胍丁胺对大鼠心室肌细胞L—钙通道电流的影响

目的:观察胍丁胺(Agm)对大鼠心室肌细胞L-型钙通道电流(I_(Ca-L))的影响.方法:以酶解法制备单个心室肌细胞.应用全细胞膜片箝技术记录大鼠单个心室肌细胞钙通道电流.结果:(1)Agm(0.5,1,2mmol/L)可浓度依赖性地降低电压依赖性激活I_(Ca-L)(pA)峰值,其值从1451±236 (对照组)到937±105(n=8,P<0.05),585±74(n=8,P<0.01),和301±156(n=8,P<0.01).(2)Agm 1 mmol/L使用依赖性地阻滞I_(Ca-L)·1 Hz时抑制率为53％±12％(P<0.05),3Hz时为69％±11％(P<0.01).(3)Agm使I-V曲线上移,但对I_(Ca-L)的电压依赖特征、最大激活电压以及I_(Ca-L)稳态激活无明显影响.在Agm 1 mmol/L作用下,半数激活电压(V_(0.5)和斜率参数(k)与对照组相比均无显著性差异.V_(0.5)分别为(-20.2±2.5)mV和(-20.5±2.7)mV,k分别为(3.2±0.4)mV和(3.0±0.5)mV.(4)Agm 1 mmol/L可明显使钙电流稳态失活曲线左移,加速钙通道电压依赖性稳态失活.V_(0.5)分别为(-32±6)mV和(-40±5)mV,k分别为(7.6±O.9)mV和(12.5±1.1)mV(P<0.05).(5)Agm 1mmol/L还使I_(Ca)从失活状态下恢复明显减慢.结论:Agm抑制I_(Ca-L),并主要作用于L-型钙通道的失活状态,表现为钙通道失活加速和从失活状态下恢复减慢.     

Short- and long-term amiodarone treatments regulate Cav3.2 low-voltage-activated T-type Ca2+ channel through distinct mechanisms

Low-voltage-activated T-type Ca2+ channels have been recognized recently in the mechanisms underlying atrial arrhythmias. However, the pharmacological effects of amiodarone on the T-type Ca2+ channel remain unclear. We investigated short- and long-term effects of amiodarone on the T-type (Cav 3.2) Ca2+ channel. The Cav3.2 alpha1H subunit derived from human heart was stably transfected into cells [human embryonic kidney (HEK)-Cav3.2] cultured with or without 5 muM amiodarone. Patch-clamp recordings in the conventional whole-cell configuration were used to evaluate the actions of amiodarone on the T-type Ca2+ channel current (ICa.T). Amiodarone blockade of ICa.T occurred in a dose- and holding potential-dependent manner, shifting the activation and the steady-state inactivation curves in the hyperpolarization direction, when amiodarone was applied immediately to the bath solution. However, when the HEK-Cav3.2 cells were incubated with 5 microM amiodarone for 72 h, ICa.T density was significantly decreased by 31.7+/-2.3% for control,-93.1+/-4.3 pA/pF (n=8), versus amiodarone,-56.5+/-3.2 pA/pF (n=13), P<0.001. After the prolonged administration of amiodarone, the activation and the steady-state inactivation curves were shifted in the depolarization direction by -7.1 (n=41) and -5.5 mV (n=37), respectively, and current inactivation was significantly delayed [time constant (tau): control, 13.3+/-1.1 ms (n=6) versus amiodarone, 39.6+/-5.5 ms (n=6) at -30 mV, P<0.001)]. Nevertheless, short-term inhibitory effects of amiodarone on the modified T-type Cav3.2 Ca2+ channel created by long-term amiodarone treatment were functionally maintained. We conclude that amiodarone exerts its short- and long-term inhibitory actions on ICa.T via distinct blocking mechanisms.     

Effect of progesterone on calcium activated potassium currents and intracellular calcium in guinea pig colon myocytes

AIMS: To study the effects of progesterone on contractile activity of smooth muscle strips and on ion currents and intracellular Ca2+ ([Ca2+]i) intensity in single colonic myocytes in guinea pig proximal colons. METHODS: Strips and single cells were dissected from female guinea pig proximal colon. Contraction of strips through an isotonic transducer was assessed and the responsible currents to progesterone were recorded with EPC-9 amplifier in nystatin perforated whole-cell configuration. Detection of [Ca2+]i fluorescence loading fura-2 acetoxymethylester (fura-2/AM) was measured with confocal microscope. RESULTS: Progesterone significantly inhibited contraction of guinea pig colon strips in a dose-dependent pattern. Inhibitory concentration 50 (IC50) of progesterone in longitudinal strips and circular strips was, respectively, 9.7 microM and 1.0 nM. Iberiotoxin (IbTX) partially blocked inhibition of progesterone in both oriented smooth muscle strips. Ca2+ activated K+ (K(Ca)) channel currents recorded by depolarizing pulse protocol were enhanced by progesterone to 138% +/- 13% (n = 9, p < 0.01), and to 143% +/- 12% (n = 8, p < 0.01) when perfused with 10 mcM onapristone. Progesterone reduced L-Ca2+ currents to 67% +/- 6% (n = 7, p < 0.01) and had no effect with 5 microM nicardipine in bath solution. [Ca2+]i fluorescence was reduced by progesterone to 75% +/- 12% (n = 8, p < 0.01). CONCLUSION: Progesterone decreases the contraction of colonic smooth muscles by enhancing K(Ca) currents and reducing Ca2+ influx.     

Recovery of the slow action potential is hastened by the calcium slow channel agonist, Bay-K-8644

Effects of the positive inotropic drug, Bay-K-8644, were studied on the slow action potential (AP) parameters and diastolic recovery of Vmax in K+ (22 mM)-depolarized rabbit papillary muscles. Bay-K-8644 (10(-6) M) increased the amplitude, maximum rate of rise (Vmax) and duration of the slow APs. Diastolic recovery of Vmax, examined by a paired-pulse protocol, was approximated by a single exponential function, both in control and in drug-treated muscles. The time constant of the recovery for drug-treated preparations was 171 +/- 20 ms (n = 9), and was significantly smaller than that for control: 414 +/- 45 ms (n = 12) (P less than 0.001). The diastolic intervals which allow 90% recovery of Vmax (T90%) were: 752 +/- 106 ms (n = 12) for control and 364 +/- 53 ms (n = 9) in the presence of drug, the latter being significantly shorter (P less than 0.01). The extent of the reductions in Vmax, at driving frequencies higher than 0.5 Hz, was minimal in the presence of the drug compared to the control. It was concluded that Bay-K-8644 not only enhanced the slow inward current, but also accelerated the reactivation process of the slow inward current and Ca2+ slow channel.     

蜂毒肽对豚鼠心室肌细胞钾电流和动作电位的影响(英文)

目的:研究蜂毒肽(Melittin,Mel)对豚鼠心室肌细胞钾电流和动作电位的影响.方法:全细胞膜片箝记录.结果:蜂毒肽可呈浓度依赖性促进延迟整流钾电流(I_k),在测定电压为40 mv时,0.05,0.1,0.2μmol·L~(-1)蜂毒肽分别使I_k从(295±109)增大到(371±142)(n=5 P<0.05),(467±180)(n=5,P<0.05),(552±248)pA(n=5,P<0.05).但药物在三个浓度时对内向整流钾电流(I_(k1))均无显著影响.蜂毒肽0.05,0.1,0.2 μmol·L~(-1)分别使动作电位APD_(50)由(520±55)减小到(459±91)(n=5,P>0.05),(385±102)(n=5,P<0.01),(281±81)ms(n=5,P<0.01),使APD_(90)由(613±96)减小到(536±93)(n=5,P>0.05),(467±96)(n=5,P<0.01),(354±95)ms(n=5,P<0.01).结论:蜂毒肽促进延迟整流钾电流,缩短动作电位时程.     

大黄素对豚鼠单个心室肌细胞胞浆游离钙浓度及L-型钙电流的影响

目的研究大黄素(emodin)对豚鼠心室肌细胞钙信号的影响。方法酶解法分离豚鼠单个心室肌细胞,应用激光扫描共聚焦显微镜联合全细胞膜片钳技术测量豚鼠心室肌细胞钙信号的变化。结果在静息状态下,1～100 μmol·L^-1大黄素对［Ca²⁺］i均无影响;对60 mmol·L^-1 KCl诱导的外钙内流引起的胞浆钙升高有不同的影响,1 μmol·L^-1表现为促进作用;10 μmol·L^-1无作用;100 μmol·L^-1则表现为抑制作用。膜片钳研究结果表明,1 μmol·L^-1大黄素可明显促进L-型钙电流,10 μmol·L^-1对L-型钙电流无影响;100 μmol·L^-1明显抑制L-型钙电流。结论大黄素对心肌细胞内钙及L-型钙电流具有双向调节作用。     

内皮素对慢性低氧大鼠肺动脉平滑肌细胞膜钾通道活性的影响

目的观察内皮素(ET-1)对慢性低氧大鼠肺动脉平滑肌细胞(PASMCs)膜电压门控钾通道(K_V)活性的影响。方法将12只Wistar大鼠随机分为对照组和慢性低氧组，每组6只。用全细胞膜片钳记录方法研究ET-1对两组大鼠PASMCs膜电位(E_m)、膜电容(C_m)及电压门控钾电流(IK_V)的影响。结果ET-1可引起两组大鼠PASMCs去极化，且对两组大鼠IK_V均有明显的浓度依赖性抑制作用。在慢性低氧组，高浓度ET-1对IK_V的抑制作用强于对照组。结论低氧并未改变ET-1引起PASMCs去极化及浓度依赖性抑制IK_V的特性，且慢性低氧可能改变了PASMCs对ET-1的敏感性，内皮素和低氧对PASMCs IK_V的抑制作用有协同性。     

未成年人心房肌细胞瞬间外向钾电流和内向整流钾电流的特性

AIM: To study the properties of transient outward K+ current (Ito) and inward rectifier K+ current (IKl) in immature human heart. METHODS: Ito and IKl were recorded using whole-cell patch-clamp technique in atrial myocytes isolated from 12 immature (aged from 6 months to 5 a) human hearts. RESULTS: Ito was voltage-dependent, activated and inactivated rapidly. The IC50 (95% confidence limits) of 4-AP on Ito was 0.64 (0.48-0.87) mmol.L-1. 4-AP 1 mmol.L-1 shifted V1/2 of activation from (6.6 +/- 2.0) mV to (19.8 +/- 3.0) mV (n = 4-10, P < 0.01). 4-AP 0.3 mmol.L-1 changed V1/2 of inactivation from (-49 +/- 4) mV to (-61.4 +/- 2.1) mV (n = 3, P < 0.01), but there were no obvious influence on voltage-dependent activation of Ito (P > 0.05). At the same concentration, the recovery time constant (tau value) was prolonged from (108 +/- 16) ms to (220 +/- 67) ms (n = 3-12, P < 0.01). IKl was also voltage-dependent. Its reverse potential was -40 mV. CONCLUSION: Both Ito and IKl are important K+ channel currents in immature human atrial myocytes. 4-AP can affect the inactivation and recovery of Ito at low concentration (0.3 mmol.L-1) and affect its activation at high concentration (1 mmol.L-1).     

肿瘤坏死因子α对大鼠心室肌细胞钙转运的影响

AIM: To study the effects of tumor necrosis factor-alpha (TNF-alpha) on calcium movement in rat ventricular myocytes. METHODS: Intracellular free Ca2+ concentration was measured with calcium fluorescent probe Fluo-3/AM and laser confocal microscope. L-type calcium current (ICa,L) was recorded with the whole-cell configuration of the patch-clamp techniques. RESULTS: At 2, 20 and 200 microg/L, TNF-alpha was found to increase intracellular free Ca2+ concentration in a dose-dependent manner illustrated by the increment of calcium fluorescence density with laser confocal microscope. Nicardipine 0.5 micromol/L slightly attenuated TNF-alpha-induced response. When the cardiac myocytes were exposed to caffeine (100 mmol/L) for 30 min, TNF-alpha failed to induce any change of intracellular free calcium. However, it was found that TNF-alpha inhibited I(Ca,L) in whole-cell patch-clamp experiments. At 2, 20, and 200 microg/L, TNF-alpha decreased peak I(Ca,L) by 3.9 % (-5.1 pA/pF+/-0.3 pA/pF vs -4.9 pA/pF+/-0.2 pA/pF, n=9, P>0.05), 15.7 % (-5.1 pA/pF+/-0.3 pA/pF vs -4.3 pA/pF+/-0.3 pA/pF, n=9, P<0.05) and 19.6 % (-5.1 pA/pF+/-0.3 pA/pF vs -4.1 pA/pF+/-0.4 pA/pF, n=9, P<0.01), respectively. It shifted the steady-state inactivation curve of I(Ca,L) to the left (V1/2 shifted from -28.7 mV+/-0.3 mV to -37.8 mV+/-1.4 mV, n=7, P<0.05), while it took no effects on steady-state activation and recovery from inactivation. CONCLUSION: TNF-alpha inhibited I(Ca,L) in rat ventricular myocytes, while increasing the intercellular free Ca2+ level due to the release of Ca2+ from intracellular stores.     

Raloxifene acutely suppresses ventricular myocyte contractility through inhibition of the L-type calcium current

1. The selective oestrogen (ER) receptor modulator, raloxifene, is widely used in the treatment of postmenopausal osteoporosis, but may also possess cardioprotective properties. We investigated whether it directly suppresses myocyte contractility through Ca(2+) channel antagonism in a similar way to 17beta-oestradiol. 2. Cell shortening and Ca(2+) transients were measured in single guinea-pig ventricular myocytes field-stimulated (1 Hz, 37 degrees C) in a superfusion chamber. Electrophysiological recordings were performed using single electrode voltage-clamp. 3. Raloxifene decreased cell shortening (EC(50) 2.4 microm) and the Ca(2+) transient amplitude (EC(50) 6.4 microm) in a concentration-dependent manner. At a concentration of 1 microm, raloxifene produced a 33+/-2% (mean+/-s.e.m) and 24+/-2% reduction, respectively (P<0.001, n=14 for both parameters). 4. These inhibitory actions were not observed in myocytes that had been incubated with the specific antagonist, ICI 182,780 (10 microm) (n=11). 5. Raloxifene (1 microm) shortened action potential durations at 50 and 90% repolarisation (P<0.05 and <0.001, respectively; n=27) and decreased peak L-type Ca(2+) current by 45%, from -5.1+/-0.5 pA/pF to -2.8+/-0.3 pA/pF (P<0.001, n=18). 6. Raloxifene did not significantly alter sarcoplasmic reticulum Ca(2+) content, as assessed by integrating the Na(+)/Ca(2+) exchanger currents following rapid caffeine application. 7. The present study provides evidence for direct inhibitory actions of raloxifene on ventricular myocyte contractility, mediated through Ca(2+) channel antagonism.