养心颗粒含药血清对豚鼠心室肌细胞外向延迟整流K电流的影响

目的观察养心颗粒含药血清对单个豚鼠心室肌细胞动作电位、外向延迟整流K电流的影响,探讨养心颗粒在离子通道水平的药理机制。方法胶原酶急性酶解法分离单个豚鼠心室肌细胞,实验分为空白对照组、养心颗粒含药血清2%、4%、8%组,采用全细胞膜片钳的方法,记录养心颗粒对动作电位、外向延迟整流K电流的影响。结果养心颗粒4%、8%组可延长APD 50和APD 90,其对应峰值钾电流分别为(15.33±1.56)、(14.33±1.11)PA/PF,与对照组比较,差异有统计学意义。养心颗粒2%组则无明显影响。结论养心颗粒通过抑制延迟整流K+通道来延长ADP50,ADP90,从而延长有效不应期,发挥其抗心律失常的作用,且可能有一定的浓度依赖性。     

蛋白激酶C对血管内皮功能的影响

【目的】研究血管内皮蛋白激酶C对内皮通透性及粘附功能的影响。【方法】以血管内皮细胞为研究对象,以流式分析法测定细胞间黏附分子-1（ICAM-1）,血管细胞黏附分子-1（VCAM-1）的表达;以同位素标记法测定HUVECs的粘附率;双室培养法测定HUVECs的通透率。【结果】10ng／mlPMA处理HUVECs后,HUVECs的通透性于30min时开始明显增高;VCAM-1,ICAM-1表达增强,对血小板粘附率2h达到最大值。【结论】PKC活化能其上调血管内皮细胞VCAM-1、ICAM-1的表达,并增强其粘附性。HUVECs的PKC活化与血管内皮细胞通透性增加有关。     

氯胺酮在细胞膜内对大鼠皮层细胞延迟整流钾通道的影响

[目的]测定高浓度氯胺酮在细胞膜内对大鼠皮层细胞延迟整流钾通道电导和动力学的影响.[方法]酶解法分离新生SD大鼠大脑皮层细胞,利用自行建立的膜片箝单离子通道检测系统内面向外模式检测其钾通道的特性,挑选出单个的延迟整流钾通道,并观测高浓度氯胺酮经细胞膜内通道内口对该通道的电导及动力学的影响.[结果]钳制电压 80 mV条件下,高浓度(0.5 mg/ml)氯胺酮在细胞膜内面作用前后其延迟整流钾通道的电流,开放、关闭常数及平均开放、关闭时间分别为:(-12.12±1.49)pA、5.490 mS、7.711mS、(12.50±21.71)mS、(8.91±10.34)mS和(-5.01±0.75)PA、0.686mS、3.252mS、(1.74±2.39)mS、(3.72±3.83)mS.[结论]高浓度氯胺酮在细胞膜内面直接使SD大鼠皮层细胞延迟整流钾通道的电导降低,开放、关闲常数及平均开放、关闭时间均减小.     

心房颤动患者超速激活延迟整流性钾电流重构的研究

目的:探讨心房颤动(房颤)患者超速激活延迟整流性钾电流(ⅠKur)的改变及意义。方法:采用膜片钳全细胞法分别记录窦性心律(窦律)患者心房肌细胞(窦律组)和房颤患者心房肌细胞(房颤组)ⅠKur密度,并进行对比。结果:指令电位+20～+50mV时,房颤组ⅠKur密度均较窦律组明显降低(P<0.05)。结论:ⅠKur密度下调是房颤心房电生理重构的重要离子基础,可能对心房动作电位时程的缩短具有一定代偿意义。     

葛根素对卵母细胞内向整流钾电流通道表达的影响

目的:探讨有扩张冠状动脉作用的葛根素对内向整流钾电流离子通道影响的电生理学机制。方法:①实验于2000-01/2002-12在解放军第四军医大学西京医院心内科实验室完成。通过对内向整流钾通道内向整流钾电流cDNA的体外转录得到内向整流钾电流的mRNA,以钠升为单位注射到非洲爪蟾卵母细胞内进行异源性表达,培养卵母细胞;通过双微电极电压钳记录内向整流钾电流通道的电流。②实验随机分6组(n=5):对照组Ⅰ犤未表达内向整流钾电流mRNA的卵母细胞(仅注射等离子水)犦;对照组Ⅱ犤用标准用液(mmol/L)2KCl,5HEPES,3MgCl2和0.2Niflumic酸做灌流液、已表达内向整流钾电流mRNA的卵母细胞犦;1.2,2.4,4.8mmo1/L葛根素组;9.6mmo1/L葛根素组(分别用1.2,2.4,4.8和9.6mmo1/L的葛根素加入标准用液来灌流的、已表达内向整流钾电流mRNA的卵母细胞)。使用pCLAMP的Clampfit软件测量电流大小。③采用加药前后的自身配对t检验,组间比较采用团体t检验。结果:注射等离子水的细胞未能记录出典型的电流,而注射4.5～5.5kb内向整流钾电流的细胞则记录出典型的内向整流钾电流。细胞外1.2mmol/L的葛根素即对内向整流钾电流的内向电流和外向电流都有抑制作用(P<0.05),随着药物浓度成倍递增,阻断作用亦增强。以标准用液记录的已表达内向整流钾电流mRNA的卵母细胞(对照组Ⅱ)电流峰值为100%,加不同浓度葛根素后记录的卵母细胞电流值为对照组Ⅱ电流的百分数,葛根素9.6mmol/L时,对内向整流钾电流的阻断率达(58.6±4.9)%(P<0.01)。结论:葛根素对卵母细胞表达的内向整流钾通道内向整流钾电流有显著的抑制作用,且抑制作用呈浓度依赖性。     

二乙酰基莲心碱对心室肌细胞内向整流钾电流和瞬时外向钾电流的影响

目的:探讨二乙酰基莲心碱(diacetyl-linesinine)对家兔心室肌细胞瞬时外向钾电流和内向整流钾电流的影响。方法:选取5只体重1.5～2.0kg的健康新西兰大耳白兔,酶解法分离单个心室肌细胞,应用全细胞膜片钳技术观察10、30、100μmol/L的二乙酰基莲心碱对心室肌细胞膜瞬时外向钾电流和内向整流钾电流的作用。结果:二乙酰基莲心碱浓度依赖性减少瞬时外向钾电流和内向整流钾电流,10、30、100μmol/L的二乙酰基莲心碱可使峰值瞬时外向钾电流降低14.7%、26.7%和36.6%;使内向整流钾电流降低13.7%、25.3%和31.1%。结论:二乙酰基莲心碱可浓度依赖性阻滞兔心室肌细胞的瞬时外向钾电流和内向整流钾电流。     

心肌缺血预适应延迟保护作用和诱导型一氧化氮合酶及蛋白激酶C的关系

缺血预适应（IPc）是体内普遍存在的一种强大的自身的保护机制。随着研究的不断深入，发现心肌缺血预适应存在具有更广泛临床意义的延迟保护作用，并且这种延迟保护作用与诱导型一氧化氮合酶（iNOS）的活化及蛋白激酶C（PKC）通路有关。文章就心肌缺血预适应延迟保护作用机制与iNOS及PKC的关系研究进展作一综述。     

血管紧张素Ⅱ及其受体拮抗剂对豚鼠心肌细胞电生理及L-型钙电流的作用

目的 探讨血管紧张素Ⅱ及其受体拮抗剂对豚鼠心肌细胞动作电位间期及L-型钙电流的作用。方法 分离豚鼠乳头肌的单个心室肌细胞，采用内充3mol／LKCl的玻璃微电极记录心肌动作电位。采用膜片钳全细胞技术，钳制电位-40mV，保持时间200ms，指令电位为0，并记录L-型钙电流的最大峰电流。结果 灌注血管紧张素Ⅱ可致多种机制的心律失常。灌注1min，动作电位振幅、动作电位复极90％的间期（APD90）、静息膜电位（RMP）较对照状态显著降低或缩短；灌注3min，动作电位复极30％和50％的间期（APD30和APD50）及有效不应期均较对照状态显著缩短。膜片钳研究示血管紧张素Ⅱ灌注5min，L-型钙电流较对照状态显著增加，氯沙坦灌注1min L-型钙电流显著降低，灌注3min较1min进一步降低，电压-电流关系曲线形状均无显著变化。结论 血管紧张素Ⅱ降低动作电位幅度，缩短动作电位时程及有效不应期，电压依赖性增加L-型钙电流最大峰电流，具有致心律失常作用，氯沙坦电压依赖性地降低L-型钙电流。     

蛋白激酶Cβ抑制剂LY333531对糖尿病大鼠造影剂肾病的保护作用

目的探讨蛋白激酶Cβ（PKCβ）抑制剂LY333531对造影剂诱导的糖尿病大鼠急性肾损伤的保护作用及其机制。 方法将40只大鼠分为健康对照组（C组）、糖尿病组（D组）、糖尿病造影剂组（DC组）以及糖尿病+ Y333531 +造影剂组（DCL组）,每组各10只。对D组、DC组及DCL组大鼠腹腔注射2%链脲佐菌素（60 mg / kg）建立糖尿病模型;对DCL组大鼠给予LY333531灌胃预处理（10 mg·kg^-1·d^-1）,持续14 d;对DC组与DCL组大鼠进行尾静脉注射76%复方泛影葡胺（10 ml / kg）,建立造影剂肾病模型。苏木素-伊红（HE）染色观察各组大鼠肾组织病理变化;检测各组大鼠血糖、血肌酐、尿微量白蛋白（mAlb）和N-乙酰-β-D-葡萄糖苷酶（NAG）水平;采用实时荧光定量PCR法检测转化生长因子β1（TGFβ1）、Smad3、Smad7、Bax、Caspase3和Bcl2的信使RNA（mRNA）表达水平;采用Western-blotting检测TGFβ1、Bax、Caspase3、Bcl2蛋白及磷酸化PKCβ（pPKCβ） / PKCβ、磷酸化p38（p-p38） / p38的比值。 结果HE结果显示,C组大鼠肾小球、肾小管结构正常;D组大鼠肾组织内肾小球及肾小管结构形态较为完整,间质周围极少量炎症细胞;DC组肾小管上皮细胞脱落、周围炎症细胞浸润明显;DCL组大鼠肾小管上皮细胞脱落程度明显降低,肾小管及肾小球周围间质少量炎症细胞浸润。各组大鼠间血糖、血肌酐、尿mAlb、尿NAG水平、TGFβ1蛋白及其mRNA、Smad3 mRNA、Smad7 mRNA、Bax蛋白及其mRNA、Caspase3蛋白及其mRNA、Bcl2蛋白及其mRNA表达水平、pPKCβ / PKCβ和p-p38 / p38比值间比较,差异均有统计学意义（F = 67.976,P < 0.001;F = 27.155,P < 0.001;F = 41.201,P < 0.001;F = 59.635,P < 0.001;F = 21.073,P < 0.001;F = 28.365,P < 0.001;F = 15.215,P < 0.001;F = 36.273,P < 0.001;F = 14.489,P < 0.001;F = 23.172,P < 0.001;F = 17.103,P < 0.001;F = 29.916,P < 0.001;F = 12.026,P < 0.001;F = 13.368,P < 0.001;F = 6.126,P = 0.002;F = 6.434,P = 0.002）。进一步两两比较发现,D组、DC组以及DCL组大鼠的血糖水平较C组大鼠均明显升高（P均< 0.05）;与DC组大鼠比较,DCL组大鼠的的血肌酐、尿mAlb、尿NAG水平、TGFβ1蛋白及其mRNA、Smad3 mRNA、Bax蛋白及其mRNA、Caspase3蛋白及其mRNA、pPKCβ / PKCβ和p-p38 / p38比值均显著降低,而Smad7 mRNA、Bcl2蛋白及其mRNA表达水平均显著升高（P均< 0.05）。 结论PKCβ抑制剂LY333531能通过抑制PKCβ-TGFβ-p38-Caspase3通路改善造影剂诱导的糖尿病大鼠急性肾损伤。     

Thioridazine lengthens repolarization of cardiac ventricular myocytes by blocking the delayed rectifier potassium current

Proarrhythmia has been observed with the antipsychotic agent thioridazine (THIO). The mechanisms underlying these effects are unknown. The objectives of this study were 1) to characterize the effects of THIO on cardiac repolarization and 2) to determine whether lengthening of the Q-T interval could be explained by blocking major K+-repolarizing currents. Isolated, buffer-perfused guinea pig hearts (n = 32) were stimulated at various pacing cycle lengths (150-250 ms) and exposed to THIO at concentrations ranging from 300 nM to 3 microM. THIO increased monophasic action potential duration at 90% repolarization (MAPD90) in a concentration-dependent manner from 14.9 +/- 1.8 at 300 nM to 37.1 +/- 3.2 ms at 3 microM. Increase in MAPD90 was also reverse frequency-dependent; THIO (300 nM) increased MAPD90 by 14.9 +/- 1.8 ms at a pacing cycle length of 250 ms, but by only 7.7 +/- 1.2 ms at a pacing cycle length of 150 ms. Patch-clamp experiments demonstrated that THIO decreases the time-dependent outward K+ current elicited by short depolarizations (250 ms; IK250) in a concentration-dependent manner. Estimated IC50 for IK250, which mostly underlies IKr, was 1.25 microM. Time-dependent outward K+ current elicited in tsA201 cells expressing high levels of HERG protein was also decreased approximately 50% by 1.25 microM THIO. On the other hand, THIO was less potent (IC50 of 14 microM) to decrease time-dependent K+ current elicited by long pulses (5000 ms; IK5000). Under the latter conditions, IK5000 corresponds mainly to IKs. Thus, these results demonstrate block of K+ currents and lengthening of cardiac repolarization by THIO in a concentration-dependent manner. This may provide an explanation of Q-T prolongation observed in some patients treated with THIO.     

Differential modulation by beta adrenoceptors of inward calcium and delayed rectifier potassium current in single ventricular cells of guinea pig heart

This study was designed to investigate the modulation of the high-threshold (L-type) inward calcium current (ICa) and the delayed rectifier potassium current (IK) by beta adrenoceptor stimulation in single ventricular cells of guinea pig heart. Single ventricular cells were prepared by the collagenase dispersion procedure, and membrane currents were recorded with a patch electrode by use of the whole-cell voltage clamp method. ICa was obtained by intra- and extracellular perfusion with the Cs(+)-solutions that suppressed potassium currents. IK was evaluated in Co+(+)-Tyrode's solution in which 0.9 mM Co+(+) was substituted for equimolar Ca+(+) to abolish ICa. Isoproterenol, a nonselective beta adrenoceptor agonist, increased not only ICa but also IK at the same threshold concentration (1 nM). In contrast, the threshold concentration of T-1583, a selective beta-1 adrenoceptor agonist, for increasing ICa (1 nM) was distinctly lower than that for increasing IK (100 nM). These results suggest that ICa and IK can be differentially modulated by beta adrenoceptors.     

Histamine modulates calcium current in guinea pig ventricular myocytes

In the present work we have examined the effects of histamine on electrophysiological parameters of isolated cardiocytes acutely dissociated from adult guinea pigs. The whole cell patch clamp technique was used to examine action potential and currents. Stable recordings of calcium current (average peak amplitude of 563 +/- 256 pA per cell, n = 83) were obtained. Application of histamine (5 nM-10 microM) resulted in marked modification of action potentials and calcium current. Calcium current was enhanced by histamine in a concentration-dependent manner (half-maximal dose, 10 nM) up to 315% of the control level. Action potentials were prolonged by small doses of histamine whereas larger doses caused voltage instabilities and spontaneous arrhythmic bursts. Histamine effects were reduced by cimetidine and ranitidine (1 microM), two H2-receptor antagonists. After the calcium current was increased by intracellular perfusion of micromolar cyclic AMP saturating doses of histamine had no effect. Micromolar concentrations of acetylcholine rapidly antagonized the histamine effect. The latter results indicate that the response to histamine is based on H2-receptor-mediated activation of adenylate cyclase and cyclic AMP regulation of phosphorylation of calcium channel.     

Pharmacological activation of rapid delayed rectifier potassium current suppresses bradycardia-induced triggered activity in the isolated guinea pig heart

Recently, attention has been drawn to compounds that activate the human ether-a-go-go channel potassium channel (hERG), which is responsible for the repolarizing rapid delayed rectifier potassium current (I(Kr)) in the mammalian myocardium. The compound NS3623 [N-(4-bromo-2-(1H-tetrazol-5-yl)-phenyl)-N'-(3'-trifluoromethylphenyl) urea] increases the macroscopic current conducted by the hERG channels by increasing the time constant for channel inactivation, which we have reported earlier. In vitro studies suggest that pharmacological activation is an attractive approach for the treatment of some arrhythmias. We present here data that support that NS3623 affects native I(Kr) and report the effects that activating this potassium current have in the intact guinea pig heart. In Langendorff-perfused hearts, the compound showed a concentration-dependent shortening of action potential duration, which was also detected as concentration-dependent shorter QT intervals. There was no sign of action potential triangulation or reverse use dependence. NS3623 decreased QT variability and distinctly decreased the occurrence of extrasystoles in the acutely bradypaced hearts. Taken together, the present data strongly support the concept of using hERG activators as a treatment for certain kinds of arrhythmias and suggest further investigation of this new approach.     

Amiodarone blocks calcium current in single guinea pig ventricular myocytes

Ca++ current (lca) block by amiodarone and the underlying mechanisms thereof were investigated in guinea pig single ventricular myocytes using the single suction pipette whole cell voltage clamp method. The dose-response curve revealed a 1:1 stoichiometry for binding of amiodarone to its receptor with an apparent dissociation constant of 5.8 microM in the resting state. Amiodarone, 5 microM did not significantly alter the time course of ICa decay, but did shift the steady-state inactivation curve for lca in the hyperpolarizing direction by 9.2 +/- 3.1 mV. Development of block at depolarized potentials was voltage-dependent between -20 and 10 mV with time constants of 112 +/- 33 and 755 +/- 212 msec at 10 mV. In the presence of 0.2 microM amiodarone, recovery from inactivation was fitted by a double exponential most likely indicating rapid recovery of the drug-free Ca++ channels and slow recovery of the drug-associated Ca++ channels with time constants of 44 +/- 12 and 108 +/- 403 msec, respectively, at -80 mV. The proportion of the current recovering via the slow phase was 36 +/- 7%. By using this value, we estimated the dissociation constant in the inactivated state to be 0.36 microM. Amiodarone's marked use-dependent block of lca is explicable in terms of its high affinity for, and slow dissociation from, Ca++ channels in the inactivated state. These results suggest that amiodarone blocks lca in both the resting and inactivated states.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lactate activates ATP-sensitive potassium channels in guinea pig ventricular myocytes.

The functional significance of cardiac ATP-sensitive potassium channels remains controversial because of the discrepancy between the low levels of ATP at which activation of the channels occurs and the much higher levels of ATP maintained during myocardial ischemia. We studied the effects of (+)-lactate, which accumulates in large quantity as a result of increased glycolysis during ischemia, on ATP-sensitive potassium channels in adult guinea pig ventricular myocytes using the whole-cell patch-clamp technique. Lactate at 20-40 mM in the internal solution activated ATP-sensitive potassium channels and shortened action potential duration. Activation of the channels occurred even in the presence of 2-5 mM ATP in the internal solution and was dependent on intracellular free magnesium levels. Our results suggest that intracellular lactate may play a significant role in activating cardiac ATP-sensitive potassium channels and shortening action potential duration even at ATP levels similar to those resulting from moderate to severe myocardial ischemia.     

Effect of ambasilide, a new class III agent, on plateau currents in isolated guinea pig ventricular myocytes: block of delayed outward potassium current.

The actions of ambasilide (LU-47110) on the action potential and membrane currents of isolated guinea pig ventricular myocytes were studied using voltage clamp techniques. Ambasilide (1 microM) prolonged the action potential (APD) at 20, 50 and 90% repolarization by 11.2 +/- 4.3, 13.8 +/- 3.9 and 13.6 +/- 3.7%, respectively, compared to control (n = 10). APD prolongation was attributed to the block of delayed rectifier outward current (Ik) in a concentration-dependent fashion (0.01-10 microM). The effects on the APD and Ik were both partially reversed after perfusion with drug-free Tyrode's solution. The block of Ik by ambasilide was compared to that by E-4031 (5 microM), a putative selective blocker of that fast, inwardly rectifying component of Ik identified in guinea pig ventricle. E-4031 produced about 65% block of Ik for pulse durations between 80 and 420 msec, but the block decreased as the pulse duration increased further, the block accounting for 34 +/- 5% of Ik at 6.3 sec. In contrast, the percentage of reduction of Ik by 10 microM ambasilide did not produce a consistent magnitude of block over a similar range of short depolarizations, but rather progressively decreased Ik as the pusle duration lengthened. Block at the end of a 2-sec pulse was about 48 +/- 8%, more block than could be attributed to an E-4031-sensitive current block alone. Whereas E-4031 (5 microM) shifted the activation curve of Ik 10 mV toward positive potentials and decreased the slope factor, k, by about 4 mV, ambasilide (5 microM) had no effect on these parameters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of beta-adrenoceptor mediated increase in delayed rectifier potassium current

Experiments were carried out in single ventricular cells of the guinea-pig heart. Isoproterenol, forskolin, intracellularly applied cyclic AMP and 3-isobutyl-1-methylxanthine increased the delayed rectifier potassium current (IK). The effect of isoproterenol was abolished by intracellularly applied guanosine 5'-O-(3-thio-triphosphate). These results indicate that isoproterenol stimulates beta-adrenoceptors to activate adenylate cyclase by mediation through the stimulatory GTP-binding protein, and causes an increase in intracellular cyclic AMP levels. Then IK is probably increased by phosphorylation of the IK-channel protein by cyclic AMP-dependent protein kinase.     

Effects of mitoxantrone on excitation-contraction coupling in guinea pig ventricular myocytes

The mechanisms of the inotropic effect of mitoxantrone (MTO), a synthetic dihydroxyanthracenedione derivative with antineoplastic activity, was investigated in guinea pig ventricular myocytes using whole-cell patch-clamp methods combined with fura-2 fluorescence and cell-edge tracking techniques. In right ventricular papillary muscles, 30 microM MTO increased isometric force of contraction as well as action potential duration (APD) in a time-dependent manner. The force of contraction was increased approximately 3-fold within 4 h. This positive inotropic effect was accompanied by a prolongation of time to peak force and relaxation time. In current-clamped single myocytes treated with 30 microM MTO for 30 min, an increase of cell shortening by 77% and a prolongation of APD by 19% was observed. Peak amplitude of the intracellular Ca(2+) transients was also increased by 10%. The contribution of APD prolongation to the enhancement of cell shortening induced by MTO was assessed by clamping control myocytes with action potentials of various duration. Prolongation of APD(90) (ADP measured at 90% of repolarization) by 24% led to an increase of cell shortening by 13%. When the cells were clamped by an action potential with constant APD, MTO still caused an increase of cell shortening by 59% within 30 min. No increase of the peak intracellular Ca(2+) transients, however, was observed under this condition. We conclude that both the APD prolongation and a direct interaction with the contractile proteins contributed to the positive inotropic effect of MTO.