Ionic basis for action potential prolongation by phenylephrine in canine epicardial myocytes

INTRODUCTION: In canine ventricle, alpha-adrenergic agonists prolong action potential duration (APD) without any effect on the action potential notch, suggesting that, in this species, the effect on repolarization might be independent of inhibition of I(to). The present study investigated the action of the alpha-adrenergic agonist phenylephrine on the action potential and the repolarizing currents I(to) and I(K) in isolated canine epicardial myocytes. METHODS AND RESULTS: Isolated cells from canine epicardial tissue, and Purkinje fibers, were studied with the whole cell, voltage clamp method. Phenylephrine 0.1 microM increased APD by 13% +/- 4% at 90% repolarization without affecting the notch or amplitude. Under voltage clamp, concentrations of phenylephrine as high as 10 microM had no effect on I(to) in canine epicardial myocytes. However, I(to) of isolated canine Purkinje myocytes was reduced to 69% +/- 7% of control by 1 microM phenylephrine. Further studies in canine epicardial myocytes revealed an action of phenylephrine to inhibit I(K), and in particular I(Ks). Using a voltage protocol that included a two-step repolarization to separate I(Ks) and I(Kr) tail components, the largely I(Kr) component was not significantly affected by 1 microM phenylephrine, whereas the largely I(Ks) component was reduced to 81% +/- 5% of control value. CONCLUSION: Alpha-adrenergic prolongation of repolarization in canine epicardium does not result from inhibition of I(to). Rather, it appears that reduction of I(Ks) contributes to the action of phenylephrine. The unresponsiveness of epicardial I(to) is not a general characteristic of the canine heart, because Purkinje myocyte I(to) was inhibited, suggesting regional differences in the molecular basis of I(to) and/or alpha-adrenergic signaling in the canine heart.     

Acceleration-Induced Action Potential Prolongation and Early Afterdepolarizations

Acceleration-Induced Early Afterdepolarizations. Introduction: Precipitation of torsades de pointes (TdP) has been shown to he associated with acceleration of heart rate in both experimental and clinical studies. To gain insight into the cellular mechanism(s) responsible for the initiation of acceleration-induced TdP, we studied the effect of acceleration of pacing rate in canine left ventricular epicardial, M region, endocardial, and Purkinje fiber preparations pretreated with E-4031, an I_Kr blocker known to induce the long QT syndrome and TdP. Methods and Results: Standard microelectrode techniques were used. E-4031 (1 to 2 μM) induced early afterdepolarization (EAD) activity in 31 of 36 M cell, 0 of 10 epicardial, 0 of 10 endocardial, and 9 of 12 Purkinje fiber preparations at basic cycle lengths (BCLs) ≥ 800 msec. In 30 of 36 M cells, sudden acceleration from a BCL range of 900 to 4,000 msec to a range of 500 to 1,500 msec induced transient EAD activity if none existed before or increased the amplitude of EADs if already present. Acceleration-induced augmentation of EAD activity was far less impressive and less readily demonstrable in Purkinje fibers (4/12). In M cells, appearance of EAD activity during acceleration usually was accompanied by an abbreviation of action potential duration (APD). Within discrete ranges of rates in the physiologic range, acceleration caused a transient prolongation of APD in 38% of M cells, whether or not a distinct EAD was generated. Acceleration produced still more dramatic APD prolongation and EADs in M cells after the BCL was returned to the original slow rate. Epicardium and endocardium APD showed little change immediately after acceleration. A decrease of BCL as small as 10% and, in some cases, a single premature heat could promote EAD activity and APD prolongation in some M cells. Ryanodine (1 μM, 10/10), flunarizine (10 μM, 3/6), and low Na (97 vs 129 mM, 5/5) abolished the acceleration-induced EAD activity and APD prolongation as well as the EAD activity observed at slow rates in M cells pretreated with E-4031. Conclusion: Our results suggest that acceleration from an initially slow rate or a single premature beat can induce or facilitate transient EAD activity and APD prolongation in canine ventricular M cell preparations pretreated with an I_Kr blocker via a mechanism linked to intracellular calcium loading. Our data provide evidence in support of an important contribution of electrogenic Na/Ca exchange current to this process. These acceleration-induced changes can result in the development of triggered activity as well as a marked dispersion of repolarization in ventricular myocardium and, thus, may contribute to the precipitation of TdP in patients with the congenital (HERG defect) and acquired (drug-induced) long QT syndrome.     

Alpha1-Adrenergic Receptor Modulation of Repolarization in Canine Purkinje Fibers

Alpha-Agonists and Repolarization. Introduction: Alpha-adrenergic receptor stimulation increases contractility and prolongs repolarization. These effects are modulated by α₁-adrenergic receptor-mediated inhibition of transsarcolemmal potassium currents.
Methods and Results: We used standard microelectrode techniques to study the actions of 4-aminopyridine (4-AP), which blocks the transient outward current, I_to, and WAY-123,398, which blocks the delayed rectifier, I_k, on canine Purkinje fiber action potential prolongation induced by phenylephrine. At a basic cycle length of 1 second, phenylephrine (0.1 to 10 μ) dose-dependently prolonged action potential duration at 90% repolarization (APD₉₀) from 331 ± 10 msec to 400 ± 12 msec (P < 0.05) at phenylephrine, 10 μ. Phenylephrine did not change phase 1 or plateau height. 4-AP (0.1 mM) decreased phase 1 magnitude, shifted plateau height to more positive potentials (from 0.1 ± 1.8 mV to 14.3 ± 1.1 mV [P < 0.05]), and shortened APD₉₀ from 318 ± 9 msec to 294 ± 8 msec (P < 0.05). 4-AP did not block phenylephrine effects on APD₉₀, which increased, at 10 μ phenylephrine, from 294 ± 8 msec to 342 ± 6 msec (P < 0.05). In contrast, WAY-123,398 (0.1 μ) prolonged APD₉₀ from 360 ± 6 msec to 452 ± 6 msec (P < 0.05), and had no effect on plateau height. In the presence of WAY-123,398, phenylephrine no longer increased APD_9o.
Conclusion: (1) Agents that block I_to shorten APD in Purkinje fibers; and (2) the α-agonist mediated increase of APD in canine Purkinje fibers can be explained by inhibition of I_k.     

Block of I(Ks) does not induce early afterdepolarization activity but promotes beta-adrenergic agonist-induced delayed afterdepolarization activity

INTRODUCTION: An early afterdepolarization (EAD)-induced triggered beat is thought to precipitate torsade de pointes (TdP) in the long QT syndrome (LQTS). Previous studies demonstrated the development of EAD activity and dispersion of repolarization under LQT2 (reduced I(Kr)) and LQT3 (augmented late I(Na)), but not LQT1 (reduced I(Ks)), conditions. The present study examines these electrophysiologic characteristics during I(Ks) block. METHODS AND RESULTS: Canine epicardial (Epi), M, and endocardial (Endo) tissues and Purkinje fibers isolated from the canine left ventricle were studied using standard microelectrode recording techniques. The I(Ks) blocker chromanol 293B (293B, 30 microM), produced a homogeneous rate-independent prolongation of action potential duration (APD) in Epi, M, and Endo, but little to no APD prolongation in Purkinje. Chromanol 293B 1 to 30 microM failed to induce EADs or delayed afterdepolarizations (DADs) in any of the four tissue types. Isoproterenol (ISO, 0.1 to 1.0 microM) in the presence of 293B 30 microM significantly prolonged the APD of the M cell (basic cycle length > or = 1 sec), abbreviated that of Purkinje, and caused little change in that of Epi and Endo. The combination of 293B 30 microM and ISO 0.2 microM did not induce EADs in any of the four tissue types, but produced DAD activity in 4 of 8 Epi, 7 of 10 M cells, and 3 of 8 Endo. CONCLUSION: Our results indicate that I(Ks) block alone or in combination with beta-adrenergic stimulation does not induce EADs in any of the four canine ventricular tissue types, but that the combination of the two induces DADs as well as accentuated dispersion of repolarization.     

Effects of Alpha Adrenergic Stimulation and Blockade on Early Afterdepolarizations Induced by Cesium in Canine Cardiac Purkinje Fibers

Alpha Adrenergic Stimulation and EADs. The effects of alpha adrenergic stimulation and three alpha adrenoceptor blockers on early afterdepolarizations (EADs) were examined in canine card diac Purkinje fibers. In the first group of 18 preparations, EADs were induced by superfusion with 7.5 mM cesium (Cs) dissolved in low potassium (2.7 mM KCl) Tyrode's solution. During alpha adrenoceptor stimulation (norepinephrine 1 μM and propranolol 1 μM) to enhance EADs, the effects of phentotamine and two new alpha 1 adrenoceptor antagonists, benoxathian and WB 4101 (1, 3, and 10 μM), were examined. WB 4101 (1 μM) suppressed EADs in all six preparations. Benoxathian required 3 μM in five preparations and 10 μM in one to suppress EADs. Phentolamine (10 μM) suppressed EADs in two of six preparations. In the second group of 21 preparations, the effects on cesium-induced EADs of the three alpha adrenoceptor hlockers (10 μM) were examined without alpha adrenoceptor stimulation. WB 4101 (10 μM) suppressed EADs in seven of seven preparations, while benoxathian (10 μM) suppressed EADs in five of seven. Phentolamine (10 μM) enhanced EADs in six of seven preparations. In the third group of 24 Purkinje fibers, the direct effects (without alpha adrenoceptor stimulation) of these three antagonists on the characteristics of normal Purkinje fiber action potentials superfused with normal Tyrode's solution were examined. Phentolamine (1, 3, and 10 μM, n = 8) prolonged action potential duration at 90% repolarization (APD90) by 5.3%± 1.3%, 10.0%± 1.8%, and 14.3%± 2.7%, respectively. Benoxathian (n = 8) and WB 4101 (n = 8) shortened APD90 equally: 6.8%± 1.1% vs 7.7%± 1.3% at 1 μM, 13.6%± 1.0% vs 14.5%± 1.6% at 3 μM, and 21.1%± 1.2% vs 20.8%± 2.1% at 10 μM, respectively. We conclude that in cesium treated Purkinje fibers: (1) Alpha adrenoceptor stimulation enhanced EADs; (2) Phentolamine was least effective in suppressing EADs, probably because of a direct effect that prolonged MM)90,: and (3) Although benoxathian and WB 4101 had similar etTects on APD90, WB 4101 was more effective in suppressing EADs at lower concentrations than henoxathian.     

Sodium channel block produces opposite electrophysiological effects in canine ventricular epicardium and endocardium.

Using microelectrode techniques we compared the effects of tetrodotoxin (TTX, 2-3 microM), DL-propranolol (1-3 micrograms/ml), and flecainide acetate (10-15 microM) on isolated canine ventricular epicardial (epicardium) and endocardial (endocardium) tissues. Propranolol, TTX, and flecainide decreased Vmax and phase 0 amplitude in a use-dependent manner in both tissues. The effects of propranolol were slow to develop and wash out. TTX and propranolol always abbreviated action potential duration in endocardium. Action potential duration was abbreviated by 23.8 +/- 5.6 msec after propranolol (1 microgram/ml, basic cycle length [BCL] = 1,000 msec) and 10.8 +/- 12.9 msec after TTX (2 microM, BCL = 1,000 msec). In epicardium, the reduction of phase 0 and 1 amplitudes led to a slowing of the second action potential upstroke and an increase in the amplitude of phase 2. This accentuation of the notch resulted in a paradoxical prolongation of the epicardial action potential. Action potential duration was prolonged 34.4 +/- 11.3 msec after 4 hours of exposure to propranolol (1 microgram/ml, BCL = 1,000 msec), 11.1 +/- 6.3 msec after 15 minutes of exposure to TTX (2 microM, BCL = 1,000 msec), and 19.9 +/- 8.2 msec after 25-45 minutes of exposure to flecainide (15 microM, BCL = 500 msec). With stronger sodium block, phase 1 terminated at more negative potentials, the second upstroke often failed to appear, and an all-or-none repolarization ensued causing a marked abbreviation of the epicardial action potential. In some epicardial preparations, we observed marked abbreviation at some sites but prolongation at other sites after sodium blockade with flecainide. The dispersion of repolarization was often attended by reentrant activity. The differential response of epicardium and endocardium to sodium blockade was not observed when the preparations were pretreated with 4-aminopyridine or ryanodine, agents known to diminish the transient outward current and epicardial notch. Acceleration-induced prolongation of refractoriness was observed after sodium blockade in epicardium but not in endocardium. Postrepolarization refractoriness also occurred in epicardium but not in endocardium after TTX, propranolol, or flecainide exposure. The data indicate that propranolol, TTX, and flecainide, via their action to block sodium current, may exert opposite effects on action potential duration and refractoriness in cells spanning the ventricular wall. The presence of the transient outward current in epicardium but not in endocardium appears to contribute importantly to these differences.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alpha 1-adrenergic agonists selectively suppress voltage-dependent K+ current in rat ventricular myocytes.

The effects of alpha 1-adrenergic agonists on the waveforms of action potentials and voltage-gated ionic currents were examined in isolated adult rat ventricular myocytes by the whole-cell patch-clamp recording technique. After "puffer" applications of either of two alpha 1 agonists, phenylephrine and methoxamine, action-potential durations were increased. In voltage-clamped cells, phenylephrine (5-20 microM) or methoxamine (5-10 microM) reduced the amplitudes of Ca2+-independent voltage-activated outward K+ currents (Iout); neither the kinetics nor the voltage-dependent properties of Iout were significantly affected. The effects of phenylephrine or methoxamine on Iout were larger and longer-lasting at higher concentrations and after prolonged or repeated exposures; in all experiments, however, Iout recovered completely when puffer applications were discontinued. The suppression of Iout is attributed to the activation of alpha 1-adrenergic receptors, as neither beta- nor alpha 2-adrenergic agonists had measurable effects on Iout; in addition, the effect of phenylephrine was attenuated in the presence of the alpha antagonist phentolamine (10 microM), but not in the presence of the beta antagonist propranolol (10 microM). Voltage-gated Ca2+ currents, in contrast, were not altered measurably by phenylephrine or methoxamine and no currents were activated directly by these agents. Suppression of Iout was also observed during puffer applications of either of two protein kinase C activators, phorbol 12-myristate 13-acetate (10 nM-1 microM) and 1-oleoyl-2-acetylglycerol (60 microM). We conclude that the activation of alpha 1-adrenergic receptors in adult rat ventricular myocytes leads to action-potential prolongation as a result of the specific suppression of Iout and that this effect may be mediated by activation of protein kinase C.     

Prominent I(Ks) in epicardium and endocardium contributes to development of transmural dispersion of repolarization but protects against development of early afterdepolarizations

INTRODUCTION: Previous studies from our laboratory demonstrated (1) a much larger I(Ks) and (2) inability to induce early afterdepolarization (EAD) activity in epicardial and endocardial cells versus M cells. This study tests the hypothesis that these two characteristics are interrelated. METHODS AND RESULTS: Standard and floating microelectrode techniques were used to record transmembrane activity from the canine left ventricular epicardial, M, and endocardial regions in isolated tissue slices and arterially perfused wedge preparations. The I(Kr) blocker E-4031 (1 to 10 microM) caused prominent prolongation of action potential duration (APD) and induced EADs in tissues isolated from the M region, but not those from epicardium or endocardium, causing a large transmural dispersion of APD. In contrast, the I(Ks) blocker chromanol 293B (10 to 30 microM) produced moderate prolongation of APD without EADs in all three tissue types. The combination of E-4031 (1 microM) and chromanol 293B (30 microM) resulted in profound prolongation of APD and the development of EADs in all three tissue types. In the perfused wedge, neither E-4031 nor chromanol 293B alone could induce EADs. In combination, the two drugs caused significant prolongation of APD and EADs in all three transmural regions. CONCLUSION: Our results support the hypothesis that a prominent I(Ks) is responsible for the ability of epicardium and endocardium to resist some but not all of the arrhythmogenic effects of I(Kr) block. The data highlight the critical importance of I(Ks) in the canine heart and the significant role of electrotonic interactions in minimizing the development of an arrhythmogenic substrate when repolarization reserve is reduced.     

Electrophysiological responses of canine atrial endocardium and epicardium to acetylcholine and 4-aminopyridine.

OBJECTIVES: Prior studies demonstrated marked electrophysiological and pharmacological differences between canine ventricular epicardium and endocardium. For atrium, however, it has been assumed that, because of the thin wall, electrical properties of epicardium and endocardium are similar. The aim of the present study was to compare the action potential (AP) characteristics in epicardial and endocardial atrial cells before and following addition of acetylcholine (ACh) and 4-aminopyridine (4-AP). METHODS AND RESULTS: Microelectrode techniques were used to study the effects of ACh (10(-7)-10(-5) M) and 4-AP (0.5 mM) on epicardial and endocardial AP of canine right atrial free wall at cycle lengths (CL) of 250 to 2000 ms. ACh hyperpolarized epicardial and endocardial cells (by 5-8 mV at 10(-5) M). In control, AP duration to 90% repolarization (APD90) was longer in endocardium at all CL. ACh shortened APD90 in either tissue with more prominent effect in endocardium (at 10(-5) M and CL = 2000 ms, from 179 +/- 10 to 90 +/- 11 ms in epicardium and from 209 +/- 10 to 65 +/- 6 ms in endocardium, P < 0.05). As a result, at 10(-5) M, APD90 in endocardium was shorter than in epicardium at all CL 4-AP effects on AP duration were similar in both tissue types. No effects of 4-AP was seen at CL = 250 ms and at long CL, the compound shortened APD90 and prolonged AP duration to 50% repolarization. CONCLUSIONS: (1) ACh exerts direct effects on atrial epicardial and endocardial AP; (2) 4-AP-sensitive transient outward current (Itol) is expressed both in canine atrial epicardial and endocardial cells; (3) differential response of epicardial and endocardial APD to ACh may alter the gradient of repolarization across the atrial wall and contribute to vagally induced atrial flutter and fibrillation.     

Alpha 1-adrenoceptor regulation of delayed afterdepolarizations and triggered activity in subendocardial Purkinje fibers surviving 1 day of myocardial infarction.

Alpha 1-adrenoceptor agonists were shown to induce delayed afterdepolarizations (DADs) and triggered activity in the presence of elevated extracellular Ca2+. We investigated the effects of alpha 1-adrenoceptor stimulation on DADs and triggered activity in canine Purkinje fibers that survived 1-day of myocardial infarction. Endocardial preparations were studied using standard microelectrode techniques. In quiescent preparations showing no DADs and in presence of propranolol (2 x 10(-7) M), phenylephrine (10(-6) M), an alpha 1-adrenoceptor agonist induced DADs (n = 6) and differentially induced triggered activity in ischemic but not in normal Purkinje fibers (n = 4). In 8 preparations that showed subthreshold DADs, phenylephrine increased the DAD amplitude from 4.0 +/- 2.5 mV to 8.0 +/- 3.3 mV (P less than 0.03) and from 3.2 +/- 1.5 mV to 6.5 +/- 3.7 mV (P less than 0.05) at paced cycle lengths of 800 and 400 ms, respectively. Phenylephrine caused subthreshold DADs to reach threshold and result in triggered activity (n = 6). The effects of phenylephrine were abolished by 10(-6) M prazosin, an alpha 1-adrenoceptor blocker. Our results suggest that alpha 1-adrenoceptor stimulation regulates DADs and triggered activity seen in subendocardial Purkinje fibers surviving 1 day of myocardial infarction and may contribute to the spontaneous ventricular tachycardia seen in vivo at this stage.     

奎尼丁对吡那地尔诱导的犬右心室跨壁复极离散的影响

目的　由吡那地尔诱导犬右心室肌细胞产生“全或无”复极,观察奎尼丁对这种跨壁复极离散的影响。方法　应用标准玻璃微电极技术在1000ms刺激周长下,记录犬右心室肌细胞不同部位(外膜下、M区、内膜下)在不同情况[正常对照、吡那地尔(2 5μmol/L)、吡那地尔( 2 5μmol/L) +奎尼丁(5μmol/L) ]的动作电位。结果　吡那地尔( 2 5μmol/L)在3层细胞产生“全或无”复极,使跨壁复极离散增大,动作电位时程跨壁复极离散由(48 .5±9 .2)ms升为(128. 7±13. 5)ms(P<0. 01),进一步灌注奎尼丁(5μmol/L)后,减为(54 .3±10 .8)ms(P<0. 01)。奎尼丁部分恢复动作电位2相平台,延长了被吡那地尔缩短的动作电位时程。结论　在犬右心室肌组织,奎尼丁(5μmol/L)减小了由吡那地尔造成的跨壁复极离散,维持了跨壁电稳定性。     

Contrasting roles of a novel K+ channel blocker and a K+ channel opener on electro-mechanical activity in canine heart tissue

We tested the effects of a potassium channel opener diazoxide on the action potential duration (APD) and contractile force changes in canine Purkinje tissue induced by a novel class III anti-arrhythmic agent (C3A), KCB-328 (0.5 microM) with 3,4-dimethoxyphenethyl ring structure (0.5 microM). KCB-328 shortened APD(25) by 8.3+/-2.1%, prolonged APD(50) and APD(90) by 31.2+/-5.3 and 50.0+/-7.1%, respectively. Diazoxide (0.1 mM) shortened APD at all levels by 58.3+/-8.1, 54.1+/-6.1, and 42.8+/-5.8%, respectively. In the presence of diazoxide, KCB-328 still prolonged APD(50) and APD(90) (12.5+/-3.8 and 26.8+/-5.9%, respectively). KCB-328 increased force of contraction in a dose-dependent manner. KCB-328 increased force less in the presence of diazoxide. Administration of diazoxide only, reduced force of contraction. We conclude that APD prolongation by KCB-328 may occur even in the presence of diazoxide. It is not sufficient for the restoration of already diminished contractile force and that such an APD prolongation may be unrelated to the restoration of force of contraction even though both are most often seen to occur simultaneously.     

Effects of alpha-adrenergic stimulation on intracellular sodium activity and automaticity in canine Purkinje fibers

Approximately 60% of adult canine Purkinje fibers respond to alpha 1-adrenergic stimulation with a decrease in automaticity. Recent studies of disaggregated Purkinje myocytes have suggested that this negative chronotropic effect results from alpha 1-adrenergic activation of the Na-K pump. In this study we evaluated 1) whether Na-K pump activation is associated with the negative chronotropic effect of alpha 1-adrenergic stimulation in adult canine Purkinje fibers and 2) if the effect of alpha-agonists on the pump is direct or mediated by an increase in intracellular sodium activity (aNai). We used sodium selective microelectrodes to determine the effects of 5 x 10(-9) and 5 x 10(-8) M phenylephrine on aNai. Phenylephrine decreased automaticity in five of eight Purkinje fibers while an increase occurred in the other three. The rate decrease was always accompanied by a decrease in aNai (-3.9 mM; p less than 0.05), whereas in fibers showing an increase in rate, aNai was unchanged. To evaluate the effect of phenylephrine in the absence of changes in automaticity, 10 Purkinje fibers were studied during pacing. A clear-cut reduction in aNai (-2.8 mM) was present in six fibers; no change was seen in the other four. The effect of phenylephrine was blocked by prazosin but not by propranolol. We conclude that the effect of alpha 1-adrenergic stimulation to reduce aNai is consistent with activation of the Na-K pump. Moreover, this action of alpha 1-adrenergic stimulation is closely linked to its negative chronotropic effect.     

Cellular electrophysiologic responses of isolated neonatal and adult cardiac fibers to d-sotalol

The short-term cellular electrophysiologic actions of d-sotalol on isolated neonatal and adult canine ventricular myocardium and Purkinje fibers were evaluated using standard microelectrode techniques. d-Sotalol, 10(-6) to 10(-4)M, had no effects on action potential amplitude, maximal diastolic potential or action potential upstroke velocity (Vmax) in any neonatal or adult preparation. In five adult myocardial preparations, d-sotalol produced concentration-dependent increases in action potential duration at 50% (APD50) and 90% (APD90) repolarization and effective refractory period. In six neonatal myocardial preparations, d-sotalol produced a biphasic response; APD50, APD90 and effective refractory period decreased at 10(-6) and 10(-5)M. At 10(-4)M, these values increased significantly but to a lesser extent compared with values in adults. In seven adult Purkinje fibers, d-Sotalol significantly increased APD50, APD90 and effective refractory period in a concentration-dependent manner. All six neonatal Purkinje fibers responded in a biphasic manner, with values for APD50, APD90 and effective refractory period being less than control at 10(-6)M and near control values at 10(-5)M. At 10(-4)M, these variables were significantly increased, but to a lesser extent than in adult preparations. Our data confirm the typical class III effects of d-sotalol in adult cardiac tissues. The shortening of repolarization and refractoriness at lower drug concentrations in developing cardiac tissues may relate to age-dependent differences in cellular ionic function and basic electrophysiology.     

Alpha 1 adrenoceptor subtype mediates noradrenaline induced contraction of the human internal mammary artery: radioligand and functional studies.

STUDY OBJECTIVE--The aim was to evaluate the characteristics of alpha adrenergic binding sites on human internal mammary arteries and the alpha adrenoceptor mediated vasoconstrictor response to catecholamines. DESIGN--Human internal mammary arteries were cut longitudinally, the intimal layer was scraped, and the arteries homogenised and centrifuged at 50,000 g to obtain a membrane pellet. Saturation isotherms with [3H]-prazosin were done with 50-100 micrograms plasma membranes per tube and increasing concentrations of [3H]-prazosin (non-specific binding: 2.5 mM noradrenaline plus superoxide dismutase and catalase). Kinetic isotherms were done with 100 micrograms plasma membranes and 1-5 nM [3H]-prazosin for time periods ranging from 1 to 90 min; at the equilibrium, dissociation of [3H]-prazosin was achieved by 10 microM prazosin. alpha 2 Adrenoceptor density on internal mammary artery membranes was assessed with [3H]-rauwolscine (non-specific binding: 1 microM yohimbine). Separation of membrane bound radioactivity was achieved by rapid vacuum filtration through Whatman GF/C fibre filters. Saturation isotherms were evaluated by Scatchard plots and kinetic data, and competition isotherms by Enzfitter analysis. Contractility studies were done with helical strips of artery (without adventitial layer) placed in a thermostated perfusion bath. Data were obtained in the presence of different concentrations of agonists and antagonist to obtain Schild plots. Antagonist drugs were employed at only one concentration for each preparation. SUBJECTS--Mammary arteries were collected from 51 patients (age range 42-65 years) undergoing surgery for coronary grafting. MEASUREMENTS AND MAIN RESULTS--The binding of [3H]-prazosin to arterial plasma membrane was rapid and reversible. The K + 1 was 0.13 (SD 0.03) X 10(9) M.min-1 (n = 5) and the Kd, determined as a ratio between k-1/K + 1, was 0.34(0.01) nM (n = 5). [3H]-Prazosin binding, displaceable by 2.5 mM (-)-noradrenaline, was saturable and disclosed an alpha 1 adrenoceptor density of 30(3) fmol.mg-1 protein with a dissociation constant (Kd) of 215(50) pM (n = 18). The adrenergic agonists competed with [3H]-prazosin in the following order of potency: (-)-adrenaline [Ki = 0.6(0.1) microM; n = 5] greater than (-)-noradrenaline [Ki = 1.05(0.015) microM; n = 12] much greater than (-)-isoprenaline [Ki = 150(10) microM; n = 4]. Specific binding of [3H]-rauwolscine to IMA plasma membranes was negligible (about 2 fmol.mg-1 protein) (n = 15) with an unfavourable ratio of non-specific v specific binding. Catecholamines induced a dose dependent contractile response in arterial strips; (-)-noradrenaline: EC50 = 0.48(0.12) microM, n = 20; (-)-adrenaline: EC50 = 0.15(0.16) microM, n = 10; and methoxamine, a selective alpha 1 adrenergic agonist: EC50 0.67(0.15) microM, n = 10. The alpha 2 adrenoceptor agonists BHT-933, BHT-920, and guanabenz did not contract the arterial strips (up to 10 mM). Prazosin (0.03-0.1 microM) produced concentration dependent right shifts of the (-)-noradrenaline [pA2 = 9.83(0.11), n = 19], (-)-adrenaline [pA2 = 9.50(0.31), n = 10], and methoxamine [pA2 = 8.96(0.18), n = 10] concentration-response curve. CONCLUSIONS - Internal mammary artery plasma membranes possess alpha 1 adrenoceptors which are involved in the vasoconstrictor response to catecholamines. alpha 2 Adrenoceptors seem not to be involved.     

Lidocaine and Nisoldipine Attenuate Almokalant-Induced Dispersion of Repolarization and Early Afterdepolarizations In Vitro

Attenuation of Almokalant-Induced Proarrhythmias In Vitro. Introduction: Treatment with Class III antiarrhythmic agents may lead to increased dispersion or repolarization and early afterdepolarizations (EADs), which are both likely substrates for torsades de pointes. Recent studies in vivo have shown that the prevalence of proarrhythmias induced by Class III agents may be reduced by Na⁺ or Ca²⁺-blocking agents. In the present study, tentative mechanisms for this protective effect were investigated in vitro. Methods and Results: Transmembrane action potentials were recorded simultaneously from rabbit isolated ventricular muscle (VM) and Purkinje fibers (PF). At a basic cycle length (BCL) of 500 msec, the Class III agent almokalant (0.1 μM) increased the dispersion by prolonging the action potential duration (APD) significantly more in the PF (33%± 4.2%, n = 18) than in the VM (17%± 5.9%, n = 18. P < 0.05). In six of the preparations, addition of 1, 5, and 25 μM lidocaine reduced the almokalant-induced prolongation in a concentration-dependent manner mainly in the PF, thereby decreasing the dispersion. At 5 μM lidocaine, the remaining prolongation was 7%± 12.2% (P < 0.05 vs time controls) in the PF and 14%± 6.4% in the VM, respectively. In six other preparations, the addition of 0.01, 0.05, and 0.25 μM nisoldipine did not reduce the almokalant-induced prolongation in the PF and VM, hut attenuated the spike-and-dome appearance of the action potential in the PF. In separate experiments performed at a BCL of 1000 msec, EADs developed in 2 of 6 and 5 of 6 PF during superfusion with almokalant (0.3 and 1 μM, respectively) at an API) of 828 ± 41.4 msec. In six separate preparations pretreated with lidocaine (5 μ) the almokalant-induced prolongation in the PF was less pronounced and EADs were not observed. Pretreatment with nisoldipine (0.05 μM) did not influence the response to almokalant, and in 4 of 6 preparations the APD exceeded 1000 msec. Despite this extensive prolongation, EADs did not appear. Conclusion: At concentrations that did not affect the APD in the VM hut reduced the APD in the PE. lidocaine suppressed almokalant-induced dispersion and the development of EADs. Nisoldipine, (m the other hand, inhibited almokalant-induced EADs directly. Hence, (he primary APD-prolonging effect of a Class III agent may he preserved, but the risk of proarrhythmary reduced, during concomitant treatment with low concentrations of a Na⁺- or Ca²⁺ blocking agent.     

犬心室肌M细胞钙电流特性的研究

用膜片钳全细胞方法研究犬心室肌内膜、中层 (M细胞 )及外膜细胞的L型钙电流的特性 ,并观察nifedipine(1μM)对三层心肌单个细胞动作电位的影响。结果 :心室肌内膜、M及外膜细胞在去极电压为 +10mv时 ,ICa ,L的峰电流pA/pF分别为 - 4.2 9± 1.2 4、- 5 .30± 0 .38、- 4.0 3± 1.19,P <0 .0 5。Nifedipine(1μM)对三层细胞动作电位均缩短。心室肌内膜、M及心外膜细胞动作电位复极化达 90 %时程分别由用药前的 44 7.3± 47.0 ,5 2 4.2± 5 5 .2 ,438.2±39.3ms缩短至用药后的 30 2 .4± 42 .7,30 5 .4± 37.2 ,30 3.3± 37.6ms ,缩短率 32 .8% ,41.5 % ,33 .6 % (P <0 .0 5 )。表明犬心室肌内膜、M及外膜细胞的L型钙电流分布的差异性 ,可能是M细胞易于产生后除极及触发活动的离子基础     

Prolonged action potential durations,increased dispersion of repolarization,and polymorphic ventricular tachycardia in a mouse model of proarrhythmia

Introduction: In the congenital long QT syndrome, inhomogeneously prolonged action potentials, bradycardia, and hypokalemia can cause afterdepolarizations and torsade de pointes. Other genetic factors may contribute to similar forms of ventricular tachycardias in hypertrophied or failing hearts, especially if the outward current I_Kr is blocked pharmacologically. We sought to develop a mouse heart model for such arrhythmias in order to identify the proarrhythmic potential in transgenic animals. Methods and results: Hearts of adult wild-type (CD1) mice were isolated and the aorta was retrogradely perfused. Three monophasic action potentials and a volume-conducted ECG were simultaneously recorded. Sotalol (10^-5M and 2 × 10^-5M) prolonged action potential duration (APD) in a concentration-dependent and reverse frequency-dependent fashion (from 34 ± 1 to 48 ± 2 ms at 100 ms basic cycle length (BCL), from 38 ± 2 to 54 ± 3 ms at 180 ms BCL for APD90, p < 0.05). Sotalol did not alter the relation between refractoriness and APD (ERP/APD ratio = 0.76 - 0.93). AV nodal block caused ventricular bradycardia and doubled dispersion of APD (APD70max-min: 11 ± 1 vs. 4 ± 1 ms, APD90max-min: 12 ± 1 vs. 5 ± 1 ms, p < 0.05). If combined with hypokalemia, afterdepolarizations induced polymorphic ventricular tachycardias in 1 of 8 hearts at K⁺ =3.0 mM and in 10 of 12 hearts at K⁺ = 2.0 mM. Prior to polymorphic ventricular tachycardia, dispersion of APD further increased (APD70max-min: 17 ± 3 ms; APD90max-min: 25 ± 3 ms; p < 0.05). Conclusions: This isolated beating mouse heart model can be used to study drug-induced action potential prolongation and repolarization-related ventricular arrhythmias provoked by bradycardia and hypokalemia. It may be suitable to identify a genetic predisposition to ventricular arrhythmias that may only become apparent under such proarrhythmic conditions.     

Arrhythmogenic and electrophysiological effects of alpha adrenoceptor stimulation during myocardial ischaemia and reperfusion

To examine possible arrhythmogenic effects of alpha adrenoceptor stimulation, we studied the effects of methoxamine 10(-6) M on arrhythmias and cellular electrophysiology during global myocardial ischaemia and reperfusion in isolated Langendorff perfused guinea-pig hearts. To avoid interference from release of endogenous catecholamines during ischaemia or reperfusion, experiments were performed using catecholamine depleted hearts (myocardial noradrenaline = 11% of control). Catecholamine depletion markedly reduced the incidence of VT and VF during ischaemia and reperfusion and perfusion with methoxamine significantly reversed this. This arrhythmogenic effect of methoxamine was only observed during ischaemia or reperfusion, was independent of beta adrenoceptor blockade and H2 receptor blockade but was abolished by alpha adrenoceptor blockade with phentolamine. Catecholamine depletion blunted the ischaemia induced fall in action potential amplitude and Vmax and prolonged action potential duration and refractory period. Perfusion with methoxamine either partially or completely reversed these effects. Thus, alpha adrenoceptor stimulation has little effect on normally perfused myocardium, but may induce VT or VF during ischaemia or reperfusion.     

Species plays an important role in drug-induced prolongation of action potential duration and early afterdepolarizations in isolated Purkinje fibers

INTRODUCTION: Although isolated Purkinje fibers (PFs) often are used to evaluate the electrophysiologic effects of new drugs in terms of prolongation of action potential duration (APD) and induction of early afterdepolarizations (EADs), species differences in this respect remain elusive. We evaluated potential species-specific differences in drug-induced prolongation of APD and EADs in isolated PF from various species. METHODS AND RESULTS: Using a microelectrode technique, PFs (n = 7 to 11 per species) were isolated from hearts of rabbits, guinea pigs, dogs, swine, goats, or sheep, superperfused in Tyrode's solution with dofetilide (1 x 10(-8) M) or quinidine (1 x 10(-5) M) for 25 minutes, and stimulated at 1 Hz for 20 minutes and at 0.2 Hz for another 5 minutes. Dofetilide increased APD at 90% repolarization (APD90) at 1 Hz by 83% (rabbit), 24% (guinea pig), 65% (dogs), 18% (swine), 61% (goat), and 30% (sheep), and prolonged APD90 at 0.2 Hz by 187% (rabbit), 31% (guinea pig), 154% (dog), 17% (swine), 61% (goat), and 8% (sheep). Similarly, quinidine changed APD90 by 93% (rabbit), 0% (guinea pig), 16% (dog), -3% (swine), 0% (goat), and -24% (sheep) at 1 Hz, and by 124% (rabbit), 15% (guinea pig), 53% (dog), 17% (swine), 11% (goat), and -39% (sheep) at 0.2 Hz in PF. During superfusion of dofetilide or quinidine, EADs occurred in most preparations in rabbit PFs at 0.2 Hz, but not in any of the PFs from other species at 0.2 Hz. CONCLUSION: Our study demonstrates that species plays an important role in the response of PF to drug-induced prolongation of APD and EADs. Rabbit PFs constitute the most sensitive model for detecting drug-induced, potential long APD and proarrhythmogenic effects in vitro.