Delayed rectifier potassium current in undiseased human ventricular myocytes

OBJECTIVE: The purpose of the study was to investigate the properties of the delayed rectifier potassium current (IK) in myocytes isolated from undiseased human left ventricles. METHODS: The whole-cell configuration of the patch-clamp technique was applied in 28 left ventricular myocytes from 13 hearts at 35 degrees C. RESULTS: An E-4031 sensitive tail current identified the rapid component of IK (IKr) in the myocytes, but there was no evidence for an E-4031 insensitive slow component of IK (IKs). When nifedipine (5 microM) was used to block the inward calcium current (ICa), IKr activation was fast (tau = 31.0 +/- 7.4 ms, at +30 mV, n = 5) and deactivation kinetics were biexponential and relatively slow (tau 1 = 600.0 +/- 53.9 ms and tau 2 = 6792.2 +/- 875.7 ms, at -40 mV, n = 7). Application of CdCl2 (250 microM) to block ICa altered the voltage dependence of the IKr considerably, slowing its activation (tau = 657.1 +/- 109.1 ms, at +30 mV, n = 5) and accelerating its deactivation (tau = 104.0 +/- 18.5 ms, at -40 mV, n = 8). CONCLUSIONS: In undiseased human ventricle at 35 degrees C IKr exists having fast activation and slow deactivation kinetics; however, there was no evidence found for an expressed IKs. IKr probably plays an important role in the frequency dependent modulation of repolarization in undiseased human ventricle, and is a target for many Class III antiarrhythmic drugs.     

Heterogeneous distribution of the two components of delayed rectifier K+ current: a potential mechanism of the proarrhythmic effects of methanesulfonanilideclass III agents.

OBJECTIVE: To elucidate the regional difference of the K+ current blocking effects of methanesulfonanilide class III agents. METHODS: Regional differences in action potential duration (APD) and E-4031-sensitive component (IKr) as well as -insensitive component (IKs) of the delayed rectifier K+ current (IK) were investigated in enzymatically isolated myocytes from apical and basal regions of the rabbit left ventricle using the whole-cell clamp technique. RESULTS: At 1 Hz stimulation, APD was significantly longer in the apex than in the base (223.1 +/- 10.6 vs. 182.7 +/- 14.5 ms, p < 0.05); application of 1 microM E-4031 caused more significant APD prolongation in the apex than in the base (32.5 +/- 6.4% vs. 21.0 +/- 8.8%, p < 0.05), resulting in an augmentation of regional dispersion of APD. In response to a 3-s depolarization pulse to +40 mV from a holding potential of -50 mV, both IK tail and IKs tail densities were significantly smaller in apical than in basal myocytes (IK: 1.56 +/- 0.13 vs. 2.09 +/- 0.21 pA/pF, p < 0.05; IKs: 0.40 +/- 0.15 vs. 1.43 +/- 0.23, p < 0.01), whereas IKr tail density was significantly greater in the apex than in the base (1.15 +/- 0.13 vs. 0.66 +/- 0.11 pA/pF, p < 0.01). The ratio of IKs/IKr for the tail current in the apex was significantly smaller than that in the base (0.51 +/- 0.21 vs. 3.09 +/- 0.89; p < 0.05). No statistical difference was observed in the voltage dependence as well as activation and deactivation kinetics of IKr and IKs between the apex and base. Isoproterenol (1 microM) increased the time-dependent outward current of IKs by 111 +/- 8% during the 3-s depolarizing step at +40 mV and its tail current by 120 +/- 9% on repolarization to the holding potential of -50 mV, whereas it did not affect IKr. CONCLUSIONS: The regional differences in IK, in particular differences in its two components may underlie the regional disparity in APD, and that methanesulfonanilide class III antiarrhythmic agents such as E-4031 may cause a greater spatial inhomogeneity of ventricular repolarization, leading to re-entrant arrhythmias.     

Isoproterenol antagonizes prolongation of refractory period by the class III antiarrhythmic agent E-4031 in guinea pig myocytes. Mechanism of action

The mechanism by which isoproterenol (ISO) prevents the prolongation of action potential duration (APD) and refractory period (RP) by the class III antiarrhythmic agent E-4031 was studied. E-4031 (1 microM) increased RP by 50% with no effect on contractile force in papillary muscles isolated from guinea pig heart. ISO (1 microM) increased force of contraction more than fivefold and decreased RP by 25%. The prolongation of RP by E-4031 was prevented by pretreatment of muscles with ISO. The prolongation of APD in isolated guinea pig ventricular myocytes by 5 microM E-4031 also was antagonized by prior exposure of the cells to 1 microM ISO. Instantaneous currents and delayed rectifier K+ currents, IK, were measured in isolated myocytes using the suction microelectrode voltage-clamp technique. Currents were measured in response to 225-msec depolarizing pulses from a holding potential of -40 mV. Previous studies have demonstrated that IK in these cells results from activation of two distinct outward K+ currents, IKs and IKr (specifically blocked by E-4031). ISO doubled the magnitude of IKs without significant effect on IKr. The instantaneous current, putatively identified as a Cl- current, also was doubled by ISO but was unaffected by E-4031. The augmented conductance of IKs and instantaneous current by ISO results in a decrease in RP. The small effect of E-4031 on APD and RP in the presence of ISO results from the smaller contribution of IKr relative to the augmented repolarizing currents.     

Differential effects of the new class III antiarrhythmic agents almokalant, E-4031 and D-sotalol, and of quinidine, on delayed rectifier currents in guinea pig ventricular myocytes.

OBJECTIVES: The effects of almokalant (4-[3-ethyl[3-(propylsulphinyl)propyl]-amino]-2-hydroxy-propoxy]- benzonitrile), E-4031 (1-[2-(6-methyl-2-pyridyl)-ethyl]-4-(4-methylsulphonyl-amino- benzoyl)piperidine), d-sotalol, and quinidine were investigated on the delayed K+ rectifier current IK. The aim of the study was to compare the drug action on the two components of this current. METHODS: Membrane currents were measured in ventricular myocytes from guinea pig hearts with the whole cell voltage clamp technique. IK was activated during clamp steps from a holding potential of -40 mV to test potentials -30 and +50 mV. The tail current Itail was measured upon stepping back to holding potential. RESULTS: In control experiments. IK and Itail declined spontaneously ("run down"). With 300 ms long test pulses to +50 mV, only d-sotalol (10(-4) M) caused a significant further decrease in IK, whereas all four agents significantly reduced Itail (almokalant 10(-6) M, E-4031 10(-7) M, quinidine 10(-5) M). When tested with 1 s long clamp steps at various potentials almokalant (3 x 10(-6) M), E-4031 (10(-6) M), quinidine (10(-5) M), and d-sotalol (10(-4) M) reduced IK in the potential range between -20 and +40 mV, yielding a bell shaped inward rectifying drug sensitive current. Itail was reduced by almokalant and E-4031 over the whole voltage range with saturation of block positive to +20 mV. Similar reductions with quinidine but not with d-sotalol were also significant. With rest pulses to +50 mV of increasing duration (25 ms-4000 ms), Itail developed with a faster time course than IK and therefore the ratio of Itail/IK declined with pulse duration. With almokalant and E-4031, this ratio became independent of test pulse duration. For 250 ms pulses, Itail/IK was also significantly reduced by d-sotalol and quinidine. CONCLUSION: Inhibition of the rapidly activating inwardly rectifying component of IK is prominent with almokalant and E-4031 and less pronounced with d-sotalol and quinidine. Since inhibition of this component prolongs the cardiac action potential, it should contribute to the antiarrhythmic properties of the agents.     

Differential block of cardiac delayed rectifier current by class Ic antiarrhythmic drugs: evidence for open channel block and unblock.

OBJECTIVE: The aim was to compare the effects of the class Ic antiarrhythmic drugs flecainide, encainide, and recainam on the delayed rectifier current, IK. METHODS: Membrane currents were studied using the single suction pipette voltage clamp technique in freshly dissociated cat ventricular myocytes bathed in HEPES buffered physiological saline at 32 degrees C. RESULTS: Flecainide and encainide decreased IK with IC50 values of 2.1 microM and 6 microM, respectively. Recainam (100 microM) reduced IK by only 7 (SEM 3)% after 20-30 min exposure and by 19% after an 80 min exposure (IC50 > 400 microM). None of the compounds blocked the inward rectifier, IK1. Block of IK by flecainide and encainide increased with depolarisation following a voltage dependence similar to that describing channel activation. Flecainide and encainide also slowed the time course of the IK tail currents, consistent with drug dissociating from open channels. CONCLUSIONS: The observed voltage dependence for IK block by flecainide and encainide resembles the interaction reported between these agents and the excitatory sodium channel, ie, depolarisation enhances block while repolarisation leads to removal of block. The results further suggest that the electrophysiological profile of class Ic agents can have a markedly different ionic basis, ie, K+ channel block by flecainide and encainide is balanced by a potent block of sodium channels, while recainam appears to be a weak but relatively specific blocker of sodium channels only. These differences are not readily accommodated by the current Harrison-Vaughan-Williams classification scheme, and suggest the possibility that potentially important drug specific differences can exist within the same antiarrhythmic drug class.     

A functional role of the erg-like inward-rectifying K+ current in prolactin secretion from rat lactotrophs

The functional role of the inward-rectifying erg-like K+ current in rat lactotrophs was studied by the use of a selective blocker, the class III antiarrhythmic agent E-4031. The erg-like current was measured as drug-sensitive current in physiological K+ gradient. In the range of the normal resting membrane potential of rat lactotrophs (around -45 mV) the erg-like current constituted a steady outward current. A selective block of this current by E-4031 resulted in a moderate (5 mV) depolarization of the membrane potential in 64% of the lactotroph cells. Measurements of basal prolactin secretion with the reverse hemolytic plaque assay showed that the number of prolactin secreting cells and the amount of prolactin secreted from single lactotrophs was significantly increased in the presence of E-4031. The data show that the contribution of the erg-like K+ current to the maintenance of the resting membrane potential is functionally important for the regulation of prolactin secretion.     

The mechanism of atrial antiarrhythmic action of RSD1235

INTRODUCTION: RSD1235 is a novel drug recently shown to convert AF rapidly and safely in patients.(1) Its mechanism of action has been investigated in a rat model of ischemic arrhythmia, along with changes in action potential (AP) morphology in isolated rat ventricular myocytes and effects on cloned channels. METHODS AND RESULTS: Ischemic arrhythmias were inhibited with an ED50 of 1.5 micromol/kg/min, and repolarization times increased with non-significant effects on PR and QRS durations. AP prolongation was observed in rat myocytes at low doses, with plateau elevation and a reduction in the AP overshoot at higher doses. RSD1235 showed selectivity for voltage-gated K+ channels with IC50 values of 13 microM on hKv1.5 (1 Hz) versus 38 and 30 microM on Kv4.2 and Kv4.3, respectively, and 21 microM on hERG channels. RSD1235 did not block IK1 (IC50 > 1 mM) nor ICa,L (IC50= 220 microM) at 1 Hz in guinea pig ventricular myocytes (n = 4-5). The drug displayed mild (IC50= 43 microM at 1 Hz) open-channel blockade of Nav1.5 with rapid recovery kinetics after rate reduction (10-->1 Hz, 75% recovery with tau= 320 msec). Nav1.5 blocking potency increased with stimulus frequency from an IC50= 40 microM at 0.25 Hz, to an IC50= 9 microM at 20 Hz, and with depolarization increasing from 107 microM at -120 mV to 31 microM at -60 mV (1 Hz). CONCLUSIONS: These data suggest that RSD1235's clinical selectivity and AF conversion efficacy result from block of potassium channels combined with frequency- and voltage-dependent block of INa.     

Reduced Inward Rectifying and Increased E-4031-Sensitive K+ Current Density in Arrhythmogenic Subendocardial Purkinje Myocytes from the Infarcted Heart

Outward Currents in Purkinje Cells from 48-Hour Infarcted Heart. Introduction : Subendocardial Purkinje myocytes from the 4K-hour infarcted heart (IZPCs) have reduced resting potentials, possibly due to altered inwardly rectifying K⁺ currents I_KI. Abnormal depolarization-activated outward K⁺ currents could contribute to long triangularly shaped action potentials of IZPCs.
Methods and Results : We used whole cell patch recordings to compare cesium-sensitive I_KI and 4-aminopyridine (4-AP)-resistant, noninactivating sustained I_K between normal Purkinje myocytes (NZPCs) and IZPCs. IZPCs showed decreased net membrane currents. Two IZPC groups were distinguished, based on 4-AP-resistant outward K⁺ currents. IZPC-I had isochronal I_KI current-voltage relations similar to NZPCs whereas IZPC-II showed significantly reduced I_KI and increased outward plateau currents. To study the sustained I_K in the presence of the Class III antiarrhythmic agent E-4031, a two-pulse protocol was used to inactivate transient outward currents, followed by step depolarizations. E-4031-sensitive currents were significantly greater in IZPCs at depolarized potentials (> 0 mV). Similar to NZPCs, IZPC E-4031 currents showed time dependence during depolarization, lack of rectification at positive steps, and voltage-dependent recovery from block.
Conclusion : Decreased I_KI may account for reduced resting potentials in IZPCs. E-4031-sensitive currents in NZPCs, unlike those in canine ventricular myocytes, are sensitive to 4-AP and are larger in IZPCs.     

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.     

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.     

Comparison of the Rate-Dependent Properties of the Class III Antiarrhythmic Agents Azimilide (NE-10064) and E-4031

Rate Dependence of Azimilide and E-4031. Introduction : Reverse rate-dependence, a lessening in Class III antiarrhythmic agent action potential duration (APD) prolongation as heart rate is increased, has been proposed to be related to an incomplete deactivution of the slow component (I_Ks) of the delayed rectifier K⁺ current (I_K). The rate-dependent properties of block of I_K by azimilide were compared to E-4031, which selectively blocks the rapid component (I_Kr) of I_k, in guinea pig ventricular muscle.
Methods and Results : Azimilide prolonged APD in isolated papillary muscles in a concentration-dependent manner and to a greater degree than E-4031. Both agents prolonged APD less at fast than slow rates, consistent with a similar reverse rate-dependent effect. Isolation of azimilide block of I_Ks by subtraction of APD during E-4031 plus azimilide from E-4031 alone revealed rate-independent prolongation of APD. In voltage clamp experiments on single ventricular myocytes, activation of I_ks was similar following 30 seconds of conditioning pulses of physiological duration (125 to 200 msec) with either a fast (cycle length 250 msec) or slow (cycle length 2000 msec) rate. The block of I_Ks by azimilide 3 μM was greater after a fast conditioning pulse train.
Conclusions : Selective block of I_ks prolongs APD in a rate-independent manner. In voltage clamped myocytes, no evidence of a rate-dependent accumulation of I_Ks was observed. These findings support a mechanism of reverse rate-dependent APD prolongation by Class III antiarrhythmic agents that block I_Kr independent of I_Ks.     

Antiarrhythmic drugs preferentially produce conduction block at the area of slow conduction in the re-entrant circuit of canine atrial flutter: comparative study of disopyramide, flecainide, and E-4031

STUDY OBJECTIVE--The aim was to test whether antiarrhythmic drugs preferentially suppressed conduction in the area of slow conduction in the re-entrant circuit. DESIGN--Intravenous disopyramide [n = 8, plasma concentrations: 1.4 (SEM 0.2) micrograms.ml-1], flecainide [n = 8, 0.6(0.1) micrograms.ml-1], and E-4031, a new class III antiarrhythmic drug [n = 8, 5.6(1.0) ng.ml-1], were investigated for their effects on atrial flutter due to re-entry in dogs with intercaval crush. In three dogs, detailed atrial activation sequence during atrial flutter was determined with a hand held bipolar electrode and an epicardial isochronal map was drawn. EXPERIMENTAL MATERIAL--24 anaesthetised adult mongrel dogs were used. MEASUREMENTS AND MAIN RESULTS--There was an area of slow conduction during atrial flutter in the low right atrium. Atrial flutter was terminated in all dogs except for one treated with flecainide. In 92% of the dogs, conduction block occurred in the low right atrium in which the area of slow conduction was located. Increase in local conduction time was greater in the area of slow conduction than other parts of the atria (percent ratio to the increase in cycle length of atrial flutter: 63% with disopyramide, 52% with flecainide, and 99% with E-4031). CONCLUSION--These data suggested antiarrhythmic drugs preferentially suppressed conduction at the area of slow conduction in the re-entrant circuit leading to termination of atrial flutter in this canine model, irrespective of electrophysiological effects of antiarrhythmic drugs.     

Potassium channel openers antagonize the effects of class III antiarrhythmic agents in canine Purkinje fiber action potentials. Implications for prevention of proarrhythmia induced by class III agents

We studied the effects of potassium channel openers (PCOs) on frequency dependent prolongations of action potential duration (APD), triggered activities and oscillatory action potentials (OSC) induced by E-4031 and dofetilide. The action potentials of canine Purkinje fibers were recorded by a glass microelectrode technique. The effects of E-4031 (10(-6)M) as well as that of additional nicorandil (2 x 10(-5) M) on the APD were examined. When abnormal automaticity was observed under perfusion of E-4031 (10(-5) M) or dofetilide (10(-5) M), action potentials were recorded continuously to estimate the sequential effects of additional perfusion of nicorandil (6 x 10(-5) M) or Y-26763 (10(-5) M) on triggered activities and OSC. APD prolongation by E-4031 at slower stimulation rates (cycle lengths > or = 1,000 msec) was suppressed by nicorandil in a dose dependent manner. Both nicorandil and Y-26763 abolished the train of early afterdepolarization (EAD) due to E-4031 or dofetilide with a shifting of the resting membrane potential to a more negative level. PCOs also normalized dofetilide induced abnormal automaticities (EAD, OSC). The antagonistic actions of PCOs on changes in action potential induced by class III antiarrhythmic agents may prevent the development of proarrhythmias produced by these agents.     

Comparative studies of ATP sensitive potassium channels in heart and pancreatic beta cells using Vaughan-Williams class Ia antiarrhythmics.

OBJECTIVE: Actions of cibenzoline and disopyramide, agents with Vaughan-Williams class Ia antiarrhythmic action, on ATP sensitive K+ (KATP) channels were examined in heart and pancreatic beta cells. METHODS: Single ventricular myocytes and beta cells were prepared enzymatically from adult Wistar rat hearts and pancreatic islets. Using patch clamp techniques, KATP channel activities were recorded in whole cell and single channel modes. In whole cell experiments, myocytes were bathed with Tyrode's medium (34 degrees C); inside out patches were bathed with internal solutions (22-24 degrees C) containing 1 microM ATP and varying concentrations of cibenzoline or disopyramide. Myocytes were voltage clamped at -40 mV and glibenclamide blockade conductance was produced by cromakalim. RESULTS: Micromolar concentrations of both cibenzoline and disopyramide suppressed cromakalim induced conductance. When applied to the cytosolic surface of the cell membrane in inside out configuration, both drugs reversibly inhibited single KATP channel activities. Neither unitary conductance nor intraburst fast kinetics was affected by the compounds. At a holding potential of -40 mV under symmetrical approximately 150 mM K+ conditions, half maximum doses (IC50) were 0.9 microM [Hill coefficient (h) = 1.3] for cibenzoline induced block of cardiac KATP channels and 1.8 microM (h = 1.0) for disopyramide block. At +40 mV, IC50 for cibenzoline block was 1.4 microM (h = 0.9). Thus there was little voltage dependence in cibenzoline induced channel block. A similar IC50 value of 2.5 microM (h = 1.2 at -60 mV under symmetrical approximately 150 mM K+) was observed for cibenzoline induced block of KATP channels. CONCLUSIONS: Near therapeutic concentrations of cibenzoline and disopyramide inhibit KATP channel activities in both heart and pancreatic beta cells. This may be causally related to the fasting hypoglycaemia which is sometimes reported in patients receiving the drugs. These antiarrhythmic agents may also modulate myocardial electrical properties during hypoxia or ischaemia.     

The slow component of the delayed rectifier potassium current in undiseased human ventricular myocytes

OBJECTIVE: The purpose of this study was to investigate the properties of the slow component of the delayed rectifier potassium current (I(Ks)) in myocytes isolated from undiseased human left ventricles. METHODS: The whole-cell configuration of the patch-clamp technique was applied in 58 left ventricular myocytes from 15 hearts at 37 degrees C. Nisoldipine (1 microM) was used to block inward calcium current (I(Ca)) and E-4031 (1-5 microM) was applied to inhibit the rapid component of the delayed rectifier potassium current (I(Kr)). RESULTS: In 31 myocytes, an E-4031 insensitive, but L-735,821 and chromanol 293B sensitive, tail current was identified which was attributed to the slow component of I(K) (I(Ks)). Activation of I(Ks) was slow (tau=903+/-101 ms at 50 mV, n=14), but deactivation of the current was relatively rapid (tau=122.4+/-11.7 ms at -40 mV, n=19). The activation of I(Ks) was voltage independent but its deactivation showed clear voltage dependence. The deactivation was faster at negative voltages (about 100 ms at -50 mV) and slower at depolarized potentials (about 300 ms at 0 mV). In six cells, the reversal potential was -81.6+/-2.8 mV on an average which is close to the K(+) equilibrium potential suggesting K(+) as the main charge carrier. CONCLUSION: In undiseased human ventricular myocytes, I(Ks) exhibits slow activation and fast deactivation kinetics. Therefore, in humans I(Ks) differs from that reported in guinea pig, and it best resembles I(Ks) described in dog and rabbit ventricular myocytes.     

Ionic currents in single smooth muscle cells of the canine renal artery.

Membrane currents from single smooth muscle cells enzymatically isolated from canine renal artery were recorded using the patch-clamp technique in the whole-cell and cell-attached configurations. These cells exhibited a mean resting potential, input resistance, membrane time constant, and cell capacitance of -51.8 +/- 2.1 mV, 5.2 +/- 0.98 G omega, 116.2 +/- 16.4 msec, and 29.1 +/- 2.0 pF, respectively. Inward current, when elicited from a holding potential of -80 mV, activated near -50 mV, reached a maximum near 0 mV and was sensitive to the dihydropyridine agonist Bay K 8644 and dihydropyridine antagonist nisoldipine. Two components of macroscopic outward current were identified from voltage-step and ramp depolarizations. The predominant charge carrier of the net outward current was identified as K+ by tail-current experiments (reversal potential, -61.0 +/- 0.8 mV in 10.8 mM [K+]o 0 mM [K+]i). The first component was a small, low-noise, voltage- and time-dependent current that activated between -40 and -30 mV (IK(dr)), and the second component was a larger, noisier, voltage- and time-dependent current that activated at potentials positive to +10 mV (IK(Ca)). Both IK(dr) and IK(Ca) displayed little inactivation during long (4-second) voltage steps. IK(Ca) and IK(dr) could be pharmacologically separated by using various Ca2+ and K+ channel blockers. IK(Ca) was substantially inhibited by external NiCl2 (500 microM), CdCl2 (300 microM), EGTA (5 mM), tetraethylammonium (Ki at +60 mV, 307 microM), and charybdotoxin (100 nM) but was insensitive to 4-aminopyridine (0.1-10 mM). IK(dr) was inhibited by 4-aminopyridine (Ki at +10 mV, 723 microM) and tetraethylammonium (Ki at +10 mV, 908 microM) but was insensitive to external NiCl2 (500 microM), CdCl2 (300 microM), EGTA (5 mM), and charybdotoxin (100 nM). Two types of single K+ channels were identified in cell-attached patches. The most abundant K+ channel that was recorded exhibited voltage-dependent activation, was blocked by external tetraethylammonium (250 microM), and had a large single-channel conductance (232 +/- 12 pS with 150 mM K+ in the patch pipette, 130 +/- 17 pS with 5.4 mM K+ in the patch pipette). The second channel was also voltage dependent, was blocked by 4-aminopyridine (5 mM), and exhibited a smaller single-channel conductance (104 +/- 8 pS with 150 mM K+ in the patch pipette, 57 +/- 6 pS with 5.4 mM K+ in the patch pipette). These results suggest that depolarization of canine renal artery cells opens dihydropyridine-sensitive Ca2+ channels and at least two K+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Suppression of time-dependent outward current in guinea pig ventricular myocytes. Actions of quinidine and amiodarone.

Prolongation of cardiac action potentials may mediate some of the arrhythmia-suppressing and arrhythmia-aggravating actions of antiarrhythmic agents. In this study, suppression of time-dependent outward current by quinidine and amiodarone was assessed in guinea pig ventricular myocytes. The net time-dependent outward current contained at least two components: a slowly activating, La(3+)-resistant delayed rectifier current (IK) and a rapidly activating, La(3+)-sensitive current. Quinidine block of total time-dependent outward current during clamp steps to positive potentials was relieved as a function of time, whereas that induced by amiodarone was enhanced. In contrast, at negative potentials, suppression of current, whereas amiodarone reduced IK but not the La(3+)-sensitive current, suggesting that differential block of the two components of time-dependent current underlies the distinct effects of the two agents. In contrast to these disparate effects on total time-dependent outward current, steady-state reduction of IK by both drugs increased at positive voltages and saturated at approximately +40 mV; the voltage dependence of block by quinidine (17% per decade, +10 to +30 mV) was steeper than that by amiodarone (5% per decade, +10 to +20 mV). Block by quinidine was time dependent at negative potentials: on stepping from +50 to -30 mV, block initially increased very rapidly, and subsequent deactivation of IK was slowed. This effect was not seen with amiodarone. At -80 mV, quinidine block was relieved with a time constant of 40 +/- 15 msec (n = 4, twin-pulse protocol). The effects of quinidine on IK were compatible with neither a purely voltage-dependent model of quinidine binding nor a model incorporating both voltage- and state-dependent binding of quinidine to delayed rectifier channels having only one open state. The voltage- and time-dependent features of quinidine block were well described by a model in which quinidine has greater affinity for one of two open states of the channel. We conclude that the effects of quinidine and amiodarone on time-dependent outward current reflects block of multiple channels. Quinidine block of IK was far more voltage dependent than that produced by amiodarone, suggesting the drugs act by different mechanisms.     

Phase 2 prolongation, in the absence of instability and triangulation, antagonizes class III proarrhythmia

OBJECTIVE: To evaluate whether prolongation of the plateau of the action potential duration, in the absence of instability and triangulation, can reverse the proarrhythmia elicited by a class III antiarrhythmic agent. METHODS: The effects of almokalant, erythromycin and their combination, on cardiac electrophysiological parameters (action potential duration (APD), instability, triangulation and ectopics) were evaluated in isolated hearts from female albino rabbits. In this study, proarrhythmia was estimated quantitatively by number of ectopic beats. RESULTS: Erythromycin lengthened the APD primarily by a prolongation of the plateau, while having only minor effects upon phase 3 repolarization. The prolongation did not induce much instability, triangulation or reverse use dependence and, as expected, erythromycin did not induce significant proarrhythmia. Almokalant also lengthened APD, but it did not lengthen the plateau; instead, it prolonged phase 3 repolarization. The prolongation markedly triangulated the action potential, elicited much instability and marked reverse use dependence. This combination of effects induced very marked proarrhythmia. When almokalant and erythromycin were combined, their effects upon APD appeared additive: both the plateau and the repolarization phase were prolonged. However, the larger prolongation of APD did not lead to more proarrhythmia; this suggests that a prolongation of APD is not proarrhythmic per se. On the contrary, proarrhythmia as a function of APD prolongation was reduced in the presence of erythromycin (P<0.05). CONCLUSION: Instability plus triangulation consistently lead to serious proarrhythmia especially when combined with reverse use dependence, but prolongation of APD in itself is not necessarily proarrhythmic. In fact, APD prolongation in the absence of instability and triangulation can be antiarrhythmic.     

Suppression of repolarization-related arrhythmias in vitro and in vivo by low-dose potassium channel activators.

Marked prolongation of cardiac action potentials and of QT intervals has been associated with early afterdepolarizations and triggered activity in vitro and with ventricular tachycardia in vivo. Because the antihypertensive potassium channel activators pinacidil and cromakalim are known to accelerate repolarization in cardiac tissues, we performed in vitro and in vivo experiments to test the hypothesis that these agents would block the arrhythmogenic effects of delayed repolarization. Early afterdepolarizations and triggered activity were elicited in canine cardiac Purkinje fibers driven at cycle lengths of 4 seconds or more (K0, 2.7 mM) during superfusion with quinidine, cesium, or sematilide, a methylsulfonylamino parasubstituted analogue of procainamide with class III antiarrhythmic activity. The potassium channel activators invariably (17 of 17) abolished this form of abnormal automaticity. This effect was observed at low concentrations that did not alter action potential characteristics at shorter cycle lengths. Intravenous Cs+ (total dose, 4.5 mM/kg) was used to produce ventricular arrhythmias in anesthetized rabbits randomly pretreated in a double-blind fashion with either low-dose pinacidil (0.2 mg/kg) or vehicle. Pinacidil pretreatment resulted in significantly fewer total ventricular ectopic beats (168 +/- 157 versus 582 +/- 448, p less than 0.005) and episodes of ventricular tachycardia (four of nine versus nine of nine, p = 0.057). At this dose, pinacidil did not alter mean blood pressure before Cs+ and maximal hypertensive response after Cs+. In summary, the potassium channel activators pinacidil and cromakalim suppressed triggered activity related to prolonged repolarization at concentrations that did not affect action potential characteristics at normal rates in vitro; pinacidil blunted arrhythmias produced by cesium administration in vivo without lowering blood pressure.(ABSTRACT TRUNCATED AT 250 WORDS)