Effects of unsaturated fatty acids on calcium-activated potassium current in gastric myocytes of guinea pigs

AIM: To investigate the effects of exogenous unsaturated fatty acids on calcium-activated potassium current [IK(Ca)] in gastric antral circular myocytes of guinea pigs. METHODS: Gastric myocytes were isolated by collagenase from the antral circular layer of guinea pig stomach. The whole-cell patch clamp technique was used to record /K(Ca) in the isolated single smooth muscle cells with or without different concentrations of arachidonic acid (AA), linoleic acid (LA), and oleic acid (OA). RESULTS: AA at concentrations of 2,5 and 10 μmol/L markedly increased IK(Ca) in a dose-dependent manner. LA at concentrations of 5, 10 and 20 μmol/L also enhanced /K(Ca) in a dose-dependent manner. The increasing potency of AA, LA, and oleic acid (OA) on /K(Ca)at the same concentration (10μmol/L) was in the order of AA>LA>OA. AA (10 μmol/L)-induced increase of Ik(ca) was not blocked by H-7 (10 μmol/L), an inhibitor of protein kinase C (PKC), or indomethacin (10 μmol/L), an inhibitor of the cyclooxygenase pathway, and 17-octadecynoic acid (10 μmol/L), an inhibitor of the cytochrome P450 pathway, but weakened by nordihydroguaiaretic acid (10μmol/L), an inhibitor of the lipoxygenase pathway. CONCLUSION: Unsaturated fatty acids markedly increase Ik(Ca), and the enhancing potencies are related to the number of double bonds in the fatty acid chain. The lipoxygenase pathway of unsaturated fatty acid metabolism is involved in the unsaturated fatty acid-induced increase of IK(Ca) in gastric antral circular myocytes of guinea pigs.     

牛磺酸对低氧条件下豚鼠心室肌细胞内向整流钾电流的影响

目的:研究牛磺酸对低氧条件下豚鼠心室肌细胞内向整流钾电流(Ik1)的影响。方法:酶解法分离单个豚鼠心室肌细胞和建立离体低氧模型,采用全细胞膜片钳记录技术记录牛磺酸对急性低氧条件下豚鼠心室肌细胞Ik1的作用。结果:在钳制电压-40 mV下,给予20 mmol/L牛磺酸使Ik1的电流峰值降低,抑制率(45.61±10.70)%;洗脱后,Ik1能够部分恢复(P<0.05)。牛磺酸影响了Ik1的电流-电压曲线。结论:牛磺酸可明显阻滞低氧条件下豚鼠心室肌细胞膜上Ik1电流,这可能是其抗心律失常作用的电生理机制之一。     

盐酸关附甲素对豚鼠和大鼠心肌细胞钾通道的阻断作用

目的用膜片钳全细胞记录法观察盐酸关附甲素(GFA)对分离的单个豚鼠心室肌细胞缓慢激活型延迟整流钾电流(IKs),大鼠内向整流钾电流(IK1)、瞬时外向钾电流(Ito)的影响。方法用急性酶解法分离获得单个豚鼠和大鼠心室肌细胞。用标准的全细胞膜片钳技术记录IKs、IK1、Ito离子通道电流,观察不同浓度的GFA对豚鼠心室肌细胞IKs,大鼠心室肌细胞IK1、Ito的影响。结果50,150,500μmol/LGFA使IKs尾电流(IKs,tail)最大峰值电流密度分别降低11.4%±3.32%、23.3%±7.36%、36.7%±4.99%,P<0.05;使IK1稳态电流密度分别降低5.1%±0.6%、7.5%±0.9%、7.2%±0.9%;50,500μmol/LGFA使Ito最大峰值电流密度分别降低6.1%±0.64%、8.6%±1.13%。结论GFA对IKs具有浓度依赖性阻滞作用,这可能是其延长动作电位时程而对静息电位影响不大的电生理基础,是其抗心律失常作用的机制之一。     

卡托普利对豚鼠心室肌细胞动作电位及外向延迟整流钾电流的作用

目的 探讨卡托普利对豚鼠心室肌细胞动作电位及外向延迟整流钾电流的作用。方法 采用内充3M KCL的标准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-50mV，持续时间100ms，指令电位 40mV，记录外向延迟整流钾电流(Ik)最大峰电流。结果 与缺血组比较，卡托普利组APD30、APD50及ERP显著延长，APD50无显著变化。缺血组Ik幅度显著增高，而卡托普利组及卡托普利 缺血组显著降低。各组电流—电压关系曲线形态虽无显著变化，但缺血组显著上移，而卡托普利组、卡托普利 缺血组比缺血组下移。结论卡托普利降低外向钾电流及延长APD30、APD50和ERP。     

Open-state unblock characterizes acute inhibition of I potassium current by amiodarone in guinea pig ventricular myocytes

INTRODUCTION: The aim of the present study was to investigate the acute action of amiodarone on the slow component of delayed rectifier K+ current (IKs) under basal conditions and during beta-adrenoceptor stimulation in guinea pig ventricular myocytes. METHODS AND RESULTS: Using the whole-cell patch-clamp method, IKs was evoked by depolarizing voltage-clamp steps, during superfusion with the Na+-, K+-, and Ca2+-free solution supplemented with 0.4 microM nisoldipine and 5 microM E-4031. The acute effect of amiodarone was evaluated, within approximately 10 minutes after starting the bath application, by the amplitude of deactivating tail currents at -50 mV. Amiodarone concentration dependently blocked I(Ks) and exerted a more potent effect on IKs when activated by shorter pulse durations; the degree of block by 30 microM amiodarone on IKs activated by 200 ms, 500 ms, and 2000 ms depolarizing pulses to +30 mV was 55.9 +/- 5.8%, 38.6 +/- 6.0%, and 27.1 +/- 4.0% (n = 5 each), respectively. An envelope of tails test conducted at +10, +30, and +60 mV demonstrated that the degree of IKs block by amiodarone was gradually attenuated during membrane depolarization, which can be described by a monoexponential function, thus supporting the presence of open channel unblock. Amiodarone also blocked IKs maximally stimulated by 1 microM isoprenaline, to an extent similar to control, when IKs was activated by pulse durations of < or =2000 ms. CONCLUSION: We propose that amiodarone acutely blocks native IKs with characteristics associated with open channel unblock, and that the protein kinase A-mediated phosphorylation of channel proteins only minimally affects the amiodarone block.     

Blockade of 2,4-dinitrophenol induced ATP sensitive potassium current in guinea pig ventricular myocytes by class I antiarrhythmic drugs.

OBJECTIVE: The aim was to assess the effects of various antiarrhythmic drugs on 2,4-dinitrophenol (DNP) induced outward current (IDNP), presumably the ATP sensitive K+ current (IK,ATP) of isolated cardiac cells and to discuss mechanisms involved in the hypoglycaemia which occurs in patients on these drugs. METHODS: The quasi-steady state current-voltage relationship from the isolated guinea pig ventricular cells was measured using whole cell voltage clamp techniques with a ramp pulse programme. The effects of seven different antiarrhythmic drugs on IDNP were examined. Action potentials were elicited at a rate of 0.2 Hz by an intracellular current injection. RESULTS: DNP (50 mumol.litre-1) increased the quasi-steady state outward current at potentials positive to about -60 mV. This current (IDNP) was completely inhibited by the subsequent application of glibenclamide (1 mumol.litre-1), thereby suggesting that the IDNP is probably IK,ATP. Cibenzoline (10 mumol.litre-1, class Ia), disopyramide (30 mumol.litre-1, class Ia), and procainamide (100 mumol.litre-1, class Ia) significantly inhibited the IDNP by 95.5(SD 11.3)%, 77.8(21.2)%, and 76.4(23.9)% respectively. Flecainide (class 1c) inhibited the IDNP by 66.9(23.9)% at 10 mumol.litre-1 but not at 2 mumol.litre-1. Mexiletine (30 mumol.litre-1, class Ib), pilsicainide (50 mumol.litre-1, class Ic), and E4031 (10 mumol.litre-1, class III) at concentrations as high as approximately fivefold the clinically effective blood levels, did not suppress IDNP. Except for 10 mumol.litre-1 flecainide, all the concentrations listed above which blocked IDNP were within twofold of the clinical blood concentrations documented to be effective for suppression of arrhythmias. Cibenzoline, disopyramide, and procainamide, but not flecainide, belong to class Ia antiarrhythmic drugs. All these class Ia antiarrhythmic drugs "shortened" the action potential duration of guinea pig ventricular cells, an opposite change to that noted for multicellular preparations, eg, guinea pig papillary muscles. CONCLUSIONS: Class Ia antiarrhythmic drugs (cibenzoline, disopyramide, and procainamide) inhibit IDNP (presumably IK,ATP) in guinea pig ventricular cells within a range of therapeutic concentrations. This inhibitory effect of IK,ATP can probably explain the hypoglycaemia which occurs in some patients receiving these drugs, and the prolongation of the action potential duration alleged to occur in "superfused" papillary muscles.     

Effect of luminal acidification on guinea pig gastric mucosa

H⁺ secretion was studied in guinea pig fundic mucosa incubated in (A) bicarbonate-Ringer's gassed with 95% O₂-5% CO₂, or (B) HEPES gassed with 100% O₂ before and after luminal pH was lowered to 2.0 for periods up to 90 min. At pH 2.0 for 60 min, H⁺ secretion in group A tissues fell by 35±4% (P=0.02) from a control rate of 1.35±0.09 eq/cm²/hr and in group B tissues by 50±11% (P=0.01) from a control rate of 1.59±0.08 eq/cm²/hr. After 90 min at pH 2.0, H⁺ secretion in group A fell by 53±8% (P=0.02) from a control rate of 1.47±0.07 eq/cm²/hr and in group B fell by 44±6% (P=0.01) from a control rate of 1.38±0.07 eq/cm²/hr. Histamine 1×10^–4 M stimulation following exposure to pH 2.0 for 90 min increased secretion in group A tissues from 0.80 ±0.14 to 1.06±0.13 eq/cm²/hr (P<0.05), compared with an increase in nonacidified controls from 1.15±0.22 to 1.80±0.20 eq/cm²/hr (P<0.05) and in group B tissues from 1.27±0.10 to 1.56±0.19 eq/cm²/hr (P<0.05) compared with nonacidified controls from 1.43±0.22 to 2.23±0.41 eq/cm²/hr (P<0.05). Secretory function and electrical characteristics were adversely affected by luminal acidification to pH 2.0 and suggested a breach in the mucosal barrier with damage to parietal cells.This study was funded by the National Institute of Health Grant No. AM 15681.     

Effects of sea anemone toxin anthopleurin-Q on potassium currents in rats and guinea pig ventricular myocytes

To investigate the effect of sea anemone toxin anthopleurin-Q (AP-Q) on potassium currents in isolated rats and guinea pig ventricular myocytes.Methods The ventricular cells of guinea pigs and SD rats were obtained by enzymatic dissociation method.Whole cell patch clamp technique was used to record potassium currents (Ito,IK,and IK1).Results AP-Q 3-100 nmol/L increased Ito in a concentration-dependent manner,with an EC50 value of 12.7 nmol/L.At a potential of +50mV,AP-Q 10nmol/L increased Ito from (13.3±3.4) pA pF-1 to (19.46±4.3) pA pF-1.AP-Q 0.1-100 nmol/L increased IK and IK tail in a concentration-dependent manner with EC50 values of 4.7 nmol/L and 5.0 nmol/L,respectively.AP-Q 1 pmol/L-100 nmol/L increased IK1 in dose-dependent manner,with an EC50 of 0.2 nmol/L.Conclusions The effect of AP-Q on Ito,IK and IK1 may partly explain its mechanism in shortening APD and increasing RP.(J Geriatr Cardiol 2008;5:243-247)     

豚鼠M细胞缓慢激活型延迟整流钾电流对缺血的反应

目的：研究缺血对豚鼠心室M细胞缓慢激活型延迟整流钾通道电流（Iks)的影响。方法：利用全细胞膜片钳技术观察豚鼠心室M细胞Iks，利用不同成分浴槽液模拟细胞正常及缺血坏死，观察Iks尾电流锋值的变化情况。结果：指令电压≥0mV时，缺血组电流显小于正常组，P＜0．05；指令电压小于0mV时，两组间无显性差异，P＞0．05。结论：缺血时M细胞Iks显减弱，可能会增加心室壁电活动不均一性，促使心律失常的发生。     

Heterogeneity of sodium current in atrial vs epicardial ventricular myocytes of adult guinea pig hearts

The different sodium channel currents (I(Na)) were reported in myocardium, neuron, and skeletal muscles. To study whether I(Na) is homogeneous within the heart, we applied whole-cell voltage clamp technique to evaluate fast voltage-gated I(Na) in atrial and ventricular myocytes isolated from guinea pig heart. It was found that the density of inward I(Na) was 50% greater at -35 mV in atrial (-42.6+/-2.9 pA/pF) than in ventricular (-27.5+/-1.8 pA/pF, P<0.01) myocytes. The half activation and inactivation voltages (V(0.5)) of I(Na) in atrial myocytes were shifted 4.5+/-0.2 and 9.6+/-0.3 mV negative to those of ventricular myocytes. Time constants for I(Na) activation (tau(m)) and inactivation (tau(h)) were twice as rapid in atrial as in ventricular myocytes. The tau(m) and tau(h) were 0.34+/-0.03 and 1.36+/-0.07 ms for atrial myocytes, and 0.69+/-0.05 and 3.27+/-0.23 ms for ventricular myocytes, respectively. Recovery of I(Na) from inactivation was slower in atrial than in ventricular myocytes, whereas the development of resting state inactivation was more rapid in atrial (tau=67.5+/-4.3 ms) than in ventricular (152.8+/-7.5 ms, P<0.01) myocytes. The results reveal marked heterogeneity of I(Na) in the density and biophysical properties in atrial and ventricular myocytes, and the study suggests the potential possibility of tissue specific cardiac sodium channel isoforms.     

西洛他唑对大鼠右心室肌细胞瞬间外向钾电流的影响

目的 :观察西洛他唑对大鼠右心室肌细胞瞬间外向钾电流 (Ito1)的影响 ,探讨西洛他唑抗心律失常作用的确切机制。方法 :酶解法分离SD大鼠右心室肌细胞 ,采用全细胞膜片钳技术记录右心室肌细胞Ito1。结果 :5 0 μmol/L西洛他唑显著降低Ito1,使Ito1幅值由加药前 (12 .7± 0 .5 ) pA/ pF降至 (7.6± 0 .4 ) pA/ pF ,降低 (4 0 .1± 3.0 ) % (P <0 .0 1,n =9)。在 1～ 5 0 μmol/L范围内 ,西洛他唑的作用呈浓度依赖性 ,半抑制浓度为 (17.2±2 .2 ) μmol/L。此外 ,该药对Ito1电压依赖性的激活和失活曲线无显著影响。结论 :西洛他唑浓度依赖性地阻滞大鼠右心室肌细胞的Ito1。     

卡维地洛对豚鼠心室肌细胞的电生理作用

目的:观察卡维地洛对豚鼠心室肌细胞膜离子流及跨膜动作电位的影响,探讨卡维地洛对心室肌细胞的直接电生理作用.方法:①应用膜片钳全细胞记录技术记录单个豚鼠心室肌细胞膜L型钙内流(ICa-L)、内向整流性钾流(IJ1)、延迟整流性钾流(IK)和快钠内流(INa),观察不同浓度的卡维地洛对各离子流的影响.②用标觋准微电极技术记录豚鼠右室乳头肌细胞动作电位,观察不同浓度卡维地洛对动作电位各参数的影响.结果:①卡维地洛浓度依赖性地抑制ICa-L、INa,半数抑制浓度(IC50)分别为3.18,0.16 μmol/L.②卡维地洛25μmol/L对IK1最大内向和外向电流差异无统计学意义(n=5,P＞0.05),但使其整流范围由-20～ 40 mV增大至-20～ 80 mV.③卡维地洛0.3μmol/L以上即可浓度依赖性抑制IK尾电流(IK.tail),且对-10 mV钳制电压下IK.tail的抑制作用明显大于对 50 mV下的抑制,在高浓度还抑制缓慢激活型IK(IKs),IC50为12.43μmol/L.④卡维地洛呈反转频率依赖性延长动作电位时程(APD90),还降低动作电位幅度(APA)和0相最大去极化速率(Vmax).结论:卡维地洛低浓度下即可抑制心肌细胞INa、快速激活相IK,高浓度下抑制ICa-L、IKs,还降低Vmax、APA,呈反转频率依赖性延长APD90.     

Late sodium current and its contribution to action potential configuration in guinea pig ventricular myocytes

We used the patch clamp technique to study the nature of the late sodium current in guinea pig ventricular myocytes. In a cell attached mode of single channel recording at room temperature (22-24 degrees C) two kinds of late (100 msec or more after beginning of the depolarizing pulse) sodium channel activities were recognized. One is isolated brief openings appearing once for about 120 depolarizations per channel (background type), while the other type is sustained openings with rapid interruptions (burst type) that occurred only once for 2,700 depolarizations per channel. The time constant obtained from the open time histogram of the burst type (1.05 msec) was about five times longer than that of background type (0.18 msec, measured at the potential 10 mV above the threshold). Magnitude of the late sodium current flowing through the entire surface of a myocyte was estimated with tetrodotoxin (60 microM), a specific inhibitor of sodium channels, in whole-cell clamp experiments. The steady tetrodotoxin-sensitive current of 12 to 50 pA was registered at -40 mV (26 +/- 14 pA, mean +/- SD, n = 5), in good agreement with the late sodium current calculated from the single channel recording. Tetrodotoxin produced small (congruent to 10%) but significant decreases in the action potential duration. These results suggest the presence of a small but significant late sodium current with slow inactivation kinetics and that this current probably plays a significant role in maintaining the action potential plateau and the duration in guinea pig ventricular myocytes.     

模拟缺血对心室肌细胞钙内流和钾外流的影响

目的 观察模拟缺血对心室肌细胞L—型钙通道和ATP敏感钾通道的影响。方法 实验用胶原配药解法急性分离豚鼠心室肌细胞,利用全细胞膜片钳的方法记录心室肌细胞的L—型钙电流(Ica.L)和ATP敏感钾电流(IKATP)。采用低氧、无糖、高钾、高乳酸和酸中毒综合方式模拟缺血灌流,造成细胞的模拟缺血。结果 模拟缺血时,Ica.L明显受到抑制,IKATP增加,后一效应能被优降糖所阻断。结论 模拟缺血时Ica.L明显受到抑制,IKATP增加,可能是导致心室肌细胞动作电位时程缩短的主要因素之一。     

低浓度强心甙对豚鼠心室肌细胞Na+/K+泵电流的激活作用

目的 :解释治疗水平 （低浓度 ）强心甙的正性肌力作用。方法 :用全细胞膜片钳技术 ,在分离的豚鼠心室肌细胞观察到双氢哇巴因 （DHO）对 Na+/ K+泵电流 （Ip ）的激活作用。结果 :DHO在 10 - 8～ 10 - 1 0 m ol· L- 1范围内可增加外向电流。当从细胞外液去除 K+或从电极内液去除 Na+或胞内应用 vanadate抑制了 Ip后 ,外向电流消失。结论 :DHO可使 Na+/ K+泵兴奋 ,产生了外向泵电流。Ip激活是低浓度强心甙对心脏 Na+/ K+泵的直接作用。     

Quinidine delays IK activation in guinea pig ventricular myocytes

A major action of the antiarrhythmic agent quinidine is prolongation of cardiac repolarization. In these experiments, the time-dependent effects of quinidine on the delayed rectifier potassium current, IK, a current contributing to cardiac repolarization, were investigated in acutely disaggregated guinea pig ventricular myocytes using the whole-cell recording configuration of the patch-clamp method. The effect of quinidine on IK was dependent on the duration of depolarization. After long (2,000 msec) pulses, IK was reduced by 30 +/- 27% (SD; n = 8, paired) by 10 microM quinidine; in contrast, after short (100 msec) pulses, the drug decreased IK 65 +/- 35% (p less than 0.05). This effect was found both in paired experiments as well as when quinidine-pretreated cells were compared to non-pretreated cells. Quinidine significantly delayed IK activation (9 +/- 20 msec at baseline vs. 44 +/- 25 msec in drug, p less than 0.05), but did not alter the subsequent time course of activation (time constant 659 +/- 118 msec). These findings are consistent with the hypothesis that quinidine promotes occupancy of a channel state from which opening does not occur.     

A novel,voltage-dependent nonselective cation current activated by insulin in guinea pig isolated ventricular myocytes

Insulin regulates cardiac metabolism and function by targeting metabolic proteins or voltage-gated ion channels. This study provides evidence for a novel, voltage-dependent, nonselective cation channel (NSCC) in the heart. Under voltage clamp at 37 degrees C and with major known conductances blocked, insulin (1 nmol/L to 1 micromol/L) activated an outwardly rectifying current (Iinsulin) in guinea pig ventricular myocytes. Iinsulin could be carried by Cs+, K+, Li+, and Na+ ions but not by NMDG+. It was inhibited by the NSCC blockers gadolinium and SKF96365 but not flufenamic acid. Iinsulin was largely blocked by the insulin receptor tyrosine kinase inhibitor HNMPA-(AM)3 and by the phospholipase C inhibitor U73122 but not by its inactive analogue U73433. Staurosporine, a potent blocker of protein kinase C, did not prevent the activation of Iinsulin. Application of an analogue of diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol, mimicked the effect of insulin. This activated an outwardly rectifying NSCC that could be carried by Cs+, K+, Li+, or Na+ and that was blocked by gadolinium but not by flufenamic acid or staurosporine. We conclude that the intracellular pathway leading to activation of this novel cardiac NSCC involves phospholipase C, is protein kinase C-independent, and may depend on direct channel activation by diacylglycerol.     

Inositol-1,4,5-trisphosphate increases contractions but not L-type calcium current in guinea pig ventricular myocytes.

OBJECTIVE: We studied the effects of intracellularly applied inositol-1,4,5-trisphosphate (InsP3) to test the hypothesis that InsP3 is a messenger for stimulation of L-type calcium current (ICa(L)) and contractions by muscarinic agonists. METHODS: Voltage clamp pulses elicited ICa(L) that evoked contractions recorded with an edge detector in single guinea pig ventricular myocytes superfused with Tyrode's solution (36 degrees C). InsP3 or cyclic AMP (cAMP) was dialyzed into the cell at selected times via the patch electrode. RESULTS: InsP3 (1-10 microM) transiently increased isotonic contractions when applied for 4-5 min; higher concentrations (50-300 microM) caused a sustained decrease in contractions. InsP3 had no effect on ICa(L) at any concentration tested. Caffeine (10 mM)-induced contractures were increased and decreased, respectively, at 3 and 100 microM InsP3. Pentosan polysulfate (50 micrograms/ml), an InsP3 receptor antagonist, opposed the increased contractions by InsP3. Intrapipette cyclic AMP (10-300 microM) caused sustained increases of ICa(L) and contractions. Cyclic AMP, but not InsP3, also increased ICa(L) when intrapipette Cs+ suppressed K+ currents. CONCLUSIONS: Increased myocyte shortening at low InsP3 concentrations accords with receptor-initiated sarcoplasmic reticulum Ca2+ release. The transient stimulation of contractions at low concentrations and the sustained reduction of contractions at high concentrations are not consistent with a role for InsP in the persistent increase of contractions by muscarinic agonist in ventricular muscle and myocytes. The failure of InsP3 to change ICa(L) when contractions were increased or decreased militates against the L-type calcium channel being an effector of InsP3.