However, the amplitude and current-voltage relation of the Ca2+ current were similar in endocardial and epicardial cells at test potentials of -30 to 60 mV elicited from a holding potential of -40 mV. The time course of inactivation of the Ca2+ current also was identical in the two cell types. After 15 minutes of superfusion with glucose-free Tyrode's solution containing 1 mM CN-, action potential duration was reduced by 13 +/- 7% in endocardial cells and by 80 +/- 9% in epicardial cells (p less than 0.01). The peak Ca2+ current was reduced by 21 +/- 9% in endocardial cells and by 37 +/- 6% in epicardial cells (p less than 0.01). CONCLUSIONS. We conclude that enhanced depression of the Ca2+ current may account in part for the greater action potential shortening in epicardial cells during ischemia and metabolic inhibition.     

Regional and frequency-dependent changes in action potentials and transient outward K<Superscript>+</Superscript> currents in ventricular myocytes from J-2-K cardiomyopathic hamsters

Abstract. Objectives Although lengthening of action potential duration (APD) and decreased transient outward K⁺ currents (I_to) have been observed in ventricular myocytes from cardiomyopathic hamsters, epi- and endo-cardial differences in I_to and their roles in frequency-dependent changes in APD have not been claried. Methods The patch-clamp technique of whole-cell conguration was used to record membrane potentials and currents in epicardial and endocardial myocytes of the J-2 hamster germline without (control) and with cardiomyopathy (CM). Results In control, APD in endocardial myocytes was longer than that in epicardial myocytes at 0.1 Hz. APD significantly lengthened with increased frequencies of stimulation from 0.1 to 6.0 Hz in both groups with the longer APD in endocardial myocytes. In CM, APD lengthened in epicardial myocytes exceeding the endocardial APD without a frequency-dependent prolongation. Pretreatment with 4 mM 4-aminopyridine completely abolished the frequency-dependent changes and abolished APD differences between epicardial and endocardial myocytes, and between control and CM hamsters. The transient outward K⁺ current (I_to) significantly decreased in epicardial myocytes from CM hamsters compared with that of control (17.5 ± 1.5 pA/pF in control vs. 9.5 ± 2.5 pA/pF in CM at +60 mV) with altered recovery from inactivation, without changes in the endocardial I_to. Moreover, the inward rectifier K⁺ current decreased in epicardial myocytes from CM hamsters and the L-type Ca2⁺ current reduced in both regions from CM compared to control. Conclusion Results indicate that differences in APD between epi- and endocardial myocytes in CM hamsters are mainly caused by a decreased current density and altered recovery from inactivation of I_to in epicardial myocytes.     

Electrophysiological properties and responses to simulated ischemia in cat ventricular myocytes of endocardial and epicardial origin

In multicellular preparations, there are differences in action potential configuration between endocardium and epicardium, and electrophysiological alterations induced by ischemia are more drastic in epicardium than in endocardium. The present study was designed to examine electrophysiological properties of single cardiac myocytes enzymatically isolated from the endocardial and epicardial surfaces of the cat left ventricle and to determine whether the differential responses to ischemia of intact tissue occur in single cells. Action potentials recorded from the isolated single cells of epicardial surface had lower action potential amplitude and a prominent notch between phase 1 and phase 2, compared with those of the cells isolated from the endocardial surface; these findings are similar to those in intact endocardial and epicardial preparations. Resting membrane potentials recorded from both endocardial and epicardial single cells were sensitive to the change in extracellular K+ concentration and had properties of a K+ electrode. Action potential duration was frequency dependent in both cell types and was shorter in epicardial cells than in endocardial cells at a stimulation rate of 3 Hz. When the cells were superfused with Tyrode's solution that was altered to mimic an ischemic environment in vivo (PO2, 30-40 mm Hg; pH 6.8; [K+], 10 mM; and glucose free), resting membrane potential, action potential amplitude, and action potential duration were reduced, and the refractory period was shortened in both endocardial and epicardial single cells, but there were no differences in the degree of changes in action potentials and refractory periods induced between the two cell types. Action potential changes induced by L-alpha-lysophosphatidylcholine (5-40 mg/l) were also similar in endocardial and epicardial single cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of the transient outward current in rabbit sino-atrial node cells

The electrophysiological properties of the transient outward current were investigated in voltage-clamped single cells from the rabbit sino-atrial node. To make a regional comparison, some experiments were repeated in atrial myocytes. The current-voltage relationship showed a characteristic outward rectification with an activation threshold of -30 mV. External 4-aminopyridine (0.01-5 mM) inhibited this current in a dose-dependent manner (IC50 = 0.28 mM, Hill coefficient = 1.38). The steady-state inactivation exhibited a half-maximum voltage of -35 mV and a slope factor of -.4 mV. The current density of the transient outward current was 6.3 +/- 0.5 pA/pF in sino-atrial node cells and 12.3 +/- 1.2 pA/pF in atrial cells. The inactivation time constant was faster in sino-atrial node cells (time constants 4.2 +/- 0.5 and 26.0 +/- 0.6 ms, respectively, for the fast and slow components) than in atrial cells (9.7 +/- 1.2 and 44.8 +/- 3.2 ms, respectively). Recovery from inactivation was much faster in sino-atrial node cells (time constants 44.7 +/- 9.0 ms) than in atrial cells (time constants 1.39 +/- 0.32 and 6.70 +/- 0.1 s, respectively, for the fast and slow components). These results suggest that the kinetic properties, as well as the current density, of the transient outward current differs between sino-atrial node and atrial cells. Taking the current density of Ito at +10 mV as 2.5 +/- 0.3 pA/pF gives a total Ito of approximately 100 pA at the peak of the action potential in rabbit sino-atrial node cells. The action potential duration was increased by 24.8 +/- 1.3% by 0.5 mM 4-AP. Thus, Ito may contribute significantly to the repolarization phase in mammalian sino-atrial node cells.     

Endocardial versus epicardial differences in L-type calcium current in canine ventricular myocytes studied by action potential voltage clamp

OBJECTIVES: The aim of the present study was to assess and compare the dynamics of L-type Ca(2+) current (I(Ca,L)) during physiologic action potential (AP) in canine ventricular cardiomyocytes of epicardial (EPI) and endocardial (ENDO) origin. METHODS: I(Ca,L) was recorded on cells derived from the two regions of the heart using both AP voltage clamp and conventional whole cell voltage clamp techniques. RESULTS: AP voltage clamp experiments revealed that the decay of I(Ca,L) is monotonic during endocardial AP, whereas the current is double-peaked (displaying a second rise) during epicardial AP. The amplitude of the first peak was significantly greater in ENDO (-4.6+/-0.8 pA/pF) than in EPI cells (-2.8+/-0.3 pA/pF). Application of epicardial APs as command pulses to endocardial cells yielded double-peaked I(Ca,L) profiles, and increased the net charge entry carried by I(Ca,L) during the AP from 0.187+/-0.059 to 0.262+/-0.056 pC/pF (n=5, P<0.05). No differences were observed in current densities and inactivation kinetics of I(Ca,L) between EPI and ENDO cells when studied under conventional voltage clamp conditions. Nisoldipine shortened action potentials and eliminated the dome of the epicardial AP. CONCLUSION: I(Ca,L) was shown to partially inactivate before and deactivate during phase-1 repolarization and reopening of these channels is responsible for the formation of the dome in canine EPI cells. The transmural differences in the profile of I(Ca,L) could be well explained with differences in AP configuration.     

Transient outward current prominent in canine ventricular epicardium but not endocardium

Previous studies have denied the presence of a transient outward current (Ito) in ventricular myocardium of dog, sheep, and calf. Using conventional microelectrode techniques, we provide evidence for a significant contribution of Ito to epicardial, but not endocardial, activity of canine ventricular myocardium. The epicardial action potential when compared with that of endocardium shows a smaller phase 0 amplitude, a much more prominent phase 1, and a phase 2 amplitude that is greater than that of phase 0. Epicardial action potentials, unlike those of endocardium, display a "spike and dome" morphology that becomes progressively more accentuated at slower stimulation rates. Using the restitution of phase 1 amplitude as a marker for the process responsible for the spike and dome phenomenon, we were able to delineate two exponential components: 1) a slow component that recovers with a time constant of 350-570 msec and 2) a fast component with a time constant of 41-85 msec. The slow component was largely abolished by 1-5 mM 4-aminopyridine, an Ito blocker. The fast component was diminished by 4-aminopyridine, but it was also inhibited by ryanodine and by Sr2+ replacement of Ca2+, which are interventions known to inhibit the Ca2+-activated component of Ito. Following 4-aminopyridine and Sr2+ or ryanodine treatment, the epicardial responses more closely resembled those of endocardium. In summary, the data demonstrate a marked heterogeneity of active membrane properties in canine ventricular muscle. These observations may aid in understanding the basis for rate-dependent changes in the T wave of the ECG, supernormal conduction in ventricular muscle, the greater sensitivity of epicardium to ischemia, and the rate dependence of some cardiac arrhythmias.     

L型钙通道在LQTl发病机制中作用的实验研究

目的 分析L型钙电流(IcaL)在犬三层心室肌细胞中的特点,探讨其在LQTl发病机制中的作用.方法 成年杂种犬14只,体重13～15 kg,雌雄不拘.分离犬三层心室肌细胞,采用全细胞膜片钳技术记录动作电位(AP)和ICaL,依次用Chromanol 293B(50ìμmoL/L)阻断慢激活延迟整流性钾电流(IKs)模拟LQTl,用异丙肾上腺素(100 nmo/L)激活13肾上腺素受体(β-AR),观察AP和,ICaL的变化.分三层取少量心室肌组织,采用实时定量逆转录聚合酶链反应(RT-PCR)技术,检测各层L型钙通道a1C亚单位的mRNA含量.结果 正常情况下,犬三层心室肌细胞ICaL电流密度差异无统计学意义[外层(4.253±0.782)pA/pF,中层(4.392±0.714)pA/pF,内层(4.182±0.665)pA/pF,P＞0.05],而中层心室肌细胞动作电位时限(APD)较内层和外层的长[外层(721.48±26.59)ms,中层(911.80±31.24)ms,内层(783.52±25.27)ms,P＜0.05];阻断IKs后ICaL电流密度没有变化,而APD均明显延长[(外层(835.21±27.34)ms,中层(1089.21±30.55)ms,内层(830.64±27.12)ms,与阻断IKs前相比,P＜0.05)];β-AR兴奋使三层心室肌细胞ICaL显著增加,且三者变化差异无统计学意义[(外层(5.654±0.756)pA/pF,中层(5.458±0.702)pA/pF,内层(5.600±0.819)pAZpF,P＞0.05].但β-AR兴奋使外层和内层心室肌细胞APD缩短,中层心室肌细胞APD延长,三者变化差异有统计学意义[外层(792.63±26.71)ms,中层(1127.85±32.10)ms,内层(811.32±27.52)ms,P＜0.05].实时定量RT-PCR结果显示,三层心室肌细胞中alC亚单位的mRNA含量差异无统计学意义(外层0.112±0.019,中层0.077±0.018,内层0.109±0.012,P＞0.05).结论 L型钙通道在犬三层心室肌中的分布没有差异,在LQTl模型中,Iso使三层心室肌细胞,ICaL均匀增加,推测ICsL本身没有引起LQTl复极不稳定.     

Potassium rectifier currents differ in myocytes of endocardial and epicardial origin.

Whole-cell voltage-clamp experiments and single-channel current recordings in cell-attached patch mode were performed on enzymatically dissociated single ventricular myocytes harvested from feline endocardial and epicardial surfaces. The studies were designed to compare the characteristics of inward rectifier K+ current (IK1) and delayed rectifier K+ current (IK) between endocardial and epicardial cells and to test the hypothesis that the differential characteristics of IK1 and/or IK are responsible for the differences in action potential configuration between the two cell types. IK1 in endocardial cells displayed a distinct N-shaped current-voltage (I-V) relation, with a prominent outward current at potentials between -80 and -30 mV. In epicardial cells, an outward current region was much smaller, and the I-V relation demonstrated a blunted N-shaped I-V relation. In single-channel current recordings in cell-attached patch mode, neither unitary current amplitude of IK1 nor probability of channel opening was different between endocardial and epicardial cells, suggesting that the difference in the number of functional channels might be responsible for the differential IK1 I-V relations. The characteristics of IK also differed between endocardial and epicardial cells. The time course of growth of tail current of IK (IK,tail) (activation of IK) was significantly enhanced and that of IK,tail deactivation was delayed in epicardial cells compared with endocardial cells. The time constant of the slow component of IK activation at +20 mV was 3,950 +/- 787 msec in endocardial cells and 2,746 +/- 689 msec in epicardial cells (p less than 0.05); the corresponding values for IK deactivation at -50 mV were 1,041 +/- 387 msec and 1,959 +/- 551 msec, respectively (p less than 0.01). The voltage dependence of steady-state activation of IK,tail was similar between endocardial and epicardial cells, suggesting that the probability of channel opening at any potential was not different in the two cell types. The amplitude and density of fully activated IK (IK,full) were significantly greater in epicardial cells than in endocardial cells. At repolarization to -20 mV, IK,full amplitude was 452 +/- 113 pA in endocardial cells and 578 +/- 135 pA in epicardial cells (p less than 0.05), and the corresponding values for IK,full density were 2.86 +/- 0.73 and 4.21 +/- 0.83 microA/cm2, respectively (p less than 0.05). A nonstationary fluctuation analysis revealed that the amplitude of IK unitary current was similar between endocardial and epicardial cells (0.23 +/- 0.07 versus 0.22 +/- 0.03 pA, p = NS).(ABSTRACT TRUNCATED AT 400 WORDS)     

动作电位参数作为区分狗心室肌三种细胞的可行性研究

应用细胞膜片钳技术测定狗心室肌细胞的电生理特征、动作电位(AP)以及收缩的参数,并用统计学方法评价所测量的参数是否可将心内、外膜及中层(M)细胞有效地区分开。结果:获取52个混合的有3种心肌细胞的AP,以AP穹隆的高度以及AP切迹的膜电位水平和穹隆上升的速度将其分三组细胞。以穹隆的膜电位水平能有效区分心外膜与其他两种细胞(P<0.05),但不能有效区分心内膜和M细胞(P>0.05)。以AP时程(APD)可以有效区分M细胞与其他两种细胞(P<0.05),但不能有效区分心内膜和心外膜细胞(P>0.05)。尖峰切迹时间以及收缩参数均不能有效区分三种心室肌细胞(P>0.05)。结论:狗心室单个细胞AP的一些参数,可以作为辨别心内、外膜,M细胞的依据。     

犬右室瞬间外向钾电流异质性的研究

应用全细胞钳制技术对犬右室心外膜下 (epi)细胞、中层 (M)细胞和心内膜下 (endo)细胞复极 1期瞬间外向钾电流 (Ito1)的强度、密度和动力学过程进行系统定量研究 ,以期从复极 1期主要离子流的角度 ,探讨右室复极 1期跨越室壁的电异质性。结果发现 :犬右室epi细胞和M细胞存在强大的Ito1离子流 ,在刺激频率为 0 .2Hz、37℃和去极化试验电压为 +70mV时 ,epi细胞和M细胞峰值Ito1离子流的强度分别为 46 5 0± 176 0 ,36 2 0± 1880pA ,其激活和失活动力学过程符合Boltzmann分布。与epi细胞和M细胞相比 ,endo细胞Ito1离子流微小 ,同样条件下其平均峰值Ito1离子流仅为 480± 130pA。表明在右室跨越室壁的三层细胞间 ,特别是epi细胞与endo细胞之间、M细胞与endo细胞之间 ,复极 1期存在明显的Ito1离子流强度差异和强大的Ito1离子流梯度 ,此为右室电异质性的一种突出表现 ,它可能是Brugada综合征等疾病所致恶性心律失常的重要离子基础之一     

兔界嵴细胞离子通道特性研究

目的研究生理状态下及异丙肾上腺素灌流对兔界嵴(CT)与梳状肌(PM)细胞动作电位(AP)及钠电流(INa)、短暂外向钾电流(Ito)、L型钙电流(ICa-L)、延迟整流钾电流(IK)及内向整流性钾电流(IK1)的影响,探讨CT与房性心律失常的关系。方法酶解法分离兔CT及PM细胞,利用全细胞膜片钳技术,记录生理状态下及异丙肾上腺素灌流后CT与PM细胞AP及INa、Ito、ICa-L、IK及IK1的变化。结果①生理状态下,CT细胞动作电位时程(APD)较长,可见明显的平台期;PM细胞AP形态与普通心房肌细胞相似,1期复极迅速,平台期短,类似三角形。②生理状态下,CT细胞Ito电流密度比PM细胞明显降低(7.13±0.38 pA/pF vs 10.70±0.62 pA/pF,n=9,P<0.01),而INa、Ito、ICa-L、IK及IK1则无明显差别。③异丙肾上腺素灌流时CT与PM细胞APD20、APD50、APD90均延长(n=8,P<0.01);指令电位+50 mV时,CT与PM细胞Ito电流密度均减少(n=9,P<0.01)而IK均增加(n=8,P<0.05);指令电位+10 mV时,CT与PM细胞ICa-L电流密度均增加(n=9,P<0.01);IK1在两种心肌细胞均无明显差异。结论 CT与PM细胞AP差异与Ito有关。异丙肾上腺素灌流时ICa-L与IK增强,Ito抑制使CT与PM细胞APD延长,触发机制可能是CT参与房性心律失常的机制之一。     

Age-related appearance of outward currents may contribute to developmental differences in ventricular repolarization.

Ventricular repolarization significantly influences contractility, refractoriness, and ion channel state. Factors affecting repolarization will thus affect these secondary phenomena. To understand the influence of age on ventricular repolarization, we studied neonatal, young, and adult dogs using electrocardiogram, action potential, and whole-cell voltage-clamp recordings from single epicardial myocytes. Hearts of neonatal and 57-58-day-old dogs require a significantly longer time for repolarization than those of adult dogs, as determined by analysis of rate-corrected QT and JT (QT minus QRS) intervals. Epicardial action potentials of neonates are significantly longer than those of adults, as determined by measurements of duration at 50% and 90% repolarization. The adult action potential is characterized by a large phase 1 notch that is absent from neonatal recordings. This notch develops between 58 and 64 days of age, and by 64-68 days of age, it is equal to that in adults. In addition, action potentials recorded from adult and young epicardial muscle are more greatly affected by rapid pacing and superfusion of 2 mM 4-aminopyridine than are potentials recorded from neonatal tissue. Whole-cell voltage-clamp recordings reveal a 4-aminopyridine-sensitive transient outward current in adult myocytes that is absent from neonatal myocytes. The correlation between developmental changes in the 4-aminopyridine-sensitive current, the action potential, and the QT interval suggests that the transient outward current may be an important determinant in the relation between age and repolarization.     

Diminished Kv4.2/3 but not KChIP2 levels reduce the cardiac transient outward K+ current in spontaneously hypertensive rats

OBJECTIVE: A reduction of the Ca(2+)-independent transient outward potassium current (I(to)) in epicardial but not in endocardial myocytes of the left ventricle has been observed in cardiac hypertrophy and is thought to contribute to the electrical vulnerability associated with this pathology. METHODS: In the present study we investigated the molecular mechanisms underlying regional alterations in I(to) in hypertrophied hearts of spontaneously hypertensive rats (SHR) using the whole-cell patch-clamp technique, quantitative RT-PCR and heterologous expression of underlying ion channel subunits. RESULTS: I(to) was significantly smaller in epicardial myocytes of SHR than in Wistar-Kyoto (WKY) controls (11.1+/-0.9 pA/pF, n=20 vs. 16.8+/-1.7 pA/pF, n=20, p<0.01), but not different in endocardial myocytes from both groups. Quantitative RT-PCR analysis of the genes encoding I(to) revealed significantly lower levels of Kv4.2 and Kv4.3 mRNA in the epicardial region of SHR rats compared to WKY rats. In contrast, mRNA expression levels of all three splice variants of the beta-subunit KChIP2 were significantly higher in both endo- and epicardial myocytes from SHR than from WKY rats. In parallel, inactivation of I(to), which is negatively modulated by KChIP2, was slowed down in SHR while recovery from inactivation remained unchanged. Heterologous co-expression of increasing amounts of KChIP2b together with a fixed amount of Kv4.2 in Xenopus laevis oocytes revealed a hyperbolic relation of recovery from inactivation and inactivation time constant, demonstrating that KChIP2 preferentially affects inactivation, if its expression level is high. CONCLUSION: These results suggest that downregulation of I(to) in the left ventricle of SHR is mediated by a reduced expression of Kv4.2 and Kv4.3 (but not of KChIP2), whereas the slower inactivation of I(to) can be explained by increased expression levels of KChIP2 in SHR.     

A role for a glibenclamide-sensitive, relatively ATP-insensitive K+ current in regulating membrane potential and current in rat aorta

OBJECTIVE: ATP-sensitive K+ channels have been classified based on their inhibition by cytoplasmic ATP. Recent evidence in vascular smooth muscle has suggested that these channels show weak sensitivity to intracellular ATP. However, it is not known whether these channels regulate the resting K+ conductance in vascular smooth muscles. Therefore, the aim of the present investigation was to characterize this current in rat aorta myocytes and to examine whether it contributes to setting the membrane potential. METHODS: The conventional and nystatin-permeablised whole cell patch clamp techniques were used to characterize the effect of glibenclamide on membrane potential and K+ current in enzymatically dispersed rat aorta myocytes. RESULTS: The mean resting potential measured in current clamp mode using the permeabilized patch approach was -54 +/- 5 mV (n = 8). Glibenclamide (10 microM) caused a reversible 24-mV depolarization in these cells. In symmetrical K+ (135 mM) solution an inward glibenclamide-sensitive (10 microM) current (-4.1 +/- 0.7 pA/pF; n = 5), hereafter termed Iglib, was observed at a membrane potential of -80 mV when cells held at -60 mV were ramped from -80 to +80 mV. In the absence of any nucleotide in the pipette solution, Iglib measured by the conventional whole-cell method was -23.69 +/- 4.65 pA/pF (n = 9). With 1 and 3 mM ATP in the pipette, the average current density was -25 +/- 6.3 pA/pF (n = 8), and -9.4 +/- 2.7 pA/pF (n = 9), respectively. In the absence of ATP, 1 mM GDP significantly (P < 0.01) increased Iglib (-44.8 +/- 8.4 pA/pF; n = 13). Inclusion of 1 mM ATP in the GDP-containing pipette solution had no significant effect on the current amplitude (-56.4 +/- 10.7 pA/pF; n = 7). Iglib fell to -11.0 +/- 2.9 pA/pF (n = 10) if 1 mM GDP and 3 mM ATP were present. In symmetrical K+, the Iglib observed in the presence of 1 mM ATP in the pipette was increased by more than two-fold in the presence of 10 microM levcromakalim. In PSS containing 5 mM K+, a significant glibenclamide-sensitive current was observed at -45 mV membrane potential when cells dialyzed with 1 mM ATP were ramped between -80 to 30 mV. CONCLUSION: These results demonstrate that Iglib channels in rat aorta myocytes differ from classical KATP channels, being relatively insensitive to intracellular ATP. Iglib therefore appears to have an important role in contributing to the maintenance of the resting potential in rat aortic smooth muscle.     

山莨菪碱对兔缺血再灌注后心室肌细胞瞬间外向钾通道电流的影响

目的建立兔心肌缺血再灌注动物模型,研究山莨菪碱对兔在体缺血再灌注后心室肌细胞Ito的影响,探讨山莨菪碱抗再灌注心律失常的细胞学离子机制。方法45只新西兰大耳白兔随机分为3组：缺血再灌注动物模型组（I—R组,结扎冠脉左前降支30min后再开放120min）,山莨菪碱治疗组（Ani组,手术前1min动物耳缘静脉注射山莨菪碱5mg／kg）和假手术对照组（只开胸不结扎血管）。观察缺血再灌注期间室性心律失常（室早、室速和室颤）的发生率及持续时间。采用酶解的方法分离缺血部位心室肌外膜单个心室肌细胞,应用全细胞膜片钳技术记录Ito结果与I—R组比较,Alli组兔室速、室颤发生率及持续时间明显下降,其心律失常的评分明显低于I—R组（2．6±0．7比3．6±0．8,P〈0．05）。对照组、I—R组和Ani组Ito电流密度（＋60mV时）分别为（17．41±3．13）pA／pF（n=15）、（9．49±1．91）pA／pF（n=11）和（16．55±2．86）pA／pF（n=10）,I—R组与对照组相比显著下降（P〈0．01）,Ani组与I—R组相比显著升高（P〈0．01）。结论山莨菪碱可降低心肌缺血再灌注期间心律失常的发生率。心肌缺血再灌注后Ito明显下降,山莨菪碱预处理可使下降的Ito上调,逆转电重构,可能为山莨菪碱降低心律失常发生率的细胞学离子机制。     

缬草单萜氧化物对兔单个心室肌细胞L-型钙电流的影响

利用全细胞膜片钳记录技术研究30μg/L和100μg/L缬草单萜氧化物(VMO)对兔单个心室肌细胞L型钙电流(ICaL)和动作电位的影响。结果:30μg/L和100μg/L的VMO使兔心室肌细胞ICaL峰值由6.04±0.59pA/pF分别减至3.99±0.31pA/pF和2.31±0.24pA/pF(n=8,P<0.01);VMO使ICaL的电流电压曲线上移,但不改变其激活电位、电位峰值和反转电位;VMO还使钙电流失活曲线左移。30μg/LVMO可使动作电位时程(APD)明显缩短,APD50和APD90分别缩短了50.3%和29.6%(n=16,P<0.05),而静息电位和动作电位幅值无明显改变。结论:VMO对LCaL具有浓度依赖性阻滞作用。这可能是其对心血管作用的重要机制之一。     

人右心室心外膜下细胞瞬间外向钾电流的初步研究

两例终末期心脏来源于心脏移植的受体病人 ,用酶解分离法获得心外膜下 (Epi)细胞 ,在全细胞钳制条件下观察人右心室Epi细胞瞬间外向钾电流 (Ito1 )的电生理特性。结果发现 :①Epi细胞具有强大的Ito1 ,在 0 .2Hz、+70mV和 37℃条件下 ,Epi细胞的峰值Ito1 离子流强度和密度分别达 1 940± 440pA、1 2 .9± 2 .6pA/pF ;②温度对Ito1 强度的影响明显 ,在 +70mV、0 .2Hz、2 2℃和 37℃条件下 ,Epi细胞峰值Ito1 强度和密度分别为 1 1 90± 31 0pA和 7.7±1 .8pA/pF、1 960± 465pA和 1 3 .1± 2 .8pA/pF ,差异有显著性 (P均 <0 .0 1 ) ;③Epi细胞Ito1 离子流强度表现出明显的频率依赖性 ,在 37℃和 +70mV、刺激频率分别为 0 .2 ,0 .5 ,1和 2Hz时 ,Epi细胞Ito1 离子流强度分别为 1 91 0±42 0 ,1 660± 360 ,1 4 1 0± 2 50 ,830± 1 4 0pA ,差异均有显著性 (P均 <0 .0 1 )。结论 :人右心室Epi细胞存在强大的Ito1 ,此为人右心室Epi细胞复极 1期一个突出的电生理特点 ,可能是Brugada综合征等疾病所致恶性心律失常的重要离子基础之一。     

异丙肾上腺素对兔右心室心外膜下心肌细胞电生理机制的影响

目的文献报道一些Brugada综合征（Brugadasyndrome,BS）患者使用异丙肾上腺素（isoproterenol,Iso）能够减轻耵段抬高,抑制窒性心律失常和电风暴的发生。本文对Iso这一作用的细胞电生理机制进行研究。方法酶解法分离兔右心室外膜下心肌（sub-epieardium,Epi）细胞。采用全细胞膜片钳技术记录1μmol Iso作用前后动作电传（actionpotential,AP）、L型钙电流（L—typecalciumeH／TeRt,ICaL）及短暂外向钾电流（transient outward potassiumcurrent,Ito）的变化。结果①Iso显著延长动作电位时限（APD）;②Iso使Ito峰值从（11．4±1．7）pA／pF减少到（6．3±0．5）pA／pF（n=16,P〈0．05）,使Ito I—V曲线下移,稳态失活曲线左移及失活后恢复曲线右移;③Iso使ICaL峰值从（-6．1±0．6）pA／pF增加到（-8．64-0．9）pA／pF（n=10,P〈0．05）,使I—V曲线下移。结论Iso呈电压依赖性抑制Ito和增加ICaL,延长APD,这可能与Iso能够减轻BS患者ST段抬高,抑制Bs患者窄性心律失常和电风暴的发生有关。