Effects of β-adrenoceptor stimulation on delayed rectifier K(+) currents in canine ventricular cardiomyocytes

BACKGROUND AND PURPOSE

While the slow delayed rectifier K⁺ current (I_Ks) is known to be enhanced by the stimulation of β-adrenoceptors in several mammalian species, phosphorylation-dependent regulation of the rapid delayed rectifier K⁺ current (I_Kr) is controversial.

EXPERIMENTAL APPROACH

In the present study, therefore, the effect of isoprenaline (ISO), activators and inhibitors of the protein kinase A (PKA) pathway on I_Kr and I_Ks was studied in canine ventricular myocytes using the whole cell patch clamp technique.

KEY RESULTS

I_Kr was significantly increased (by 30–50%) following superfusion with ISO, forskolin or intracellular application of PKA activator cAMP analogues (cAMP, 8-Br-cAMP, 6-Bnz-cAMP). Inhibition of PKA by Rp-8-Br-cAMP had no effect on baseline I_Kr. The stimulating effect of ISO on I_Kr was completely inhibited by selective β₁-adrenoceptor antagonists (metoprolol and CGP-20712A), by the PKA inhibitor Rp-8-Br-cAMP and by the PKA activator cAMP analogues, but not by the EPAC activator 8-pCPT-2''-O-Me-cAMP. In comparison, I_Ks was increased threefold by the activation of PKA (by ISO or 8-Br-cAMP), and strongly reduced by the PKA inhibitor Rp-8-Br-cAMP. The ISO-induced enhancement of I_Ks was decreased by Rp-8-Br-cAMP and completely inhibited by 8-Br-cAMP.

CONCLUSIONS AND IMPLICATIONS

The results indicate that the stimulation of β₁-adrenoceptors increases I_Kr, similar to I_Ks, via the activation of PKA in canine ventricular cells.     

Inositol trisphosphate-dependent Ca2+ stores and mitochondria modulate slow wave activity arising from the smooth muscle cells of the guinea pig prostate gland

Background and purpose

Changes in smooth muscle tone of the prostate gland are involved in aetiology of symptomatic prostatic hyperplasia, however the control mechanisms of prostatic smooth muscle are not well understood. Here, we have examined the role of internal Ca²⁺ compartments in regulating slow wave activity in the guinea pig prostate.

Experimental approach

Standard intracellular membrane potential recording techniques were used.

Key results

The majority (89%) of impaled cells displayed ‘slow wave’ activity. Cyclopiazonic acid (10 µmol·L⁻¹) transiently depolarized (3–9 mV) the membrane potential of the prostatic stroma and transiently increased slow wave frequency. Thereafter, slow wave frequency slowly decreased over 20–30 min. Ryanodine transiently increased slow wave frequency, although after 30 min exposure slow wave frequency and time course returned to near control values. Caffeine (1 mmol·L⁻¹) reduced slow wave frequency, accompanied by membrane depolarization of about 8 mV. Blockade of inositol trisphosphate receptor (IP₃R)-mediated Ca²⁺ release with 2-aminoethoxy-diphenylborate (60 µmol·L⁻¹) or Xestospongin C (3 µmol·L⁻¹) or inhibiting phospholipase C and IP₃ formation using {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (5 µmol·L⁻¹) or neomycin (1 and 4 mmol·L⁻¹) reduced slow wave frequency, amplitude and duration. The mitochondrial uncouplers, p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (1–10 µmol·L⁻¹), carbonyl cyanide m-chlorophenylhydrazone (1–3 µmol·L⁻¹) or rotenone (10 µmol·L⁻¹), depolarized the membrane (8–10 mV) before abolishing electrical activity.

Conclusion and implications

These results suggest that slow wave activity was dependent on the cyclical release of Ca²⁺ from IP₃-controlled internal stores and mitochondria. This implies that intracellular compartments were essential in the initiation and/or maintenance of the regenerative contractile activity in the guinea pig prostate gland.     

Slow delayed rectifier K+ current block by HMR 1556 increases dispersion of repolarization and promotes Torsades de Pointes in rabbit ventricles

Background and purpose:

The slow delayed rectifier K⁺ current (I_Ks) contributes to ventricular repolarization when the action potential (AP) is prolonged. I_Ks block during drug-induced AP prolongation may promote Torsades de Pointes (TdP), but whether this is due to additional AP prolongation is uncertain.

Experimental approach:

In bradycardic perfused rabbit ventricles, the incidence of spontaneous TdP, monophasic AP duration at 90% repolarization (MAPD₉₀) and ECG interval between the peak and the end of T wave (T_peak−end) (index of dispersion of repolarization) were measured after the administration of veratridine (125 nM, slows Na⁺ channel inactivation), dofetilide (7.5 or 10 nM, a rapid delayed rectifier blocker) and HMR 1556 (HMR, 100 nM, an I_Ks blocker), alone or in combinations (n=6 each).

Key results:

HMR did not prolong MAPD₉₀, whereas veratridine or 7.5 nM dofetilide prolonged MAPD₉₀ (P<0.01) without inducing TdP. Veratridine+7.5 nM dofetilide additively prolonged MAPD₉₀ (P<0.05), induced 4±6 TdP per heart and prolonged T_peak−end by 12±10 ms. Subsequent addition of HMR did not further prolonged MAPD₉₀, but increased the number of TdP to 22±18 per heart and increased T_peak−end by 39±21 ms (P<0.05). Increasing dofetilide concentration from 7.5 to 10 nM (added to veratridine) produced a longer MAPD₉₀, but fewer TdP (5±5 per heart) and less T_peak−end prolongation (17±8 ms) compared to the veratridine+7.5 nM dofetilide+HMR group (P<0.05).

Conclusions and implications:

Adding I_Ks block markedly increases TdP incidence in hearts predisposed to TdP development by increasing the dispersion of repolarization, but without additional AP prolongation.     

High potency inhibition of hERG potassium channels by the sodium-calcium exchange inhibitor KB-R7943

BACKGROUND AND PURPOSE

KB-R7943 is an isothiourea derivative that is used widely as a pharmacological inhibitor of sodium–calcium exchange (NCX) in experiments on cardiac and other tissue types. This study investigated KB-R7943 inhibition of hERG (human ether-à-go-go-related gene) K⁺ channels that underpin the cardiac rapid delayed rectifier potassium current, I_Kr.

EXPERIMENTAL APPROACH

Whole-cell patch-clamp measurements were made of hERG current (I_hERG) carried by wild-type or mutant hERG channels and of native rabbit ventricular I_Kr. Docking simulations utilized a hERG homology model built on a MthK-based template.

KEY RESULTS

KB-R7943 inhibited both I_hERG and native I_Kr rapidly on membrane depolarization with IC₅₀ values of ∼89 and ∼120 nM, respectively, for current tails at −40 mV following depolarizing voltage commands to +20 mV. Marked I_hERG inhibition also occurred under ventricular action potential voltage clamp. I_hERG inhibition by KB-R7943 exhibited both time- and voltage-dependence but showed no preference for inactivated over activated channels. Results of alanine mutagenesis and docking simulations indicate that KB-R7943 can bind to a pocket formed of the side chains of aromatic residues Y652 and F656, with the compound''s nitrobenzyl group orientated towards the cytoplasmic side of the channel pore. The structurally related NCX inhibitor SN-6 also inhibited I_hERG, but with a markedly reduced potency.

CONCLUSIONS AND IMPLICATIONS

KB-R7943 inhibits I_hERG/I_Kr with a potency that exceeds that reported previously for acute cardiac NCX inhibition. Our results also support the feasibility of benzyloxyphenyl-containing NCX inhibitors with reduced potential, in comparison with KB-R7943, to inhibit hERG.     

Piceatannol, a derivative of resveratrol, moderately slows INa inactivation and exerts antiarrhythmic action in ischaemia-reperfused rat hearts

Background and purpose:

Piceatannol is more potent than resveratrol in free radical scavenging in association with antiarrhythmic and cardioprotective activities in ischaemic-reperfused rat hearts. The present study aimed to investigate the antiarrhythmic efficacy and the underlying ionic mechanisms of piceatannol in rat hearts.

Experimental approach:

Action potentials and membrane currents were recorded by the whole-cell patch clamp techniques. Fluo-3 fluorimetry was used to measure cellular Ca²⁺ transients. Antiarrhythmic activity was examined from isolated Langendorff-perfused rat hearts.

Key results:

In rat ventricular cells, piceatannol (3–30 µmol·L⁻¹) prolonged the action potential durations (APDs) and decreased the maximal rate of upstroke (V_max) without altering Ca²⁺ transients. Piceatannol decreased peak I_Na and slowed I_Na inactivation, rather than induced a persistent non-inactivating current, which could be reverted by lidocaine. Resveratrol (100 µmol·L⁻¹) decreased peak I_Na without slowing I_Na inactivation. The inhibition of peak I_Na or V_max was associated with a negative shift of the voltage-dependent steady-state I_Na inactivation curve without altering the activation threshold. At the concentrations more than 30 µmol·L⁻¹, piceatannol could inhibit I_Ca,L, I_to, I_Kr, Ca²⁺ transients and Na⁺-Ca²⁺ exchange except I_K1. Piceatannol (1–10 µmol·L⁻¹) exerted antiarrhythmic activity in isolated rat hearts subjected to ischaemia-reperfusion injury.

Conclusions and implications:

The additional hydroxyl group on resveratrol makes piceatannol possessing more potent in I_Na inhibition and uniquely slowing I_Na inactivation, which may contribute to its antiarrhythmic actions at low concentrations less than 10 µmol·L⁻¹.     

Novel blockers of hyperpolarization-activated current with isoform selectivity in recombinant cells and native tissue

BACKGROUND AND PURPOSE

Selective hyperpolarization activated, cyclic nucleotide-gated channel (HCN) blockers represent an important therapeutic goal due to the wide distribution and multiple functions of these proteins, representing the molecular correlate of f- and h-current (I_f or I_h). Recently, new compounds able to block differentially the homomeric HCN isoforms expressed in HEK293 have been synthesized. In the present work, the electrophysiological and pharmacological properties of these new HCN blockers were characterized and their activities evaluated on native channels.

EXPERIMENTAL APPROACH

HEK293 cells expressing mHCN1, mHCN2 and hHCN4 isoforms were used to verify channel blockade. Selected compounds were tested on native guinea pig sinoatrial node cells and neurons from mouse dorsal root ganglion (DRG) by patch-clamp recordings and on dog Purkinje fibres by intracellular recordings.

KEY RESULTS

In HEK293 cells, EC18 was found to be significantly selective for HCN4 and MEL57A for HCN1 at physiological membrane potential. When tested on guinea pig sinoatrial node cells, EC18 (10 µM) maintained its activity, reducing I_f by 67% at −120 mV, while MEL57A (3 µM) reduced I_f by 18%. In contrast, in mouse DRG neurons, only MEL57A (30 and 100 µM) significantly reduced I_h by 60% at −80 mV. In dog cardiac Purkinje fibres, EC18, but not MEL57A, reduced the amplitude and slowed the slope of the spontaneous diastolic depolarization.

CONCLUSIONS

Our results have identified novel and highly selective HCN isoform blockers, EC18 and MEL57A; the selectivity found in recombinant system was maintained in various tissues expressing different HCN isoforms.     

Blockade of I(Ca) suppresses early afterdepolarizations and reduces transmural dispersion of repolarization in a whole heart model of chronic heart failure

BACKGROUND AND PURPOSE

Chronic heart failure (CHF) is associated with action potential prolongation and Ca²⁺ overload, increasing risk of ventricular tachyarrhythmias (VT). We therefore investigated whether I_Ca blockade was anti-arrhythmic in an intact perfused heart model of CHF.

EXPERIMENTAL APPROACH

CHF was induced in rabbits after 4 weeks of rapid ventricular pacing. Hearts from CHF and sham-operated rabbits were isolated and perfused (Langendorff preparation), with ablation of the AV node. VT was induced by erythromycin and low [K⁺] (1.5mM). Electrophysiology of cardiac myocytes, with block of cation currents, was simulated by a mathematical model.

KEY RESULTS

Repolarization was prolonged in CHF hearts compared with sham-operated hearts. Action potential duration (APD) and overall dispersion of repolarization were further increased by erythromycin (300 µM) to block I_Kr in CHF hearts. After lowering [K⁺] to 1.5mM, CHF and sham hearts showed spontaneous episodes of polymorphic non-sustained VT. Additional infusion of verapamil (0.75 µM) suppressed early afterdepolarizations (EAD) and VT in 75% of sham and CHF hearts. Verapamil shortened APD and dispersion of repolarization, mainly by reducing transmural dispersion of repolarization via shortening of endocardial action potentials. Mathematical simulations showed that EADs were more effectively reduced by verapamil assuming a state-dependent block than a simple block of I_Ca.

CONCLUSIONS AND IMPLICATIONS

Blockade of I_Ca was highly effective in suppressing VT via reduction of transmural dispersion of repolarization and suppression of EAD. Such blockade might represent a novel therapeutic option to reduce risk of VT in structurally normal hearts and also in heart failure.

LINKED ARTICLE

This article is commented on by Stams et al., pp. 554–556 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01818.x     

Spontaneous release of acetylcholine from autonomic nerves in the bladder

Background and purpose:

Bladder contractility is regulated by intrinsic myogenic mechanisms interacting with autonomic nerves. In this study, we have investigated the physiological role of spontaneous release of acetylcholine in guinea pig and rat bladders.

Experimental approach:

Conventional isotonic or pressure transducers were used to record contractile activity of guinea pig and rat bladders.

Key results:

Hyoscine (3 µmol·L⁻¹), but not tetrodotoxin (TTX, 1 µmol·L⁻¹), reduced basal tension, distension-evoked contractile activity and physostigmine (1 µmol·L⁻¹)-evoked contractions of the whole guinea pig bladder and muscle strips in vitro. ω-Conotoxin GVIA (0.3 µmol·L⁻¹) did not affect physostigmine-induced contractions when given either alone or in combination with ω-agatoxin IVA (0.1 µmol·L⁻¹) and SNX 482 (0.3 µmol·L⁻¹). After 5 days in organotypic culture, when extrinsic nerves had significantly degenerated, the ability of physostigmine to induce contractions was reduced in the dorso-medial strips, but not in lateral strips (which have around 15 times more intramural neurones). Most muscle strips from adult rats lacked intramural neurones. After 5 days in culture, physostigmine-induced or electrical field stimulation-induced contractions of the rat bladder strips were greatly reduced. In anaesthetized rats, topical application of physostigmine (5–500 nmol) on the bladder produced a TTX-resistant tonic contraction that was abolished by atropine (4.4 µmol·kg⁻¹ i.v.).

Conclusions and implications:

The data indicate that there is spontaneous TTX-resistant release of acetylcholine from autonomic cholinergic extrinsic and intrinsic nerves, which significantly affects bladder contractility. This release is resistant to blockade of N, P/Q and R type Ca²⁺ channels.British Journal of Pharmacology (2009) 157, 607–619; doi:10.1111/j.1476-5381.2009.00166.x; published online 3 April 2009     

Reduced signal transduction by 5-HT4 receptors after long-term venlafaxine treatment in rats

BACKGROUND AND PURPOSE

The 5-HT₄ receptor may be a target for antidepressant drugs. Here we have examined the effects of the dual antidepressant, venlafaxine, on 5-HT₄ receptor-mediated signalling events.

EXPERIMENTAL APPROACH

The effects of 21 days treatment (p.o.) with high (40 mg·kg⁻¹) and low (10 mg·kg⁻¹) doses of venlafaxine, were evaluated at different levels of 5-HT₄ receptor-mediated neurotransmission by using in situ hybridization, receptor autoradiography, adenylate cyclase assays and electrophysiological recordings in rat brain. The selective noradrenaline reuptake inhibitor, reboxetine (10 mg·kg⁻¹, 21 days) was also evaluated on 5-HT₄ receptor density.

KEY RESULTS

Treatment with a high dose (40 mg·kg⁻¹) of venlafaxine did not alter 5-HT₄ mRNA expression, but decreased the density of 5-HT₄ receptors in caudate-putamen (% reduction = 26 ± 6), hippocampus (% reduction = 39 ± 7 and 39 ± 8 for CA1 and CA3 respectively) and substantia nigra (% reduction = 49 ± 5). Zacopride-stimulated adenylate cyclase activation was unaltered following low-dose treatment (10 mg·kg⁻¹) while it was attenuated in rats treated with 40 mg·kg⁻¹ of venlafaxine (% reduction = 51 ± 2). Furthermore, the amplitude of population spike in pyramidal cells of CA1 of hippocampus induced by zacopride was significantly attenuated in rats receiving either dose of venlafaxine. Chronic reboxetine did not modify 5-HT₄ receptor density.

CONCLUSIONS AND IMPLICATIONS

Our data indicate a functional desensitization of 5-HT₄ receptors after chronic venlafaxine, similar to that observed after treatment with the classical selective inhibitors of 5-HT reuptake.     

Primary afferents with TRPM8 and TRPA1 profiles target distinct subpopulations of rat superficial dorsal horn neurones

Background and purpose:

The transient receptor potential (TRP) channels, transient receptor potential melastatin-1 (TRPM8) and transient receptor potential ankyrin-1 (TRPA1), are expressed in subpopulations of sensory neurones and have been proposed to mediate innocuous and noxious cold sensation respectively. The aim of this study was to compare TRPM8 and TRPA1 modulation of glutamatergic afferent transmission within the spinal dorsal horn.

Experimental approach:

Whole cell patch clamp recordings were made from rat spinal cord slices in vitro to examine the effect of TRP agonists and temperature on glutamatergic excitatory postsynaptic currents (EPSCs).

Key results:

Icilin (3 or 100 µmol·L⁻¹), menthol (200 µmol·L⁻¹) and capsaicin (1 µmol·L⁻¹) reduced the amplitude of primary afferent evoked EPSCs in subpopulations of lamina I and II neurones. In a subpopulation of superficial neurones, innocuous cold (threshold 29°C), 3 µmol·L⁻¹ icilin (EC₅₀ 1.5 µmol·L⁻¹) and menthol (EC₅₀ 263 µmol·L⁻¹) increased the rate of spontaneous miniature EPSCs. In the majority of lamina I and II neurones, 100 µmol·L⁻¹ icilin (EC₅₀ 79 µmol·L⁻¹), allyl isothiocyanate (EC₅₀ 226 µmol·L⁻¹), cinnamaldehyde (EC₅₀ 38 µmol·L⁻¹) and capsaicin (1 µmol·L⁻¹) increased miniature EPSC rate. The response to 100 µmol·L⁻¹, but not 3 µmol·L⁻¹ icilin, was abolished by ruthenium red, while neither was affected by iodoresiniferatoxin. Responsiveness to 3 µmol·L⁻¹, but not to 100 µmol·L⁻¹ icilin, was highly predictive of innocuous cold responsiveness. Neurones responding to 3 µmol·L⁻¹ icilin and innocuous cold were located more superficially than those responding to 100 µmol·L⁻¹ icilin.

Conclusions and implications:

Activation of TRPM8 and TRPA1 presynaptically modulated glutamatergic transmission onto partially overlapping but distinct populations of superficial dorsal horn neurones. Spinal TRPM8 and TRPA1 channels may therefore provide therapeutic targets in cold hyperesthesia.     

Toxicodynamic effects of ciclosporin are reflected by metabolite profiles in the urine of healthy individuals after a single dose

AIMS

The immunosuppressant ciclosporin is an efficient prophylaxis against transplant organ rejection but its clinical use is limited by its nephrotoxicity. Our previous systematic studies in the rat indicated urine metabolite pattern changes to be sensitive indicators of the negative effects of ciclosporin on the kidney. To translate these results, we conducted an open label, placebo-controlled, crossover study assessing the time-dependent toxicodynamic effects of a single oral ciclosporin dose (5 mg kg⁻¹) on the kidney in 13 healthy individuals.

METHODS

In plasma and urine samples, ciclosporin and 15-F_2t-isoprostane concentrations were assessed using HPLC-MS and metabolite profiles using ¹H-NMR spectroscopy.

RESULTS

The maximum ciclosporin concentrations were 1489 ± 425 ng ml⁻¹ (blood) and 2629 ± 1308 ng ml⁻¹ (urine). The increase in urinary 15-F_2t-isoprostane observed 4 h after administration of ciclosporin indicated an increase in oxidative stress. 15-F_2t-isoprostane concentrations were on average 2.9-fold higher after ciclosporin than after placebo (59.8 ± 31.2 vs. 20.9 ± 19.9 pg mg⁻¹ creatinine, P < 0.02). While there were no conclusive changes in plasma 15-F_2t-isoprostane concentrations or metabolite patterns, non-targeted metabolome analysis using principal components analysis and partial least square fit analysis revealed significant changes in urine metabolites typically associated with negative effects on proximal tubule cells. The major metabolites that differed between the 4 h urine samples after ciclosporin and placebo were citrate, hippurate, lactate, TMAO, creatinine and phenylalanine.

CONCLUSION

Changes in urine metabolite patterns as a molecular marker are sufficiently sensitive for the detection of the negative effects of ciclosporin on the kidney after a single oral dose.     

Does terfenadine-induced ventricular tachycardia/fibrillation directly relate to its QT prolongation and Torsades de Pointes?

BACKGROUND AND PURPOSE

Terfenadine has been reported to cause cardiac death. Hence, we investigated its pro-arrhythmic potential in various in vitro models.

EXPERIMENTAL APPROACH

Pro-arrhythmic effects of terfenadine were investigated in rabbit isolated hearts and left ventricular wedge preparations. Also, using whole-cell patch-clamp recording, we examined its effect on the human ether-à-go-go-related gene (hERG) current in HEK293 cells transfected with hERG and on the I_Na current in rabbit ventricular cells and human atrial myocytes.

KEY RESULTS

Terfenadine concentration- and use-dependently inhibited I_Na in rabbit myocytes and in human atrial myocytes and also inhibited the hERG. In both the rabbit left ventricular wedge and heart preparations, terfenadine at 1 µM only slightly prolonged the QT- and JT-intervals but at 10 µM, it caused a marked widening of the QRS complex, cardiac wavelength shortening, incidences of in-excitability and non-TdP-like ventricular tachycardia/fibrillation (VT/VF) without prolongation of the QT/JT-interval. At 10 µM terfenadine elicited a lower incidence of early afterdepolarizations versus non- Torsades de Pointes (TdP)-like VT/VF (100% incidence), and did not induce TdPs. Although the concentration of terfenadine in the tissue-bath was low, it accumulated within the heart tissue.

CONCLUSION AND IMPLICATIONS

Our data suggest that: (i) the induction of non-TdP-like VT/VF, which is caused by slowing of conduction via blockade of I_Na (like Class Ic flecainide), may constitute a more important risk for terfenadine-induced cardiac death; (ii) although terfenadine is a potent hERG blocker, the risk for non-TdP-like VT/VF exceeds the risk for TdPs; and (iii) cardiac wavelength (λ) could serve as a biomarker to predict terfenadine-induced VT/VF.     

Rosiglitazone selectively inhibits K(ATP) channels by acting on the K(IR) 6 subunit

BACKGROUND AND PURPOSE

Rosiglitazone is an anti-diabetic drug acting as an insulin sensitizer. We recently found that rosiglitazone also inhibits the vascular isoform of ATP-sensitive K⁺ channels and compromises vasodilatory effects of β-adrenoceptor activation and pinacidil. As its potency for the channel inhibition is in the micromolar range, rosiglitazone may be used as an effective K_ATP channel inhibitor for research and therapeutic purposes. Therefore, we performed experiments to determine whether other isoforms of K_ATP channels are also sensitive to rosiglitazone and what their sensitivities are.

EXPERIMENTAL APPROACH

K_IR6.1/SUR2B, K_IR6.2/SUR1, K_IR6.2/SUR2A, K_IR6.2/SUR2B and K_IR6.2ΔC36 channels were expressed in HEK293 cells and were studied using patch-clamp techniques.

KEY RESULTS

Rosiglitazone inhibited all isoforms of K_ATP channels in excised patches and in the whole-cell configuration. Its IC₅₀ was 10 µmol·L⁻¹ for the K_IR6.1/SUR2B channel and ∼45 µmol·L⁻¹ for K_IR6.2/SURx channels. Rosiglitazone also inhibited K_IR6.2ΔC36 channels in the absence of the sulphonylurea receptor (SUR) subunit, with potency (IC₅₀= 45 µmol·L⁻¹) almost identical to that for K_IR6.2/SURx channels. Single-channel kinetic analysis showed that the channel inhibition was mediated by augmentation of the long-lasting closures without affecting the channel open state and unitary conductance. In contrast, rosiglitazone had no effect on K_IR1.1, K_IR2.1 and K_IR4.1 channels, suggesting that the channel inhibitory effect is selective for K_IR6.x channels.

CONCLUSIONS AND IMPLICATIONS

These results suggest a novel K_ATP channel inhibitor that acts on the pore-forming K_IR6.x subunit, affecting the channel gating.

LINKED ARTICLE

This article is commented on by Dart, pp. 23–25 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.01990.x     

Modulation of the late sodium current by ATX-II and ranolazine affects the reverse use-dependence and proarrhythmic liability of IKr blockade

BACKGROUND AND PURPOSE

Drug-induced torsades de pointes (TdP) often occurs during bradycardia due to reverse use-dependence. We tested the hypothesis that inhibition or enhancement of late sodium current (I_Na,L) could modulate the drug-induced reverse use-dependence in QT and T_p-e (an index of dispersion of repolarization), and therefore the liability for TdP.

EXPERIMENTAL APPROACH

Arterially perfused rabbit left ventricular wedge preparations were used. Action potentials from the endocardium were recorded simultaneously with a transmural ECG. The effects of Anemonia sulcata toxin (ATX-II) (an I_Na,L enhancer), d,l-sotalol, clarithromycin and ranolazine (an I_Na,L blocker) on rate-dependent changes in QT, T_p-e and proarrhythmic events were tested, either alone or in combination. Rate-dependent QT and T_p-e slopes and TdP score (a combined index of TdP liability) were calculated at control and during drug infusion.

KEY RESULTS

ATX-II (30 nM) and sotalol (300 µM) caused a marked increase in QT and T_p-e intervals, steeper QT-basic cycle length (BCL) and T_p-e-BCL slopes (i.e. reverse use-dependence), and TdP. Addition of ranolazine (15 µM) to ATX-II or sotalol significantly attenuated QT-BCL, T_p-e-BCL slopes and the increased TdP scores. In contrast, clarithromycin (100 µM) moderately prolonged QT and T_p-e without causing R-on-T extrasystole or TdP, but addition of ATX-II (1 nM) to clarithromycin markedly amplified the QT-BCL and T_p-e-BCL slopes and further increased TdP score.

CONCLUSION AND IMPLICATIONS

Modulation of I_Na,L altered drug-induced reverse use-dependence related to QT as well as T_p-e, indicating that inhibition of I_Na,L can markedly reduce the TdP liability of agents that prolong QT intervals.     

Quercetin potentiates insulin secretion and protects INS-1 pancreatic ��-cells against oxidative damage via the ERK1/2 pathway

BACKGROUND AND PURPOSE

Quercetin lowers plasma glucose, normalizes glucose tolerance tests and preserves pancreatic β-cell integrity in diabetic rats. However, its mechanism of action has never been explored in insulin-secreting β-cells. Using the INS-1 β-cell line, the effects of quercetin were determined on glucose- or glibenclamide-induced insulin secretion and on β-cell dysfunctions induced by hydrogen peroxide (H₂O₂). These effects were analysed along with the activation of the extracellular signal-regulated kinase (ERK)1/2 pathway. N-acetyl-L-cysteine (NAC) and resveratrol, two antioxidants also known to exhibit some anti-diabetic properties, were used for comparison.

EXPERIMENTAL APPROACH

Insulin release was quantified by the homogeneous time resolved fluorescence method and ERK1/2 activation tested by Western blot experiments. Cell viability was estimated by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) colorimetric assay.

KEY RESULTS

Quercetin (20 µmol·L⁻¹) potentiated both glucose (8.3 mmol·L⁻¹)- and glibenclamide (0.01 µmol·L⁻¹)-induced insulin secretion and ERK1/2 phosphorylation. The ERK1/2 (but not the protein kinase A) signalling pathway played a crucial role in the potentiation of glucose-induced insulin secretion by quercetin. In addition, quercetin (20 µmol·L⁻¹), protected β-cell function and viability against oxidative damage induced by 50 µmol·L⁻¹ H₂O₂ and induced a major phosphorylation of ERK1/2. In the same conditions, resveratrol or NAC were ineffective.

CONCLUSION AND IMPLICATIONS

Quercetin potentiated glucose and glibenclamide-induced insulin secretion and protected β-cells against oxidative damage. Our study suggested that ERK1/2 played a major role in those effects. The potential of quercetin in preventing β-cell dysfunction associated with diabetes deserves further investigation.     

Analysis of the contribution of I(to) to repolarization in canine ventricular myocardium

BACKGROUND AND PURPOSE

The contribution of the transient outward potassium current (I_to) to ventricular repolarization is controversial as it depends on the experimental conditions, the region of myocardium and the species studied. The aim of the present study was therefore to characterize I_to and estimate its contribution to repolarization reserve in canine ventricular myocardium.

EXPERIMENTAL APPROACH

Ion currents were recorded using conventional whole-cell voltage clamp and action potential voltage clamp techniques in canine isolated ventricular cells. Action potentials were recorded from canine ventricular preparations using microelectrodes. The contribution of I_to to repolarization was studied using 100 µM chromanol 293B in the presence of 0.5 µM HMR 1556, which fully blocks I_Ks.

KEY RESULTS

The high concentration of chromanol 293B used effectively suppressed I_to without affecting other repolarizing K⁺ currents (I_K1, I_Kr, I_p). Action potential clamp experiments revealed a slowly inactivating and a ‘late’ chromanol-sensitive current component occurring during the action potential plateau. Action potentials were significantly lengthened by chromanol 293B in the presence of HMR 1556. This lengthening effect induced by I_to inhibition was found to be reverse rate-dependent. It was significantly augmented after additional attenuation of repolarization reserve by 0.1 µM dofetilide and this caused the occurrence of early afterdepolarizations. The results were confirmed by computer simulation.

CONCLUSIONS AND IMPLICATIONS

The results indicate that I_to is involved in regulating repolarization in canine ventricular myocardium and that it contributes significantly to the repolarization reserve. Therefore, blockade of I_to may enhance pro-arrhythmic risk.     

Mechanisms of U46619-induced contraction of rat pulmonary arteries in the presence and absence of the endothelium

Background and purpose:

Thromboxane A₂ and endothelial dysfunction are implicated in the development of pulmonary hypertension. The receptor-transduction pathway for U46619 (9,11-dideoxy-9α, 11α-methanoepoxy prostaglandin F_2α)-induced contraction was examined in endothelium-intact (E+) and denuded (E−) rat pulmonary artery rings.

Experimental approach:

Artery rings were mounted on a wire myograph under a tension of 7–7.5 mN at 37°C and gassed with 95% O₂/5% CO₂. Isometric recording was made by using Powerlab data collection and Chart 5 software.

Key results:

Both E+ and E− contractile responses were sensitive to Rho-kinase inhibition and the chloride channel blocker NPPB [5-nitro-2-(3-phenylpropylamino)benzoic acid]. The E+ response was sensitive to the store-operated calcium channel blockers SKF-96365 {1-[B-[3-(4-methoxyphenyl)propoxy]-4-methoxy-phenethyl]-1H-imidazole hydrochloride} and 2-APB (2-amino ethoxy diphenylborate) (75–100 µmol·L⁻¹). The E− response was sensitive to 2-APB (10–30 µmol·L⁻¹), a putative IP₃ receptor antagonist, and the calcium and chloride channel blockers nifedipine, DIDS (4,4′-diisothiocyanostilbene-2,2′-disulphonic acid) and niflumic acid but was insensitive to SKF-96365. Inhibiting K_V with 4-AP in E+ rings exposed a contraction sensitive to nifedipine, DIDS and niflumic acid, whereas inhibiting BK_Ca exposed a contraction sensitive to mibefradil, DIDS and niflumic acid. This indicates that removal of the endothelium allows the TP receptor to inhibit K_V, which may involve coupling to phospholipase C, because inhibition of phospholipase C with {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-y]amino]hexyl]– 1H-pyrrole-2,5-dione) switched the E− pathway to the E+ pathway.

Conclusions and implications:

The results from this study indicate that distinct transduction pathways can be employed by the TP receptor to produce contraction and that the endothelium is able to influence the coupling of the TP receptor.British Journal of Pharmacology (2009) 157, 581–596; doi:10.1111/j.1476-5381.2008.00084.x; published online 22 April 2009This article is part of a themed section on Endothelium in Pharmacology. For a list of all articles in this section see the end of this paper, or visit: http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009     

Cannabinoid CB1 receptors mediate the effects of corticotropin-releasing factor on the reinstatement of cocaine seeking and expression of cocaine-induced behavioural sensitization

BACKGROUND AND PURPOSE

The endocannabinoid and corticotropin-releasing factor (CRF) systems have been implicated in several long-lasting behavioural effects of prior cocaine experience. The present experiments were designed to probe functional interactions between endocannabinoids and CRF by testing the role of cannabinoid CB₁ receptors in cocaine-related behaviours induced or mediated by CRF.

EXPERIMENTAL APPROACH

In Experiment 1, rats trained to self-administer cocaine were pretreated with the CB₁ receptor antagonist, AM251 (0, 10, 100 or 200 µg, i.c.v.), before tests for reinstatement in response to CRF (0, 0.5 µg, i.c.v.), intermittent footshock stress (0, 0.9 mA) or cocaine (0, 10 mg·kg⁻¹, i.p.). In Experiment 2, rats pre-exposed to cocaine (15–30 mg·kg⁻¹, i.p.) or saline for 7 days were pretreated with AM251 (0, 10 or 100 µg, i.c.v.) before tests for locomotion in response to CRF (0.5 µg, i.c.v.), cocaine (15 mg·kg⁻¹, i.p.) or saline (i.c.v.).

KEY RESULTS

Pretreatment with AM251 selectively interfered with CRF-, but not footshock- or cocaine-induced reinstatement. AM251 blocked the expression of behavioural sensitization induced by challenge injections of both CRF and cocaine.

CONCLUSIONS AND IMPLICATIONS

These findings reveal a mediating role for CB₁ receptor transmission in the effects of CRF on cocaine-related behaviours.     

Inhibitory effects of sevoflurane on pacemaking activity of sinoatrial node cells in guinea-pig heart

BACKGROUND AND PURPOSE

The volatile anaesthetic sevoflurane affects heart rate in clinical settings. The present study investigated the effect of sevoflurane on sinoatrial (SA) node automaticity and its underlying ionic mechanisms.

EXPERIMENTAL APPROACH

Spontaneous action potentials and four ionic currents fundamental for pacemaking, namely, the hyperpolarization-activated cation current (I_f), T-type and L-type Ca²⁺ currents (I_Ca,T and I_Ca,L, respectively), and slowly activating delayed rectifier K⁺ current (I_Ks), were recorded in isolated guinea-pig SA node cells using perforated and conventional whole-cell patch-clamp techniques. Heart rate in guinea-pigs was recorded ex vivo in Langendorff mode and in vivo during sevoflurane inhalation.

KEY RESULTS

In isolated SA node cells, sevoflurane (0.12–0.71 mM) reduced the firing rate of spontaneous action potentials and its electrical basis, diastolic depolarization rate, in a qualitatively similar concentration-dependent manner. Sevoflurane (0.44 mM) reduced spontaneous firing rate by approximately 25% and decreased I_f, I_Ca,T, I_Ca,L and I_Ks by 14.4, 31.3, 30.3 and 37.1%, respectively, without significantly affecting voltage dependence of current activation. The negative chronotropic effect of sevoflurane was partly reproduced by a computer simulation of SA node cell electrophysiology. Sevoflurane reduced heart rate in Langendorff-perfused hearts, but not in vivo during sevoflurane inhalation in guinea-pigs.

CONCLUSIONS AND IMPLICATIONS

Sevoflurane at clinically relevant concentrations slowed diastolic depolarization and thereby reduced pacemaking activity in SA node cells, at least partly due to its inhibitory effect on I_f, I_Ca,T and I_Ca,L. These findings provide an important electrophysiological basis of alterations in heart rate during sevoflurane anaesthesia in clinical settings.