Effects of azimilide on the muscarinic acetylcholine receptor-operated K+ current and experimental atrial fibrillation in guinea-pig hearts

Effects of azimilide, a class III antiarrhythmic drug, on the acetylcholine (ACh) receptor-operated K+ current (I K.ACh) and the delayed rectifier K+ current (IK) were examined in guinea-pig atrial cells using patch-clamp techniques. Effects of azimilide on experimental atrial fibrillation (AF) were also examined in isolated guinea-pig hearts. In single atrial myocytes, azimilide inhibited both the rapid (IKr) and slow component of IK (IKs). Azimilide inhibited the I K.ACh induced by carbachol (CCh, 1 microM), adenosine (10 microM), and intracellular loading of GTPgammaS (100 microM) in a concentration-dependent manner. The IC50 values of azimilide for inhibiting the CCh-, adenosine-, and GTPgammaS-induced I K.ACh were 1.25, 29.1, and 20.9 microM, respectively, suggesting that azimilide inhibits I K.ACh mainly by blocking the muscarinic receptors. Azimilide concentration-dependently (0.3 - 10 microM) prolonged the action potential duration (APD) in the absence and presence of muscarinic stimulation. In isolated hearts, perfusion of CCh shortened the duration of the monophasic action potential (MAP) and effective refractory period (ERP) of the left atrium and lowered the atrial fibrillation threshold (AFT). Addition of azimilide inhibited the induction of AF by prolonging the duration of MAP and ERP. The I K.ACh inhibition by azimilide may at least in part contribute to the effectiveness to prevent parasympathetic-type AF.     

Inhibitory effects of dronedarone on muscarinic K+ current in guinea pig atrial cells

Dronedarone (SR33589), an amiodarone-like noniodinated antiarrhythmic agent, is undergoing clinical trials in atrial fibrillation. Because vagal activation plays a role in the pathophysiology of supraventricular arrhythmias, we have assessed the ability of dronedarone (0.01, 0.1, and 1 microM), compared with amiodarone (0.1, 1, and 10 microM) to inhibit the muscarinic acetylcholine receptor-operated K+ current (I(K(ACh))) in single cells isolated from guinea pig atria (patch-clamp technique). I(K(ACh)) was activated by extracellular application of carbachol (10 microM) or by intracellular loading with GTP-gamma-S (100 microM). Dronedarone and amiodarone reduced the carbachol-induced I(K(ACh)) with an IC50 (concentration required for 50% inhibition) slightly above 10 nM and 1 microM, respectively. Dronedarone also inhibited the GTP-gamma-S induced K+ current by 28% and 58% at 0.01 and 0.1 microM, respectively. These data suggest that dronedarone inhibits I(K(ACh)) by depressing the function of K(ACh) channel itself or associated GTP-binding proteins. Compared with amiodarone, dronedarone is approximately 100 times more potent on I(K(ACh)) and seems more selective in inhibiting I(K(ACh)) with respect to its antagonism of other inward and outward currents reported in the literature. This relative high potency of dronedarone to reduce I(K(ACh)) may be involved, at least in part, in the antiarrhythmic action of dronedarone against atrial fibrillation.     

Histamine H1-receptor-mediated modulation of the delayed rectifier K+ current in guinea-pig atrial cells: opposite effects on IKs and IKr

1. Histamine receptor-mediated modulation of the rapid and slow components of the delayed rectifier K+ current (IK) was investigated in enzymatically-dissociated atrial cells of guinea-pigs using the whole cell configuration of the patch clamp technique. 2. Histamine at a concentration of 10 microM enhanced IK recorded during strong depolarization to potentials ranging from +20 to +40 mV and inhibited IK recorded during mild depolarization to potentials ranging from -20 to -10 mV. The increase of IK was more prominent with longer depolarizing pulses, whereas the inhibition of IK was more marked with shorter depolarizing pulses, suggesting that histamine enhances IKs (the slow component of IK) and inhibits IKr (the rapid component of IK). 3. The histamine-induced enhancement of IKs and inhibition of IKr were abolished by 3 microM chlorpheniramine but not by 10 microM cimetidine, suggesting that these opposite effects of histamine on IKr and IKs are mediated by H1-receptors. 4. In the presence of 5 microM E-4031, an IKr blocker, histamine hardly affected IK during mild depolarization although it enhanced IK during strong depolarization in a concentration-dependent manner. Histamine increased IKs with EC50 value of 0.7 microM. In the presence of 300 microM indapamide, an IKs blocker, histamine hardly affected IKs but inhibited IKr in a concentration-dependent manner. Histamine decreased IKr with IC50 value of 0.3 microM. 5. Pretreatment with 100 nM calphostin C or 30 nM staurosporine, protein kinase C inhibitors, abolished the histamine-induced enhancement of IKs, but failed to affect the histamine-induced inhibition of IKr. 6. We conclude that in guinea-pig atrial cells H1-receptor stimulation enhances IKs and inhibits IKr through different intracellular mechanisms.     

Inhibitory effect of thiopental on ultra-rapid delayed rectifier K+ current in H9c2 cells

Using the whole-cell voltage clamp technique, we investigated the effects of thiopental on membrane currents in H9c2 cells, a cell line derived from embryonic rat heart. Thiopental blocked a rapidly activating, very slowly-inactivating ultra-rapid type I(Kur)-like outward K(+) current in a concentration-dependent manner. The half-maximal concentration (IC(50)) of thiopental was 97 microM with a Hill coefficient of 1.2. The thiopental-sensitive current was also blocked by high concentrations of nifedipine (IC(50) = 9.1 microM) and 100 microM chromanol 293B, a blocker of slowly activating delayed rectifier K+ current (I(Ks)), but was insensitive to E-4031, an inhibitor of rapidly activating delayed rectifier K(+) current (I(Kr)). TEA (tetraethylammonium) at 5 mM and 4-AP (4-aminopiridine) at 1 mM reduced the K(+) current to 30.8 +/- 12.2% and 20.5 +/- 6.5% of the control, respectively. Using RT-PCR, we detected mRNAs of Kv2.1, Kv3.4, Kv4.1, and Kv4.3 in H9c2 cells. Among those, Kv2.1 and Kv3.4 have I(Kur)-type kinetics and are therefore candidates for thiopental-sensitive K(+) channels in H9c2 cells. This is the first report showing that thiopental inhibits I(Kur). This effect of thiopental may be involved in its reported prolongation of cardiac action potentials.     

Anti-cholinergic effect of verapamil on the muscarinic acetylcholine receptor-gated K+ channel in isolated guinea-pig atrial myocytes

Summary Effects of verapamil on the acetylcholine (ACh)-induced K⁺ current were examined in single atrial cells, using the tight-seal whole-cell clamp technique. The pipette solution contained guanosine-5-triphosphate (GTP) or guanosine-5-O-(3-thiotriphosphate) (GTP-S, a non-hydrolysable GTP analogue). In GTP-loaded cells, ACh induced a specific K⁺ current, which is known to be mediated by pertussis toxin-sensitive GTP-binding (G) proteins. Verapamil (0.1–100 M) depressed the ACh-induced K⁺ current in a concentration-dependent fashion. In GTP-S-loaded cells, the K⁺ current remained persistently after wash-out of ACh, probably due to irreversible activation of G proteins by GTP-S. Verapamil (0.1–100 M) also depressed the intracellular GTP-S-induced K⁺ current. However, the magnitude of verapamil-depression of the K⁺ current in GTP-S-loaded cells was significantly smaller than that in GTP-loaded cells at concentrations between 1 and 10 M of the drug. From these results, it is suggested that verapamil may block not only the function of muscarinic ACh receptors but also of G proteins and/or the K⁺ channel itself and thereby depress the ACh-induced K⁺ current in isolated atrial myocytes.Supported by grants from the Ministry of Education, Science and Culture of Japan and the Research Program on Ca Signal Control Send offprint requests to Y. Kurachi at the above address     

Osmosensitive properties of rapid and slow delayed rectifier K+ currents in guinea-pig heart cells

1. Changes in cell volume affect a variety of sarcolemmal transport processes in the heart. To study whether osmotically induced cell volume shrinkage has functional consequences for K+ channel activity, guinea-pig cardiac preparations were superfused with hyperosmotic Tyrode's solution (1.2-2-fold normal osmolality). Membrane currents and cell surface dimensions were measured from whole-cell patch-clamped ventricular myocytes and membrane potentials were recorded from isolated ventricular muscles and non-patched myocytes. 2. Hyperosmotic treatment of myocytes quickly (< 3 min to steady state) shrank cell volume (approximately 20% reduction in 1.5-fold hyperosmotic solution) and depressed the delayed rectifier K+ current (IK). Analysis using different activation protocols and a selective inhibitor (5 micro mol/L E4031) indicated that the IK inhibition was due to osmolality and cell volume-dependent changes in the two subtypes of the classical cardiac IK (rapidly activating IKr and slowly activating IKs); 1.5-fold hyperosmotic treatment depressed the amplitudes of IKr and IKs by approximately 30 and 50%, respectively. 3. Superfusion of muscles and myocytes with 1.5-fold hyperosmotic solution lengthened the action potentials by 14-17%. Hyperosmotic treatment also caused 6-7 mV hyperpolarization that is most likely due to a concentrating of intracellular K+. 4. The inhibition of IK helps explain the lengthening of action potentials observed in osmotically stressed heart cells. These results, together with the reported IK stimulation by hyposmotic cell swelling, provide further support for cell volume-sensitive properties of cardiac electrical activity.     

雌二醇抑制豚鼠心室肌细胞内向整流和延迟整流钾通道电流

研究雌二醇对心室肌细胞动作电位,内向整流钾通道电流及延迟整流钾通道电流的影响。方法：全细胞膜片箝技术。结果：ＥＳＴ１０μｍｏｌ．Ｌ＾－１使豚鼠心室肌细胞ＡＰ时程明显缩短,ＡＰＤ５０由给药前（４７４±７１）ｍｓ缩短至（３３０±７５）ｍｓ（Ｐ〈０．０５）,Ｅｓｔ１００μｍｏｌ．Ｌ＾－１使ＡＰＤ５０缩短至（２２９±６７）ｍｓ,ＡＰＤ９０由（５８７±６０）ｍｓ缩短至（４１８±７９）ｍｓ,Ｅｓｔ浓度依赖性地     

Stimulatory action of protein kinase C(epsilon) isoform on the slow component of delayed rectifier K+ current in guinea-pig atrial myocytes

BACKGROUND AND PURPOSE: Protein kinase C (PKC) comprises at least twelve isoforms and has an isoform-specific action on cardiac electrical activity. The slow component of delayed rectifier K(+) current (I (Ks)) is one of the major repolarizing currents in the hearts of many species and is also potentiated by PKC activation. Little is known, however, about PKC isoform(s) functionally involved in the potentiation of I (Ks) in native cardiac myocytes. EXPERIMENTAL APPROACH: I (Ks) was recorded from guinea-pig atrial myocytes, using the whole-cell configuration of patch-clamp method. KEY RESULTS: Bath application of phenylephrine enhanced I (Ks) concentration-dependently with EC(50) of 5.4 microM and the maximal response (97.1+/-11.9% increase, n=16) was obtained at 30 microM. Prazosin (1 microM) almost totally abolished the potentiation of I (Ks) by phenylephrine, supporting the involvement of alpha(1)-adrenoceptors. The stimulatory action of phenylephrine was significantly, if not entirely, inhibited by the general PKC inhibitor bisindolylmaleimide I but was little affected by G?-6976, G?-6983 and rottlerin. Furthermore, this stimulatory effect was significantly reduced by dialyzing atrial myocytes with PKCepsilon-selective inhibitory peptide epsilonV1-2 but was not significantly affected by conventional PKC isoform-selective inhibitory peptide betaC2-4. Phorbol 12-myristate 13-acetate (PMA) at 100 nM substantially increased I (Ks) by 64.2+/-1.3% (n=6), which was also significantly attenuated by an internal dialysis with epsilonV1-2 but not with betaC2-4. CONCLUSIONS AND IMPLICATIONS: The present study provides experimental evidence to suggest that, in native guinea-pig cardiac myocytes, activation of PKC contributes to alpha(1)-adrenoceptor-mediated potentiation of I (Ks) and that epsilon is the isoform predominantly involved in this PKC action.     

An M2-like muscarinic receptor enhances a delayed rectifier K+ current in rat sympathetic neurones

BACKGROUND AND PURPOSE: Resting superior cervical ganglion (SCG) neurones are phasic cells that switch to a tonic mode of firing upon muscarinic receptor stimulation. This effect is partially due to the muscarinic inhibition of the M-current. Because delayed rectifier K+ channels are essential to sustain tonic firing in central neurones, we asked whether the delayed rectifier current IKV in SCG neurones was modulated by the muscarinic receptors expressed in these cells. EXPERIMENTAL APPROACH: Whole-cell patch-clamp records of M-current and IKV were done in cultured or acutely dissociated rat SCG neurones. To characterize the receptor that regulates IKV, cells were bathed with muscarinic agonists and antagonists, relatively specific for receptor subtypes. KEY RESULTS: The muscarinic agonist oxotremorine-M (Oxo-M) enhanced IKV by approximately 46% relative to its basal value. This effect remained unaltered when M-current was suppressed by linopirdine or Ba2+. Enhancement of IKV was insensitive to the M1-antagonist pirenzepine, whereas it was inhibited (approximately 60%) by the M2/4-antagonist himbacine. Further, the relatively specific M2-agonist bethanechol was as potent as Oxo-M in enhancing IKV. The modulation of IKV was insensitive to pertussis toxin (PTX), but was severely attenuated when internal ATP was replaced by its non-hydrolysable analogue AMP-PNP. CONCLUSIONS AND IMPLICATIONS: These results suggest that an M2-like muscarinic receptor couples to a PTX-insensitive G-protein and to an ATP-dependent pathway to enhance IKV. Modulation of IKV must be taken into consideration in order to understand more precisely how muscarinic receptors acting on different ion channels regulate sympathetic excitability.     

Inhibition of the delayed rectifier K current in guinea-pig cardiomyocytes by thiamine tetrahydrofurfuryl disulfide

We examined effect of thiamine tetrahydrofurfuryl disulfide on electrophysiological characteristics of single atrial myocytes, obtained by digestion of guinea-pig heart, using collagenase. Membrane potential and ion channel current in the atrial myocytes were recorded by the patch clamp method. Thiamine tetrahydrofurfuryl disulfide prolonged action potentials at cycle lengths from 250 to 10,000 ms. The degree of thiamine tetrahydrofurfuryl disulfide-induced prolongation was similar among these cycle lengths. Thiamine tetrahydrofurfuryl disulfide inhibited the delayed rectifier K⁺ current, without affecting Ca²⁺ current and inward-rectifier K⁺ current. Thiamine tetrahydrofurfuryl disulfide blocked the delayed rectifier K⁺ current in voltage- and time-independent manner, indicating that thiamine tetrahydrofurfuryl disulfide blocked both subtypes of the delayed rectifier K⁺ current (rapid and slow components). Thiamine, the parent molecule of thiamine tetrahydrofurfuryl disulfide, blocked the delayed rectifier K⁺ current only when thiamine was applied intracellularly. Thiamine tetrahydrofurfuryl disulfide may be converted to thiamine in the cytoplasm, and then may block the the delayed rectifier K⁺ channel from the intracellular side. Although thiamine tetrahydrofurfuryl disulfide (or thiamine) has some of the properties of class III antiarrhythmics agents, thiamine tetrahydrofurfuryl disulfide did not exhibit reverse use-dependent prolongation of action potential. Received: 18 August 1997 / Accepted: 10 February 1998     

Effects of trimebutine maleate on delayed rectifier K+ currents in guinea-pig ventricular myocytes

The effects of trimebutine maleate, a drug commonly used to regulate motility in the gastrointestinal tract, on the delayed rectifier K+ current (I(K)) were evaluated in guinea-pig ventricular myocytes to determine whether the drug has a proarrhythmic effect through blockade of I(K). Trimebutine decreased I(K) in a concentration-dependent manner. To investigate the effects of trimebutine on two components of I(K) (I(Kr) and I(Ks); rapidly activated and slowly activated components, respectively), we performed the envelope-of-tails test. Trimebutine-sensitive I(K) was determined by digital subtraction of I(K) during exposure to trimebutine from control I(K) for each duration of the test pulse over the range 50 ms-2 s. The ratio of deltaI(K,tail)/deltaI(K) plotted against pulse duration for trimebutine-sensitive I(K) gradually decreased to a steady-state value as the duration of the test pulse was lengthened. This finding suggested a weak inhibitory effect of trimebutine on both I(Kr) and I(Ks). The effects of trimebutine on the inward rectifier K+ current (I(K1)) responsible for the resting potential and final repolarization phase of the action potential were investigated by applying voltage clamp ramps over a broad range of potentials. No significant effects were observed at 10 or 100 microM. We next investigated the effects of the drug on the L-type Ca2+ current (I(Ca)). Significant inhibition of I(Ca) was observed at trimebutine concentrations greater than 10 microM. These results suggested that trimebutine maleate has weak inhibitory effects on I(Kr), I(Ks) and I(Ca) at concentrations much higher than those in clinical use.     

Inhibitory effect of aprindine on Na+/Ca2+ exchange current in guinea-pig cardiac ventricular myocytes

1. Using the whole-cell voltage clamp technique, the effect of aprindine on Na+/Ca2+ exchange current (I(NCX)) was examined in guinea-pig single cardiac ventricular myocytes and CCL39 fibroblasts expressing a dog cardiac Na+/Ca2+ exchanger (NCX1). 2. I(NCX) was recorded by ramp pulses from the holding potential of -60 mV with the external solution containing 140 mM Na+ and 1 mM Ca2+, and the pipette solution containing 20 mM Na+, 20 mM BAPTA and 13 mM Ca2+ (433 nM free Ca2+). 3. External application of aprindine suppressed I(NCX) in a concentration-dependent manner. The IC50 values of outward (measured at 50 mV) and inward (measured at -100 mV) I(NCX) components were 48.8 and 51.8 microM with Hill coefficients of 1.3 and 1, respectively. 4. Intracellular application of trypsin via the pipette solution did not change the blocking effect of aprindine, suggesting that aprindine does not affect the exchanger from the cytoplasmic side. 5. Aprindine inhibited I(NCX) of a mutant NCX1 with a deletion of amino acids 247 - 671 in the large intracellular domain between the transmembrane segments 5 and 6 in a similar manner to that of the wild-type, suggesting that the site of aprindine inhibition is not in the large intracellular domain of NCX1. 6. A kinetic study indicated that aprindine was cooperatively competitive with KB-R7943, another inhibitor of NCX and that aprindine was a competitive inhibitor with respect to external Ca2+. 7. We conclude that aprindine may modestly inhibit I(NCX) in a therapeutic range of concentrations (around 2.5 approximately 6.9 microM) possibly at an external or intra-membranous site of the exchanger.     

充血性心衰病人心房肌细胞瞬间外向钾电流和超快速延迟整流钾电流的特征

AIM: To study the properties of transient outward K+ current (Ito) and ultra-rapid delayed rectifier K+ current (IKur) in isolated human atrial myocytes from patients with congestive heart failure (CHF). METHODS: Single cells were isolated from CHF patients with collagenase and protease. Ito and IKur were recorded using whole cell patch-clamp technique. RESULTS: The activation and inactivation of I(to) were voltage-dependent and time-dependent. The half-activation and half-inactivation voltage were (15 +/- 12) mV and (-45 +/- 4) mV respectively. When membrane potential went up from -40 mV to +60 mV, the activation time constant means decreased from (6.9 +/- 2.3) ms to (1.40 +/- 0.20) ms, while the inactivation time constant means decreased from (69 +/- 17) ms to (21 +/- 14) ms. Otherwise, the mean reactivation time constants was (125 +/- 65) ms when the membrane potential was held at -80 mV, but the recovery was not complete during the interval observed. Ito showed less frequency-dependent reduction at test frequency between 0.2-2 Hz. Compared with Ito, the activation of IKur only showed voltage-dependence, without time-dependence. Its mean current densities was (3.4 +/- 0.7) pA/pF when test potential was +60 mV. The half activation voltage of IKur was (23 +/- 14) mV. No clear frequency-dependence was observed at the same frequency range of Ito either. CONCLUSION: I(to) and IKur are important outward potassium channel currents in isolated human atrial myocytes from CHF patients and they have different kinetic properties.     

DDPH对豚鼠心室肌细胞延迟整流钾电流两种成分的抑制作用

目的:研究1-(2,6-二甲基苯氧基)-2-(3,4-二甲氧基苯乙氨基)丙烷盐酸盐(DDPH)对豚鼠心室肌细胞快激活(I_(Kr))和慢激活(I_(Ks))延迟整流钾电流的作用.方法:全细胞膜片箝技术.结果:DDPH 0.1-100μmol/L浓度依赖性抑制I_(Kr),I_Kr-tail[IC_(50)(μmol/L)为6.1,95％可信限为(2.8—13.5)].DDPH同时浓度依赖性抑制 I_(Ks),I_(Ks-tail[IC_(50)(μmol/L)为12.5,95％可信限为(4.8-32.2)].DDPH(10 μmol/L)不影响I_(Kr)和I_(Ks)的电压依赖性激活过程,给药前I_(Kr)的半激活电压(V_(1/2),mV)和斜率因子(k,mV)分别为(-21.7±0.8)和(5.9±0.8),给药后分别为(-23.5±2.4)和(8.1±2.2),无统计学意义(P>0.05).用药前后I_(Ks)的半激活电压和斜率因子的差异亦无统计学意义(P>0.05),用药前分别为(27.0±0.8)和(14.9± 0.9),用药后分别为(27.1±0.7)和(16.6±0.8).DDPH(<10μmol/L)可抑制 I_(Kr)和 I_(Ks)的去激活过程,并且加快I_(Kr)的失活.结论:DDPH抑制I_(Kr)和I_(Ks)无选择性.且主要作用于其去激活过程,而非激活过程.DDPH进一步通过加速其失活过程抑制I_(Kr).     

Effects of polyamines on the muscarinic receptor-operated cation current in guinea-pig ileal smooth muscle myocytes

The effects of extracellular and intracellular polyamines (PAs), spermine and putrescine, on the cation current (mI(CAT)) evoked either by activating muscarinic receptors with carbachol or by intracellularly applied GTPgammaS (in the absence of carbachol) were studied using patch-clamp recording techniques in single guinea-pig ileal myocytes. Extracellular spermine and putrescine rapidly and reversibly inhibited mI(CAT) in a concentration- and voltage-dependent manner with the IC(50) values at -40 mV of about 1 and 5 mM, respectively. Membrane depolarization relieved the blocking action of PAs although cation conductance activation curve remained N-shaped. The inhibition was similar for both carbachol- and GTPgammaS-evoked currents, suggesting that the cation channel rather than the muscarinic receptor was the primary site of the PA action. In outside-out membrane patches, both cation channel unitary conductance and open probability were reduced. In perforated-patch experiments used to retain cytoplasmic PAs sustained 100 microM carbachol-induced mI(CAT) was significantly smaller (478 +/- 76 pA, n = 7) compared to that recorded using conventional whole-cell configuration with nominally PA-free pipette solution (1314 +/- 76 pA, n = 12), but comparable in size to mI(CAT) with 0.3 mM spermine in the pipette solution (509 +/- 41 pA, n = 19). Intracellular putrescine inhibited mI(CAT) less potently compared to spermine. In conclusion, these results show a novel role of intestinal PAs in mI(CAT) inhibition, which can contribute to their well-known suppressing effect on the gastrointestinal smooth muscle excitability and contractility.     

Sevoflurane inhibition of the slowly activating delayed rectifier K+ current in guinea pig ventricular cells

Single ventricular cells were enzymatically isolated from guinea pig hearts and the effects of sevoflurane on the delayed rectifier K(+) current were investigated by the patch clamp method. The rapidly (I(Kr)) and slowly activating delayed rectifier K(+) current (I(Ks)) were isolated using chromanol 293B, a selective blocker for I(Ks) or E4031 (N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl]-4-piperidinyl]carbonyl]phenyl]methanesulfonamide dihydrochloride), a blocker for I(Kr). Sevoflurane and halothane decreased I(Ks) in a concentration-dependent manner with an IC(50) value of 0.38 mM for sevoflurane and 1.05 mM for halothane. I(Ks) inhibition was characterized by suppression of maximum conductance with little effect on activation kinetics. Inhibition occurred immediately after anesthetic application and recovered upon wash-out. In contrast to the marked inhibition of I(Ks), I(Kr) was hardly affected by sevoflurane. Under the current clamp, sevoflurane prolonged the action potential duration in a reversible manner and this effect was more marked when I(Kr) was inhibited by E4031. The results suggest that sevoflurane inhibits I(Ks), and not I(Kr), in a concentration-dependent manner at clinically relevant concentrations. The resulting prolongation of ventricular repolarization may partly account for the clinical observation of excessive QT prolongation by these anesthetics.     

Effects of JTV-519, a novel anti-ischaemic drug, on the delayed rectifier K+ current in guinea-pig ventricular myocytes

We studied the effects of a newly synthesized anti-ischaemic agent, 4-[3-(4-benzylpiperidin-1-yl) propionyl]-7-methoxy-2, 3, 4, 5-tetrahydro-1, 4-benzothiazepine monohydrochloride (JTV-519) on the delayed rectifier potassium current (IK), using guinea-pig ventricular myocytes and whole-cell voltage-clamp techniques, under blockade of the L-type calcium current (ICa,L) by D600 (1 microM) or nitrendipine (5 microM). The IK in guinea-pig ventricular cells consists of two different components; the rapidly activating, E4031-sensitive component (IKr) and the slowly activating E4031-resistant component (IKs). Under steady-state conditions, JTV-519 (1 and 5 microM) did not change the amplitude of IKs remaining after blockade of IKr with 5 microM E4031. The effect of JTV-519 on IKr was assessed using short (50 ms) pulses which evoked a tail current that was sensitive to E4031 but not to chromanol 293B, a specific blocker of IKs. JTV-519 suppressed the IKr with a half-maximal inhibitory concentration of 1.2 microM. Selective inhibition of IKr by this agent was confirmed by using the "envelope of tails" test. These results suggest that the blockade of IKr may underlie the prolongation of action potential duration in ventricular muscle and QT-intervals alleged to occur in animal as well as human hearts.     

Time-dependent block of the slowly activating delayed rectifier K(+) current by chromanol 293B in guinea-pig ventricular cells

The slowly activating delayed rectifier K(+) current (I(Ks)) was recorded in single myocytes dissociated from guinea-pig ventricles and the mechanism underlying the block of I(Ks) by a chromanol derivative, 293B, was investigated. In the presence of 1 - 100 microM 293B, activation phase of I(Ks) was followed by a slower decay during 10 s depolarizing pulses. Both the rate and extent of the decay were increased in a concentration-dependent manner. The relationship between the concentration of 293B and the block showed a Hill's coefficient of approximately 1. The half-inhibitory concentration was approximately 3.0 microM and did not differ significantly at various membrane potentials from +20 to +80 mV. A mathematical model for the 293B block was constructed on the basis of multiple closed and open states for the I(Ks) channels, and the blocking rate was calculated by fitting the model to the original current traces. The blocking rate constant showed a linear function with the 293B concentration, indicating 1 : 1 binding stoichiometry. At +80 mV the blocking rate was 4x10(4) M(-1) s(-1) and the unblocking rate was 0.2 s(-1). The results indicate that 293B is an open channel blocker with relatively smaller blocking rate than those reported so far for time-dependent blockade of various ionic channels.     

Blocking action of chromanol 293B on the slow component of delayed rectifier K(+) current in guinea-pig sino-atrial node cells

1. In guinea-pig sino-atrial (SA) node cells the delayed rectifier K(+) current (I(K)) is composed of rapidly and slowly activating components of I(K) (I(Kr) and I(Ks), respectively). The present study was undertaken to characterize the blocking action of the chromanol derivative 293B on I(Ks) in guinea-pig SA node cells using whole-cell patch-clamp technique. 2. Bath application of 293B blocked I(Ks), elicited by 4-s depolarizing voltage pulses from a holding potential of -50 mV, under conditions in which the L-type Ca(2+) current (I(Ca,L)) and I(Kr) were inhibited; the effect was concentration-dependent with an IC(50) of 5.3 microM, when evaluated by the decrease in the amplitude of I(Ks) tail current following 4-s depolarizing voltage steps to +50 mV. 3. The 293B block of I(Ks) progressed with time during depolarizing voltage steps with a more rapid block at higher concentrations. 4. The block of I(Ks) by 293B was fully reversed within a few minutes after washing off the drug, even when a maximal effect (a nearly full block) was achieved at high drug concentration (50 microM). 5. Bath application of 293B at 50 microM greatly and reversibly reduced the amplitude of I(Ks) which is maximally stimulated by beta-adrenergic agonist isoprenaline (1 microM), while the degree of 293B block of the isoprenaline-stimulated I(Ks) was slightly but significantly smaller than that of non-stimulated I(Ks) (94.0+/-0.98% block, n=6 vs 99.4+/-0.45% block, n=6; P<0.01). 6. We conclude that, in guinea-pig SA node cells (i) 293B is a potent and fully reversible blocker of I(Ks) in control and during beta-adrenergic stimulation and (ii) block with 293B occurs in a time-dependent manner during depolarizing voltage steps.     

碘化N-正丁基氟哌啶醇对豚鼠心房肌细胞乙酰胆碱敏感性钾通道的影响

目的研究碘化N正丁基氟哌啶醇(F2)对豚鼠心房肌细胞乙酰胆碱敏感性钾通道(KACh)的影响,探讨其对KACh的作用机制。方法采用膜片钳全细胞记录方法,测定F2对原代培养的豚鼠心房肌细胞乙酰胆碱敏感性钾电流IK(ACh)的影响。结果细胞外给予F2对豚鼠心房肌细胞IK(ACh)呈可逆性、浓度依赖性的阻断作用。细胞内添入抗水解的GTP类似物GTPγS后,结果同前。细胞内给予50μmol·L-1F2对IK(ACh)无作用。结论F2是豚鼠心房肌细胞KACh的一种快速通道阻断剂,发挥作用部位在细胞膜外侧,作用位点在钾通道本身,与乙酰胆碱受体无关。