Inhibition Of IK Research Articles

SYMPOSIUM: EVOLVING PICTURE OF CARDIAC ARRHYTHMIAS - ON BEHALF OF THE WORKING GROUP ON CARDIAC CELLULAR ELECTROPHYSIOLOGY

# 777 Cardiac inward rectifier potassium current IK1 is inhibited by acetaldehyde at clinically relevant concentrations: a role in arrhythmogenesis related to alcohol consumption? {#article-title-2} Purpose: Alcohol intoxication may induce electrocardiographic changes and arrhythmias. Modifications of cardiac inward rectifier potassium currents including IK1 are known to play an important role in the pathogenesis of various types of arrhythmias including those observed under alcohol intoxication. We have recently published a study describing a significant effect of ethanol at clinically relevant concentrations on the ventricular IK1. In the current study, we aimed to analyse changes of the ventricular IK1 in the presence of acetaldehyde, the primary metabolite of ethanol. Methods: Experiments were performed on enzymatically isolated rat ventricular myocytes by the whole cell patch clamp technique at 23 ± 1 °C. IK1 was analysed as the current sensitive to 100 μM Ba2+. Solutions were applied into the near surrounding of the measured cell with a rapid perfusion system. Results: Acetaldehyde (0.3–300 μM) induced a reversible inhibition of IK1 with the concentration causing 50% inhibition of 53.7 ± 7.7 μM at −100 mV. A significant inhibition was documented even at clinically relevant concentrations; namely 3 μM acetaldehyde reduced IK1 by 13.1 ± 3.0% of. Both development and wash-out of the acetaldehyde effect on IK1 showed a single exponential time course with average time constants of 24.5 ± 3.5 s and 41.5 ± 3.6 s at 3 μM, respectively. The inhibition was voltage-dependent between −120 and −80 mV; at −80 mV and more positive voltages, the inhibition was by ~10% lower than at −100 mV. Conclusions: We conclude that the observed changes of IK1 under clinically relevant concentrations of acetaldehyde might contribute to the alcohol-induced alterations of the cardiac electrophysiology, namely in people with a genetic defect of aldehyde dehydrogenase and, thus, higher plasma levels of acetaldehyde who are often native to Asia.

Synergistic modulation of KCNQ1/KCNE1 K+ channels (IKs) by phosphatidylinositol 4,5-bisphosphate (PIP2) and [ATP

Cardiac KCNQ1/KCNE1 channels (IKs) are dependent on the concentration of membrane phosphatidylinositol-4,5-bisphosphate (PIP2) and on cytosolic ATP by two distinct mechanisms. In this study we measured IKs and FRET between PH-PLCδ-based fluorescent PIP2 sensors in a stable KCNQ1/KCNE1 CHO cell line. Effects of activating either a muscarinic M3 receptor or the switchable phosphatase Ci-VSP on IKs were analyzed. Recovery of IKs from inhibition induced by muscarinic stimulation was incomplete despite full PIP2 resynthesis. Recovery of IKs was completely suppressed under ATP-free conditions, but partially restored by the ATP analog AMP-PCP, providing evidence that depletion of intracellular ATP inhibits IKs independent of PIP2-depletion.Simultaneous patch-clamp and FRET measurements in cells co-expressing Ci-VSP and the PIP2-FRET sensor revealed a component of IKs inhibition directly related to dynamic PIP2-depletion. A second component of inhibition was independent of acute changes in PIP2 and could be mimicked by ATP-free pipette solution, suggesting that it results from intracellular ATP-depletion. The reduction of intracellular ATP upon Ci-VSP activation appears to be independent of its activity as a phosphoinositide phosphatase. Our data demonstrate that ATP-depletion slowed IKs activation but had no short-term effect on PIP2 regeneration, suggesting that impaired PIP2-resynthesis cannot account for the rapid IKs inhibition by ATP-depletion. Furthermore, the second component of IKs inhibition by Ci-VSP was reduced by AMP-PCP in the pipette filling solution, indicating that direct binding of ATP to the KCNQ1/KCNE1 complex is required for voltage activation of IKs. We suggest that fluctuations of the cellular metabolic state regulate IKs in parallel with Gq-coupled PLC activation and PIP2-depletion.

Combined inhibition of key potassium currents has different effects on cardiac repolarization reserve and arrhythmia susceptibility in dogs and rabbits.

A reliable assessment of the pro-arrhythmic potential for drugs in the development phase remains elusive. Rabbits and dogs are commonly used to create models of pro-arrhythmia, but the differences between them with respect to repolarizing potassium currents are poorly understood. We investigated the incidence of drug-induced torsades de pointes (TdP) and measured conventional ECG parameters and the short-term variability of the QT interval (STVQT) following combined pharmacological inhibition of IK1+IKs and IK1+IKr in conscious dogs and anesthetized rabbits. A high incidence of TdP was observed following the combined inhibition of IK1+IKs in dogs (67% vs. 14% in rabbits). Rabbits exhibited higher TdP incidence after inhibition of IK1+IKr (72% vs. 14% in dogs). Increased TdP incidence was associated with significantly larger STVQT in both models. The relatively different roles of IK1 and IKs in dog and rabbit repolarization reserve should be taken into account when extrapolating the results from animal models of pro-arrhythmia to humans. A stronger repolarization reserve in dogs (likely due to stronger IK1 and IKs), and the more human-like susceptibility to arrhythmia of rabbits argues for the preferred use of rabbits in the evaluation of adverse pro-arrhythmic effects.

Nociceptin/orphanin FQ-induced inhibition of delayed rectifier potassium currents by calcium/calmodulin-dependent protein kinase type II.

The neuropeptide nociceptin/orphanin FQ (N/OFQ) has been shown to inhibit delayed rectifier potassium current (IK) in acutely dissociated rat parietal cortical neurons. However, the detailed mechanism of N/OFQ-induced inhibition on IK is not clear. This study is the first to explore an involvement of calcium/calmodulin (CaM)-dependent protein kinase type II (CaMKII) in mediating N/OFQ-induced responses. Utilizing pharmacological inhibitors of CaM and CaMKII, we have investigated the contribution of CaMKII in N/OFQ-induced effects using the whole-cell patch-clamp technique. In whole-cell voltage clamp, W-7 (100 μM), an antagonist of CaM, as well as KN-62, an inhibitor of CaMKII activity, attenuated the inhibitory effects of N/OFQ on IK. Activation and inactivation analysis indicated that the kinetics of IK were altered by N/OFQ, with decreased activation and promoted inactivation of IK. W-7 and KN-62 (10 μM) partly abolished the activation and inactivation curves shift of IK induced by N/OFQ. These findings show that CaMKII plays a critical role in N/OFQ-induced inhibition of IK in acutely dissociated rat parietal cortical neurons.

Differential effects of genetically-encoded Gβγ scavengers on receptor-activated and basal Kir3.1/Kir3.4 channel current in rat atrial myocytes

Opening of G-protein-activated inward-rectifying K+ (GIRK, Kir3) channels is regulated by interaction with βγ-subunits of Pertussis-toxin-sensitive G proteins upon activation of appropriate GPCRs. In atrial and neuronal cells agonist-independent activity (Ibasal) contributes to the background K+ conductance, important for stabilizing resting potential. Data obtained from the Kir3 signaling pathway reconstituted in Xenopus oocytes suggest that Ibasal requires free Gβγ. In cells with intrinsic expression of Kir3 channels this issue has been scarcely addressed experimentally. Two Gβγ-binding proteins (myristoylated phosducin — mPhos — and Gαi1) were expressed in atrial myocytes using adenoviral gene transfer, to interrupt Gβγ-signaling. Agonist-induced and basal currents were recorded using whole cell voltage-clamp. Expression of mPhos and Gαi1 reduced activation of Kir3 current via muscarinic M2 receptors (IK(ACh)). Inhibition of IK(ACh) by mPhos consisted of an irreversible component and an agonist-dependent reversible component. Reduction in density of IK(ACh) by overexpressed Gαi1, in contrast to mPhos, was paralleled by substantial slowing of activation, suggesting a reduction in density of functional M2 receptors, rather than Gβγ-scavenging as underlying mechanism. In line with this notion, current density and activation kinetics were rescued by fusing the αi1-subunit to an Adenosine A1 receptor. Neither mPhos nor Gαi1 had a significant effect on Ibasal, defined by the inhibitory peptide tertiapin-Q. These data demonstrate that basal Kir3 current in a native environment is unrelated to G-protein signaling or agonist-independent free Gβγ. Moreover, our results illustrate the importance of physiological expression levels of the signaling components in shaping key parameters of the response to an agonist.

Differential Regulation of Slow and Rapid Delayed Rectifier Potassium Currents by cGMP Dependent Nitric Oxide Signalling Pathways in Isolated Adult Guinea Pig Ventricular Myocytes

Nitric oxide (NO) signalling pathways are reported to regulate cardiac repolarisation. This was investigated using BAY 60-2770, which activates soluble guanylyl cyclase (sGC) in a NO and haem independent manner to generate cGMP. Action potentials (APs) and currents were recorded from isolated guinea pig ventricular myocytes at 37oC using the perforated patch clamp technique. APs were stimulated at 2 Hz. The slow and rapid delayed rectifier potassium currents, IKs and IKr were measured as tail currents elicited by a voltage step from +40 mV to −50 mV. The following experiments were carried out in the presence of 100 µM IBMX (3-Isobutyl-1-methylxanthine) a non-selective phosphodiesterase (PDE) inhibitor. 1 µM BAY 60-2770 lengthened APD90 by 15.7 ms (p<0.05, n=7), suggesting that sGC activation and an increase in cGMP inhibits repolarising currents. Further experiments showed that IKs was inhibited by BAY 60-2770. IBMX alone enhanced IKs by 77 ± 9 % (p<0.001, n=8) and subsequent addition of BAY 60-2770 reduced the IBMX dependent enhancement of IKs by 68 ± 2 % (p<0.001, n=8). This suggests that PDE inhibition permits IKs to be inhibited by cGMP dependent NO signalling pathways. The sGC mediated inhibition of IKs by BAY 60-2770 is unlikely to be due to phosphorylation by protein kinase G (PKG) because the inhibition remained in the presence of 100 nM KT-5823, a PKG inhibitor and because PKG activation with 100 µM 8-bromo-cGMP had no effect on IKs. IKr was not regulated by PDE inhibition, sGC activation or PKG activation. Overall, these findings suggest that cGMP dependent NO signalling pathways regulate IKs, but not IKr, via a PKG independent mechanism.

Ionic mechanisms limiting cardiac repolarization reserve in humans compared to dogs

The species-specific determinants of repolarization are poorly understood. This study compared the contribution of various currents to cardiac repolarization in canine and human ventricle. Conventional microelectrode, whole-cell patch-clamp, molecular biological and mathematical modelling techniques were used. Selective IKr block (50-100 nmol l(-1) dofetilide) lengthened AP duration at 90% of repolarization (APD90) >3-fold more in human than dog, suggesting smaller repolarization reserve in humans. Selective IK1 block (10 μmol l(-1) BaCl2) and IKs block (1 μmol l(-1) HMR-1556) increased APD90 more in canine than human right ventricular papillary muscle. Ion current measurements in isolated cardiomyocytes showed that IK1 and IKs densities were 3- and 4.5-fold larger in dogs than humans, respectively. IKr density and kinetics were similar in human versus dog. ICa and Ito were respectively ~30% larger and ~29% smaller in human, and Na(+)-Ca(2+) exchange current was comparable. Cardiac mRNA levels for the main IK1 ion channel subunit Kir2.1 and the IKs accessory subunit minK were significantly lower, but mRNA expression of ERG and KvLQT1 (IKr and IKs α-subunits) were not significantly different, in human versus dog. Immunostaining suggested lower Kir2.1 and minK, and higher KvLQT1 protein expression in human versus canine cardiomyocytes. IK1 and IKs inhibition increased the APD-prolonging effect of IKr block more in dog (by 56% and 49%, respectively) than human (34 and 16%), indicating that both currents contribute to increased repolarization reserve in the dog. A mathematical model incorporating observed human-canine ion current differences confirmed the role of IK1 and IKs in repolarization reserve differences. Thus, humans show greater repolarization-delaying effects of IKr block than dogs, because of lower repolarization reserve contributions from IK1 and IKs, emphasizing species-specific determinants of repolarization and the limitations of animal models for human disease.

Procainamide and lidocaine produce dissimilar changes in ventricular repolarization and arrhythmogenicity in guinea‐pig

Procainamide is class Ia Na(+) channel blocker that may prolong ventricular repolarization secondary to inhibition of IK r , the rapid component of the delayed rectifier K(+) current. In contrast to selective IN a blockers such as lidocaine, procainamide was shown to produce arrhythmogenic effects in the clinical setting. This study examined whether pro-arrhythmic responses to procainamide may be accounted for by drug-induced repolarization abnormalities including impaired electrical restitution kinetics, spatial gradients in action potential duration (APD), and activation-to-repolarization coupling. In perfused guinea-pig hearts, procainamide was found to prolong the QT interval on ECG and left ventricular (LV) epicardial monophasic APD, increased the maximum slope of electrical restitution, enhanced transepicardial APD variability, and eliminated the inverse correlation between the local APD and activation time values determined at distinct epicardial recording sites prior to drug infusion. In contrast, lidocaine had no effect on electrical restitution, the degree of transepicardial repolarization heterogeneities, and activation-to-repolarization coupling. Spontaneous episodes of monomorphic ventricular tachycardia were observed in 57% of procainamide-treated heart preparations. No arrhythmia was induced by lidocaine. In summary, this study suggests that abnormal changes in repolarization may contribute to pro-arrhythmic effects of procainamide.

Propafenone blocks human cardiac Kir2.x channels by decreasing the negative electrostatic charge in the cytoplasmic pore

Human cardiac inward rectifier current (IK1) is generated by Kir2.x channels. Inhibition of IK1 could offer a useful antiarrhythmic strategy against fibrillatory arrhythmias. Therefore, elucidation of Kir2.x channels pharmacology, which still remains elusive, is mandatory. We characterized the electrophysiological and molecular basis of the inhibition produced by the antiarrhythmic propafenone of the current generated by Kir2.x channels (IKir2.x) and the IK1 recorded in human atrial myocytes. Wild type and mutated human Kir2.x channels were transiently transfected in CHO and HEK-293 cells. Macroscopic and single-channel currents were recorded using the patch-clamp technique. At concentrations >1μM propafenone inhibited IKir2.x the order of potency being Kir2.3∼IK1>Kir2.2>Kir2.1 channels. Blockade was irrespective of the extracellular K+ concentration whereas markedly increased when the intracellular K+ concentration was decreased. Propafenone decreased inward rectification since at potentials positive to the K+ equilibrium potential propafenone-induced block decreased in a voltage-dependent manner. Importantly, propafenone favored the occurrence of subconductance levels in Kir2.x channels and decreased phosphatidylinositol 4,5-bisphosphate (PIP2)-channel affinity. Blind docking and site-directed mutagenesis experiments demonstrated that propafenone bound Kir2.x channels at the cytoplasmic domain, close to, but not in the pore itself, the binding site involving two conserved Arg residues (residues 228 and 260 in Kir2.1). Our results suggested that propafenone incorporated into the cytoplasmic domain of the channel in such a way that it decreased the net negative charge sensed by K+ ions and polyamines which, in turn, promotes the appearance of subconductance levels and the decrease of PIP2 affinity of the channels.

Inhibitory Effects of Glycyrrhetinic Acid on the Delayed Rectifier Potassium Current in Guinea Pig Ventricular Myocytes and HERG Channel

Background. Licorice has long been used to treat many ailments including cardiovascular disorders in China. Recent studies have shown that the cardiac actions of licorice can be attributed to its active component, glycyrrhetinic acid (GA). However, the mechanism of action remains poorly understood. Aim. The effects of GA on the delayed rectifier potassium current (I K), the rapidly activating (I Kr) and slowly activating (I Ks) components of I K, and the HERG K+ channel expressed in HEK-293 cells were investigated. Materials and Methods. Single ventricular myocytes were isolated from guinea pig myocardium using enzymolysis. The wild type HERG gene was stably expressed in HEK293 cells. Whole-cell patch clamping was used to record I K (I Kr, I Ks) and the HERG K+ current. Results. GA (1, 5, and 10 μM) inhibited I K (I Kr, I Ks) and the HERG K+ current in a concentration-dependent manner. Conclusion. GA significantly inhibited the potassium currents in a dose- and voltage-dependent manner, suggesting that it exerts its antiarrhythmic action through the prolongation of APD and ERP owing to the inhibition of I K (I Kr, I Ks) and HERG K+ channel.

Sex differences in repolarization and slow delayed rectifier potassium current and their regulation by sympathetic stimulation in rabbits

Slow delayed rectifier potassium current (IKs) is important in action potential (AP) repolarization and repolarization reserve. We tested the hypothesis that there are sex-specific differences in IKs, AP, and their regulation by β-adrenergic receptors (β-AR's) using whole-cell patch-clamp. AP duration (APD90) was significantly longer in control female (F) than in control male (M) myocytes. Isoproterenol (ISO, 500 nM) shortened APD90 comparably in M and F, and was largely reversed by β1-AR blocker CGP 20712A (CGP, 300 nM). Inhibition of IKs with chromanol 293B (10 μM) resulted in less APD prolongation in F at baseline (3.0 vs 8.9 %, p < 0.05 vs M) and even in the presence of ISO (5.4 vs 20.9 %, p < 0.05). This suggests that much of the ISO-induced APD abbreviation in F is independent of IKs. In F, baseline IKs was 42 % less and was more weakly activated by ISO (19 vs 68 % in M, p < 0.01). ISO enhancement of IKs was comparably attenuated by CGP in M and F. After ovariectomy, IKs in F had greater enhancement by ISO (72 %), now comparable to control M. After orchiectomy, IKs in M was only slightly enhanced by ISO (23 %), comparable to control F. Pretreatment with thapsigargin (to block SR Ca release) had bigger impact on ISO-induced APD shortening in F than that in M (p < 0.01). In conclusion, we found that there are sex differences in IKs, AP, and their regulation by β-AR's that are modulated by sex hormones, suggesting the potential for sex-specific antiarrhythmic therapy.

Effects of hERG openers and the influence of IKS inhibition in an isolated rabbit heart model

Effects of hERG openers and the influence of IKS inhibition in an isolated rabbit heart model

Atrial-selective Prolongation of Refractory Period With AVE0118 is Due Principally to Inhibition of Sodium Channel Activity

The action of AVE0118 to prolong effective refractory period (ERP) in atria but not in ventricles is thought to be due to its inhibition of IKur. However, in nonremodeled atria, AVE0118 prolongs ERP but not action potential duration (APD70-90), which can be explained with the inhibition of sodium but not potassium channel current. ERP, APD, and the maximum rate of increase of the AP upstroke (Vmax) were measured in the canine-isolated coronary-perfused right atrial and in superfused ventricular tissue preparations. Whole-cell patch-clamp techniques were used to measure sodium channel current in HEK293 cells stably expressing SCN5A. AVE0118 (5-10 μM) prolonged ERP (P < 0.001) but not APD70 and decreased Vmax (by 15%, 10 μM, P < 0.05; n = 10 for each). Ventricular ERP, APD90, and Vmax were not changed significantly by 10 μM AVE0118 (all P = ns; n = 7). AVE0118 effectively suppressed acetylcholine-mediated persistent atrial fibrillation. AVE0118 (10 μM) reduced peak current amplitude of SCN5A-WT current by 36.5% ± 6.6% (P < 0.01; n = 7) and shifted half-inactivation voltage (V0.5) of the steady-state inactivation curve from -89.9 ± 0.5 to -96.0 ± 0.9 mV (P < 0.01; n = 7). Our data suggest that AVE0118-induced prolongation of atrial, but not ventricular ERP, is due largely to atrial-selective depression of sodium channel current, which likely contributes to the effectiveness of AVE0118 to suppress atrial fibrillation.

Effects of ketamine and its metabolites on ion currents in differentiated hippocampal H19-7 neuronal cells and in HEK293T cells transfected with α-hslo subunit

Ketamine (KT), a dissociative anesthetic, is known to induce schizophrenia-like psychosis. The percentage of KT abuse has recently grown fast despite KT being a controlled drug. The mechanism of KT actions is related to the inhibition of NMDA receptors. Whether KT produces other effects on ion currents in hippocampal neurons remains unclear. In this study, we attempted to evaluate the possible effects of KT and other related compounds on ion currents in hippocampal neuron-derived H19-7 cells. This drug exerted an inhibitory effect on Ca2+-activated K+ current (IK(Ca)) in these cells with an IC50 value of 274μM. Pimaric acid (30μM) or abietic acid (30μM), known to stimulate large-conductance Ca2+-activated K+ channels, reversed KT-induced inhibition of IK(Ca). In HEK293T cells expressing α-human slowpoke, KT-induced inhibition of IK(Ca) still existed. Dehydronorketamine (300μM) had little or no effect on the IK(Ca) amplitude, while norketamine (300μM) slightly but significantly suppressed it. In inside–out configuration, KT applied to the intracellular face of the membrane did not alter single-channel conductance of large-conductance Ca2+-activated K+ (BKCa) channels; however, it did significantly reduce the probability of channel openings. Addition of KT was effective in depressing the peak amplitude of voltage-gated Na+ current. Moreover, the presence of KT was noted to enhance the amplitude of membrane electroporation-induced inward currents (IMEP) in differentiated H19-7 cells. KT-stimulated IMEP was reversed by further application of LaCl3 (100μM), but not by NMDA (30μM). The modulations by this compound of ion channels may contribute to the underlying mechanisms through which KT and its metabolites influence the electrical behavior of hippocampal neurons if similar findings occur in vivo.

Evidence for aconitine-induced inhibition of delayed rectifier K+ current in Jurkat T-lymphocytes

Aconitine (ACO) is a highly toxic diterpenoid alkaloid and known to exert the immunomodulatory action. However, whether it has any effects on ion currents in immune cells remains unknown. The effects of ACO and other related compounds on ion currents in Jurkat T-lymphocytes were investigated in this study. ACO suppressed the amplitude of delayed-rectifier K+ current (IK(DR)) in a time- and concentration-dependent manner. Margatoxin (100nM), a specific blocker of KV1.3-encoded current, decreased the IK(DR) amplitude in these cells and the ACO-induced inhibition of IK(DR) was not reversed by 1-ethyl-2-benzimidazolinone (30μM) or nicotine (10μM). The IC50 value for ACO-mediated inhibition of IK(DR) was 5.6μM. ACO accelerated the inactivation of IK(DR) with no change in the activation rate of this current. Increasing the ACO concentration not only reduced the IK(DR) amplitude, but also accelerated the inactivation time course of the current. With the aid of minimal binding scheme, the inhibitory action of ACO on IK(DR) was estimated with a dissociation constant of 6.8μM. ACO also shifted the inactivation curve of IK(DR) to a hyperpolarized potential with no change in the slope factor. Cumulative inactivation for IK(DR) was enhanced in the presence of ACO. In Jurkat cells incubated with amiloride (30μM), the ACO-induced inhibition of IK(DR) remained unaltered. In RAW 264.7 murine macrophages, ACO did not modify the kinetics of IK(DR), although it suppressed IK(DR) amplitude. Taken together, these effects can significantly contribute to its action on functional activity of immune cells if similar results are found in vivo.

Propofol inhibited the delayed rectifier potassium current (Ik) via activation of protein kinase C epsilon in rat parietal cortical neurons

Propofol has been shown to exert neuroprotective effects. Delayed rectifier potassium current (IK) was reported to be closely related to neuronal damage. This study was designed to test the effects of propofol on IK in rat parietal cortical neurons and the involvement of PKC in this activity. Whole-cell patch-clamp recordings were performed in rat parietal cortical neurons. The amplitudes of IK were recorded before and after the addition of different concentrations of propofol. Propofol concentration-dependently inhibited IK with an IC50 value of 36.3±2.7μM. Moreover, propofol caused a downward shift of the I–V curve of IK in a concentration dependent manner. The kinetics of IK was altered by propofol, with decreased activation and delayed recovery of IK. Pretreatment with calphostin-C (a non-selective inhibitor of PKC) or PKC epsilon translocation inhibitor peptide (PKC epsilon inhibitor) abrogated the inhibition of IK by propofol. In conclusion, propofol inhibited IK via the activation of PKC epsilon in rat cerebral parietal cortical neurons.

Antiarrhythmic effects and ionic mechanisms of oxymatrine from Sophora flavescens

Accumulating evidence indicates that oxymatrine may exert protective effects on the cardiovascular system. This study was designed to evaluate the antiarrhythmic effects as well as the electrophysiological properties of oxymatrine. The antiarrhythmic activity of oxymatrine was observed in a rat model of arrhythmia induced by coronary ligation. Action potential duration (APD), L-type calcium current (I(Ca-L) ), transient outward potassium current (I(to) ) and inward rectifier potassium current (I(K1)) in rat ventricular myocytes were recorded by utilizing the whole cell patch-clamp technique. The results showed that administration of oxymatrine significantly delayed the onset of ventricular arrhythmia, decreased the duration of ventricular arrhythmia and reduced the arrhythmia score of arrhythmic rats. The beneficial effects of oxymatrine may be related to the shortening of APD through reduction of I(Ca-L) , enhancement of I(to) and inhibition of I(K1).

2-Aminoethoxydiphenyl borate blocks electrical coupling and inhibits voltage-gated K+ channels in guinea pig arteriole cells

2-Aminoethoxydiphenyl borate (2-APB) analogs are potentially better vascular gap junction blockers than others widely used, but they remain to be characterized. Using whole cell and intracellular recording techniques, we studied the actions of 2-APB and its potent analog diphenylborinic anhydride (DPBA) on vascular smooth muscle cells (VSMCs) and endothelial cells in situ of or dissociated from arteriolar segments of the cochlear spiral modiolar artery, brain artery, and mesenteric artery. We found that both 2-APB and DPBA reversibly suppressed the input conductance (G(input)) of in situ VSMCs (IC(50) ≈ 4-8 μM). Complete electrical isolation of the recorded VSMC was achieved at 100 μM. A similar gap junction blockade was observed in endothelial cell tubules of the spiral modiolar artery. Similar to the action of 18β-glycyrrhetinic acid (18β-GA), 2-APB and DPBA depolarized VSMCs. In dissociated VSMCs, 2-APB and DPBA inhibited the delayed rectifier K(+) current (I(K)) with an IC(50) of ∼120 μM in the three vessels but with no significant effect on G(input) or the current-voltage relation between -140 and -40 mV. 2-APB inhibition of I(K) was more pronounced at potentials of ≤20 mV than at +40 mV and more marked on the fast component than on the slow component, which was mimicked by 4-aminopyridine but not by tetraethylammonium, nitrendipine, or charybdotoxin. In contrast, 18β-GA caused a linear inhibition of I(K) between 0 to +40 mV, which was similar to the action of tetraethylammonium or charybdotoxin. Finally, the 2-APB-induced inhibition of electrical coupling and I(K) was not affected by the inositol 1,4,5-trisphosphate receptor antagonist xestospongin C. We conclude that 2-APB analogs are a class of potent and reversible vascular gap junction blockers with a weak side effect of voltage-gated K(+) channel inhibition. They could be gap junction blockers superior to 18β-GA only when Ca(2+)-actived K(+) channel inhibition by the latter is a concern but inositol 1,4,5-trisphosphate receptor and voltage-gated K(+) channel inhibitions are not.

Synthesis and cytotoxicity evaluation of some 8-hydroxyquinoline derivatives.

Interest in Mannich bases of 8-hydroxyquinoline stems from reports of their high potency against human cancer cells. In the search for potential anticancer drug candidates, Mannich bases of 8-hydroxyquinoline (7-pyrrolidinomethyl-8-hydroxyquinoline, 7-morpholinomethyl-8-hydroxyquinoline, 7-piperidinomethyl-8-hydroxyquinoline and 7-diethylaminomethyl-8-hydroxyquinoline) were synthesised by reaction with various secondary amines and formaldehyde. They were prepared as hydrochlorides. The cytotoxic activity of 7-pyrrolidinomethyl-8-hydroxyquinoline, 7-morpholinomethyl-8-hydroxyquinoline and 7-diethylaminomethyl-8-hydroxyquinoline compounds in the National Cancer Institute in-vitro cancer cell line panel was determined. It was found that they exhibited substantial cytotoxic activity against leukaemia. The log concentration of 7-pyrrolidinomethyl-8-hydroxyquinoline, 7-morpholinomethyl-8-hydroxyquinoline and 7-diethylaminomethyl-8-hydroxyquinoline that inhibited 50% of 60 cell lines' growth were -4.81 M, -5.09 M and -5.35 M, respectively. Compound 7-pyrrolidinomethyl-8-hydroxyquinoline was selected for further in-vivo testing. The electrophysiological effect of 7-pyrrolidinomethyl-8-hydroxyquinoline also was tested in human myeloma cells (RPMI 8226). The outward current was voltage dependent, activating at -40 mV and believed to be the voltage-activated K+ current I(K(V)). 7-Pyrrolidinomethyl-8-hydroxyquinoline (1-30 microM) caused the inhibition of I(K(V)) in a concentration-dependent manner. The IC50 value of 7-pyrrolidinomethyl-8-hydroxyquinoline-induced inhibition of I(K(V)) is 23 microM. The GI50 value of 7-pyrrolidinomethyl-8-hydroxyquinoline-induced inhibition of cell growth is 14 microM. The results suggest that at least part of the cytotoxicity effect of 7-pyrrolidinomethyl-8-hydroxyquinoline on myeloma cells could be related to blockade of voltage-activated K+ channels.

Inhibition of IK,ACh current may contribute to clinical efficacy of class I and class III antiarrhythmic drugs in patients with atrial fibrillation

Inward rectifier potassium currents I(K1) and acetylcholine activated I(K,ACh) are implicated in atrial fibrillation (AF) pathophysiology. In chronic AF (cAF), I(K,ACh) develops a receptor-independent, constitutively active component that together with increased I(K1) is considered to support maintenance of AF. Here, we tested whether class I (propafenone, flecainide) and class III (dofetilide, AVE0118) antiarrhythmic drugs inhibit atrial I(K1) and I(K,ACh) in patients with and without cAF. I(K1) and I(K,ACh) were measured with voltage clamp technique in atrial myocytes from 58 sinus rhythm (SR) and 35 cAF patients. The M-receptor agonist carbachol (CCh; 2 microM) was employed to activate I(K,ACh). In SR, basal current was not affected by either drug indicating no effect of these compounds on I(K1). In contrast, all tested drugs inhibited CCh-activated I(K,ACh) in a concentration-dependent manner. In cAF, basal current was confirmed to be larger than in SR (at -80 mV, -15.2 +/- 1.2 pA/pF, n = 88/35 vs. -6.5 +/- 0.4 pA/pF, n = 194/58 [myocytes/patients]; P < 0.05), whereas CCh-activated I(K,ACh) was smaller (-4.1 +/- 0.5 pA/pF vs. -9.5 +/- 0.6 pA/pF; P < 0.05). In cAF, receptor-independent constitutive I(K,ACh) contributes to increased basal current, which was reduced by flecainide and AVE0118 only. This may be due to inhibition of constitutively active I(K,ACh) channels. In cAF, all tested drugs reduced CCh-activated I(K,ACh). We conclude that in cAF, flecainide and AVE0118 reduce receptor-independent, constitutively active I(K,ACh), suggesting that they may block I(K,ACh) channels, whereas propafenone and dofetilide likely inhibit M-receptors. The efficacy of flecainide to terminate AF may in part result from blockade of I(K,ACh).